acute rejection ( ar ) is a major risk factor for chronic allograft nephropathy and renal allograft failure after kidney transplantation .
the banff classification criteria divided ar into acute cellular rejection ( acr ) and acute humoral rejection .
t cell mediated rejection ,  as it is associated with cytotoxic t cell infiltration .
however , long - term allograft survival has not been completely improved by controlling factors that affect the t cell pathway [ 35 ] .
in addition to t cells , other inflammatory cells , including cd20-positive b cells [ 68 ] , plasma cells [ 911 ] , macrophages [ 1215 ] , eosinophils [ 1618 ] , and nk cells , can also infiltrate grafts in acr and may affect the severity of rejection and the therapeutic response .
reported the presence of cd20-positive b lymphocytes in the graft interstitium of pediatric recipients with acr for the first time , and they concluded that it was strongly associated with clinical glucocorticoid resistance and graft loss .
classified acr biopsies into the cd20-positive group if they demonstrated strong and diffuse staining characteristics , while trace or rare cd20-positivity was recognized as the cd20-negative group .
other studies also suggested a correlation between cd20 graft infiltration and steroid - resistant rejection [ 20 , 21 ] .
however , relevant studies have argued that cd20-positive b cells exhibited in biopsies have no effect on clinical outcome [ 22 , 23 ] .
clatworthy et al . conducted a clinical trial comparing rituximab ( an anti - cd20 monoclonal antibody ) with daclizumab ( an anti - cd25 monoclonal antibody ) as induction therapy in nonsensitive kidney transplant recipients , but this trial was suspended because of an excess incidence of acr in the rituximab group .
the authors surmised that this anti - cd20 monoclonal antibody might have cleared immunoregulatory b cells , including b cells in the allograft tissue , which led to a marked increase in acr .
disagreements that exist in published studies may be because of a relatively small sample size and lack of a unified standard for the definitions of what is cd20-positive and cd20-negative .
the aim of this study was to determine the effects of cd20-positive b cell graft infiltration during acr on allograft outcome in a chinese population .
this is a retrospective study of patients who underwent kidney transplantation between september 2001 and december 2014 at the kidney disease center of the first affiliated hospital of zhejiang university ( hangzhou , china ) .
this study was approved by the committee of ethics in biomedical research of zhejiang university .
pathological records , clinical data , test results , and follow - up information of all patients were collected from the electronic medical record system and the kidney transplantation database of our center .
altogether , 217 patients were identified with biopsy - proven acr ( grade i or ii ) according to the banff 2005 criteria and were negative for c4d staining .
excluding one patient who was lost of follow - up after antirejection treatment , 216 patients were included in this analysis .
all patients were followed up until june 30 , 2015 . according to the presence of cd20-positive b cell infiltration
, 83 recipients were classified into the cd20-negative group , and 133 were classified into the cd20-positive group .
most of the recipients received calcineurin inhibitor ( cni ) ( cyclosporine or tacrolimus ) in combination with mycophenolate mofetil ( mmf ) and steroids as maintenance immunosuppressive regimen , while some received rapamycin in place of cni .
cyclosporine ( csa ) was initiated at 5  mg / kg / d and tacrolimus ( fk506 ) at 0.100.15 
g / l and 812  g / l , respectively , during the first month after transplantation ; 200250  g / l and 610  g / l , respectively , from the second to the third month ; 150200  g / l and 48  g
/ l , respectively , from the fourth to the sixth month ; and around 150  g / l and 36  g / l , respectively , after the sixth month .
mmf was started at 1.52  g / d for half a month and maintained at 1 
since then , starting from 80  mg / d , with a daily reduction of 10  mg , prednisone was maintained at 1015  mg / d .
once acute rejection was proven by allograft biopsy , intravenous methylprednisolone was administrated at 610  mg / kg daily for 3 days as pulse therapy .
if the serum creatinine ( cr ) level decreased more than 50% or went back to the baseline level within 1 - 2 weeks , the treatment was considered effective . if not , acr was further treated with okt3 at 510  mg / d or atg at 100200  mg / d for 57 days .
response to therapy was determined by comparing the serum cr level measured two weeks after completion of the antirejection treatment to the baseline serum cr level measured before rejection .
response was considered complete if a decrease in serum cr level was maximally 125% of baseline and partial if the cr was 125%175% of baseline , and it is no - response if cr was still more than 175% of the baseline or graft loss ( back to dialysis or nephrectomy ) .
kidney allograft pathology diagnosis was made by an experienced renal pathologist ( w. h. ) according to the banff 2005 criteria .
vascular rejection refers to the existence of intimal arteritis in the acr sample , that is , grades iia , iib , iia + ia , iia + ib , iib + ia , and iib + ib , according to the banff 2005 criteria .
immunohistochemical staining for cd20 and c4d was routinely performed on paraffin sections using the cd20 monoclonal antibody from zhongshan ( cat number za0549 , beijing , china ) and the c4d polyclonal antibody from abcam ( cat number ab36075 , cambridge , uk ) .
, cd20-positive was defined as strong and diffuse staining characteristics , while trace or rare cd20-positivity was assigned to cd20-negative .
the cd20-positive specimens were further assessed independently by two authors ( w. r. and w. h. ) and defined as mild - positive if cd20-positive cells accounted for less than 25% of the inflammatory cells , moderate - positive if cd20-positive cells were 26%49% of the inflammatory cells , and severe - positive if cd20-positive cells were more than 50% of the inflammatory cells .
continuous variables are expressed as mean  standard deviation ( sd ) or median ( range ) .
normally distributed continuous variables were analyzed using student 's t - test or one - way anova , and nonnormally distributed continuous variables were analyzed using mann 
graft / patient survival was analyzed with kaplan - meier method and differences between survival curves were calculated by the log - rank test .
factors associated with graft loss on the univariate analysis with p < 0.1 were included into a cox multivariate analysis .
eighty - three patients were assigned to the cd20-negative group , and 133 patients were classified as cd20-positive .
no significant differences in age , gender , cold / warm ischemia time , donor type , primary disease , induction regimen , and prerejection immunosuppressive drugs were observed between these two groups .
acr was diagnosed earlier after kidney transplantation in the cd20-negative group compared with cd20-positive group ( median time to acr , 29 days versus 142 days , p = 0.016 ) . as presented in table 1
there was significantly more vascular rejection ( iia , iib , iia + ia , iia + ib , iib + ia , and iib + ib ) in the cd20-negative group ( 50/83 patients , 60.2% ) , compared with the cd20-positive group ( 55/133 patients , 41.4% ) ( p = 0.005 ) . in general , cd20-negative patients presented with higher prerejection serum cr levels , compared with the cd20-positive group ( 180.1  128.4 versus 130.6  68.1  mol / l , p = 0.002 ) .
the same results were obtained with peak cr at rejection ( 352.7  242.3 versus 274.1  265.6  mol / l , p = 0.027 ) .
no significant differences were observed at any other time point during follow - up ( table 3 ) .
corresponding to this , worse gfr was observed in the cd20-negative group before rejection ( 47.2  21.3 versus 60.4  21.6  ml / min , p < 0.001 ) , and the same results were obtained at the time of rejection ( 25.0  15.0 versus 30.6  13.3  ml / min , p = 0.005 ) .
no significant differences between cd20-positive and cd20-negative groups were observed at any other time point during follow - up ( table 4 ) .
patients in the cd20-positive and cd20-negative groups received similar maintenance immunosuppressive regimen after rejection ( table 5 ) .
after acr , significantly more cd20-negative patients ( 49/83 , 59.0% ) received steroid plus antibody therapy ( defined as steroid - resistant rejection ) compared with the cd20-positive group ( 52/133 , 39.1% ) ( p = 0.004 ) .
more patients in the cd20-negative group ( 27/83 , 32.5% ) experienced graft loss compared with the cd20-positive group ( 25/133 , 18.8% ) , which reached a significant difference ( p = 0.022 ) .  
figure 2(a ) displayed the graft survival over time analyzed by the kaplan - meier death - censored method for cd20-positive and cd20-negative groups .
we further divided the cd20-positive group into cd20 mild - positive subgroup ( n = 76 ) , cd20 moderate - positive subgroup ( n = 36 ) , and cd20 severe - positive subgroup ( n = 31 ) according to the percentage of cd20-positive b cells found in the inflammatory cell population .  
figure 3(a ) showed that the cd20 severe - positive subgroup tended to have better graft survival compared to the other three groups , but this difference was not significant .
univariate analysis showed that the cd20-positive infiltration , prerejection immunosuppressive regimen , antirejection therapy , and antirejection response were the factors influencing renal allograft loss .
further multivariate cox regression analysis revealed that cd20 infiltration was a protective factor for graft loss .
the adjusted risk ratio of graft loss for steroid plus antibody treatment was 2.316 compared with steroid alone . compared with the complete response ,
the adjusted risk ratio of graft loss was 2.538 for partial - response and 13.847 for no - response , as exhibited in table 6 .
the prerejection immunosuppressive regimen , which was significant in the univariate analysis , did not reach significance in the multivariate analysis .
our study demonstrated that cd20-positive infiltration in the biopsy specimens from the allografts with acr was associated with less steroid - resistant rejection and better allograft survival .
further exploration of the infiltration degree suggested that it tended to be positively related with graft survival , but without statistical significance .
multivariate cox regression revealed that cd20-positive infiltration , antirejection therapy , and antirejection response were independent predictors of graft loss .
b cells and plasma cells in pathological tissues have been considered as nonspecific effector cells in the past [ 26 , 27 ] . in 2003 ,
first demonstrated the presence of cd20-positive b cells in the graft interstitium of pediatric transplant recipients experiencing acr .
their study suggested that cd20-positive infiltration was associated with steroid - resistant rejection and worse graft survival .
since then , the role of cd20-positive b cells in acr has attracted more attention . in adult
kidney transplantation , 22% of acute rejection ( ia , ib ) specimens had cd20-positive infiltration , and it was associated with worse outcome .
a mouse model of acute cardiac allograft rejection demonstrated that recipients ' b cells participate in indirect alloantigen presentation and play an important role in the progression of acute vascular rejection .
studies analyzing the cd20-positive b cells ' infiltration pattern in adult kidney transplantation reported higher serum cr levels in recipients with clusters of b cells .
some studies have suggested that once recruited into injured grafts , b cells can act as antigen - presenting cells to promote t cell mediated rejection , which is resistant to conventional steroid therapy .
different result was observed , suggesting that the early infiltration of b cells can be beneficial [ 23 , 31 ] .
performed immunostaining for cd20 on 54 biopsy - proven acr samples , and no correlation was found between the number of cd20 cells , in clusters or in a scattered pattern , and clinical outcome .
increased peripheral blood b cells were detected in the steady recipients , instead of those experiencing rejection .
cd20 transcription was found to be increased in tolerant kidney transplant recipients [ 3335 ] .
kayler et al . suggested that b cells were not indicators of graft loss or steroid resistance in their analysis of 120 acr biopsies .
one of the possible explanations for this is the lack of a unified standard on the definition of cd20-positive and cd20-negative .
sarwal and her colleagues defined cd20-positive as more than 275 cd20-positive cells in a single high power field ( hpf ) and came to the conclusion that cd20-positive cells were strongly associated with severe graft rejection .
hippen et al . used a qualitative method in their research and defined biopsies with strong and diffuse staining characteristics of cd20-positive cells as the cd20-positive group , while trace or rare cd20-positive cells were recognized as the cd20-negative group .
their research indicated that cd20-positive infiltration was more likely to have steroid - resistant rejection and worse graft survival . in bagnasco 's research , cd20-positive patients ( at least one cluster containing more than 100/hpf cd20-positive cells ) and cd20-negative patients ( the count was below 50/hpf ) were compared .
cd20-positive lymphoid clusters , which were defined as any dense cluster of lymphoid cells containing more than 15 cd20-positive b cells by kayler et al . , did not predict steroid resistance or worse graft survival .
the definition of cd20-positive and cd20-negative in our study is in line with hippen et al .
's research . however , our study 's findings that cd20-positive infiltration is associated with better clinical outcomes are not consistent with hippen et al . 's results .
first , our study included a larger number of acr biopsies than any published studies so far .
a total of 216 cases of biopsy - proven acr were included in our analysis ; 83 samples were assigned to the cd20-negative group , and 133 were classified as cd20-positive .
's study cohort contained 27 patients and only 6 biopsies were classified as cd20-positive . as mentioned in the discussion by the authors , the relative small sample sizes in their analysis may limit the generalization of their conclusions .
's research was conducted in biopsies with proven banff ia or ib rejection within the first year after transplantation .
our study included patients with acr grade i and grade ii at any time point after kidney transplantation .
the differences in the inclusion criteria of study population may contribute to the different results .
thus , besides the lack of a uniformed definition of cd20-positive and cd20-negative , a relatively small sample size , different study populations ( pediatrics versus adults ; different subgroups of acr ) , differences in the induction and maintenance immunosuppression therapy , different follow - up durations , and other factors can contribute to the inconsistency in the studies concerning the role of cd20-positive infiltration in acr .
the prognostic study of cd20-positive cells in acr helps to select future treatment , in particular , whether or not to apply b - cell - depleting agents in steroid - resistant rejection .
rituximab , an anti - cd20 monoclonal antibody , was first applied in the treatment of lymphoma .
it has been proven effective in treatment of a number of hematological diseases and autoimmune diseases [ 3840 ] . in the aspect of transplantation
, rituximab has been used for desensitization of panel reactive antibody , anti - hla antibody , and anti - abo antibody before transplantation as induction regimen and treatment of humoral rejection after transplantation [ 4147 ] . in 2002 ,
a cardiac transplant patient with vascular rejection refractory to plasmapheresis was successfully treated with rituximab , which was the first documented application of an anti - cd20 monoclonal antibody in the aspect of rejection .
later , other case reports confirmed that cd20 monoclonal antibody was effective in the treatment of vascular rejection in heart and pancreas transplantation [ 4951 ] . at present
the cd20-positive b cells in graft are significantly reduced after treatment , but the studies of the reconstruction of b cells in grafts are rather rare .
our results showed that cd20-positive infiltration in patients with acr appeared to have a protective effect on graft outcome .
additionally , a clinical trial which planned to use rituximab in nonsensitive kidney transplant recipients was forced to stop because of increases in rejection .
thus , the administration of anti - cd20 monoclonal antibody in patients with acr requires careful consideration .
cd20-positive b cell infiltration in renal allograft biopsies with acr is associated with less steroid - resistant rejection and better graft survival .
 
 background . it is controversial whether lymphocyte infiltration exhibited in biopsy specimens is associated with transplant outcomes . 
 this study focused on the effect of cd20-positive b cell infiltration in biopsy specimens from allografts with acute cellular rejection ( acr ) in a chinese population . 
 methods . altogether , 216 patients transplanted from sep . 
 2001 to dec . 
 2014 with biopsy - proved acr ( banff i or banff ii ) were included in the analysis . 
 biopsies were immunostained for cd20 and c4d . 
 baseline information , serum creatinine and gfr before and after treatment , steroid resistance , response to treatment , graft loss , and survival were analyzed . 
 results . 
 eighty - three patients were classified into cd20-negative group , and 133 patients were classified into cd20-positive group . significantly more cd20-negative patients ( 49/83 , 59.0% ) received steroid plus antibody therapy compared with the cd20-positive group ( 52/133 , 39.1% ) ( p = 0.004 ) . the response to treatment for acr did not differ between these two groups . 
 the cd20-positive group had less graft loss ( 18.8% versus 32.5% , p = 0.022 ) and a better graft survival rate . 
 further exploration of the infiltration degree suggested that it tended to be positively related to graft survival , but this did not reach statistical significance . conclusion . 
 cd20-positive b cell infiltration in renal allograft biopsies with acr is associated with less steroid resistance and better graft survival . 
 the presence of cd20-positive b cells is protective for renal allografts . 
1. Introduction 
2. Patients and Methods
3. Results 
4. Discussion 
5. Conclusion