- Email: [email protected]
Permissible and immunogenic HLA-A mismatches: cytotoxic T-cell precursor frequencies reflect graft survival data
Permissible and Immunogenic HLA-A Mismatches: Cytotoxic T-Cell Precursor Frequencies Reflect Graft Survival Data Dave L. Roelen, Inge Stobbe, Neil T. Young, Simone P. M. J. van Bree, Ilias I. N. Doxiadis, M. Oudshoorn, Peter J. Morris, Kathryn J. Wood, and Frans H. J. Claas ABSTRACT: Analysis of the in vivo immunogenicity of single HLA mismatches, in the context of a patient’s own human leukocyte antigen (HLA) phenotype, has been used to define permissible and immunogenic HLA mismatches. Kidney graft survival in the case of permissible mismatches was similar to that of completely HLA matched combinations, whereas immunogenic mismatches lead to a significantly poorer graft survival. The present study tested whether such permissible and immunogenic HLA mismatches are reflected in the in vitro cytotoxic T-lymphocyte (CTL) allorepertoire. Limiting dilution experiments were performed to analyze the number of precursor CTL directed against individual HLA class I antigens. In general, the frequency of CTLp directed against permissible HLA-A antigens (n ⫽ 70, mean frequency 27 CTLp per million peripheral blood lymphocytes [PBL]) was found to be significantly lower compared with the CTLp directed against immunogenic HLA-A antigens (n ⫽ 73, mean frequency 59 CTLp per ABBREVIATIONS CTLp cytotoxic T-lymphocyte precursor GVHD graft-versus-host disease HLA human leukocyte antigen LDA limiting dilution analysis NIH National Institutes of Health
million PBL). The difference was found both in healthy individuals and a population of renal transplant candidates. These results were confirmed by a retrospective analysis of CTLp frequencies performed between partly mismatched unrelated bone marrow donors and their potential recipients. In conclusion, on the population level the permissible and immunogenic HLA-A mismatches are indeed reflected in the CTL allorepertoire. However, due to the big overlap of the CTLp frequencies in these populations, the permissible or immunogenic nature of a mismatch for a particular patient should be determined on an individual basis. Human Immunology 62, 661– 667 (2001). © American Society for Histocompatibility and Immunogenetics, 2001. Published by Elsevier Science Inc. KEYWORDS: HLA; transplantation; cytotoxic T lymphocytes; permissible; immunogenic
PBL PCRSSP SBT
peripheral blood lymphocytes polymerase chain reaction–single-strand polymorphism sequence-based typing
INTRODUCTION Matching for human leukocyte antigens (HLA) is associated with increased graft and patient survival [1]. Additional studies have reported that matching for the products of the individual HLA class I loci does not have
equal importance in this respect. For instance, HLA-A mismatches were found to have less influence on renal allograft survival, compared with HLA-B mismatches [2,
From the Deparment of Immunohaematology and Bloodbank (D.L.R., I.S., S.P.M.J.vB., I.I.N.D., M.O., F.H.J.C.), Leiden University Medical Centre, Leiden, The Netherlands; and the Nuffield Department of Surgery (N.T.Y., P.J.M., K.J.W.), University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
Address reprint requests to: Dr. Dave Roelen, Deparment of Immunohaematology and Bloodbank, Leiden University Medical Centre, PO Box 9600, 2300 RC, Leiden, The Netherlands; Tel: ⫹31 (71) 5263982; Fax: ⫹31 (71) 5216751; E-mail: [email protected]. Received January 26, 2001; revised April 12, 2001; accepted April 17, 2001.
Human Immunology 62, 661– 667 (2001) © American Society for Histocompatibility and Immunogenetics, 2001 Published by Elsevier Science Inc.
0198-8859/01/$–see front matter S0198-8859(01)00263-4
662
D. L. Roelen et al.
TABLE 1 Listing of permissible and immunogenic donor HLA antigens Recipient antigen A1 A2 A3 A9 (23,24) A10 (25,26) A11 A28
Permissible mismatch A3 A1 A1 A3 A3 A3 A1
Immunogenic mismatch
A23 A24 A25 A26 A11 A29 A30 A31 A3 A23 A24 A28 A29 A30 A31 A34 A2 A26 A11 A28 A29 A30 A31 A28 A30 A33 A34 A23 A24 A28 A30 A23 A24 A25 A26 A29 A30 A34 A11 A31
A2 A32 A25 A26 A23 A24 A1 A2 A25 A26 A11 A29 A31 A32 A1 A2 A11 A34 A2 A28 A2 A23 A24 A25 A26 A30 A34
Permissible and immunogenic donor HLA antigens as defined by Maruya et al. [10].
3]. In vitro analyzes reported that this different matching effect is reflected in the cytotoxic T-cell allorepertoire for HLA-A and HLA-B antigens; in general, lower frequencies of cytotoxic T-lymphocyte precursor (CTLp) are found for HLA-A mismatches compared with HLA-B mismatches [4 – 6]. The observation that some mismatched grafts function well for long periods [7, 8] has led to attempts to determine the immunogenicity of individual HLA mismatches in classical transplantation. Analysis of kidney graft survival in donor/recipient combinations with a single HLA mismatch did not provide evidence that any single HLA antigen was more immunogenic than another [9]. However, by analyzing the effect of a donor mismatch in the context of a patient’s own HLA antigens, Maruya et al. [10] could identify permissible mismatches, i.e., cadaver-donor transplants mismatched for a single HLA antigen that had graft survival rates equivalent to zero HLA-A,B,DR mismatched grafts. This study considered the immunogenicity of the mismatch always in the context of the responder’s HLA phenotype. Maruya et al. [10] approached this matter studying by observing living-related donor transplants and defining those mismatch combinations that resulted in ⬎ 85% 1-year graft survival rate as “permissible.” For simplification of the analysis, only one HLA-A,B,DR mismatched kidneys were considered and the study focused on HLA-A and -B antigens that occurred in high frequencies. A separate file of cadaver-donor transplant patients was then divided into those with permissible and those with immunogenic mismatches. After 3 years, kidneys with one permissible mismatch had a similar graft survival to zero-A, B, DR mismatched transplants, whereas those with immunogenic mismatches had significantly lower graft survival rates [10]. These data were confirmed in the Eurotransplant data set [11]. The present study has analyzed whether such permissible and immunogenic mismatches are also reflected in the in vitro cytotoxic T-cell allorepertoire on the population level. If this study can detect a difference on the population level, then it may be of use to analyze the correlation on an individual basis. For this analysis the
terms permissible and immunogenic HLA mismatches are based on the definition of the graft survival data of Maruya et al. [10]. MATERIALS AND METHODS Permissible Mismatches HLA mismatches were classified as permissible or immunogenic mismatches, according to Maruya et al. [10] (Table 1). The current study was restricted to HLA-A mismatches as the immunogenic and detrimental nature of the HLA-A antigens was better defined than that of the HLA-B antigens [10]. In some patients the mismatched HLA-A antigen was a permissible mismatch for one HLA-A antigen in the responder’s HLA phenotype, and at the same time an immunogenic mismatch for the other (e.g., HLA-A2 as a mismatch for an HLA-A1⫹ and A3⫹ individual, whereas HLA-A2 is permissible for HLA-A3 but immunogenic for HLA-A1). The CTLp frequencies in this group were compared with combinations of only permissible and only immunogenic mismatches to analyze the “dominance” of either the immunogenic or permissible mismatch. Subjects and Patients Healthy volunteers were HLA-typed for class I and class II by conventional serologic methods using the standard NIH complement-dependent cytotoxicity assay. The renal patients were typed with low-medium resolution DNA typing (phototyping, in the second center of John Radcliffe Hospital [Oxford, United Kingdom]), whereas the hematologic patients were typed with high resolution DNA typing (polymerase chain reaction–singlestrand polymorphism [PCR-SSP] and sequence-based typing [SBT]). Homozygosity for a particular HLA allel was confirmed by DNA analysis and/or family studies. The renal patients who were analyzed were on hemodialysis for end-stage renal failure and had not previously been transfused or transplanted. These patients (14 in total) were on the waiting list from the Oxford Transplant Center. The patients (20 in total) with a hemato-
CTL and Permissible HLA Mismatches
663
logic disorder were waiting for a unrelated bone marrow transplant. The CTLp frequencies are measured between donors and recipient as part of the selection procedure (graft-versus-host disease [GVHD] direction) according to the Europdonor guidelines. Limiting Dilution Analysis For the analysis of the allospecific CTLp frequencies limiting dilution assays (LDA) were performed as described [12]. Briefly, graded numbers (from 40,000 cells per well to 312 in twofold dilutions) of single cell suspensions of peripheral blood lymphocytes (PBL) were cultured in 24 replicates (96 -well U bottom plates [Costar,Cambridge MA, USA]) with 50,000 irradiated (3000 rad) stimulator cells (PBL) in a total volume of 0.2 ml of RPMI 1640 (Gibco, Paisley, United Kingdom) supplemented with penicillin G (75 U/ml), streptomycin (45 mg/ml), kanamycin sulphate (90 mg/ml), glutamine (2 mM), and 10% heat-inactivated pooled human serum. The test medium contained 20 U/ml interleukin-2 (IL-2; Ortho Pharmaceutical Corporation, Raritan, NJ, USA). Assay I. After 7 days of culture, each well was tested individually for cytolytic activity against 5000 51Crlabeled target cells. The LDA cultures were incubated for 4 h with the target cells; 20 l of supernatant was harvested on a spot-on filtermat (Wallac, Turku, Finland) and the release of 51Cr was assayed in a Betaplate counter (Wallac). Microcultures were considered cytolytic if the observed 51Cr release exceeded the spontaneous release (stimulators only) plus 3 SD. Assay II. After 7 days of culture, each well was tested individually for cytolytic activity against 5000 europium-labeled target cells (as described by Bouma et al. [13]). The LDA cultures were incubated for 4 h with the target cells; 20 l of supernatant was transferred to a 96-well U bottom low-absorbance plate (Nunc, Rockilde, Denmark) and enhancement solution was added. Microcultures were considered cytolytic if the observed europium release exceeded the spontaneous release (stimulators only) plus 3 SD. Cells from the healthy individuals or patients were incubated with HLA-typed stimulator cells. The target cells were selected on the basis of sharing only one HLA-A antigen with the stimulator. This study was performed in two separate transplant centers. Cell-mediated lympholysis (CML) assay I was applied in Oxford (United Kingdom), while the center in Leiden (The Netherlands) used CML assay II. Anti-CD8 Inhibition Studies In order to distinguish high- and low-avidity alloreactive CTLs, LDAs were performed in the absence and presence
FIGURE 1 Cytotoxic T-lymphocyte precursor (CTLp) frequencies directed toward immunogenic human leukocyte antigen (HLA-A; right panel) and toward permissible HLA-A antigens (left panel), as performed in healthy individuals. Horizontal bars represent the median of the group. A statistical difference was found for the CTLp frequencies of the immunogenic HLA-A versus the CTLp frequencies against permissible HLA-A according to the Mann-Whitney test (p ⫽ 0.011).
of antibodies to CD8 as earlier described [14]. The anti-CD8 monoclonal antibody FK18 was used as a 1:400 dilution of ascitic fluid [15]. Before adding the targets, FK18 was added to each well and incubated with the effector cells during 1 h at room temperature. Statistical Analysis Frequencies of CTLp and the 95% confidence intervals were calculated as described [16]. Precursor frequencies were only taken into account if the goodness of fit was ⱕ 15 (jackknife method) to assure single-hit kinetics. The statistical significance of the differences between the several groups was analyzed using the Mann-Whitney test. RESULTS In a first series of experiments, CTLp frequencies (CTLpf) were determined for individual HLA-A alloantigens using PBLs from healthy individuals. Using the established classification of the University of California at Los Angeles (UCLA) Kidney Transplant Registry, the HLA-A mismatches tested were classified as permissible or immunogenic mismatches [10] (see Table 1). Overall, a large range of CTLpf was evident (Figure 1). In a group of 54 allogeneic combinations CTLpf against the permissible HLA-A antigens (n ⫽ 25, mean fre-
664
D. L. Roelen et al.
FIGURE 2 Comparison of the frequencies of cytotoxic Tlymphocyte (CTL) directed toward human leukocyte antigen (HLA-A) in the real permissible group (no conflicts permissible/immunogenic), in the true immunogenic group (no conflicts permissible/immunogenic), and in the combined permissible/immunogenic group as evident in healthy individuals. Horizontal bars represent the median of the group. A statistical difference was found for the differences in CTL precursor (CTLp) frequencies of the only immunogenic HLA-A antigens versus the only permissible HLA-A antigens according to the Mann-Whitney test (p ⫽ 0.002), and also between the only permissible group versus the permissible/immunogenic group (p ⫽ 0.042). No statistical difference was found between the only immunogenic group versus the permissible/immunogenic group (p ⫽ 0.842).
FIGURE 3 Comparison of the frequencies of cytotoxic Tlymphocyte (CTL) directed toward immunogenic and permissible human leukocyte antigen (HLA-A24; left panel) and toward immunogenic and permissible HLA-A1 (right panel) as evident in healthy individuals. Horizontal bars represent the median of the group. A statistical difference was found for the differences in CTL precursor (CTLp) frequencies of the immunogenic HLA-A24 versus the permissible HLA-A24 according to the Mann-Whitney test (p ⫽ 0.024). No statistical difference was evident for the differences in CTLp frequencies of the immunogenic HLA-A1 versus the permissible HLA-A1 (p ⫽ 0.079).
quency 31 CTLp per million PBLs) were significantly lower than those against immunogenic HLA-A antigens (n ⫽ 29, mean frequency 62 CTLp per million PBLs, p ⫽ 0.011). Next, CTLpf was determined in a group of individuals where the mismatched HLA-A antigen was a permissible mismatch for one HLA-A antigen in the responder’s HLA phenotype, while an immunogenic mismatch for the other (e.g., HLA-A2 as a mismatch for a HLA-A1⫹ and A3⫹ individual, where HLA-A2 is permissible for HLA-A3 but immunogenic for HLA-A1) (Figure 2). The mean frequencies in the real permissible group (only permissible; no conflicts permissible/immunogenic) was 8 CTLp/106 PBLs (n ⫽ 13), in the true immunogenic group (only immunogenic; no conflicts permissible/immunogenic) 35 CTLp/106 (n ⫽ 10), and in the permissible/immunogenic group 47 CTLp/106 PBLs (n ⫽ 9). CTLp frequencies in the latter two groups differed significantly from those of the permissible group and were not significantly different from each other, demonstrating that immunogenic mismatches are dominant. In subsequent analysis the data of these two groups have been combined. As some HLA-A mismatches lead to higher CTLpf compared with others (e.g., HLA-A2 induces a high
CTLpf [6]), the study checked whether the individual HLA-A antigens were equally divided among the permissible or immunogenic mismatches, which was indeed the case (data not shown). Another way to circumvent the influence of individual HLA antigens is to compare CTLp frequencies in combinations where the same antigen functions as either a permissible or an immunogenic mismatch. When such an analysis was performed for HLA-A1, HLA-A2, and HLA-A24 antigens, higher CTLp frequencies were evident for immunogenic combinations compared with permissible combinations, although these differences did not reach statistical significance in all combinations (Figure 3). The difference between permissible and immunogenic HLA-A CTLp frequencies in healthy individuals could be confirmed in a second study. Again, CTLpf against permissible HLA-A antigens (mean frequency 28 CTLp per million PBLs, n ⫽ 22) were significantly lower than those against immunogenic HLA-A antigens (mean frequency 59 CTLp per million PBLs, n ⫽ 29) (p ⫽ 0.032, data not shown). A similar analysis in a group of dialysis patients awaiting a renal transplant, indicated a mean frequency of 21 CTLp per million PBLs (n ⫽ 23) in permissible mismatches and a mean frequency of 55 CTLp per mil-
CTL and Permissible HLA Mismatches
FIGURE 4 The combined data of all four groups studied. Frequencies of cytotoxic T-lymphocyte precursor (CTLp) directed toward immunogenic human leukocyte antigens (HLA-A; right panel) and toward permissible HLA-A antigens (left panel) are illustrated. Horizontal bars represent the median of the group. A statistical difference was found for the CTLp frequencies of the immunogenic HLA-A versus the CTLp frequencies against permissible HLA-A according to the Mann-Whitney test (p ⬍ 0.001).
lion PBLs (n ⫽ 28) (p ⫽ 0.003) in immunogenic HLA-A mismatches (data not shown). Finally, we analyzed the results of CTLp tests performed between partially HLA-mismatched unrelated bone marrow donors and their potential recipients, which were performed as part of a donor selection procedure. In case of permissible HLA-A mismatches, 16 CTLp per million PBLs (n ⫽ 10) were found versus 27 CTLp per million PBLs (n ⫽ 10) for immunogenic HLA-A mismatches (p ⫽ 0.045). The combined result of these four groups analyzed together are illustrated in Figure 4 (p ⬍ 0.001). In the present study anti-CD8 antibodies were found to inhibit all HLA-A directed CTLp tested, irrespective of whether these HLA-A antigens were classified as permissible (n ⫽ 17, mean CD8 inhibition 79%) or immunogenic HLA-A mismatches (n ⫽ 31, mean CD8 inhibition 81%; data not shown). DISCUSSION The increasing number of detectable HLA alleles makes selection of an unrelated, HLA completely-matched donor more and more difficult and for many patients even impossible. Because earlier epidemiologic studies did not report any difference in immunogenicity of individual donor HLA mismatches, Maruya et al. [10] were the first to consider the immunogenicity of the donor HLA mismatch in the context of a recipient’s own HLA phenotype. This is a logical method of analysis because the peripheral T-cell repertoire is shaped by self-HLA anti-
665
gens during the processes of thymic selection. Furthermore, with increased knowledge of the molecular structure of the HLA antigens, it is apparent that certain HLA specificities share more epitopes than others, which is another reason why the recipient HLA type will influence the immunogenicity of a particular mismatch. By analysis of the immunogenicity of single HLA mismatches in the context of the patient’s own HLA phenotype, Maruya et al. [10] defined permissible and immunogenic HLA mismatches. Kidney graft survival in case of permissible mismatches was similar to that of completely HLA matched combinations, whereas immunogenic mismatches lead to a significantly poorer graft survival. A similar analysis was performed for the HLA class II antigens by Vereerstraeten et al. [17], which demonstrated differential effects of donor-recipient HLA-DR mismatches on cadaver kidney graft survival. The data of the UCLA registry (Maruya) could be confirmed by an analysis of the Eurotransplant dataset [11]. Survival of grafts with permissible mismatches was similar to zero-mismatched grafts, whereas nonpermissible (or immunogenic) mismatches lead to significantly poorer graft survival. Consequently, we examined whether the in vivo difference between permissible and immunogenic HLA class I antigens is reflected in the in vitro frequency of cytotoxic T-cell precursors directed against the individual HLA antigens. In two separate groups of healthy individuals a significantly lower CTLp frequency was observed in permissible HLA-A mismatches compared with immunogenic HLA-A mismatches. This finding was not restricted to these healthy individuals; dialysis patients awaiting renal transplantation and a group of potential bone marrow donors demonstrated a similar significantly lower frequency of CTLp directed against permissible mismatches compared with immunogenic mismatches. As the donor anti-recipient CTLpf has been reported to predict the occurrence of GVHD [18], transplantation in case of permissible mismatches might be a way to extend the pool of unrelated bone marrow transplant (BMT) donors. A recent analysis did indeed demonstrate that CTLpf assays were able to discriminate permissible from nonpermissible HLA-A, -B, or -Cw mismatches in case of T-cell depleted BMT [19]. Furthermore, CTLp assays have been demonstrated to be a useful tool (in addition to good anti-HLA antibody screening) for selection of patients that can be successfully transplanted with a repeated HLA mismatch [20]. Because it is impossible to test every possible combination, a hypothesis regarding the relative immunogenicity of alloantigens can only be formulated through the analysis of large data sets. Just as the original concept was validated in two (large) datasets [10], in the current
666
study two independent laboratories have used in vitro assays to validate the concept. An important aspect of the algorithm proposed by Maruya et al. [10] was that the immunogenicity of HLA-A mismatches was also considered in relation to the B-locus specificities (inter-locus), and to the DR-locus specificities (cross-locus) of the recipient. However, in this study group, the number of patients in the immunogenic group was too small (inter, n ⫽ 4, and cross, n ⫽ 3) to enable a proper analysis. Besides analyzing quantitative differences, qualitative differences between CTLp recognizing the permissible mismatches compared with immunogenic mismatches were analyzed. Cytotoxic T cells recognizing their ligands with high avidity, which often are primed cells, do not need CD8 molecules to stabilize the TCR-HLA interaction, in contrast to low avidity cytotoxic T cells [21]. In the present study anti-CD8 antibodies were found to inhibit all HLA-A directed CTLp, irrespective of whether these HLA-A antigens were classified as permissible or immunogenic HLA-A mismatches (data not shown). This is consistent with previous studies reporting a greater inhibition of HLA-A reactive CTLp by anti-CD8 compared with HLA-B reactive CTLp [7]. In conclusion, on the population level the permissible and immunogenic HLA-A mismatches are indeed reflected in the CTL allorepertoire. However, due to the big overlap of the CTLp frequencies in these populations, the permissible or immunogenic nature of a mismatch for a certain patient should be determined on an individual basis.
D. L. Roelen et al.
4.
5.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENTS
This work was supported by The Wellcome Trust, The British Heart Foundation, The Medical Research Council (UK), The European Society for Organ Transplantation, and the Dutch Kidney Foundation. We thank Mr. Gareth Plant for his assistance in our tissue culture facility. We would like to thank all blood donors, all physicians who cooperated in this study, and Eurotransplant. We thank Dr. G.M.T. Schreuder and Dr. F. Koning for their critical comments on the manuscript.
13.
14.
REFERENCES 1. Opelz G: CTS: Effect of HLA matching in 10,000 cyclosporine-treated cadaver kidney transplants. Transpl Proc 19:641, 1987. 2. Takiff H, Cicciarelli J, Yin L, Terasaki PI: Benefit of histocompatibility in cyclosporine-treated recipients of first cadaveric renal transplants and implications for kidney sharing. Transpl Proc 20(Suppl 1):39, 1988. 3. Thorogood J, Persijn GG, Schreuder GMTh, D’Amaro J, Zandvoort FA, Van Houwelingen JC, Van Rood JJ: The effect of HLA matching on kidney graft survival in sep-
15.
16.
17.
arate posttransplantation intervals. Transplantation 50: 146, 1990. Zhang L, Van Bree S, Van Rood JJ, Claas FHJ: The effect of individual HLA-A and -B mismatches on the generation of cytotoxic T lymphocyte precursors. Transplantation 50:1008, 1990. Breur-Vriesendorp B, Vingerhoed J, Van Twuyver E, De Waal LP, Ivanyi P: Frequency analysis of HLA-specific cytotoxic T lymphocyte precursors in humans. Transplantation 51:1096, 1991. Roelen DL, Van Bree FPMJ, Van Beelen E, Schanz U, Van Rood JJ, Claas FHJ: Cytotoxic T lymphocytes (CTLs) against HLA-B antigens are less naive than CTLs against HLA-A antigens. Transplantation 57:446, 1994. Gjertson DW, Terasaki PI, Takemoto S, Mickey MR: National allocation of cadaveric kidneys by HLA matching. N Engl J Med 324:1032, 1991. Hendriks GFJ, Schreuder GMTh, Claas FHJ, D’Amaro J, Persijn GG, Cohen B, Van Rood JJ: HLA-DRw6 and renal allograft rejection. Br Med J 286:85, 1983. Opelz G, Terasaki PI: Studies on the strength of HLA antigens in related donor kidney transplants. Transplantation 24:106, 1977. Maruya E, Takemoto S, Terasaki PI: HLA matching: identification of permissible HLA mismatches. Clin Transpl 511, 1993. Van Rood JJ, Lagaaij EL, Doxiadis I, Roelen D, Persijn G, Claas F: Permissible HLA mismatches, acceptable mismatches, and tolerance: new trends in decision making. Clin Transpl ??:285, 1993. Zhang L, Li S, Vandekerckhove BAE, Termijtelen A, Van Rood JJ, Claas FHJ: Analysis of cytotoxic T cell precursor frequencies directed against individual HLA-A and HLA-B alloantigens. J Immunol Meth 121: 39, 1989. Bouma GJ, van der Meer-Prins PM, van Bree FP, van Rood JJ, Claas FHJ: Determination of cytotoxic T-lymphocyte precursor frequencies using europium labeling as a nonradioactive alternative to labeling with chromium51. Hum Immunol 35:85, 1992. Roelen DL, Datema G, van Bree FPMJ, van Rood JJ, Claas FHJ: Evidence that antibody formation against a certain HLA alloantigen is associated not with a quantitative but with a qualitative change in the cytotoxic T cells recognizing the same antigen. Transplantation 53: 899, 1992. Koning F, Kardol M, Van de Poel J: In Rheinherz EL (ed.): Proceedings of the Second International Workshop on Human Leukocyte Differentiation Antigens. Heidelberg: Springer, 1986. Strijbosch L, Buurman W, Does R, Zinken P, Groenewegen G: Limiting dilution assays. Experimental design and statistical analysis. J Immunol Meth 97:133, 1987. Vereerstraeten P, Dupont E, Andrien M, De Pauw L, Abramowicz D, Goldman M, Kinnaert P: Influence of
CTL and Permissible HLA Mismatches
donor-recipient HLA-DR mismatches and OKT3 prophylaxis on cadaver kidney graft survival. Transplantation 60:253, 1995. 18. Kaminski E, Hows J, Man S, Brookes P, Mackinnon S, Hughes T, Avakian O, Goldman JM, Batchelor JR: Prediction of graft versus host disease by frequency analysis of cytotoxic T cells after unrelated donor bone marrow transplantation. Transplantation 48:608, 1989. 19. Van der Meer A, Joosten I, Schattenberg AV, De Witte TJ, Allebes WA: Cytotoxic T-lymphocyte precursor frequency (CTLp-f) as a tool to distinguish permissible from non-permissible class I mismatches in T-cell depleted
667
allogeneic bone marrow transplantation. Br J Haematol 111:685, 2000. 20. Van Kampen CA, Versteeg-van der Voort Maarschalk MFJ, Roelen DL, Ten Berge RJM, Claas FHJ: Rejection of a kidney transplant does not always lead to priming of cytotoxic T cells against mismatched donor HLA class I antigens. Transplantation 71:869, 2001. 21. MacDonald HR, Glasebrook AL, Bron C, Kelso A, Cerottini JC: Clonal heterogeneity in the functional requirement for Lyt-2/3 molecules on cytolytic T lymphocytes (CTL): possible implications for the affinity of CTL antigen receptors. Immunol Rev 68:89, 1982.
million PBL). The difference was found both in healthy individuals and a population of renal transplant candidates. These results were confirmed by a retrospective analysis of CTLp frequencies performed between partly mismatched unrelated bone marrow donors and their potential recipients. In conclusion, on the population level the permissible and immunogenic HLA-A mismatches are indeed reflected in the CTL allorepertoire. However, due to the big overlap of the CTLp frequencies in these populations, the permissible or immunogenic nature of a mismatch for a particular patient should be determined on an individual basis. Human Immunology 62, 661– 667 (2001). © American Society for Histocompatibility and Immunogenetics, 2001. Published by Elsevier Science Inc. KEYWORDS: HLA; transplantation; cytotoxic T lymphocytes; permissible; immunogenic
PBL PCRSSP SBT
peripheral blood lymphocytes polymerase chain reaction–single-strand polymorphism sequence-based typing
INTRODUCTION Matching for human leukocyte antigens (HLA) is associated with increased graft and patient survival [1]. Additional studies have reported that matching for the products of the individual HLA class I loci does not have
equal importance in this respect. For instance, HLA-A mismatches were found to have less influence on renal allograft survival, compared with HLA-B mismatches [2,
From the Deparment of Immunohaematology and Bloodbank (D.L.R., I.S., S.P.M.J.vB., I.I.N.D., M.O., F.H.J.C.), Leiden University Medical Centre, Leiden, The Netherlands; and the Nuffield Department of Surgery (N.T.Y., P.J.M., K.J.W.), University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
Address reprint requests to: Dr. Dave Roelen, Deparment of Immunohaematology and Bloodbank, Leiden University Medical Centre, PO Box 9600, 2300 RC, Leiden, The Netherlands; Tel: ⫹31 (71) 5263982; Fax: ⫹31 (71) 5216751; E-mail: [email protected]. Received January 26, 2001; revised April 12, 2001; accepted April 17, 2001.
Human Immunology 62, 661– 667 (2001) © American Society for Histocompatibility and Immunogenetics, 2001 Published by Elsevier Science Inc.
0198-8859/01/$–see front matter S0198-8859(01)00263-4
662
D. L. Roelen et al.
TABLE 1 Listing of permissible and immunogenic donor HLA antigens Recipient antigen A1 A2 A3 A9 (23,24) A10 (25,26) A11 A28
Permissible mismatch A3 A1 A1 A3 A3 A3 A1
Immunogenic mismatch
A23 A24 A25 A26 A11 A29 A30 A31 A3 A23 A24 A28 A29 A30 A31 A34 A2 A26 A11 A28 A29 A30 A31 A28 A30 A33 A34 A23 A24 A28 A30 A23 A24 A25 A26 A29 A30 A34 A11 A31
A2 A32 A25 A26 A23 A24 A1 A2 A25 A26 A11 A29 A31 A32 A1 A2 A11 A34 A2 A28 A2 A23 A24 A25 A26 A30 A34
Permissible and immunogenic donor HLA antigens as defined by Maruya et al. [10].
3]. In vitro analyzes reported that this different matching effect is reflected in the cytotoxic T-cell allorepertoire for HLA-A and HLA-B antigens; in general, lower frequencies of cytotoxic T-lymphocyte precursor (CTLp) are found for HLA-A mismatches compared with HLA-B mismatches [4 – 6]. The observation that some mismatched grafts function well for long periods [7, 8] has led to attempts to determine the immunogenicity of individual HLA mismatches in classical transplantation. Analysis of kidney graft survival in donor/recipient combinations with a single HLA mismatch did not provide evidence that any single HLA antigen was more immunogenic than another [9]. However, by analyzing the effect of a donor mismatch in the context of a patient’s own HLA antigens, Maruya et al. [10] could identify permissible mismatches, i.e., cadaver-donor transplants mismatched for a single HLA antigen that had graft survival rates equivalent to zero HLA-A,B,DR mismatched grafts. This study considered the immunogenicity of the mismatch always in the context of the responder’s HLA phenotype. Maruya et al. [10] approached this matter studying by observing living-related donor transplants and defining those mismatch combinations that resulted in ⬎ 85% 1-year graft survival rate as “permissible.” For simplification of the analysis, only one HLA-A,B,DR mismatched kidneys were considered and the study focused on HLA-A and -B antigens that occurred in high frequencies. A separate file of cadaver-donor transplant patients was then divided into those with permissible and those with immunogenic mismatches. After 3 years, kidneys with one permissible mismatch had a similar graft survival to zero-A, B, DR mismatched transplants, whereas those with immunogenic mismatches had significantly lower graft survival rates [10]. These data were confirmed in the Eurotransplant data set [11]. The present study has analyzed whether such permissible and immunogenic mismatches are also reflected in the in vitro cytotoxic T-cell allorepertoire on the population level. If this study can detect a difference on the population level, then it may be of use to analyze the correlation on an individual basis. For this analysis the
terms permissible and immunogenic HLA mismatches are based on the definition of the graft survival data of Maruya et al. [10]. MATERIALS AND METHODS Permissible Mismatches HLA mismatches were classified as permissible or immunogenic mismatches, according to Maruya et al. [10] (Table 1). The current study was restricted to HLA-A mismatches as the immunogenic and detrimental nature of the HLA-A antigens was better defined than that of the HLA-B antigens [10]. In some patients the mismatched HLA-A antigen was a permissible mismatch for one HLA-A antigen in the responder’s HLA phenotype, and at the same time an immunogenic mismatch for the other (e.g., HLA-A2 as a mismatch for an HLA-A1⫹ and A3⫹ individual, whereas HLA-A2 is permissible for HLA-A3 but immunogenic for HLA-A1). The CTLp frequencies in this group were compared with combinations of only permissible and only immunogenic mismatches to analyze the “dominance” of either the immunogenic or permissible mismatch. Subjects and Patients Healthy volunteers were HLA-typed for class I and class II by conventional serologic methods using the standard NIH complement-dependent cytotoxicity assay. The renal patients were typed with low-medium resolution DNA typing (phototyping, in the second center of John Radcliffe Hospital [Oxford, United Kingdom]), whereas the hematologic patients were typed with high resolution DNA typing (polymerase chain reaction–singlestrand polymorphism [PCR-SSP] and sequence-based typing [SBT]). Homozygosity for a particular HLA allel was confirmed by DNA analysis and/or family studies. The renal patients who were analyzed were on hemodialysis for end-stage renal failure and had not previously been transfused or transplanted. These patients (14 in total) were on the waiting list from the Oxford Transplant Center. The patients (20 in total) with a hemato-
CTL and Permissible HLA Mismatches
663
logic disorder were waiting for a unrelated bone marrow transplant. The CTLp frequencies are measured between donors and recipient as part of the selection procedure (graft-versus-host disease [GVHD] direction) according to the Europdonor guidelines. Limiting Dilution Analysis For the analysis of the allospecific CTLp frequencies limiting dilution assays (LDA) were performed as described [12]. Briefly, graded numbers (from 40,000 cells per well to 312 in twofold dilutions) of single cell suspensions of peripheral blood lymphocytes (PBL) were cultured in 24 replicates (96 -well U bottom plates [Costar,Cambridge MA, USA]) with 50,000 irradiated (3000 rad) stimulator cells (PBL) in a total volume of 0.2 ml of RPMI 1640 (Gibco, Paisley, United Kingdom) supplemented with penicillin G (75 U/ml), streptomycin (45 mg/ml), kanamycin sulphate (90 mg/ml), glutamine (2 mM), and 10% heat-inactivated pooled human serum. The test medium contained 20 U/ml interleukin-2 (IL-2; Ortho Pharmaceutical Corporation, Raritan, NJ, USA). Assay I. After 7 days of culture, each well was tested individually for cytolytic activity against 5000 51Crlabeled target cells. The LDA cultures were incubated for 4 h with the target cells; 20 l of supernatant was harvested on a spot-on filtermat (Wallac, Turku, Finland) and the release of 51Cr was assayed in a Betaplate counter (Wallac). Microcultures were considered cytolytic if the observed 51Cr release exceeded the spontaneous release (stimulators only) plus 3 SD. Assay II. After 7 days of culture, each well was tested individually for cytolytic activity against 5000 europium-labeled target cells (as described by Bouma et al. [13]). The LDA cultures were incubated for 4 h with the target cells; 20 l of supernatant was transferred to a 96-well U bottom low-absorbance plate (Nunc, Rockilde, Denmark) and enhancement solution was added. Microcultures were considered cytolytic if the observed europium release exceeded the spontaneous release (stimulators only) plus 3 SD. Cells from the healthy individuals or patients were incubated with HLA-typed stimulator cells. The target cells were selected on the basis of sharing only one HLA-A antigen with the stimulator. This study was performed in two separate transplant centers. Cell-mediated lympholysis (CML) assay I was applied in Oxford (United Kingdom), while the center in Leiden (The Netherlands) used CML assay II. Anti-CD8 Inhibition Studies In order to distinguish high- and low-avidity alloreactive CTLs, LDAs were performed in the absence and presence
FIGURE 1 Cytotoxic T-lymphocyte precursor (CTLp) frequencies directed toward immunogenic human leukocyte antigen (HLA-A; right panel) and toward permissible HLA-A antigens (left panel), as performed in healthy individuals. Horizontal bars represent the median of the group. A statistical difference was found for the CTLp frequencies of the immunogenic HLA-A versus the CTLp frequencies against permissible HLA-A according to the Mann-Whitney test (p ⫽ 0.011).
of antibodies to CD8 as earlier described [14]. The anti-CD8 monoclonal antibody FK18 was used as a 1:400 dilution of ascitic fluid [15]. Before adding the targets, FK18 was added to each well and incubated with the effector cells during 1 h at room temperature. Statistical Analysis Frequencies of CTLp and the 95% confidence intervals were calculated as described [16]. Precursor frequencies were only taken into account if the goodness of fit was ⱕ 15 (jackknife method) to assure single-hit kinetics. The statistical significance of the differences between the several groups was analyzed using the Mann-Whitney test. RESULTS In a first series of experiments, CTLp frequencies (CTLpf) were determined for individual HLA-A alloantigens using PBLs from healthy individuals. Using the established classification of the University of California at Los Angeles (UCLA) Kidney Transplant Registry, the HLA-A mismatches tested were classified as permissible or immunogenic mismatches [10] (see Table 1). Overall, a large range of CTLpf was evident (Figure 1). In a group of 54 allogeneic combinations CTLpf against the permissible HLA-A antigens (n ⫽ 25, mean fre-
664
D. L. Roelen et al.
FIGURE 2 Comparison of the frequencies of cytotoxic Tlymphocyte (CTL) directed toward human leukocyte antigen (HLA-A) in the real permissible group (no conflicts permissible/immunogenic), in the true immunogenic group (no conflicts permissible/immunogenic), and in the combined permissible/immunogenic group as evident in healthy individuals. Horizontal bars represent the median of the group. A statistical difference was found for the differences in CTL precursor (CTLp) frequencies of the only immunogenic HLA-A antigens versus the only permissible HLA-A antigens according to the Mann-Whitney test (p ⫽ 0.002), and also between the only permissible group versus the permissible/immunogenic group (p ⫽ 0.042). No statistical difference was found between the only immunogenic group versus the permissible/immunogenic group (p ⫽ 0.842).
FIGURE 3 Comparison of the frequencies of cytotoxic Tlymphocyte (CTL) directed toward immunogenic and permissible human leukocyte antigen (HLA-A24; left panel) and toward immunogenic and permissible HLA-A1 (right panel) as evident in healthy individuals. Horizontal bars represent the median of the group. A statistical difference was found for the differences in CTL precursor (CTLp) frequencies of the immunogenic HLA-A24 versus the permissible HLA-A24 according to the Mann-Whitney test (p ⫽ 0.024). No statistical difference was evident for the differences in CTLp frequencies of the immunogenic HLA-A1 versus the permissible HLA-A1 (p ⫽ 0.079).
quency 31 CTLp per million PBLs) were significantly lower than those against immunogenic HLA-A antigens (n ⫽ 29, mean frequency 62 CTLp per million PBLs, p ⫽ 0.011). Next, CTLpf was determined in a group of individuals where the mismatched HLA-A antigen was a permissible mismatch for one HLA-A antigen in the responder’s HLA phenotype, while an immunogenic mismatch for the other (e.g., HLA-A2 as a mismatch for a HLA-A1⫹ and A3⫹ individual, where HLA-A2 is permissible for HLA-A3 but immunogenic for HLA-A1) (Figure 2). The mean frequencies in the real permissible group (only permissible; no conflicts permissible/immunogenic) was 8 CTLp/106 PBLs (n ⫽ 13), in the true immunogenic group (only immunogenic; no conflicts permissible/immunogenic) 35 CTLp/106 (n ⫽ 10), and in the permissible/immunogenic group 47 CTLp/106 PBLs (n ⫽ 9). CTLp frequencies in the latter two groups differed significantly from those of the permissible group and were not significantly different from each other, demonstrating that immunogenic mismatches are dominant. In subsequent analysis the data of these two groups have been combined. As some HLA-A mismatches lead to higher CTLpf compared with others (e.g., HLA-A2 induces a high
CTLpf [6]), the study checked whether the individual HLA-A antigens were equally divided among the permissible or immunogenic mismatches, which was indeed the case (data not shown). Another way to circumvent the influence of individual HLA antigens is to compare CTLp frequencies in combinations where the same antigen functions as either a permissible or an immunogenic mismatch. When such an analysis was performed for HLA-A1, HLA-A2, and HLA-A24 antigens, higher CTLp frequencies were evident for immunogenic combinations compared with permissible combinations, although these differences did not reach statistical significance in all combinations (Figure 3). The difference between permissible and immunogenic HLA-A CTLp frequencies in healthy individuals could be confirmed in a second study. Again, CTLpf against permissible HLA-A antigens (mean frequency 28 CTLp per million PBLs, n ⫽ 22) were significantly lower than those against immunogenic HLA-A antigens (mean frequency 59 CTLp per million PBLs, n ⫽ 29) (p ⫽ 0.032, data not shown). A similar analysis in a group of dialysis patients awaiting a renal transplant, indicated a mean frequency of 21 CTLp per million PBLs (n ⫽ 23) in permissible mismatches and a mean frequency of 55 CTLp per mil-
CTL and Permissible HLA Mismatches
FIGURE 4 The combined data of all four groups studied. Frequencies of cytotoxic T-lymphocyte precursor (CTLp) directed toward immunogenic human leukocyte antigens (HLA-A; right panel) and toward permissible HLA-A antigens (left panel) are illustrated. Horizontal bars represent the median of the group. A statistical difference was found for the CTLp frequencies of the immunogenic HLA-A versus the CTLp frequencies against permissible HLA-A according to the Mann-Whitney test (p ⬍ 0.001).
lion PBLs (n ⫽ 28) (p ⫽ 0.003) in immunogenic HLA-A mismatches (data not shown). Finally, we analyzed the results of CTLp tests performed between partially HLA-mismatched unrelated bone marrow donors and their potential recipients, which were performed as part of a donor selection procedure. In case of permissible HLA-A mismatches, 16 CTLp per million PBLs (n ⫽ 10) were found versus 27 CTLp per million PBLs (n ⫽ 10) for immunogenic HLA-A mismatches (p ⫽ 0.045). The combined result of these four groups analyzed together are illustrated in Figure 4 (p ⬍ 0.001). In the present study anti-CD8 antibodies were found to inhibit all HLA-A directed CTLp tested, irrespective of whether these HLA-A antigens were classified as permissible (n ⫽ 17, mean CD8 inhibition 79%) or immunogenic HLA-A mismatches (n ⫽ 31, mean CD8 inhibition 81%; data not shown). DISCUSSION The increasing number of detectable HLA alleles makes selection of an unrelated, HLA completely-matched donor more and more difficult and for many patients even impossible. Because earlier epidemiologic studies did not report any difference in immunogenicity of individual donor HLA mismatches, Maruya et al. [10] were the first to consider the immunogenicity of the donor HLA mismatch in the context of a recipient’s own HLA phenotype. This is a logical method of analysis because the peripheral T-cell repertoire is shaped by self-HLA anti-
665
gens during the processes of thymic selection. Furthermore, with increased knowledge of the molecular structure of the HLA antigens, it is apparent that certain HLA specificities share more epitopes than others, which is another reason why the recipient HLA type will influence the immunogenicity of a particular mismatch. By analysis of the immunogenicity of single HLA mismatches in the context of the patient’s own HLA phenotype, Maruya et al. [10] defined permissible and immunogenic HLA mismatches. Kidney graft survival in case of permissible mismatches was similar to that of completely HLA matched combinations, whereas immunogenic mismatches lead to a significantly poorer graft survival. A similar analysis was performed for the HLA class II antigens by Vereerstraeten et al. [17], which demonstrated differential effects of donor-recipient HLA-DR mismatches on cadaver kidney graft survival. The data of the UCLA registry (Maruya) could be confirmed by an analysis of the Eurotransplant dataset [11]. Survival of grafts with permissible mismatches was similar to zero-mismatched grafts, whereas nonpermissible (or immunogenic) mismatches lead to significantly poorer graft survival. Consequently, we examined whether the in vivo difference between permissible and immunogenic HLA class I antigens is reflected in the in vitro frequency of cytotoxic T-cell precursors directed against the individual HLA antigens. In two separate groups of healthy individuals a significantly lower CTLp frequency was observed in permissible HLA-A mismatches compared with immunogenic HLA-A mismatches. This finding was not restricted to these healthy individuals; dialysis patients awaiting renal transplantation and a group of potential bone marrow donors demonstrated a similar significantly lower frequency of CTLp directed against permissible mismatches compared with immunogenic mismatches. As the donor anti-recipient CTLpf has been reported to predict the occurrence of GVHD [18], transplantation in case of permissible mismatches might be a way to extend the pool of unrelated bone marrow transplant (BMT) donors. A recent analysis did indeed demonstrate that CTLpf assays were able to discriminate permissible from nonpermissible HLA-A, -B, or -Cw mismatches in case of T-cell depleted BMT [19]. Furthermore, CTLp assays have been demonstrated to be a useful tool (in addition to good anti-HLA antibody screening) for selection of patients that can be successfully transplanted with a repeated HLA mismatch [20]. Because it is impossible to test every possible combination, a hypothesis regarding the relative immunogenicity of alloantigens can only be formulated through the analysis of large data sets. Just as the original concept was validated in two (large) datasets [10], in the current
666
study two independent laboratories have used in vitro assays to validate the concept. An important aspect of the algorithm proposed by Maruya et al. [10] was that the immunogenicity of HLA-A mismatches was also considered in relation to the B-locus specificities (inter-locus), and to the DR-locus specificities (cross-locus) of the recipient. However, in this study group, the number of patients in the immunogenic group was too small (inter, n ⫽ 4, and cross, n ⫽ 3) to enable a proper analysis. Besides analyzing quantitative differences, qualitative differences between CTLp recognizing the permissible mismatches compared with immunogenic mismatches were analyzed. Cytotoxic T cells recognizing their ligands with high avidity, which often are primed cells, do not need CD8 molecules to stabilize the TCR-HLA interaction, in contrast to low avidity cytotoxic T cells [21]. In the present study anti-CD8 antibodies were found to inhibit all HLA-A directed CTLp, irrespective of whether these HLA-A antigens were classified as permissible or immunogenic HLA-A mismatches (data not shown). This is consistent with previous studies reporting a greater inhibition of HLA-A reactive CTLp by anti-CD8 compared with HLA-B reactive CTLp [7]. In conclusion, on the population level the permissible and immunogenic HLA-A mismatches are indeed reflected in the CTL allorepertoire. However, due to the big overlap of the CTLp frequencies in these populations, the permissible or immunogenic nature of a mismatch for a certain patient should be determined on an individual basis.
D. L. Roelen et al.
4.
5.
6.
7.
8.
9.
10.
11.
12.
ACKNOWLEDGMENTS
This work was supported by The Wellcome Trust, The British Heart Foundation, The Medical Research Council (UK), The European Society for Organ Transplantation, and the Dutch Kidney Foundation. We thank Mr. Gareth Plant for his assistance in our tissue culture facility. We would like to thank all blood donors, all physicians who cooperated in this study, and Eurotransplant. We thank Dr. G.M.T. Schreuder and Dr. F. Koning for their critical comments on the manuscript.
13.
14.
REFERENCES 1. Opelz G: CTS: Effect of HLA matching in 10,000 cyclosporine-treated cadaver kidney transplants. Transpl Proc 19:641, 1987. 2. Takiff H, Cicciarelli J, Yin L, Terasaki PI: Benefit of histocompatibility in cyclosporine-treated recipients of first cadaveric renal transplants and implications for kidney sharing. Transpl Proc 20(Suppl 1):39, 1988. 3. Thorogood J, Persijn GG, Schreuder GMTh, D’Amaro J, Zandvoort FA, Van Houwelingen JC, Van Rood JJ: The effect of HLA matching on kidney graft survival in sep-
15.
16.
17.
arate posttransplantation intervals. Transplantation 50: 146, 1990. Zhang L, Van Bree S, Van Rood JJ, Claas FHJ: The effect of individual HLA-A and -B mismatches on the generation of cytotoxic T lymphocyte precursors. Transplantation 50:1008, 1990. Breur-Vriesendorp B, Vingerhoed J, Van Twuyver E, De Waal LP, Ivanyi P: Frequency analysis of HLA-specific cytotoxic T lymphocyte precursors in humans. Transplantation 51:1096, 1991. Roelen DL, Van Bree FPMJ, Van Beelen E, Schanz U, Van Rood JJ, Claas FHJ: Cytotoxic T lymphocytes (CTLs) against HLA-B antigens are less naive than CTLs against HLA-A antigens. Transplantation 57:446, 1994. Gjertson DW, Terasaki PI, Takemoto S, Mickey MR: National allocation of cadaveric kidneys by HLA matching. N Engl J Med 324:1032, 1991. Hendriks GFJ, Schreuder GMTh, Claas FHJ, D’Amaro J, Persijn GG, Cohen B, Van Rood JJ: HLA-DRw6 and renal allograft rejection. Br Med J 286:85, 1983. Opelz G, Terasaki PI: Studies on the strength of HLA antigens in related donor kidney transplants. Transplantation 24:106, 1977. Maruya E, Takemoto S, Terasaki PI: HLA matching: identification of permissible HLA mismatches. Clin Transpl 511, 1993. Van Rood JJ, Lagaaij EL, Doxiadis I, Roelen D, Persijn G, Claas F: Permissible HLA mismatches, acceptable mismatches, and tolerance: new trends in decision making. Clin Transpl ??:285, 1993. Zhang L, Li S, Vandekerckhove BAE, Termijtelen A, Van Rood JJ, Claas FHJ: Analysis of cytotoxic T cell precursor frequencies directed against individual HLA-A and HLA-B alloantigens. J Immunol Meth 121: 39, 1989. Bouma GJ, van der Meer-Prins PM, van Bree FP, van Rood JJ, Claas FHJ: Determination of cytotoxic T-lymphocyte precursor frequencies using europium labeling as a nonradioactive alternative to labeling with chromium51. Hum Immunol 35:85, 1992. Roelen DL, Datema G, van Bree FPMJ, van Rood JJ, Claas FHJ: Evidence that antibody formation against a certain HLA alloantigen is associated not with a quantitative but with a qualitative change in the cytotoxic T cells recognizing the same antigen. Transplantation 53: 899, 1992. Koning F, Kardol M, Van de Poel J: In Rheinherz EL (ed.): Proceedings of the Second International Workshop on Human Leukocyte Differentiation Antigens. Heidelberg: Springer, 1986. Strijbosch L, Buurman W, Does R, Zinken P, Groenewegen G: Limiting dilution assays. Experimental design and statistical analysis. J Immunol Meth 97:133, 1987. Vereerstraeten P, Dupont E, Andrien M, De Pauw L, Abramowicz D, Goldman M, Kinnaert P: Influence of
CTL and Permissible HLA Mismatches
donor-recipient HLA-DR mismatches and OKT3 prophylaxis on cadaver kidney graft survival. Transplantation 60:253, 1995. 18. Kaminski E, Hows J, Man S, Brookes P, Mackinnon S, Hughes T, Avakian O, Goldman JM, Batchelor JR: Prediction of graft versus host disease by frequency analysis of cytotoxic T cells after unrelated donor bone marrow transplantation. Transplantation 48:608, 1989. 19. Van der Meer A, Joosten I, Schattenberg AV, De Witte TJ, Allebes WA: Cytotoxic T-lymphocyte precursor frequency (CTLp-f) as a tool to distinguish permissible from non-permissible class I mismatches in T-cell depleted
667
allogeneic bone marrow transplantation. Br J Haematol 111:685, 2000. 20. Van Kampen CA, Versteeg-van der Voort Maarschalk MFJ, Roelen DL, Ten Berge RJM, Claas FHJ: Rejection of a kidney transplant does not always lead to priming of cytotoxic T cells against mismatched donor HLA class I antigens. Transplantation 71:869, 2001. 21. MacDonald HR, Glasebrook AL, Bron C, Kelso A, Cerottini JC: Clonal heterogeneity in the functional requirement for Lyt-2/3 molecules on cytolytic T lymphocytes (CTL): possible implications for the affinity of CTL antigen receptors. Immunol Rev 68:89, 1982.