III. Antibodies in Transplantation: The Effects of HLA and Non-HLA Antibody Binding and Mechanisms of Injury
- What are the effects of HLA antibody binding to graft vascular and other cells?
- Briefly summarize the experimental methods used to assess such outcomes.
Antibody binding depends on FC part as Fab fragment cross links the specific molecule on donor cell .Binding to endothelial and smooth muscle cells depends on complement dependent cytotoxicity and cytotoxicity of AB cell mediated .This can lead to hyper -acute and acute humoral rejection.
Target cell signaling produced by HLA AB s results in cellular proliferation and chronic rejection, cytokine and VEGF production and leukocyte recruitment.
Experimental methods
In vitro
-Binding capacity assay of HLA ABs
.Determination of intra-cellular phosphorylation cascade resulting from binding with HLA I
.Using FC to assess cellular proliferation
Evaluation .cellular migration and cytoskeleton changes
Leukocyte adhesion assay
In vivo
AMR study in animals
Human sampling
Effect of HLA antibody binding to graft
the effects of HLA antibody binding to graft vascular cells depend on both canonical antibody function mediated by Fc portion molecules and on binding via the Fab which cross links the target molecules on donor cells .
Alloantibody binding to endothelial and smooth muscle cells promote Fc dependent function such as complement cascade activation and antibody dependent cell molecule toxicity lead to hyperacute and acute rejection
chronic rejection or transplant vasculopathy is a proliferative disease in which the vessels of graft become occluded by thickened intima invaded by smooth muscle cells . DSA are strongly associated with vasculopathy as ligation of HLA I by antibodies induces intracellular signaling cascade which have an important implication for cell function changes especially cellular proliferation and cytoskeletal changes in vascular cells
The pathogensis by which HLA II antibodies promote inflammation and proliferation is less defined but they frequently accompany chronic rejection in renal transplant and correlate with AMR incidence in absence of a positive T cell cross match and its presence significantly correlates with worse graft outcomes
Experimental methods used to assess DSA outcomes
In vitro
In vivo
in vivo models of antibody mediated rejection
Clinical frequency and relevance of donor specific antibodies:
-survival varies from 83% without HLA antibodies to 49% with HLA antibodies.When desensitisation occur with reducing DSA level ,long term survival happened than if DSA persistent.
Heart allograft patients with DSA leads to low graft survival and chronic allograft vaculopathy .
-DSA is a strong risk factor for rejection attacks in small bowel transplantation.
-15% of low risk renal transplant patient without presensitisation developed DSA later after transplantation with reduction in graft survival at 10 y.
It was found that mismatch of HLA-DRB1 predictor for production of de novo DSA and non-compliance immunosuppressive recipients.
-biopsies may reveal capillaritis with or without c4d staining but graft function remains stable and any injury is subclinical .With times it will progress to clinical dysfunction and failure due to sustained microvascular injury and cellular infiltration.
In ABMR in renal transplantation:
– poor graft function.
– complement deposition C4d in preitubules of graft or DSA in circulation
In ABMR in cardiac transplantation:
-intravascular macrophages , endothelial cell swelling , C4d stain and DSA HLA antibodies.
* Experimental techniques to measure effects of Antibodies:
-In Vitro Techniques:
* Measurment of HLA antibody binding capacity by fluorescence cytometry.
* Analysis of intracellular signaling.
* determination of cell growth .
* Measurment of leukocyte adherence.
* Determination of cytoskeletal changes and cell migration.
* siRNA and pharmacological inhibitors.
-In Vivo Models of Antibody mediated rejection:
* Murine models with genetic manipulation.
* Rat immunodeficient recipients or RAG1 used .
– Patient samples:
RNA from patient biopsies evaluated by microarray or sequencing to determine expression of protein.
-Mechanism of injury of Fc Dependant effects of antibodies:
* Hyperacute and acute rejection:
– complement activation.
-Antibody Dependent Cell Mediated Cytotoxicity.
~ Mechanism of injury : Target Cell Signaling Induced by HLA I Antibodies.
* survival and accommodation:
Persistence of DSA antibodies by themselves predictive to late graft loss with no relation to AMR episodes
Clinical frequency and relevance of donor specific antibodies:
-survival varies from 83% without HLA antibodies to 49% with HLA antibodies.When desensitisation occur with reducing DSA level ,long term survival happened than if DSA persistent.
Heart allograft patients with DSA leads to low graft survival and chronic allograft vaculopathy .
-DSA is a strong risk factor for rejection attacks in small bowel transplantation.
-15% of low risk renal transplant patient without presensitisation developed DSA later after transplantation with reduction in graft survival at 10 y.
It was found that mismatch of HLA-DRB1 predictor for production of de novo DSA and non-compliance immunosuppressive recipients.
-biopsies may reveal capillaritis with or without c4d staining but graft function remains stable and any injury is subclinical .With times it will progress to clinical dysfunction and failure due to sustained microvascular injury and cellular infiltration.
In ABMR in renal transplantation:
– poor graft function.
– complement deposition C4d in preitubules of graft or DSA in circulation
In ABMR in cardiac transplantation:
-intravascular macrophages , endothelial cell swelling , C4d stain and DSA HLA antibodies.
* Experimental techniques to measure effects of Antibodies:
-In Vitro Techniques:
* Measurment of HLA antibody binding capacity by fluorescence cytometry.
* Analysis of intracellular signaling.
* determination of cell growth .
* Measurment of leukocyte adherence.
* Determination of cytoskeletal changes and cell migration.
* siRNA and pharmacological inhibitors.
-In Vivo Models of Antibody mediated rejection:
* Murine models with genetic manipulation.
* Rat immunodeficient recipients or RAG1 used .
– Patient samples:
RNA from patient biopsies evaluated by microarray or sequencing to determine expression of protein.
-Mechanism of injury of Fc Dependant effects of antibodies:
* Hyperacute and acute rejection:
– complement activation.
-Antibody Dependent Cell Mediated Cytotoxicity.
~ Mechanism of injury : Target Cell Signaling Induced by HLA I Antibodies.
* survival and accommodation:
Persistence of DSA antibodies by themselves predictive to late graft loss with no relation to AMR episodes.
Accommodation defined as clinically silent antibody-mediated graft damage which causes failure slowly.
This is due to HLA antibody mediated transplant accommodation .
At high concentration , HLA I antibodies from patients sera or monoclonal antibodies trigger endothelial cell death .While at low concentration, resistance to complement induce cell death by induce heme oxygenase 1 (HO-1) and activating cyclic AMP-dependent protein kinase A.
* chronic rejection or transplant vasculopathy is proliferation disease in which vessels of graft sever occluded by thicker intima invaded by smooth muscle cell and DSA associated with vasculopathy in heart and kidney transplantation.
Stimulation of lung epithelial carcinoma with HLA sera from patient increases proliferation and tyrosine phosphorylation which may provokes bronchitis obliterans syndromes.
* Leukocyte Recruitment through:
– Infiltration in Antibody-Mediated Rejection.
– HLA I Antibody-Induced Leukocyte Recruitment.
Therapies suggested from experimental evidence :
– Fausdil ( ROCK inhibitors) : suppress intimacy thickening.
– Rho kinase inhibitors: reduce immune cell infiltration and prevent intimal thickening.
– Rapamycin ( mTOR inhibitors) : reduce HLA I AB triggered endothelial cell proliferation.
Adhesions molecules antagonist:
Complement inhibitors and reduce leukocyte infiltration.
Eculizimab: monoclonal antibody block activation of C5 which reduced AMR incidence in patients with DSA .
-Antibodies to HLA II occur with chronic rejection in renal transplant and correlated with AMR in absence of a positive T cell cross match.
In human cardiac allografts HLA DR increased during rejection.
Non-HLA antibodies found in higher rates in renal and cardiac transplant recipients with or without rejection than normal or waiting list.
References:
1. Zhang Q, Cecka JM, Gjertson DW, Ge P, Rose ML, Patel JK, et al. HLA and MICA: targets of
antibody-mediated rejection in heart transplantation. Transplantation. 2011; 91:1153–1158. [PubMed: 21544036]
2. Cardarelli F, Pascual M, Tolkoff-Rubin N, Delmonico FL, Wong W, Schoenfeld DA, et al.
Prevalence and significance of anti-HLA and donor-specific antibodies long-term after renal
transplantation. Transpl Int. 2005; 18:532–540. [PubMed: 15819801]
3. Everly MJ, Everly JJ, Arend LJ, Brailey P, Susskind B, Govil A, et al. Reducing de novo donorspecific antibody levels during acute rejection diminishes renal allograft loss. Am J Transplant.
2009; 9:1063–1071. [PubMed: 19344434]
4. Lachmann N, Terasaki PI, Schonemann C. Donor-specific HLA antibodies in chronic renal allograft
rejection: a prospective trial with a four-year follow-up. Clin Transpl. 2006:171–199. [PubMed:
18365377]
5. Lachmann N, Terasaki PI, Budde K, Liefeldt L, Kahl A, Reinke P, et al. Anti-human leukocyte
antigen and donor-specific antibodies detected by luminex posttransplant serve as biomarkers for
chronic rejection of renal allografts. Transplantation. 2009; 87:1505–1513. [PubMed: 19461487]
6. Otten HG, Verhaar MC, Borst HP, Hene RJ, Zuilen AD. Pretransplant donor-specific HLA class-Iand -II antibodies are associated with an increased risk for kidney graft failure. Am Transplant. 2012; 12(6):1618–1623. [PubMed: 22404993]
7. Song EY, Lee YJ, Hyun J, Kim YS, Ahn C, Ha J, et al. Clinical relevance of pretransplant HLA
class II donor-specific antibodies in renal transplantation patients with negative T-cell cytotoxicity crossmatches. Ann Lab Med. 2012; 32:139–144. [PubMed: 22389881]
8. Bishara A, Brautbar C, Eid A, Scherman L, Ilan Y, Safadi R. Is presensitization relevant to liver
transplantation outcome? Hum Immunol. 2002; 63:742–750. [PubMed: 12175728]
9. Appel JZ III, Hartwig MG, Davis RD, Reinsmoen NL. Utility of peritransplant and rescue
intravenous immunoglobulin and extracorporeal immunoadsorption in lung transplant recipients
sensitized to HLA antigens. Hum Immunol. 2005; 66:378–386. [PubMed: 15866701].
Prevalence of DSA is variable from 30% to almost 85% in different studies and is associated with worse graft survival
There are two different methods to evaluate effects of Abs on allograft: in vitro and in vivo.
In vitro:
Cultured cell of endothelial, epithelial and smooth muscle are affected by anti-HLA Abs.
1-Capacity of anti HLA Abs is measured by flow cytometry.
2- Evaluation of phosphorylation caused as intracellular signaling by Western bolting.
3- Flow-cytometry to assess cell proliferation.
4- Adhesion assay for leukocyte using fluorescence microscopy.
5- Assessment of cell migration after 24h injury by fluorescent microscopy
6- Using small interfering RNA (siRNA) or inhibitors to disturb function of proteins in HLA signaling.
In vivo:
1- A fully mismatched organ is transplanted to a rat with SCID with healthy innate system and then DSA is infused and characteristics of AMR is studied.
2- Another option is transplantation of a human tissue to a murine with SCID then administer anti- HLA Abs.
3- Assessment of patient biopsy by IHC.
Mechanisms of injury:
1- Fc-dependent effects of Abs:
a. Complement activation caused hyper acute and acute or chronic humoral rejection. Single antigen assay by Luminax and assessment of complement fixing ability by C1q assay and evaluation of IgG subclasses are useful.
b. Antibody dependent cell mediated cytotoxicity (ADCC): Anti–HLA Abs can increase lytic properties of NK cells.
2- Anti-HLA I Abs can change cell function and proliferation and cause chronic rejection.
a. Sometimes this process can result in accommodation due to low titer anti-HLA I Abs which inhibit Bcl-2 expression.
b. Cell proliferation: HLA I ligation by Abs can induce cell proliferation in endothelial and smooth muscle cell of vessels in vitro. Humoral response is important in chronic rejection in vivo, too. This is shown by infusion of HLA I Ab to SC1D mice transplanted with human arteries or by evaluation of allograft from RAG1 knockout mice.
c. HLA I Abs can increase FGFR expression or production of cytokines by endothelial cells.
d. HLA I Ab can cause activation of endothelial cells and induce leukocyte recruitment which is shown by expression of vWF in AMR.
e. Therapies such as inactivation of Rho kinase by fasudil or reduction of endothelial proliferation by rapamycin or Adhesion molecules antagonists or complement inhibitor (eculizumab)can help us to understand mechanism of AMR .
3- Anti-HLA II Abs are effective in chronic rejection and there is an association between these Abs with chronic rejection.
4- Non- HLA Abs especially anti- endothelial cell Abs were associated with lower graft function probably by some homology to HLA class I. Abs against M1CA and M1CB were found in chronic rejection. But there is not much information about mechanism of non – HLA Abs.
. Abs.
Anti HLA DSAs can bing either against major HLA (class I, II), minor or non-HLA antigens. They bind to the endothelial cells and promote Fc-dependent function such as complement cascade activation and Ab-mediated cell cytotoxicity. These effects are particularly relevant in hyperacute and acute humoral rejection. diagnosis of ABMR in renal transplant can be diagnosed by poor renal function and staining of cd4 shown microscopically. this can be in conjunction with cellular rejection.
there ae may experimental techniques to measure the effects of antibodies. either in vitro measuring of HLA ab binding capacity, analysis of intracellular signaling, measurement of leukocyte adherence etc., there are in vivo models.
presence of circulating DSAs may not always indicate rejection
What are the effects of HLA antibody binding to graft vascular and other cells?؟
Fc-Dependent Effects of Antibodies
The effects of HLA antibody binding to graft vascular cells are both the antibody functions mediated by
1:the Fc portion of the molecule,
2binding via the Fab fragment.
●● Hyperacute and Acute Rejection●●
Via complement activation and ab mediated .
1●.Complement Activation—The complement cascade is a complex systemconsisting of proteases that become sequentially activated upon cleavage. There are three
pathways of complement, which mediate immunity against different immunologic threats
and are activated by different signals.
1: The classical pathway bridges the adaptive and innate
immune responses by partnering with substrate bound antibodies. 2:The lectin pathway
mediates immunity against bacterial and fungal pathogens by recognizing mannan
carbohydrate motifs.
3:The alternative complement
pathway is effective against cellular
pathogens and facilitatesopsonization by phagocytes
2:●Antibody-Dependent Cell-Mediated Cytotoxicity—Antibodies bridge the
innate and adaptive arms of the immune system by interacting with natural killer (NK) cells
through their FcγRIII (CD16), which binds the constant Fc region of the antibody.
●●Briefly summarize the experimental methods used to assess such outcomes.?
●In vitro
1.Measurement of HLA Antibody Binding CapacityUsing flouresent either fc or single bead which is more accurate and standard results.
2:Analysis of Intracellular Signaling.
3:Determination of Cell Growth:
By Radioactive study or using fc .
4:Measurement of Leukocyte Adherence—measured by an adhesion assay in which endothelial monolayers are stimulated and lymphocytes or myeloid cells which are fluorescently labeled.
5:Determination of Cytoskeletal Changes and Cell Migration
is assessed by the scratch, or wound healing, assay. Cells are cultured, a wound is introduced by scratching the plate with a pipet tip, and closure of the wound in the absence
or presence of stimulants is measured after 24 h using
microscopy.
6:siRNA and Pharmacological Inhibitors: knockdown of expression of the protein of interest by small interfering RNA (siRNA), or pharmacological antagonism of the protein with commercial
inhibitors.
●●IN VIVO : transplantation in animal models and forming ab mediated reaction via human immune cells ..
Binding of DSAs to graft vasculature triggers processes mediated through Fc ,and Fab portions of the antibody
Fc mediated functions is irrespective of target molecule , depend on Ig subclass and isotype
Fc mediated functions include complement activation and ADCC .
Complement activation occures through activation of the classic pathway, IgG and IgM binds to C1q , cleaving C2 and C4 , split products then activates C3 , again split products activates C5 with subsequent activation of MAC and cell lysis
Complement activation is mainly increminated in hyperacute and acute rejections .
ADCC is mediated through NK cells with recognise Ab coated cells causing cell lysis .
2/ experimental methods used :
In vitro methods
** Measurement of DSA binding capacity :
The flourecence detected by flow cytometry will be more intense with higher DSA Titre and higher binding capacity to target cell
**Measurement of intracellular signaling:
Intracellular signaling requires phosphorylation of kinases and proteases , this phosphorylation can be detected by Western Blotting with phosphorylation site-specific antibodies
**detection of cellular growth is achieved by various techniques including flow cytometry or using radiolabeled substrate incorporated in cell division .
**Measurement of leucocytes adherence : is done using adhesion assays, endothelial cells are stimulated , lymphocytes are labelled by flourecent dye ,and are placed on the endothelium,
Non adherent cells are washed away , adherent cells are visualized using flourecence microscopy
** detection of cytoskeletal changes and cell migration :
Scratch test in which cells are cultured and a Scratch in done on the plate , and is covered to asses the response after 24 hrs ,
Changes could be monitored using immustaining
** siRNA and Pharmacological Inhibitors:
They are used to inhibit the function of proteins produced as a result of intracellular signaling following DSA interaction with endothelial cells.
In vivo methods :
Human tissue can be transplanted into immunodeficient animal models , together with Anti HLA antibodies, human immune cells .
Then analysing the process of Ab mediated rejection
In vitro findings are confirmed in patient biopsies using immunohistochemical techniques to detect
histological changes, complement deposition, cellular infiltration, total protein expression,
or phosphorylation status of specific proteins in the graft
What are the effects of HLA antibody binding to graft vascular and other cells?
HLA antibodies bind to endothelial cells and smooth muscle cells then stimulate Fc dependent functions via: 1- complement cascade activation, 2- antibody dependent cell mediated cytotoxicity (ADCC).
1/ Complement cascade activation:
Activation of complement will result in hyper acute or acute on chronic rejection which is dependent on Fc portion of antibodies.
Consists of 3 pathways classical, alternative and lectin pathways.
C4d deposition may not be a useful predictive value for diagnosis of AMR.
HLA antibodies specificity and complement fixing ability can be determined by binding of C1q on SAB.
C1q +Ve antibodies have a predictive value for early diagnosis of AMR.
Chronic rejection lesions can develop in response to non-complement fixing MHC antibodies or in complement deficient allograft recipients.
HLA antibodies bind to NK—àantibody coated cell lysis.
HLA antibodies induce target cell signals –àgrowth of endothelial cells and rejection.
Circulating DSAs can cause either: 1- harm, 2- tolerance, survival via kinases and anti-oxidant mechanisms.
HLA antibodies increase autocrine proliferative signals–àcell proliferation.
HLA antibodies recruit leucocyte which include Infiltration of macrophages (acute and chronic rejection) and natural killer cells (NK cells) (AMR).
HLA II Antibodies are related to chronic rejection and AMR, while the exact mechanism is unknown.
HLA antibodies bind to endothelial cells—àactivation of endothelial cells–àrecruit leucocytes.
2/ Antibody-dependent cell-mediated cytotoxicity (ADCC):
HLA antibodies bind to endothelial and smooth muscles cells and promote Fc-dependent function such as complement cascade activation and antibody-dependent cell-mediated cytotoxicity (ADCC). These effects are particularly relevant in hyper acute and acute humoral rejection.
Recent studies demonstrated that NK cells have a role in MHC AMCR in the mouse and in human AMR.
AMR is frequently characterized by intravascular macrophages, which can promote both acute and chronic rejection inflammation.
HLA I antibodies low levels cause resistance to complement induced cell death by inducing oxygenase (HO-1) and activating cAMP dependent protein kinase A (PKA) and increase endothelial Nrf-2 mediated antioxidant responses, protecting endothelial cells from complement induced death via HO-1 and ferritin H as well as inactivation of the proapoptotic factor B and increased expression of apoptosis inhibitors Bcl-2 and Bcl-x.
In murine B cell deficient recipients but with T cell immune response, the graft did not progress to vasculopathy indicating that the humoral response is needed for full chronic rejection.
Other studies demonstrated that humoral response can cause rejection on its own.
MHC I antibodies mediate chronic rejection through complement-independent mechanism.
HLA I activation of endothelial cells stimulate autocrine and paracrine cell proliferation and enhance recruitment of leukocytes.
AMR is associated with intravascular macrophages infiltration leading to acute and chronic rejection.
Some reports mentioned that HLA II expression on endothelia in grafts suggests that allogeneic attack can enhance MHC II expression.
Non-HLA antibodies has been spot lighted due to occurrence of antibody-mediated rejection in the absence of HLA antibodies.
In. many murine allograft models, selectin deficiency in the donor graft prolongs graft survival and reduces cellular infiltration. so, a potential therapeutic target to reduce immune cell recruitment by HLA antibody induced P-selectin and/or vWF.
HLA antibodies may directly induce the pathogenesis of chronic rejection.
HLA increases cell surface expression of fibroblast growth factor receptor (FGFR) on endothelial cells. The proliferative response to bFGF is thus significantly enhanced by sensitization with HLA antibodies.
1. Briefly summarize the experimental methods used to assess such outcomes.
HLA antibody binding to cells of the graft can be assessed by the following:
-A system with cultured graft cells where intracellular signaling and cell–cell interactions can be dissected in detail and specific functional changes can be analyzed.
– A more complicated but more physiological system utilizes in vivo transplantation into immune deficient recipients lacking B and T cells, which are passively transferred with DSA to simulate AMR.
In Vitro Techniques:
Endothelial, smooth muscle, or epithelial cells are cultured and stimulated in vitro with HLA antibodies, and the direct effects can be analyzed in detail.
1- Measurement of HLA Antibody Binding Capacity.
The actual amount of HLA antibody on a donor cell can be measured using flow cytometry and normalized using fluorescence calibration MESF beads (Simply Cellular).
This technique is useful when comparing two monoclonal antibodies with differing affinity or two targets with differing expression on the cell surface.
Fluorescein intensity in Flow cytometry is affected by many factors as antibody titers, affinity, epitopes and expression on target cells.
2- Analysis of Intracellular Signaling:
Endothelial cells are stimulated with HLA I antibody for defined time.
Cell lysates are separated by SDS-PAGE, then transferred onto a PVDF membrane, and probed with phosphorylation specific antibodies for Western Blot (Cell Signaling is an excellent source). Endothelial cells are stimulated with HLA I antibody for a period of time ranging from 1-30 min then probed with phosphorylation specific antibodies for Western Blot
3-Determination of Cell Growth:
measured by several techniques, either: 1/ radioactive substrates incorporated during division or 2/ using flow cytometry.
4- Measurement of Leukocyte Adherence:
is measured by an adhesion assay in which endothelial monolayers are stimulated and lymphocytes or myeloid cells which are fluorescently labeled with a vital dye such as CFSE are overlaid.
Nonadherent cells are washed off and adherent cells are imaged in many fields per sample using fluorescence microscopy. by an adhesion assay in which endothelial cells and lymphocytes or myeloid cells are stimulated they are fluorescently labelled. Adherent cells are seen by fluorescence microscopy.
5 Determination of Cytoskeletal Changes and Cell Migration:
including stress fiber formation, are monitored by immunofluorescent staining of the actin cytoskeleton using phalloidin, which selectively labels F-actin, and visualized by fluorescent microscopy. assessed by the scratch, or wound healing, assay
6- siRNA and Pharmacological Inhibitors:
In order to definitively identify a role for proteins in HLA I-mediated intracellular signaling leading to functional changes, it is important to utilize strategies to inhibit the protein’s function to verify the upstream and downstream relationship to other proteins in the pathway. Two commonly used methods involve knockdown of expression of the protein of interest by small interfering RNA (siRNA), or pharmacological antagonism of the protein with commercial inhibitors. by using methods to inhibit the protein’s function to identify the upstream and downstream relationship to proteins in the pathway could be achieved by two ways including knockdown of expression of the protein of interest by small interfering RNA (siRNA), or pharmacological inhibitor of the protein
In Vivo Models of AMR:
Human tissue can be transplanted onto an immune deficient murine recipient (Murine or rat immunodeficient recipient, as severe combined immunodeficiency (SCID) or recombinase activating gene-1 (RAG1) knockout mice, is used, but retains an intact innate immune system) and anti-HLA antibodies or human immune cells are administered.
Animals lacking T cells are unable to produce a complete humoral response,
DSAs are able to elicit acute and chronic rejection.
-DSAs are directed against HLA class I and II molecules or minor histocompatibility molecules such as MICA and MICB or non-HLA antigen expressed on endothelial cells, epithelial cells, or organ-specific targets.
-Preformed or de novo DSA is associated with poor graft outcomes.
Mechanisms of injury:
-Fc-dependent effect of antibodies:
The effect of HLA antibody binding to graft vascular cells depends on canonical antibody function mediated by the Fc portion molecule and on binding via Fab fragement. Fc binding varies depending on the isotype and subclass of the DSA.
-Fc-dependent function lead to complement activation and antibody-dependent cell-mediated cytotoxicity which were relevant in acute and hyperacute humoral rejection.
Target cell signaling induced by HLA I antibodies:
It has important implication for cell functional changes ,especially cellular proliferation which is essential for chronic rejection.
-The presence of DSA may not always indicate ongoing rejection
-DSAs are associated with vasculopathy and ligation of HLA I by antibodies trigger proliferation and cytoskeletal changes in vascular cells.
-recruitment of immune cell ( macrophages, NK ) into graft facilitate AMR. the experimental methods used to assess such outcomes:
-In vitro Techniques :
1. Measurement of HLA antibody binding capacity :
Measurement of HLA antibodies depended on fluorescence intensity in flow cytometry that affected by antibody concentration, antibody, s affinity for its ligand whether epitope is monomorphic or polymorphic, and expression level on the target cell.
2. Analysing of intracellular signaling :
HLA I antibody binding to target cells induces cell signaling and function events.
3. Determination of cell growth: cell proliferation can be measured by several
Flow cytometry.
4. Measurement of leukocyte adherence: it measured by adhesion assay in which endothelial monolayers are stimulated and lymphocyte or myeloid cells are fluorescent with vital dye are overlaid.
5. Determination of cytoskeletal changes and cell migration: Migration of cells is assessed by scratch, or wound healing, assay.
6.siRNA and pharmacological inhibitors: two –methods used knockdown expression of the protein of interest by siRNA or pharmacological antagonism of the protein with commercial inhibitors.
2. In vivo models of antibody-mediated rejection :
-Antibody-secreting B cells required T cell help during maturation and isotype switching.
-Murine models are useful because they allow genetic manipulation of putative targets, using knockout or transgenic animals.
3. Patient samples :
In vitro findings are confirmed inpatient biopsies using immunohistochemical techniques to detect histological changes, complement deposition, cellular infiltration, total protein expression, or phosphorylation status of specific proteins in graft.
Antibodies to donor antigens, DSA), can be directed against polymorphic HLA class I
(HLA I), HLA class II (HLA II), or minor histocompatibility molecules such as MICA and
MICB, or against non-HLA antigens expressed on endothelial cells, epithelial cells, or
organ specific targets .
The frequency of donor specific HLA antibodies among transplant patients ranges
greatly, from as low as 4 % to more than 50 % .
Mechanisms of Injury:
Fc-Dependent Effects of Antibodies:
The effects of HLA antibody binding to graft vascular cells are manifold, and depend on
both antibody functions mediated by the Fc portion of the molecule, and on binding via
the Fab fragment, which cross-links the target molecule on donor cells.
Hyper acute and Acute Rejection:
Alloantibody binding to endothelial and smooth muscle cells promotes Fc-dependent
functions such as :
complement cascade activation.
And antibody-dependent cell-mediated cytotoxicity (ADCC).
Mechanisms of Injury:
target Cell Signaling Induced by HLA I Antibodies HLA I ligation directly induces
intracellular signaling cascades which have important implications for cell functional
changes, especially cellular proliferation which is central to the pathogenesis of chronic
rejection.
Cell Proliferation:
There is strong evidence that ligation of HLA I by antibodies triggers proliferation and
cytoskeletal changes in vascular cells.
Leukocyte Recruitment:
MHC or HLA I antibody binding to endothelial cells causes rapid endothelial cell
activation, promoting recruitment of leukocytes.
Experimental Techniques to Measure Effects of Antibodies:
In Vitro Techniques:
1-Measurement of HLA Antibody Binding Capacity—Fluorescence intensity in flow
cytometry is influenced by a variety of factors, including antibody concentration or titer,
the antibody’s affinity for its ligand, whether the epitope is monomorphic or polymorphic,
and the expression level on the target cell.
2- Analysis of I using Western Blotting with phosphorylation site-specific antibodies.
3-Determination of Cell Growth:
Using a flow cytometry assay.
4- Measurement of Leukocyte Adherence—Leukocyte adherence is measured by an
adhesion assay in which endothelial monolayers are stimulated and lymphocytes or
myeloid cells which are fluorescently labeled with a vital dye such as CFSE .
5 Determination of Cytoskeletal Changes and Cell Migration:
is assessed by the scratch, or wound healing assay.
6- SiRNA and Pharmacological Inhibitors:
Two commonly used methods involve knockdown of expression of the protein of interest
by small interfering RNA (siRNA), or pharmacological antagonism of the protein with
commercial inhibitors. A variety of inhibitors are available commercially (especially from
Tocris, Sigma, or Calbiochem) for the enzymes noted to be crucial to HLA I signaling.
Vivo Models of Antibody-Mediated Rejection:
Murine models are particularly useful because they allow genetic manipulation of
putative targets, using knockout or transgenic animals.
Russell et al. first described the model in which alloserum alone is sufficient to elicit
allograft rejection .
Patient Samples :
The final step in translational studies of the effects of HLA antibodies on the graft is to
assess changes in transplanted organs during antibody mediated rejection in humans.
Sensetization of the patient occurs when antibodies performed due to exposure to non self Ag like in case of previous transplantation, blood transfusion and pregnancy this will lead to Ab formation and attacks of both major and minor HLA also can affect on non HLA Ag which present on endothelial cells and epithelial cells.
Recipient risk arrange according to DSA level from standard risk to high risk with highly sensetization patients with CPRA more than 80% which is associated with high risk of graft loss due to either already existing DSA or denovo DSA that is highly associated with HLA- DRB1 mismatch also denova DSA can occur in non compliance patients.
Alot of morphological changes can be demonstrated in the graft biopsy in association with DSA evev in case of subclinical rejection like inflammatory cells infiltration ,vasculitis and micro vascular injury with or without C4D deposition.
Techniques that are used to measure DSA effect:
1_in vitro techniques consist of :
A_ Measurement of HLA-Ab binding capacity using fluorescence intensity in flow cytometry.
B_ analysis of intracellular signaling cell signaling and function events by HLA-I Ab binding to target cells.
C_ cell growth determinations by using either radioactive substrates incorporated during division or using flow cytometry.
D_ leukocytes adherence measurement by adhesion assay
E_ determination of cytoskeleton changes and cell migration by scratch or wound healing assay
F_siRNA and pharmacological inhibitors to determine proteins in HLA- I mediated signaling cause functional changes
2_In vivo models
3_ patient samples
Effects of HLA antibody on graft vascular cells are numerous and depend on both the antibody functions mediated by the Fc portion of the molecule and on binding via Fab fragment.
Fc dependent functions are irrespective of target proteins or receptors but depend on the isotype and subclass of the DSA.
Binding of DSAs to vascular cells promotes Fc dependent functions such as complement system activation and ADCC. (particularly in hyperacute and acute AMR)
Complement activation: A classical pathway bridges adaptive and innate immune response. In humans, IgM, IgG1, and IgG3 are effective activators of complement cascade that ultimately lead to C5a production (an important inflammatory factor) and MAC production (which causes lysis of target cells by disrupting the plasma membrane).
Complement activation is an important mediator of acute and hyper acute rejection. C4d deposition in graft may be evidence of acute AMR, although it may not have sufficiently reliable value for diagnosis of AMR.
Antibody isotype and subclass can define complement activation. For example, shifting DSA to complement fixing IgG3 predisposes the recipient to rejection or C1q positive antibodies had a positive predictive value for early episodes of antibody-mediated rejection.
Antibody dependent cell mediated cytotoxicity: Antibodies bridge the innate and adaptive arms of the immune system by interacting with NK cells through their CD16, which binds the constant Fc region of the antibody. DSAs of sensitized patients increases the lytic capacity of NK cells against renal epithelium.
Target cell signaling induced by HLA I antibodies has important implications for cellular proliferation which is central to the pathogenesis of chronic rejection. Antibody titer may be an important factor. Persistence of DSA per se is a predictor of late graft loss independent of episodes of AMR.
HLA I antibodies trigger proliferation and cytoskeletal changes in vascular cells and are strongly associated with vasculopathy in kidney transplantation and mTOR activation has a role in inducing this effect. Matrix metalloproteases/sphingolipid signaling, a stress-induced pathway, is also involved in HLA I antibody stimulation of smooth muscle cell growth, illustrating the pleiotropic effects of HLA I ligation leading to cellular proliferation. HLA I ligation causes dramatic reorganization of the actin cytoskeleton that is relevant to cell migration and proliferation.
Humoral immunity is sufficient to produce vasculopathy and full chronic rejection. MHC I antibodies mediate chronic rejection via a complement-independent mechanism.
In addition to direct activation of intracellular signaling cascades, it is revealed that HLA I antibodies increase sensitivity to and production of soluble mediators which promote autocrine proliferative signaling. For example, HLA I cross-linking rapidly increases cell surface expression of fibroblast growth factor receptor (FGFR) on endothelial cells. In addition, it also triggers endothelial cell production of cytokines which may have a secondary effect on cell growth such as VEGF.
Leukocyte recruitment: intravascular infiltration of macrophages in AMR can promote both acute and chronic rejection. MHC or HLA I antibody binding to endothelial cells causes rapid endothelial cell activation, promoting recruitment of leukocytes by externalizing vWF and increasing cell surface P-selectin.
mTOR is a central signaling molecule in HLA I-induced cellular proliferation, thus rapamycin and its analogs (everolimus, sirolimus) may have clinical therapeutic potential. mTOR inhibition in endothelial and smooth muscle cells may prevent HLA I antibody induced proliferation.
The potential therapeutic role of complement inhibitors to prevent complement-mediated tissue injury has been suggested. Use of eculizimab, a monoclonal antibody which blocks activation of C5, may reduce antibody mediated rejection incidence in patients with DSA.
HLA II antibodies
HLA II antibodies accompany chronic rejection in kidney transplant and correlate with poor graft outcome.
HLA II is not expressed on endothelial cells of most vascular beds, with the possible exception of renal microvasculature, but it can be upregulated after inflammatory insult.
HLA II-induced soluble factors such as IL-6 produced by fibroblasts could promote the proliferation of endothelial cells.
Non-HLA Antibodies
Non-HLA antibodies in transplantation can be directed against either polymorphic or nonallelic proteins. The development of antibodies against nonpolymorphic targets may be due to upregulation during inflammation, in response to transplantation. Non-HLA antibodies, such as anti-endothelial cell antibodies are commonly found in transplant recipients.
In addition, antibodies to MICA and MICB, are found in renal as well as cardiac transplant recipients, which they are associated with chronic rejection.
Other targets of antibodies in transplantation include vascular receptors, adhesion molecules, and intermediate filaments. Nucleolin, neuropilin, CD36, vimentin, cardiac myosin, and GST are other targets of non-HLA antibodies in transplant patients.
Non-HLA antibodies against cell surface markers may fix complement or mediate ADCC. Angiotensin II receptor AT1 autoantibodies are well-established agonists, and may contribute to renal allograft rejection.
Non-HLA antibodies are predominantly of the noncomplement fixing isotypes IgG2 and IgG4.
A variety of experimental models are available to test the effects of HLA antibody binding to cells of the graft.
The first is a simplified system with cultured graft cells (endothelium, smooth muscle, or airway epithelium), where intracellular signaling and cell–cell interactions can be analyzed.
A more complicated but more physiological system utilizes in vivo transplantation into immunodeficient recipients lacking B and T cells, which are passively transferred with DSA to recapitulate antibody-mediated rejection.
Finally, the mechanisms uncovered by experimental models can be confirmed in human biopsies.
In vitro techniques include measurement of HLA Antibody binding capacity, analysis of intracellular signaling, determination of cell growth, measurement of leukocyte adherence, determination of cytoskeletal changes and cell migration, siRNA and pharmacological Inhibitors.
What are the effects of HLA antibody binding to graft vascular and other cells?
Donor specific antibodies (DSA), can be directed against HLA class I, HLA class II , or minor histocompatibility molecules such as MICA and MICB, or against non-HLA antigens expressed on endothelial cells, epithelial cells, or organ specific targets.
literature clearly illustrates that DSA adversely affect the survival of a transplanted organ . Numerous studies have linked the presence of preexisting or de novo antibodies to poor graft outcome
The effects of DSA on graft survival are not restricted to renal transplantation. Heart allograft patients with DSA also experience lower graft survival, especially if the antibodies appear after 1 year post-transplant .
the presence of HLA specific DSA and the incidence of AMR correlate with chronic rejection in the heart . Even if patients are asymptomatic, HLA-DSA significantly increased chronic allograft vasculopathy (CAV) .
Role of HLA DRB1 mismatch
investigators found that a mismatch at HLA-DRB1 was an independent predictor of the production of de novo DSA, as was recipient nonadherence to immunosuppression .
The authors propose that inflammatory cytokines expressed early after transplant increase HLA expression by the graft, which in turn promotes B cell allorecognition and production of donor specific HLA antibodies. Biopsies may reveal capillaritis with or without C4d staining, but graft function remains stable and any injury is subclinical.
Over time in the presence of donor specific HLA antibodies, the graft progresses to clinical dysfunction and ultimately failure due to sustained microvascular injury and cellular infiltration.
Diagnosis of rejection in renal transplantation
1 poor graft function.
2 evidence of complement deposition (C4d) in the peritubules of the graft with or without DSA in the circulation .
Mechanism of action of the DSAs:
1 ADCC involves complement activation cascade through Fc binding.
C5a acts as a chemoattractant for neutrophils, monocytes and T cells.
C5b act on other complements gives rise to the membrane attack complex (MAC) forming transmembrane pores leading to cell lysis and death.
ADCC: CD16 of NK cells bind to Fc region of antibody leading to recognition of antibody coated cells and their lysis.
2) Signaling intracellular cascades at the target cell level: This is due to HLA I antibodies.
Anti HLA antibodies at high concentration lead to endothelial cell death, but at low concentration, may lead to increased endothelial anti-oxidant responses and by activating HO-1 and PKA (protein kinase A) may develop resistance to complement induced cell death (accommodation).
HLA I cross linking at endothelial cell surface leads to increased expression of FGFR (Fibroblast growth factor receptor) leading to increased migration, proliferation and production of cytokines like VEGF.
HLA I cross linking also increases intracellular calcium releasing von willebrand factor (vWF) and increase expression of P-selectin
Experimental Techniques to Measure Effects of Antibodies:
variety of experimental models are available to test the effects of HLA antibody binding to cells of the graft.
1 Technique used in vitro:
it is a simplified system with cultured graft cells (endothelium, smooth muscle, or airway epithelium), where intracellular signaling and cell–cell interactions can be dissected in detail and specific functional changes can be analyzed.
2 Measurement of HLA Antibody Binding Capacity:
The actual amount of HLA antibody on a donor cell can be measured using flow cytometric methods and normalized using fluorescence calibration Molecules of Soluble Fluorochrome (MESF) beads (Simply Cellular)
This technique is useful when comparing two monoclonal antibodies with differing affinity or two targets with differing expression on the cell surface
3 Analysis of Intracellular Signaling:
endothelial cells are stimulated with HLA antibody for defined time period (usually between 1 and 30 min depending on the signaling molecule of interest, as phosphorylation events are rapid).
Cell lysates are separated by SDS-PAGE, then transferred onto a PVDF membrane, and probed with phosphorylation specific antibodies for Western Blot (Cell Signaling is an excellent source).
3 Determination of Cell Growth :
can be assessed either by flowcytometry (showing shift of histogram to left) or by incorporating radioactive substrates in the cells at the stage of cell ddivision.
4 Measurement f Leukocyte Adherence:
endothelial monolayers are stimulated and lymphocytes or myeloid cells which are fluorescently labeled with a vital dye such as CFSE are overlaid adherent cells are imaged in many fields per sample using fluorescence microscopy. Cells are counted using automated quantification ssoftware.
5 determination of Cytoskeletal Changes and Cell Migration:
Cells are cultured, a wound is introduced by scratching the plate with a pipet tip, and closure of the wound in the absence or presence of stimulants is measured after 24 h using microscopy. Cytoskeletal changes, including stress fiber formation, are monitored by immunofluorescent staining of the actin cytoskeleton using phalloidin, which selectively labels F-actin, and visualized by fluorescent microscopy
6 Small interfering RNA (siRNA) and pharmacological inhibitors: These inhibit a specific protein’s function and hence identify the role of the specific protein.
2) In Vivo methods: Mutated murine models lacking T and B cells but having an intact innate immune system including complement, monocytes, neutrophils and NK cells have shown that DSAs can trigger acute and chronic rejection. But these have limitations as murine and human immune systems have many differences with respect to immunoglobulin isotypes and expression of different receptors
Final step is to confirm this changes on transplant patients by biopsies
There are different effects of HLA Abs that bind to graft vascular cells & these effects depend on Ab function mediate through both Fc portion of molecule & Fab fragment. The Ab binding to endothelial & smith muscle cells improve Fc-dependent function through complement activation & Ab dependent cell-mediated cytotoxicity which related to hyper acute & AMR. Usually these HLA Ab ( DSA ) occurs during pregnancy, blood transfusion or transplantation. DSA directed against HLA class I & II Ag, mHC ( MICA, MICB ) or non HLA Ags. Both preformed & de novo DSA found to be associated with AMR & chronic rejection leading to poor graft outcome.
As the presence of HLA Abs associated with decreased graft survival, it is important to test these effects of these Abs bind to graft cells by using of following techniques:
(A) In Vitro:
endothelial, smith muscle or epithelial cells are cultured in vitro & stimulated by adding HLA & analyze the direct effects through several methods:
(B) In Vivo
by using murine models ( murine or rat immunodeficient) recipients to assess AMR in physiological setting.
(C) Patient sample:
study the effect of HLA Ab on graft ( pathological changes seen on graft biopsy).
HLA antibody binding to graft vessels and other cells has a variety of impacts.
The Fc-Dependent Effects of Antibodies are those that will: activate complement resulting in hyperacute and acute-on-chronic inflammation;
Bind to natural killer cells (NK cells), which then lyse cells that have been coated with antibodies.
-Target Cell Signaling Induced by HLA I Antibodies, which results in endothelial cell growth and persistent rejection in the body.
There is considerable evidence that not all circulating DSA causes obvious harm, since some may cause tolerance or survival via the action of kinases and antioxidant mechanisms.
Increased expression of secondary factors that will assist autocrine proliferative signalling, resulting in increased cell proliferation.
Leukocyte Recruitment: -Infiltration of macrophages (acute and chronic rejection) and natural killer cells (NK cells) (AMR)
Inhibition of HLA I Antibody-Induced Leukocyte Recruitment by exocytosis of vesicles containing preformed von Willebrand Factor (vWF), P-selectin, and other chemokines
– HLA II Antibodies are related to chronic rejection and AMR, while the exact mechanism is unknown.
– Antibodies that are not HLA-specific (associated with some cases of AMR but not proven).
Techniques for Investigating the Effects of Antibodies in Experiments
1 Technique used in vitro:
Murine graft cells (endothelium, smooth muscle, or airway epithelium) were grown and then transplanted into the rat. The antibody binding capacity of HLA antigens was determined.
Patients’ antibodies improved the lytic ability of CD3-CD16+ (NK) peripheral blood mononuclear cells against renal epithelium when they were exposed to the antibodies.
Intracellular Signaling is being investigated.
Intracellular signalling cascades include the successive phosphorylation of proteins and kinases in the presence of a signalling molecule.
Cell Growth is Determined by a Number of Factors
Flow cytometry is used to calculate the Cellular Proliferation Index (CPI).
An adhesion test utilizing fluorescence microscopy is used to assess leukocyte adhesion in a laboratory setting.
The scratch test, also known as the wound healing assay, is used to determine cytoskeletal changes and cell migration.
Two models of antibody-mediated rejection were developed in vitro.
Models based on mice are particularly useful because they allow for genetic manipulation of potential targets, such as mutation: which is deficient in both T cells and B cells but has a fully functional innate immune system that includes complement components, monocytes, natural killer cells (NK cells), and neutrophils.
Murine immune systems vary from human immune systems in that the murine immune system lacks the activating FcRIIA (CD32A) and FcRIIC (CD32C) molecules, while the human immune system does. Inhibitory NK cell receptors are extremely diverse between mouse and human, immunoglobulin isotypes, chemokine expression varies between mouse and human.
Acute and hyperacute rejection are two types of rejection.
Allo-AB Through complement cascade activation and antibody-dependent cell-mediated cytotoxicity, binding to endothelium and smooth muscle cells increases Fc-dependent function in endothelial and smooth muscle cells (ADCC).
Activation of the Complement:
The classical route, which collaborates with substrate-bound antibodies, serves to bridge the gap between the adaptive and innate immune responses. Hyperacute rejection is severe damage to the allograft that occurs within hours of transplantation and is largely mediated by the complement system. It is caused by a high titer of preexisting HLA antibodies, or in rare instances, non-HLA antibodies, in individuals who have been previously sensitized.
1. What are the effects of HLA antibody binding to graft vascular and other cells?
inflammatory cytokines expressed early after transplant increase HLA expression by the graft, which in turn promotes B cell allorecognition and production of donor specific HLA antibodies. Biopsies may reveal capillaritis with or without C4d staining, but graft function remains stable and any injury is subclinical
Over time in the presence of donor specific HLA antibodies, the graft progresses to clinical dysfunction and ultimately failure due to sustained microvascular injury and cellular infiltration.
Antibody-mediated rejection in renal transplantation is diagnosed by poor graft function, evidence of complement deposition (C4d) in the peritubules of the graft and/or DSA in the circulation
2.Briefly summarize the experimental methods used to assess such outcomes.
Mechanisms of Injury: Fc-Dependent Effects of Antibodies
Hyperacute and Acute Rejection
a)Complement Activation
b)Antibody-Dependent Cell-Mediated Cytotoxic
Mechanisms of Injury: Target Cell Signaling Induced by HLA I Antibodies
Survival and Accommodation
Cell Proliferation
a) In Vitro Evidence
b) In Vivo Evidence
Induction of Secondary Factors
Leukocyte Recruitment
a) Infiltration in Antibody-Mediated Rejection
b) HLA I Antibody-Induced Leukocyte Recruitment
HLA II Antibodies
Association with Outcome
Antibodies to HLA II frequently accompany chronic rejection in renal transplants and correlate with AMR incidence in the absence of a positive T cell crossmatch .The presence of antibody to HLA II significantly correlates with worse graft outcome, in addition to antibody to HLA I .
Limitations to Studying HLA II in Model Systems
Non-HLA Antibodies
Frequency and Outcome
anti-endothelial cell antibodies associated with a greater rate of cellular rejection and lower graft function
Non-HLA antibodies binding to airway epithelial cells were detected in lung transplant patients with chronic rejection, bronchiolitis obliterans syndrome (BOS), but not in patients without BOS
Experimental Models
To date, little has been studied on the actions of non-HLA antibodies during the humoral alloimmune response.
Experimental Techniques to Measure Effects of Antibodies
In Vitro Techniques
Measurement of HLA Antibody Binding Capacity
Analysis of Intracellular Signaling
Determination of Cell Growth
Measurement of Leukocyte Adherence
Determination of Cytoskeletal Changes and Cell Migration
siRNA and Pharmacological Inhibitors
In Vivo Models of Antibody-Mediated Rejection
Effect of HLA binding to graft vascular and other cells:
HLA antibodies bind to target graft cells and induce intracellular signaling cascades with phosphorylation of proteins kinases.
The effect of binding depends on the antibody function mediated by Fc portion and binding via Fab fragment.
Binding to endothelium and smooth muscle cells induce Fc dependent function as complement activation and antibody dependent cell mediated cytotoxicity (ADCC)
Complement fixation and activation are relevant to hyperacute and acute rejection and are mediators of acute AMR.
ADCC by NK cells that recognize antibody coated cells and cause cell lysis.
Target Cell Signaling Induced by HLA I Antibodies cause injury through:
Pathogenesis of HLA-II antibodies is less defined due to limitations in studying its role.
Experimental models to determine the effect of HLA antibody binding to graft cells:
In vitro:
Simplified system with cultured graft cells stimulated in vitro with HLA antibodies, can analyze intracellular signaling, cell-cell interaction and intracellular functional changes.
multiple clones of murine origin against human HLA molecules can recognize monomorphic epitopes on HLA-I and murine anti-HLA antibodies with allelle specificity are available.
In vivo:
complicated system use recipients lacking B and T cells, passively transferred with DSA to resemble AMR, it showed direct evidence that DSA MHC antibodies can cause acute and chronic rejection.
The crucial differences between murine and human immune system should be considered during in vivo experiments.
What are the effects of HLA antibody binding to graft vascular and other cells?
The effect of these antibodies are manifold and depend on both of the antibody functions mediated by the FC portion of the molecule and on the binding via the Fab fragment which cross link the target molecule on donor cells.
FC dependent functions occur irrespective of the target protein or receptor, but vary depending on the isotype or subclass of the DSA.
Alloantibody binding to endothelial and smooth muscle cells promotes FC dependent functions such as complement cascade activation and antibody dependent cell mediated cytotoxicity. These effects are particularly relevant in hyperacute and acute ABMR.
HLA I antibodies directly induces intracellular signaling cascades which have important implications for cell functional changes especially cellular proliferation which is central to the pathogenesis of chronic rejection.
The pathogenesis of HLA II antibodies is less defined. They accompany chronic rejection and correlate with worse graft outcome.
Briefly summarize the experimental methods used to assess such outcomes.
In vitro techniques:
In vivo models of ABMR
Patient samples
1. What are the effects of HLA antibody binding to graft vascular and other cells?
Donor specific antibodies (DSA) can be directed against HLA class I and II, minor histocompatibility antigens like MICA and MICB as well as non-HLA antigens on endothelial cells, epithelial cells and cells on specific organs.
Presence of DSA is associated with poor transplant outcomes. Patients with de-novo DSA developed after one year post transplant have poorer graft survival. even 15% of low-risk patients develop DSA.
The mechanism involved in antibody formation is: Post transplant inflammatory markers increase expression of HLA by the graft leading to increased B cell allorecognition and production of DSAs.
Mechanism of action of the DSAs:
1) Fc dependent effects: It involves complement activation and antibody dependent cell mediated cytotoxicity (ADCC)
a) Complement activation: IgG1, IgG3 and IgM bind to C1q molecule, leading to cleavage of C2 and C4, giving rise to C2a and C4b, which in turn cleave C3 to C3b and further cleaving C5 to C5a and C5b. C3b is responsible for activation of eosinophils and mast cells leading to vasodilatation and allergy. C5a acts as a chemoattractant for neutrophils, monocytes and T cells. C5b, by acting on C6, C7, C8 and C9, gives rise to the membrane attack complex (MAC) forming transmembrane pores leading to cell lysis and death.
In hyperacute rejection, severe form this complement mediated injury occurs, due to pre-formed antibodies, within hours of transplant. In acute rejection, similar mechanism can be proved by histological staining of C4d deposition.
b) ADCC: CD16 (Fcgamma RIII) of NK cells bind to Fc region of antibody leading to recognition of antibody coated cells and their lysis. This effect can be facilitated even by non-HLA antibodies like anti-endothelial cell antibodies.
2) Signaling intracellular cascades at the target cell level: This is due to HLA I antibodies.
Anti HLA antibodies at high concentration lead to endothelial cell death, but at low concentration, may lead to increased endothelial anti-oxidant responses and by activating HO-1 and PKA (protein kinase A) may develop resistance to complement induced cell death (accommodation).
Anti HLA antibodies cause proliferation and cytoskeletal changes in vascular cells leading to smooth muscle cell invasion of intima causing vessel occlusion and transplant vasculopathy. HLA I cross linking at endothelial cell surface leads to increased expression of FGFR (Fibroblast growth factor receptor) leading to increased migration, proliferation (due to association between integrin beta4 and HLA I) and production of cytokines like VEGF.
HLA I cross linking also increases intracellular calcium, leading to Weibel-Palade bodies in endothelial cells releasing von willebrand factor (vWF) and increase expression of P-selectin
3) HLA II antibodies, by acting on B cells and APCs, increase intracellular calcium and tyrosine phosphorylation activating fibroblasts and increasing prostaglandin E, IL-6 and MCP- leading to proliferation of endothelial cells.
2. Briefly summarize the experimental methods used to assess such outcomes.
To assess the outcome of action of anti HLA antibodies on the graft both in vitro and in vivo methods have been utilized.
1) In Vitro methods: Direct effect of anti HLA antibodies (isolated from serum of sensitized patients) on cultured endothelial cells, smooth muscle cells and epithelial cells can be assessed with respect to:
a) HLA antibody binding capacity: Flowcytometry can be used to assess the actual amount of antibody on a cell. It depends on antibody concentration, its affinity to the ligand on cell, the expression of ligand on the cell and whether the epitope is monomorphic or polymorphic.
b) Intracellular signaling: Phosphorylation of proteins and kinases can be assessed using phosphorylation site-specific antibodies and western blot technique.
c) Cell growth determination: Cell growth can be assessed either by flowcytometry (showing shift of histogram to left) or by incorporating radioactive substrates in the cells at the stage of cell division.
d) Leukocyte adherence measurement: It can be done using an assay involving fluorescent dye labelled lymphocytes and endothelial cells in which fluorescence microscope is used to assess adherent cells.
e) Cytoskeletal changes and cell migration assessment: Scratch assay or wound healing assay will lead to cytoskeletal changes including stress fiber formation that can be assessed using fluorescent microscopy looking for phalloidin staining of actin.
f) Small interfering RNA (siRNA) and pharmacological inhibitors: These inhibit a specific protein’s function and hence identify the role of the specific protein.
2) In Vivo methods: Mutated murine models lacking T and B cells but having an intact innate immune system including complement, monocytes, neutrophils and NK cells have shown that DSAs can trigger acute and chronic rejection. But these have limitations as murine and human immune systems have many differences with respect to immunoglobulin isotypes and expression of different receptors.
What are the effects of HLA antibody binding to graft vascular and other cells ?
HLA antibodies bind to the endothelial and smooth muscles cells and promote Fc-dependent function such as complement cascade activation and antibody-dependent cell-mediated cytotoxicity (ADCC) . These effects are particularly relevant in hyperacute and acute humoral rejection.
In addition to antibody-induced complement deposition, antibody mediated rejection is frequently characterized by intravascular macrophages , which can promote both acute and chronic rejection inflammation .
The HLA antibody binding to endothelial cells causes rapid endothelial cell activation, promoting recruitment of leukocytes. The clinical relevance of this pathway to AMR has not been definitively elucidated . In a variety of murine allograft models, selectin deficiency in the donor graft prolongs graft survival and reduces cellular infiltration , suggesting that HLA antibody-induced P-selectin and/or vWF may be a rational therapeutic target to decrease immune cell recruitment .
HLA antibodies may directly induce intracellular signaling cascades which have important implications in cell proliferation which is central to the pathogenesis of chronic rejection.
HLA antibodies increase sensitivity to and production of soluble mediators which promote autocrine proliferative signaling.
HLA increases cell surface expression of fibroblast growth factor receptor (FGFR) on endothelial cells. The proliferative response to bFGF is thus significantly enhanced by sensitization with HLA antibodies .
Stimulation of HLA also triggers endothelial cell production ofcytokines which may have a secondary effect on cell growth .
Therefore, the progress which has been made in understanding the mechanisms of HLA I antibody-mediated graft injury has suggested rational clinical therapies in the treatment and prevention of AMR .
Briefly summarize the experimental method s used to assess such out come ?
The effects of HLA antibody binding to cells of the graft can be assessed by the following system;
-A simplified system with cultured graft cells (endothelium, smooth muscle, or airway epithelium), where intracellular signaling and cell–cell interactions can be dissected in detail and specific functional changes can be analyzed.
– The more complicated but more physiological system utilizes in vivo transplantation into immune deficient recipients lacking B and T cells, which are passively transferred with DSA to recapitulate antibody-mediated rejection.
In Vitro Techniques. Endothelial, smooth muscle, or epithelial cells are cultured and stimulated in vitro with HLA antibodies, and the direct effects can be analyzed in detail.
1- Measurement of HLA Antibody Binding Capacity. The actual amount of HLA antibody on a donor cell can be measured using flow cytometric methods and normalized using fluorescence calibration Molecules of Soluble Fluorochrome (MESF) beads (Simply Cellular). This technique is also useful when comparing two monoclonal antibodies with differing affinity or two targets with differing expression on the cell surface.
2- Analysis of Intracellular Signaling Endothelial cells are stimulated with HLA I antibody for defined time period .Cell lysates are separated by SDS-PAGE, then transferred onto a PVDF membrane, and probed with phosphorylation specific antibodies for Western Blot (Cell Signaling is an excellent source).
3-Determination of Cell Growth Cellular proliferation can be measured by several techniques, which involve either radioactive substrates incorporated during division or using flow cytometry.
4- Measurement of Leukocyte Adherence Leukocyte adherence is measured by an adhesion assay in which endothelial monolayers are stimulated and lymphocytes or myeloid cells which are fluorescently labeled with a vital dye such as CFSE are overlaid. Nonadherent cells are washed off and adherent cells are imaged in many fields per sample using fluorescence microscopy.
5 Determination of Cytoskeletal Changes and Cell Migration Cytoskeletal changes, including stress fiber formation, are monitored by immunofluorescent staining of the actin cytoskeleton using phalloidin, which selectively labels F-actin, and visualized by fluorescent microscopy.
6- siRNA and Pharmacological Inhibitors In order to definitively identify a role for proteins in HLA I-mediated intracellular signaling leading to functional changes, it is important to utilize strategies to inhibit the protein’s function to verify the upstream and downstream relationship to other proteins in the pathway. Two commonly used methods involve knockdown of expression of the protein of interest by small interfering RNA (siRNA), or pharmacological antagonism of the protein with commercial inhibitors.
In Vivo Models of Antibody-Mediated Rejection;
Human tissue can be transplanted onto an immune deficient murine recipient, and anti-HLA antibodies or human immune cells are administered .
well done.
-The effects of HLA antibody binding to graft vascular and other cells
Hyperacute and Acute Rejection
Endothelial and smooth muscle cells binding to antibody stimulates Fc-dependent functions through
· complement cascade activation
consisting of 3 pathways classical , alternative and lectin pathways ,
C4d may not be a sensitive marker, as multipe studies mentioned that C4d deposition may not be a useful predictive value for diagnosis of antibody mediated rejection
HLA antibodies specifity and complement fixing ability can be determined by binding of C1q on single antigen beads. C1q positive antibodies have a predictive value for early diagnosis of antibody-mediated rejection
Chronic rejection lesions can develop in response to non complement fixing MHC antibodies or in complement deficient allograft recipients
· antibody-dependent cell-mediated cytotoxicity (ADCC).
Recent studies demonstrated that NK cells have a role in MHC antibody-mediated chronic rejection in the mouse and in human antibody-mediated rejection
Mechanism of injury
the presence of DSA is an indicator of late graft loss apart from AMR occurrence ;so , accommodation does not means indefinite graft survival.
HLA I antibodies low levels cause resistance to complement induced cell death by inducing oxygenase (HO-1) and activating cyclic AMPdependent protein kinase A (PKA) and increase endothelial Nrf-2 mediated antioxidant responses, protecting endothelial cells from complement induced death via HO-1 and ferritin H as well as inactivation of the proapoptotic factor Bad and increased expression of apoptosis inhibitors Bcl-2 and Bcl-xL.
Cell proliferation caused by ligation of HLA I by antibodies leading to cytoskeletal changes in vascular cells.
In murine B cell deficient recipients but with T cell immune response, the graft did not progress to vasculopathy indicating that the humoral response is needed for full chronic rejection.
Other studies demonstrated that humoral response can cause rejection on its own.
MHC I antibodies mediate chronic rejection through complement-independent mechanism.
HLA I activation of endothelial cells stimulate autocrine and paracrine cell proliferation and enhance recruitment of leukocytes.
Antibody mediated rejection is associated with intravascular macrophages infiltration leading to acute and chronic rejection.
Some reports mentioned that HLA II expression on endothelia in grafts suggests that allogeneic attack can enhance MHC II expression.
Non-HLA antibodies has been spot lighted due to occurrence of antibody-mediated rejection in the absence of HLA antibodies.
-The experimental methods used to assess such outcomes
In vitro techniques
HLA antibodies stimulate cultured endothelial, smooth muscle, or epithelial cells , and the effects are analyzed. Specified human monoclonal antibodies has been developed
HLA Antibody Binding Capacity are measured
Flourescine intensity in Flow cytometery is affects by many factors as antibody titers ,affinity , epitopes and expression on target cells. This method can be used to compare two monoclonal antibodies with differing affinity or two targets with differing expression .
Analysis of Intracellular Signaling
Endothelial cells are stimulated with HLA I antibody for aperiod of time ranging from 1-30 min then probed with phosphorylation specific antibodies for Western Blot
Determination of Cell Growth
Can be done by radioactive substrates incorporated during division or using flow cytometery
Leukocyte Adherence measurement
by an adhesion assay in which endothelial cells and lymphocytes or myeloid cells are stimulated they are fluorescently labelled. Adherent cells are seen by fluorescence microscopy.
Cytoskeletal Changes and Cell Migration
assessed by the scratch, or wound healing, assay
siRNA and Pharmacological Inhibitors
by using methods to inhibit the protein’s function to identify the upstream and downstream relationship to proteins in the pathway could be achieved by two ways including knockdown of expression of the protein of interest by small interfering RNA (siRNA), or pharmacological inhibitor of the protein .
In Vivo methods
Animals lacking T cells are unable to produce a complete humoral response,
Murine or rat immunodeficient recipient, as severe combined immunodeficiency (SCID) or recombinase activating gene-1 (RAG1) knockout mice, is used, but retains an intact innate immune system.
Donor specific MHC antibodies are able to elicit acute and chronic rejection.
There are differences between mouse and humans rendering murine not always physiologically analogous to clinical settings.
Patient sampling
By biopsy and immunohistochemistry assessment as well as RNA from the biopsy assessed by microarray and/or sequencing.
good, how do you think HLA antibodies help out to select suitable donors and select immunosuppression therapy
effects of HLA antibody binding to graft vascular and other cells
1-Fc-Dependent Effects of Antibodies which will :
· Activate complement resulting in hyper-acute and AM
· Bind NK cells which in turn do lyses of cells coating the antibodies
2-Target Cell Signaling Induced by HLA I Antibodies which end with
· Endothelial cell proliferation and chronic rejection.
· Not all circulating DSA result in apparent damage as some may result in accommodation or survival through kinases and antioxidant mechanism.
· Induction of Secondary Factors which will help autocrine proliferative signaling which will result in cell proliferation
· Leukocyte Recruitment :
-Infiltration of macrophages (acute and chronic rejection) and NK (AMR)
-HLA I Antibody-Induced Leukocyte Recruitment through exocytosis of
vesicles containing preformed von Willebrand Factor (vWF), P-selectin, and others
3- HLA II Antibodies are associated with chronic rejection and AMR through un-determined mechanisms
4- Non-HLA Antibodies(associated with some cases of AMR but not proven)
Experimental Techniques to Measure Effects of Antibodies
1. In Vitro Techniques
2. In Vivo Models of Antibody-Mediated Rejection
3. Patient Samples
needs more and precise detailing
Experimental Techniques to Measure Effects of Antibodies
1. In Vitro Techniques
· Measurement of HLA Antibody Binding Capacity: assess concentration and can compare 2 antibodies with different expression on the cell surface
· Analysis of Intracellular Signaling through Western Blotting with phosphorylation site-specific antibodies
· Determination of Cell Growth through radioactive substrates incorporated during division or using flow cytometry
· Measurement of Leukocyte Adherence through an imaging assay using fluorescence or dye
· Determination of Cytoskeletal Changes and Cell Migration by the scratch, or wound healing, assay. It assess cytoskeletal stress and fiber through fluorescent
· microscopy.
· siRNA and Pharmacological Inhibitors: through either knockdown of expression of the protein of interest by small interfering RNA (siRNA), or pharmacological antagonism of the protein with commercial inhibitors.
2. In Vivo Models of Antibody-Mediated Rejection through using murine or rat lacking T and
B cells and provided with serum from sensitized animals or human tissue to assess AMR.this
techniques needs consideration of the availability of DSA containing , the difference
between rats and human in severity of chronic rejection and genetic expression
3. Patient Samples: using patients biopsies to detect
histological changes, complement deposition, cellular infiltration, total protein expression,
, phosphorylation status of specific proteins in the graft and RNA evaluation
Donor specific antibodies (DSA), can be directed against polymorphic HLA class I , HLA class II , or minor histocompatibility molecules such as MICA and MICB, or against non-HLA antigens expressed on endothelial cells, epithelial cells, or organ specific targets . The presence of preexisting or de novo antibodies associated with poor graft function , this not only in renal transplant also in heart and small bowel transplantation .
Diagnosis of Antibody-Mediated Rejection Antibody mediated rejection in renal transplantation is diagnosed by , evidence of complement deposition (C4d) in the peritubules of the graft and/or circulating DSA. Intravascular macrophages, endothelial cell swelling, C4d staining and donor specific HLA antibodies.
Experimental Techniques to Measure Effects of Antibodies
In Vitro Techniques :
Measurement of HLA Antibody Binding Capacity
Analysis of Intracellular Signaling
Determination of Cell Growth
Measurement of Leukocyte Adherence
Determination of Cytoskeletal Changes and Cell Migration
siRNA and Pharmacological Inhibitors
In Vivo Techniques:
Antibodies to donor antigens, called donor specific antibodies (DSA), can be directed against polymorphic class1 ,11 or minor-histocompatibility molecules such as MICA and MICB, or against non-HLA antigen expressed on endothelial cells, epithelial cells, or organ specific targets.
The frequency of the DSA against HLA antigens in kidney transplant are very variable, reported as low as 4% and some reports even higher than 50-%, the frequency of DSAs are higher in patients waiting second transplantation, in one report found that in more than 60%, renal recipients with positive PRA >10 % or with positive DSA have lower graft survival at 3- and 5-years post-transplant [2, 14, 15].
Both performed DSAs AB against HLA antigens and de novo AB associated with AMR and progressive graft failure.
De novo DSAs are associated with late acute antibody-mediated rejection, chronic antibody-mediated rejection, and transplant glomerulopathy, HLA-DRB1mismatch was an independent predictor of the production of denovo DSA, (23,24).
Donor specific HLA antibodies are strongly associated with vasculopathy in heart and kidney transplantation.
presence of antibody to HLA II significantly correlates with worse graft outcome, with AMR even with negative CDC-crossmatch.
Diagnosis of Antibody-Mediated Rejection
1- Progressive graft dysfunction with lower GFR
2-Histological finding of PTC with glomerulitis and PTC – C4D staining, complement mediated endovascular injuries
3- circulating DSAs AB
Experimental Techniques to Measure Effects of Antibodies
1 In Vitro Techniques:
Based on cultured graft cells (endothelium, smooth muscle, or airway epithelium by using murine rat, Measurement of HLA Antibody Binding Capacity
Antibodies from sensitized patients increased the lytic capacity of CD3-CD16+ (NK) peripheral blood mononuclear cells against renal epithelium.
analysis of Intracellular Signaling
Intracellular signaling cascades entail sequential phosphorylation of proteins and kinases.
Determination of Cell Growth
calculation of Cellular proliferation index by flow cytometry
Leukocyte adherence is measured by an adhesion assay using fluorescence microscopy.
Determination of Cytoskeletal Changes and Cell Migration by the scratch, or wound healing, assay.
2- In Vivo Models of Antibody-Mediated Rejection:
Murine models are particularly useful because they allow genetic manipulation of putative targets, mutation: which lacks both T cells and B cells, but retains an intact innate immune system, including complement components, monocytes, natural killer (NK) cells, and neutrophils.
the murine immune systems differ from human immune system, murine system lacks the activating FcγRIIA (CD32A) and FcγRIIC (CD32C) molecules. Inhibitory NK cell receptors are highly divergent between mouse and human, immunoglobulin isotypes, chemokine expression differ between mouse and human.
Hyperacute and Acute Rejection.
Allo-AB binding to endothelial and smooth muscle cells promotes Fc-dependent function through complement cascade activation and antibody-dependent cell-mediated cytotoxicity (ADCC).
Complement Activation:
Classical pathway, bridges the adaptive and innate immune responses by partnering with substrate bound antibodies. Hyperacute rejection is a predominantly complement-mediated severe injury to the allograft occurring within hours of transplantation. It is caused by high titer of preexisting HLA, or in rare cases non-HLA, antibodies in presensitazed patients.
therapies Suggested from Experimental Evidence:
mTOR like everolimus , sirolimus is a central signaling molecule in HLA I-induced cellular proliferation [77]. Pretreatment of endothelial cells with rapamycin, an mTOR inhibitor, reduced HLA I Ab-triggered endothelial proliferation, Akt phosphorylation, and Bcl-2 expression
Eculizumab anti C5A m AB used for treatment of AMR
Survival and Accommodation
graft accommodation is defined as clinically silent- antibody-mediated graft damage that slowly causes failure .
Conclusions:
DSAs AB induce injuries by multiple mechanisms, complement mediated activation results in inflammation and destruction of the graft vasculatures by stimulation of HLA I, HLA II, endothelial or epithelial cell surface markers may induce intracellular signaling leading to recruitment of immune cells, apoptosis, neutrophil infiltration, features of acute rejection, as well as cellular proliferation and vascular lesion formation,in chronic rejection .
Nicely written
1- What are the effects of HLA antibody binding to graft vascular and other cells?
A- membrane attack complex (MAC) : this is the final product of activation and
will result in cellular membranes peforation and lysis.
B- C5b : it is a pro-inflamatory mediator and exerts chemotactic effect for
Neutrophils, Monocytes, Lymphocytes ..
C- C3b : it is an anaphylaxis mediator, activating Eosinophils & Mast cells
resulting in systemic effects of vasodilatation and allergy.
2- Briefly summarize the experimental methods used to assess such outcomes.
a- measurement of antibody binding capacity e.g by Flow cytometry.
b- analysis of intracellualr signalling e.g by Western Blot.
c- determination of cell growth by flow cytometric assays.
d- measurement of leukocyte adhesion by adhesion assays & radiolabelled lymphocytes.
e- determination of cell migration e.g by induction of wounds.
f- using small interferring RNA or pharmacological inhibitors of intracellualr signalling.
very succinct and point wise explanation
DSA can be directed against polymorphic HLA class I or class II, or miHA such as MICA and MICB, or against non-HLA antigens.
Patients with AMR are likely to have preformed DSA anti-HLA antibodies. AMR secondary to preformed DSA or de novo DSA is associated with lower renal allograft survival.
Diagnosis of Antibody-Mediated Rejection
Antibody-mediated rejection in renal transplantation is diagnosed based on clinical, laboratory and histological findings. Poor graft function, evidence of C4d staining in the peritubular capillaries, and presence of DSA in the circulation define antibody mediated rejection.
Experimental Techniques to Measure Effects of Antibodies
In Vitro Techniques:
1.Measurement of HLA Antibody Binding Capacity
2.Analysis of Intracellular Signaling
3.Determination of Cell Growth
4.Measurement of Leukocyte Adherence
5.Determination of Cytoskeletal Changes and Cell Migration
6. siRNA and Pharmacological Inhibitors
In Vivo Techniques:
Murine models were used because they allow genetic manipulation of putative targets. A murine or rat immunodeficient recipient is used, that lack T cells and B cells, but retains an intact innate immune system, including complement components, monocytes, natural killers, and neutrophils.
Thank you very much. Short and sweet
the experimental methods used to assess such outcomes.
A: In Vitro methods:
1- Measuring binding capacity of HAL antibodies
2- Measuring intracellular phosphorylation cascade resulting from binding with HLA I.
3- Using flow cytometry to measure cell proliferation.
4- Leucocytes adhesion essay.
5- Cellular migration and changes in cytoskeleton
B: In vivo methods:
1- Animal models studying AMR.
2- Patient samples.
Effects of HLA antibody binding to graft vascular and other cells:
1/ The binding effect of HLA Antibodies depend on it’s FC portion and the Fab fragment which further cross-links the target molecule on donor cell.
HLA-Antibodies binding to endothelial and smooth muscle cells promotes Fc-dependent activation of:
Hyperacute and Acute Humoral Rejection are typical examples whereby injury is induced by CDC and ADCC.
2/ Target Cell Signaling Induced by HLA I Antibodies leads to:
The experimental methods used to assess measures and outcomes of the effect of HLA-Antibodies:
There are variable experimental methods for testing the effect of HLA antibodies:
In Vitro Techniques:
1.Measurement of HLA Antibody Binding Capacity: using flow cytometric methods
2.Analysis of Intracellular Signaling: using Western Blotting with phosphorylation site- specific antibodies.
3.Determination of Cell Growth: several techniques can be used for measuring Cellular proliferation, which involve either:
4.Measurement of Leukocyte Adherence : using adhesion assay, fluorescence microscopy and automated quantification software for cells counting such as CellProfiler (MIT).
5.Determination of Cytoskeletal Changes and Cell Migration: these are monitored by immunofluorescent staining and visualized by fluorescent microscopy.
6. siRNA and Pharmacological Inhibitors: small interfering RNA (siRNA), or pharmacological antagonism of the protein are utilizing
strategies to inhibit the protein’s function to verify the upstream and downstream relationship to other proteins in the pathway.
In Vivo Techniques:
Patient Samples(Human Biopsy )
Thank you very much Safi
Thanks for your appreciation Professor Ahmed
DSA can be directed to one of the following targets
What are the effects of HLA antibody binding to graft vascular and other cells?
HLA antibodies bind to endothelial cells and smooth muscle cells causing damage to endothelial cells through the following mechanisms:
Briefly summarize the experimental methods used to assess such outcomes.
In vitro techniques:
Involve culture of Endothelial cells, epithelia cells and smooth muscle cells , stimulation by HLA ab then analyzing the effect seen using the following methods
1- Measurement of HLA Antibody Binding Capacity using FCM that measure MFI
2- Analysis of Intracellular Signaling
3- Determination of Cell Growth
4- Measurement of Leukocyte Adherence
5- Determination of Cytoskeletal Changes and Cell Migration
6- siRNA and Pharmacological Inhibitors
In vivo techniques
Using murine or rat
Patient Samples
Thanks
Presence of HLA antibody will bind to HLA antigens, it is complement fixing process will initiate complement cascade and induce AMR which characterized by c4d deposition peritubular, endothelial cell swelling and intravascular macrophages, with or without presence of DSA.
Experimental techniques measure effects of antibodies:
A- in vitro methods:
1- Measurement of HLA Antibody Binding Capacity by flow cytometry measuring its intensity
2- Analysis of Intracellular Signaling
3- Determination of Cell Growth
4- Measurement of Leukocyte Adherence
5- Determination of Cytoskeletal Changes and Cell Migration
6- siRNA and Pharmacological Inhibitors
B- in vivo methods Models of Antibody-Mediated Rejection animal models
C- Patient Samples
Thanks
Experimental methods used to assess outcomes
1)In Vitro Techniques
Endothelial, smooth muscle, or epithelial cells are cultured and stimulated in vitro with HLA
antibodies, and the direct effects can be analyzed in detail.
Measurement of HLA Antibody Binding Capacity—Fluorescence intensity in flow cytometry is used.
For Analysis of Intracellular signalling Cell lysates are separated by SDS-PAGE, then transferred
onto a PVDF membrane, and probed with phosphorylation specific antibodies for Western
Blot .
Determination of Cell Growth by either radioactive substrates incorporated during division or using flow cytometry.
Leukocyte adherence is measured by an adhesion assay.
Migration of cells is assessed by the scratch, or wound healing, assay.
In order to definitively identify a role for proteins in HLA I-mediated intracellular signaling leading to functional changes, it is important to utilize strategies to inhibit the protein’s function to verify the upstream and downstream relationship to other proteins in the pathway commonly used methods involve knockdown of expression of the protein of interest by small interfering RNA
(siRNA), or pharmacological antagonism of the protein with commercial inhibitors.
2) in vivo models
murine or rat immunodeficient recipient, such as severe combined immunodeficiency (SCID) or recombinase activating gene-1 (RAG1) knockout mice, is used, which lacks T cells and B cells.
3) patient samples
In vitro findings are confirmed in patient biopsies.
Effects of the HLA antibody binding to graft vascular and other cells
Fc dependent effects of antibodies
Target Cell Signaling Induced by HLA I Antibodies
Vessels of the graft become occluded by a severely thickened intima invaded by smooth muscle cells. There is strong evidence that ligation of HLA I by antibodies triggers proliferation and cytoskeletal changes in vascular cells.
3)Induction of Secondary Factors
In addition to direct activation of intracellular signaling cascades, in vitro experiments have
revealed that HLA I antibodies increase sensitivity to and production of soluble mediators
which promote autocrine proliferative signaling.
4) leucocyte recruitment
In addition to antibody-induced complement deposition, antibody mediated rejection is frequently characterized by ntravascular macrophages which can promote both acute and chronic rejection.Endothelial cells contain intracellular rod-shaped vesicles called Weibel–Palade bodies, which contain preformed von Willebrand Factor (vWF), P-selectin, and other vascular mediators. Release of these vesicles, which fuse with the cell membrane and secrete contents into the extracellular space leading to leucocyte recruitment.
Investigation of the effects of HLA II antibodies on graft cells has been limited by its
restricted expression pattern and by constraints in experimental systems.The consequences of cross-linking of HLA II on vascular cells are not well defined.