there are 20 class i genes and in transplantation hla - a , b , and c are the classic genes referenced to when typing the recipient .
class i major histocompatibility complex ( mhc ) molecule processing and loading of peptides occurs in all nucleated cells , where class ii mhc molecule involves primarily b cells , macrophages , and dendritic cells by method of endocytosis and phagocytosis .
class ii mhc molecule nomenclature is designated by class ( d ) , family ( m , o , p , q , r ) , and chain ( a or b )
. late antibody - mediated rejection ( amr ) is a major cause of late kidney transplant failure and several developments in understanding of pathogenesis allow for improvement of diagnosis , treatment , and prevention .
this review will concentrate on reviewing the pathogenesis , diagnosis , and treatment of amr .
not only can antibodies form against hla molecules , but to endothelial - cell antigens and across abo blood group .
blood transfusions can induce humoral immunity by formation of hla alloantibodies and are more likely to occur in individuals who have been previously pregnant .
our understanding of how blood transfusions cause sensitization is incomplete , but it is not inevitable and can be attenuated by immunosuppression . in a study performed by eikmans et al .
it was found two weeks after blood transfusion in both nonsensitized and sensitized recipients , increased numbers of ifn--producing cells were produced stimulating cd4 + and nk cells . with de novo hla production ,
endothelium of donor graft peritubular and glomerular capillaries display mhc molecules which are the target for antibody production .
once the endothelium is damaged by antibody , von willebrand factor and p - selectin are released as an inflammatory response .
leukocytes adhere to glomeruli or to dilated peritubular capillaries via cytokines ( il-1  , il-8 , and chemokine ligand 2 ) allowing complement activation . in antibody rejection
, c4d is a useful marker of complement activation and is seen commonly in peritubular capillaries with proper immunohistochemical staining technique .
chemoattractants c3a and c5a are a part of the compliment cascade which activate c5b allowing for assembly of membrane attack complex ( mac ) which is composed of c5b - c9 .
the mac complex once activated will cause local necrosis allowing detachment of endothelial cells from the basement membrane , which is also a characteristic finding upon biopsy indicative of amr . if not treated properly in a timely manner , sequelae can include thrombotic microangiopathy ( tma ) causing hemorrhage with arterial wall necrosis , and ultimately graft loss . also potentially playing a role in amr pathogenesis
are polymorphic mhc - class - i - related chain a ( mica ) antigens .
it has been found that not all patients with amr have anti - hla antibodies , which suggests other possible mechanisms involved in acute or chronic graft damage . in a study conducted by worthington et al .
, only five out of 14 patients with renal graft dysfunction and pathology showing c4d deposition in peritubular capillaries were found to have anti - hla antibodies .
mica antigens are structurally similar to mhc class i proteins , closely linked to hla - b and c loci .
mica antigens can be found on fibroblasts , endothelial cells , dendritic cells , and many tumors which unfortunately makes detection difficult by standard donor and recipient crossmatching .
mica eplet repertoire has become the basis for developing hlamatchmaker program which analyzes antibody patterns with single mica alleles on luminex platform .
it has been determined so far ( duquesnoy et al . ) that there are 38 potentially immunogenic mica eplets associated with mica - reactive serum .
mica and hla class i chains are similar in configuration however none of the polymorphic residues are shared .
hlamatchmaker program is therefore used to determine the specificity of anti - mica antibodies in sensitized patients since mica is considered as a separate alloantigenic system . in a study by zhang et al .
, 52 patients transplanted who were crossmatch negative were placed on triple therapy ( cyclosporine / tacrolimus , mycophenolate , and prednisone ) . once graft dysfunction was diagnosed with positive c4d , both anti - hla and anti - mica antibodies were collected from serum and reviewed via luminex . of the 15 patients found to have anti - mica antibodies , 9 patients were positive for class i and/or class ii anti - hla antibodies .
nine out of 15 patients were found to have c4d deposition , and after reviewing patients for the follow - up period ( 1 year ) , it was found that egfr decreased in anti - mica and anti - hla antibody positive group .
this study suggested that patients with mica antibodies have a more rapid deterioration of graft function versus patients with just anti - hla antibodies .
there are several proposed mechanisms for anti - mica antibody activation involving complement or natural killer cells and studies are continuing to determine the pathogenesis . a more thorough understanding of hla typing as well as continuing studies to determine anti - mica and antibody rejection pathogenesis may allow for novel treatment strategies .
amr occurs in up to 33% of cases undergoing abo incompatible kidney transplantation . among cases in which amr is triggered in the early post - op period ,
this is opposed to the case in which gloor found patients who received desensitization to hla antibodies showed more glomerular changes versus abo incompatible transplant grafts not having clinical amr , suggesting subclinical amr may exist .
the resistance of the graft to amr in spite of the presence of antibodies against donor endothelium is called accommodation .
this is explained either by difference in antigen expression , change in repertoire / level of antibodies in recipient , or decreased susceptibility of injury to endothelium secondary to continual stimulation of antibody and complement components .
it is proposed that continued stimulation of endothelial cells by endotoxin or il-1 over time diminishes cellular responses to restimulation .
another proposed mechanism explaining accommodation could be a decrease in the recipient 's type blood - type antigen on the endothelium of the graft .
koestner et al . reported histo - blood type change of a blood type b graft after abo - mismatched heart transplantation , where the recipient 's blood - type o antigen was replaced by the donor 's type b antigen after amr graft loss .
the mechanism of replacement of the recipient 's antigens remains unclear , and several studies have been attempted to explain occurrence of blood - type chimerism .
one explanation is replacement of the recipient type antigens by stem cells derived from injured vessel walls after graft rejection .
it is speculated that during amr some blood - type antigens could be shaved off from endothelial surface from immunological reaction , resulting in loss of blood - type antigens on the transplanted mismatched organ .
in addition the presence of diffuse c4d without capillary damage or inflammation infers that accommodation may include resistance to the terminal complement cascade .
there are some noted differences between hla - sensitized and abo - incompatible renal transplantation in terms of pattern of cell injury after c4d deposition .
it has been found that there is more graft loss with positive c4d amr in hla - sensitized patients which may be due to differences in biological responses between non - self - carbohydrate antigens ( blood type antigens ) and non - self - peptide antigens ( hla antigens ) .
crossmatching not only improves long - term graft survival by identifying the presence of antidonor antibody in the recipient , but is used as a predictor of amr .
the donor cells are incubated with the recipient serum , and antihuman globulin which is fluorescent dye tagged is placed in the flow cytometer .
hla - specific antibody detects antibodies binding to individual hla antigens and allowing determination of antigens to avoid in a donor , coined  unacceptable antigens  thus avoiding rejection . of patients who are highly sensitized
hla - specific antibody detection prior to transplantation and surveillance after transplantation during a rejection episode allows for avoidance of rejection and determination of course of treatment for amr , respectively .
the 1997 banff classification was used to classify rejection prior to the meeting in 2001 , which further defined pathological classification of amr .
gorer determined the role of antibody after much debate in the early days of transplantation , and unfortunately after his death in 1961 the concept of amr was lost . in 1991 - 1992 ,
feucht and halloran described c4d as a marker for amr however this was not uniformly utilized until the report from solez et al . in 19982000 .
after international transplant meetings in 1997 , amr was an entity recognized by most of the participants and c4d staining raised hope that morphological classification of amr can be further defined .
incidence of amr has been reported as 08% in renal transplant recipients in larger centers largely due to increased recognition , detection of dsa , retransplanted patients , as well as increase in positive crossmatch and abo incompatible transplantation for highly sensitized patients .
colvin had reviewed studies from massachusetts general hospital which for the first time indicated a clear correlation of peritubular capillary c4d staining pathological features with dsa in amr .
a few studies have indicated c4d staining is around 9396% specific , but 3195% sensitive .
this raises a concern that additional evidence is needed to diagnose amr such as quantification of dsa or other morphologic feature consistent with amr on pathology .
however it should be noted that regele and colleagues presented data from 1 year after amr that kidney transplant patients with positive c4d had higher incidence of graft loss and elevation of serum creatinine . after the banff meeting in 2001
acute tubular injury ; neutrophils and/or mononuclear cells in peritubular capillaries and/or glomeruli , and/or capillary thrombosis ; or arteritis / fibrinoid necrosis in the intima along with intramural / transmural arterial inflammation .
c4d evidence for antibody action and/or immunoglobulin in peritubular capillaries , immunoglobulin and complement in arterial fibrinoid necrosis .
anti - hla antibody ( dsa ) circulation in serum or other antidonor endothelial antigens . after the banff meeting in 2009 ,
positive c4d deposition without evidence of rejection was added to criteria suggesting antibody - mediated changes .
several studies had reported this finding including mark haas ( los angeles ) who had seen c4d deposition in biopsies without evidence of rejection . in protocol biopsies from abo incompatible grafts ,
21/37 had c4d deposition without evidence of amr lesions or t - cell - mediated rejection which can suggest accommodation .
another addition to banff criteria indicating antibody - mediated changes was determined to be positive c4d with presence of circulating antidonor antibodies ( no signs of acute or chronic t - cell - mediated rejection or amr / no atn - like minimal inflammation ) . in normal kidneys , specifically in the glomerular mesangium as well as arterioles at the vascular pole , turnover of immune complexes occurs ( c4d ) .
transplanted kidneys in addition show deposition of c4d in peritubular capillaries and can follow a dynamic course .
c4d can be in a transitional state and show staining of only a few capillaries indicating either an ongoing humoral attack or resolving previous attack .
there is sometimes diffuse staining in every capillary or focal staining in only a few capillaries , but c4d is always attached to endothelial cells and basement membranes .
there has also been evidence for c4d negative amr in a couple studies , which in the past few years has brought to the forefront the limitations of c4d as a marker for amr .
in one such study by sis et al . , overexpression of 12-individual endothelial - associated transcripts genes ( endats ) upon transplant biopsies with graft dysfunction was correlated with an increased risk of graft loss .
high endat expression and endothelial activation with dsa was found to be strongly associated with the presence of transplant glomerulopathy .
interestingly , fewer than 50% of the biopsies performed ( n = 173 ) with dsa and endats had diffuse c4d staining in peritubular capillaries .
investigators also have found that c4d negative form of amr is less severe than c4d positive amr but is associated with chronic changes within the graft , such as transplant glomerulopathy .
it has been postulated that dsa binding to endothelial cells triggers natural - killer cells to release interferon- resulting in granule - associated toxicity .
dsa has been found to play a role in diagnosis of amr and can be an independent predictor of allograft loss .
it was found when patients had dsa at time of clinical rejection diagnosed by luminex , 50% reduction in dsa within 14 days of diagnosis had higher allograft survival . in a study performed by everly et al .
, acute rejection was defined as an increase in serum creatinine by 20% above the baseline upon rejection ; dsa was identified by antigen bead panels by luminex assay .
650 patients were analyzed for acute rejection , 94 of which were identified with biopsy - proven acute rejection by banff criteria .
analysis of predictors of allograft loss revealed dsa to be an independent predictor of allograft loss , with a sixfold increase in allograft loss rates .
however , it is noted in this study that dsa was present in the absence of c4d staining on biopsy .
further studies need to be conducted to determine if complete elimination of dsa is necessary .
however , from this study it should be deduced that dsa reduction should receive consideration as a potential therapeutic goal for rejection therapy .
suppression of dsa with antihumoral therapies may provide a means for improving long - term renal allograft survival .
bortezomib , a new anti - b - cell agent that targets antibody producing cells ( plasma cells ) has shown promise as an effective means for reducing dsa as well .
it has also been postulated that surveillance of dsa can be a means of preventing rejection , especially for highly sensitized patients .
it has been discussed that high - risk patients ( presensitized and retransplants ) may benefit from more dsa surveillance in the first 3 months after transplant to detect an early immune response ; however this is not rigorously proven to improve outcomes .
plasmapheresis ( pp ) removes alloantibodies from the circulation and is the fastest and most effective method for elimination of dsa .
usually 1.01.5 full - volume exchanges are performed using albumin solution daily or alternate days , continued until serum creatinine decreases 30% from the baseline value .
although pp is effective in removing alloantibodies ( dsa ) from the circulation , rebound synthesis of de novo alloantibodies can occur .
ivig is used in combination with pp to neutralize antibodies and potentially block complement activity as well as agents used for suppression of antibody ( cacineurin inhibitors , mycophenolate , or rituximab ) .
immunoadsorption ( ia ) is a substance derived from the cowan strain of staphylococcus aureus .
the protein a coating the column beads has a strong affinity for the fc portion of immunoglobulin and immune complexes .
for the past decade , protein a ia was found to remove harmful antibodies and immune complexes efficiently and has been used as treatment of amr of highly sensitized patients .
it was found in a study by qing et al . that classes i and ii panel - reactive antibody ( pra ) as well as immunoglobulin and complement ( c3 and c4 ) were reduced significantly .
however , it was found that within 6 to 8 hours rebound of antibodies was seen .
it has been postulated that ia may cause a conformation change of the structure of the antibodies and immune complexes making them more immunogenic , and immunostimulating to regulatory anti - idiotype antibodies .
these antibodies play a role in the control of autoimmune mechanisms and reverse the continuing cycle of abnormal immune balance .
the long lasting response of ia therapy may be explained by phagocytosis of the reticuloendothelial system and activation of complement .
ivig is prepared by human plasma from approximately 50,000100,000 of healthy donors , composed of 90% intact igg , a few dimers , fabs ( fragment antigen - binding ) and traces of igm and iga .
the fc region in igg allows it to interact and signal through fc - gamma receptors on phagocytes , b cells , and other cells as well as with fc - binding plasma proteins , such as components of the complement system .
immune globulin also prevents damage mediated by immune complexes containing c3b into an inactive form ic3b as well as neutralization of some cytokines .
ivig can neutralize autoantibodies and down regulate synthesis of antibodies by b cells expressing the relevant idiotype .
there are some studies with usage of ivig alone or in combination with pp , and when ivig is used alone a high dose is given ( 1 - 2  g / kg ) .
it was found ivig was effective in reversing rejection within 25 days of infusion with no recurrence in kidney transplant recipients .
the usual recommended dose is 100  mg / kg of ivig after each pp session and 300400  mg / kg for 1 - 2 days after the last pp with a cumulative dose of 1000  mg / kg .
a benefit of ivig is its ability to replenish gammaglobulin lost during pp , decreasing infection risk .
adverse effects of ivig which are common are headache , fever , chills , myalgias , and hypotension / hypertension which can be reduced by slowing the infusion rate .
serious adverse effects are rare and can include aseptic meningitis , acute renal failure , thrombotic events , and anaphylaxis . 

jin et al . had used thymoglobulin ( atg ) 0.75  mg / kg / day for 510 days in combination with plasma exchange in 7 patients with amr and showed 85% reversal in amr .
atg eliminates cd4 + t - cell and b - cell interaction causing b - cell toxicity / apoptosis and modulation of alloantibody production .
if both cellular and humoral features are seen on biopsy , atg is often used along with steroids for treatment of amr .
atg can be given in 3 - 4 doses to a cumulative dose of 6  mg / kg , and it is noted that leukopenia is a side effect which should be monitored to guide specific treatment for each patient .
the spleen is the largest lymphoid organ in the body and has been found to play a role in alloantibody generation .
splenectomy is used in desensitization protocols for abo incompatible transplants as well as positive crossmatches , however it has been found to surprisingly treat amr refractory to treatment .
there are a few case studies , one in particular where abundant clusters of cd138 + plasma cells were found upon review of pathology of the spleen . usually in traumatic splenectomy controls , cd138 + cells
she had been treated with pp / ivig and then underwent emergent splenectomy . immediately afterward
the mechanism of action is not entirely clear as to why splenectomy treats amr , but several case reports have indicated that it could be a potential treatment for refractory amr to pp / ivig .
it is not certain whether the cd138 + cells found in the spleen had produced anti - dr51 antibodies in this particular case , but provides a novel insight for further studies in the role of the spleen with amr .
of course , splenectomy has risks associated with it which include increased risk of infections and the risks associated with surgery .
therefore these risks must be discussed with the patient before proceeding , as this is not a conventional treatment for amr .
there is lifelong risk with encapsulated microorganisms ; therefore patients must receive haemophilus influenza vaccine , pneumococcal , and meningococcal vaccines .
most episodes of amr are associated by evidence of early complement activation by the presence of c4d+ staining of the peritubular capillaries .
eculizumab is a humanized monoclonal antibody with high affinity for c5 and blocks the activation of terminal complement and is fda approved for the treatment of paroxysmal nocturnal hemoglobinuria .
serious adverse effects of this medication are an increased risk of infections , particularly to encapsulated bacteria .
it is recommended to immunize patients at least two weeks before the administration of eculizumab to meningococcus and pneumococcus .
the transplant community is looking towards the use of this medication because of its highly selective mechanism of action .
there are several case reports using eculizumab for desensitization before transplant and to treat amr after transplant .
stegall and colleges reported using eculizumab in sensitized renal transplant recipients with high levels of dsa to determine if the incidence of amr in the first 3 months after transplant was decreased compared to historical controls .
these patients were immunized one month prior to transplant , received plasmapheresis treatments before and after transplant based on the crossmatch channel shifts , and received the same induction and maintenance immunosuppression .
the eculizumab dosing regimen included 600  mg on postoperative day 1 , and 600  mg weekly thereafter for 4 weeks . at week 4 , assessment of dsa levels was performed to determine if patients would continue treatment based on dsa levels .
the study found the incidence of amr was statistically significanty lower in the eculizumab group compared to the control group and reduced the need for splenectomy , reducing transplant glomerulopathy .
considering the cost of this medication , this dosing regimen and prophylactic use might be unrealistic for smaller transplant centers .
a few case reports used eculizumab in treating renal thrombotic microangiopathy ( tma ) and reversal of complement activation after abo - incompatible transplantation [ 2628 ] .
the use for tma involved a patient who underwent a living related kidney transplant with a history of lupus developed severe tma , glomerular scarring , and diffuse tubulointerstitial fibrosis with positive apl antibodies . the patient received dialysis and plasmapheresis sessions with no improvement in kidney function .
the patient received weekly infusions of eculizumab without complication and renal function improved with subsequent biopsy revealed complete resolution of tma .
the transplant community has recognized the potential use of an agent that can directly target plasma cells .
traditional treatments have been successful in removing antibodies , inhibiting antibody activity or lowering production but no treatments have been effective in targeting mature antibody production in plasma cells .
amr involves the production of high levels of dsa by plasma cells either newly made from memory or nave b cells or from those that existed prior to transplant .
bortezomib is a proteasome inhibitor which is fda approved for the treatment of multiple myeloma .
this medication has been shown to cause apoptosis of normal plasma cells which in turn decreases alloantibody production in sensitized patients .
the university of cincinnati reported the first study on the use of bortezomib in amr .
everly and colleagues treated six kidney transplant recipients with refractory amr and concomitant acr with bortezomib and found fast rejection reversal , reductions in dsa levels , improved renal allograft function , and suppression of recurrent rejection .
the same group reported two patients with amr who received bortezomib and these patients experienced prompt amr reversal and dsa elimination in 14 days .
the use of bortezomib has also been published in five patients with a combination of acr and amr .
these patients were given four doses of bortezomib and reversal of both acr and amr occurred with reduction in dsa .
other studies have shown positive and mixed results regarding the use of this medication in amr and dsa levels [ 31 , 32 ] .
rituximab is a chimeric anti - cd 20 ( anti - b cell ) monoclonal antibody that is currently fda approved for the treatment of lymphoma .
the cd 20 antigen is expressed early in b - cell cycle but is absent on mature plasma cells .
the variable region of rituximab binds to cd 20 through three different mechanisms including antibody - dependent cell cytotoxicity , complement - dependent cell killing , and induction of apoptotic cell death .
these mechanisms mark cells for destruction and leads to sustained depletion of circulating b cells [ 32 , 33 ] .
genberg and colleagues reviewed the pharmacodynamics after a single dose of rituximab given to renal transplant patients and showed b - cell elimination was rapid and occurred in the peripheral blood over 1 - 2 days .
the effect on the b - cell population was prolonged and did not reemerge for 1 year and remained suppressed for 2 years .
the first report of using rituximab in amr evaluated 27 patients with refractory rejection and received a single dose of rituximab .
three grafts were lost and the remaining grafts had good function at the time of discharge .
kaposztas and colleagues published a retrospective study involving 54 patients with amr and were split into two groups .
group a ( n = 26 ) received plasmapheresis and rituximab and group b ( n = 28 ) received plasmapheresis alone .
the two year graft survival was significantly better in the rituximab group . since these two large case reports
, there have been many case series , and so forth published on using rituximab with any of the studies for desensitization and for amr [ 3740 ] . with many of studies
the patient population is small , with incomplete criteria for amr diagnosis , and other treatments used in conjunction with rituximab including ivig , plasmapheresis , and steroids . however it should be noted in other studies particularly by pescovitz , a small number of patients failed to show a decrease in pra with rituximab alone .
 antibody - mediated rejection ( amr ) is a major cause of late kidney transplant failure . 
 it is important to have an understanding of human - leukocyte antigen ( hla ) typing including well - designed studies to determine anti - mhc - class - i - related chain a ( mica ) and antibody rejection pathogenesis . 
 this can allow for more specific diagnosis and treatment which may improve long - term graft function . 
 hla - specific antibody detection prior to transplantation allows one to help determine the risk for amr while detection of dsa along with a biopsy confirms it . 
 it is now appreciated that biopsy for amr does not have to include diffuse c4d , but does require a closer look at peritubular capillary microvasculature . although plasmapheresis ( pp ) is effective in removing alloantibodies ( dsas ) from the circulation , rebound synthesis of alloantibodies can occur . 
 splenectomy is used in desensitization protocols for abo incompatible transplants as well as being found to treat amr refractory to conventional treatment . 
 also used are agents targeted for plasma cells , b cells , and the complement cascade which are bortezomib rituximab and eculizumab , respectively . 
1. Understanding HLA
2. Pathogenesis of HLA Antibodies
3. Prevention
4. Diagnosis
5. Treatment