Running titles: Battochio et al. CD45 isoforms in renal graft rejection
Lymphocyte surface marker analysis is a powerful tool for correlating cell phenotype and function. In kidney allograft rejection, a primary role of CD8+ cells in the inflammatory infiltrate has been apparent for some time , and the expression of other antigens (such as CD57)  also appears to be correlated with acute rejection.
The CD45 molecule (leukocyte common antigen) exists as multiple isoforms which are expressed by different cell types in different functional states . The CD45RO isoform, in conjunction with CD8, has predictive value in the diagnosis of acute rejection . New monoclonal antibodies provide an even more refined tool for the study of CD45 variants; these reagents recognize additional epitopes on the different CD45 isoforms, some of which are glycosylation-dependent .
In this study, our aim was to determine whether the extended phenotype of the inflammatory infiltrate, based on expression of CD45 isoforms and other membrane antigens, is a useful adjunct in evaluation of the kidney allograft biopsy. Our findings confirm that an interstitial infiltrate rich in CD8+ and CD45RO+ cells is characteristic of acute rejection. In addition, we show that these antigens have considerably more diagnostic power than other T cell activation markers, including CD25, CD26 and CD69.
OCT-embedded frozen sections were cut at 4Ám, and stained using the panel of monoclonal antibodies and a standard two-step indirect immunoperoxidase method. In each condition, 200-500 infiltrating mononuclear cells were scored as positive or negative by an observer blinded as to the diagnosis, and the results expressed as a proportion of the total CD45+ cells.
Expression of the activation antigen CD38 showed a similar 3.5-fold increase, from 6 to 21%, in acute rejection. In both groups, approximately 30% of the CD38+ population was recognizably plasmacytoid with very strong CD38 expression (denoted CD38 bright cells). The activation antigens CD25, CD26 and CD69 were present at low levels in both groups, and were not significantly increased in rejection. HLA-DR was also expressed to a comparable degree in the non-rejection biopsies (35 ▒ 10% of interstitial cells) and in the rejection group (45 ▒ 3%).
Expression of the different CD45 isoforms differed between the two groups (Table 2). The proportion of CD45RO+ cells, mainly antigen-experienced T cells, increased markedly (5 to 11-fold) in the rejection biopsies. Expression of the isoforms CD45RA and CD45RB, which have a wider cellular distribution, generally increased to a smaller degree (2 to 4-fold). An exception was the subset of CD45RB+ cells detected by the antibody MT3, which increased by a factor of 8 in the rejection biopsies.
In T cells, the CD45RO isoform is believed to correlate with antigen experience or immunologic memory, and is expressed reciprocally to the 'naive' isoform CD45RA. We found expression of CD45RO was markedly augmented in acute rejection, with a greater increase observed when the antibody OPD4, rather than UCHL1, was used for detection. This difference may be due to the different specificities of the two antibodies: the epitope recognized by OPD4 is T cell specific, whereas UCHL1 also detects a population of monocytes .
The observed increase in CD45RA+ cells in the rejection infiltrates may reflect greater overall numbers of T cells, as well as expression of CD45RA by other cell populations including B cells and mononuclear phagocytes. Lower levels of CD45RA expression were observed using the antibody MB1.
CD45RB was detected at much lower levels using the antibody MT3 rather than 6B6. This is consistent with the known specificity of MT3 for a restricted CD45RB epitope (present only in the absence of the sequence encoded by the C exon) . In contrast, the CD45RB epitope detected by 6B6 has a wide cellular distribution and is present on most lymphocytes and mononuclear phagocytes. Using 6B6, we observed CD45RB expression in only 68% of the infiltrating leukocytes, suggesting that only a strongly staining population was detected under our conditions. In T cells, there is evidence that a strongly CD45RB+ subpopulation is responsible for secretion of the Th1-type inflammatory cytokines (reviewed in Ref. 9).
Our results support the view that the T cell antigens CD8 and CD45RO may be useful markers of acute rejection . Observations in an additional patient indicate that the surface antigen profile has independent diagnostic power. This 22-year-old woman had deteriorating renal function eight weeks after kidney transplantation; a marginally adequate biopsy was obtained which showed no definite tubulitis or vasculitis. Following further deterioration, a second biopsy was obtained 12 days later which showed mild acute rejection. Retrospective immunohistochemical staining of both biopsies showed a CD45 isoform profile typical of acute rejection (Table 3). These results suggest that the immunophenotype may provide a valuable supplement to morphology in evaluating the difficult or atypical biopsy.
|CD2 + 3||17 ▒3||59 ▒8|
|CD4||19 ▒3||23 ▒3|
|CD8||10 ▒1||39 ▒2|
|CD25||2 ▒1||3 ▒2|
|CD26||3 ▒3||6 ▒2|
|CD38 dim||4 ▒2||15 ▒3|
|CD38 bright||2 ▒1||6 ▒2|
|CD69||0.3 ▒0.3||1.2 ▒0.6|
Click here to go back.
|CD45RO||OPD4||1.5 ▒1||17 ▒5|
|CD45RO||UCHL1||5 ▒4||22 ▒5|
|CD45RA||MT2||14 ▒7||26 ▒5|
|CD45RA||MB1||3 ▒3||11 ▒4|
|CD45RB||MT3||4 ▒1||31 ▒6|
|CD45RB||6B6||36 ▒8||68 ▒8|
Click here to go back.
|Banff score||g0 i2 t0 v0||g0 i2 t2 v0|
|CD2 + 3||57%||61%|
Click here to go back.