In allo-stem cell transplantation (SCT), it is unclear whether donor-specific anti-HLA Abs (DSAs) can actually mediate graft rejection or if they are simply surrogate markers for the cellular immunity that causes graft rejection. Here, we first analyzed a case of cord blood allograft rejection in which DSA and cytotoxic T lymphocyte (CTL) specific for donor HLA-B*54:01 were detected at the time of graft rejection. Both the DSA and CTL inhibited colony formation by unrelated bone marrow mononuclear cells sharing HLA-B*54:01, suggesting that the humoral and cellular immune responses were involved in the graft rejection. Interestingly, the DSA and CTL were also detected in cryopreserved pre-transplant patient blood, raising a hypothesis that the presence of anti-HLA Abs could be an indicator for corresponding HLA-specific T cells. We then evaluated the existence of HLA-specific CD8+ T cells in other patient blood specimens having anti-HLA class I Abs. Interferon-γ enzyme-linked immunospot assays clearly confirmed the existence of corresponding HLA-specific T-cell precursors in three of seven patients with anti-HLA Abs. In conclusion, our data demonstrate that integrated humoral and cellular immunity recognizing the same alloantigen of the donor can mediate graft rejection in DSA-positive patients undergoing HLA-mismatched allo-SCT. Further studies generalizing our observation are warranted.
Graft rejection continues to be a significant complication after allo-stem cell transplantation (SCT).1, 2, 3 Recipient-derived cellular immune responses are regarded as the primary contributors of graft rejection.4, 5, 6, 7, 8 Indeed, recipient T cells directed against mismatched donor HLA alleles or minor histocompatibility Ags have been isolated from the peripheral blood of patients at the time of graft rejection.9, 10, 11, 12
Donor-specific anti-HLA Abs (DSAs) have been linked to graft rejection in solid organ transplantation.13, 14, 15, 16 In allo-SCT, however, the importance of humoral immunity-mediated graft rejection is controversial. Although recent registry data have suggested that the presence of pre-transplant DSAs is correlated with an increased risk for graft rejection,17, 18, 19, 20, 21, 22 other reports have shown no association between the presence of DSAs and engraftment.23, 24 In addition, strategies for prevention of Ab-mediated graft rejection, such as plasma exchange, platelet transfusion and rituximab, have been proposed, but their efficacy is limited.25, 26 Thus, it is unclear whether DSAs can actually mediate graft rejection or if they are simply surrogate markers for the cellular immunity that causes graft rejection.27, 28, 29
To investigate the role of DSAs in allo-SCT, we first examined a case of cord blood allograft rejection. DSA and donor HLA-specific cytotoxic T lymphocyte (CTL) recognizing the same alloantigen of the donor were found before and after transplantation, and both of them mediated graft rejection. Moreover, we further analyzed additional patients having anti-HLA Abs and showed that the presence of anti-HLA Abs could provide evidence for the existence of corresponding HLA-specific CD8+ T cells. Although these data are observational and do not allow for general conclusions, this study could explain how allograft rejection occurs after HLA-mismatched allo-SCT and provide new insight into our understanding of the mechanism of graft rejection.
Materials and methods
A 53-year-old man with recurrent AML underwent unrelated cord blood transplantation (CBT). HLA allele types are shown in Table 1. Screening for pre-transplant DSAs was not routinely performed at that time because Japanese health insurance did not cover anti-HLA Ab testing. The preparative regimen consisted of 125 mg/m2 fludarabine and 180 mg/m2 melphalan. The numbers of infused total nuclear cells and CD34+ cells were 2.7 × 107/kg and 0.7 × 105/kg, respectively. The GvHD prophylaxis consisted of tacrolimus and short-term methotrexate. The WBC count increased transiently to 300/μL on day 27, but subsequently decreased to <100/μL (lymphocytes 100%). Graft rejection was diagnosed based on severe bone marrow hypoplasia and a complete loss of donor chimerism on day 34.
Human experiments were performed under protocols approved by the institutional review board of Nagoya University. Human samples were collected with written informed consent in accordance with the Declaration of Helsinki.
Anti-HLA Ab testing
Anti-HLA Abs were detected from patient serum as described previously.30 Anti-HLA Ab positivity was defined as mean fluorescence intensity >1000.
The effect of complement-dependent cytotoxicity (CDC), Ab-dependent cellular cytotoxicity activities of DSAs, and CTLs for engraftment were evaluated by colony-forming assays using MethoCult H4034.31, 32 Colony-forming units for granulocyte-macrophage colonies were counted.
Generation of CTL clones
CTL clones were isolated from a blood sample as described previously.33 Briefly, peripheral blood mononuclear cells (PBMCs) were obtained from the patient on day 34 and cultured in interleukin-2-containing media without stimulator cells, and T-cell clones were isolated by limiting dilution. The T-cell clones were expanded by using a rapid expansion protocol.33
Chromium release assay
A cytotoxicity assay with 51Cr-labeled EBV-transformed lymphoblastoid cells (B-LCLs) was performed as previously described.34 Briefly, B-LCLs were labeled with 51Cr and incubated with CTL clones at various E:T ratios.
Determination of TCR Vβ gene and CDR3
TCR Vβ usage was assessed by reverse transcription-PCR as previously described.35, 36 Briefly, total RNA was extracted from individual CTL clones, and cDNA was synthesized. Reverse transcription-PCR reactions were performed with forward primers specific for different Vβ families and a reverse primer specific for the constant region of TCR-β. The T-cell receptor beta variable (TRBV) gene and complementarity determining region 3 (CDR3) were determined by the international ImMunoGeneTics information system software, IMGT/V-QUEST (http://www.imgt.org/).
HLA blocking assay
Donor B-LCL was incubated with anti-HLA class I Ab or anti-HLA class II Ab for 1 h at 37 °C. CTLs were cultured with the pre-incubated donor B-LCL for 24 h at 37 °C. Interferon (IFN)-γ production was measured in the supernatant by ELISA.
Transfection of COS cells and CTL stimulation assay
COS cells were transfected with HLA cDNAs using the FuGENE 6 Transfection Reagent (Promega, Madison, WI, USA). COS transfectants were co-cultured with CTL clones for 24 h at 37 °C.
PCR specific for the TCR
To determine the presence of the CTL clone-specific TCR rearrangement, semi-nested PCR was performed on cDNA extracted from the pre-transplant patient PBMC and with TCR Vβ-specific primer sets. The sequences of the primers are listed in Supplementary Table S1. PCR products were sequenced, and it was confirmed whether they were identical in sequence to the CTL-specific TCR rearrangement.
TCR-β deep sequencing
TCR-β deep sequencing was performed as previously described.37, 38 Briefly, total RNA was extracted and TRB gene products were amplified through 5’ rapid amplification of cDNA ends (RACE) PCR (Clontech, Palo Alto, CA, USA) followed by sequencing with Roche 454 sequencer (Roche, Basel, Switzerland).
Cell preparation and ELISPOT assay
The enzyme-linked immunospot (ELISPOT) assay was performed as previously described34 using PBMCs collected from patients with anti-HLA class I Abs. CD8+ T cells were isolated from PBMC by CD8 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) and expanded by anti-CD3/CD28 beads (Invitrogen, Carlsbad, CA, USA). CD8+ T cells (5 × 104 per well) were stimulated with HLA-transduced 721.221 cells (5 × 104 per well), and IFN-γ-producing cells were detected.
Detection of DSA at the time of graft rejection
We first examined whether humoral immunity could be responsible for graft rejection in a patient undergoing HLA-mismatched CBT (Table 1). The patient's serum at the time of graft rejection was screened for anti-HLA Abs. An Ab against HLA-B*54:01, which was expressed in donor cells but not in patient cells, was detected (Figure 1a). Abs against HLA-B7 cross-reactive group Ags, which shared the same public epitopes of HLA-B*54:01, were also detected (data not shown). Neither Abs against HLA-Cw*01:02, which was the other mismatched class I donor Ag, nor Abs against matched donor Ags were detected (Figure 1a). Abs against HLA class II Ags were not detected (data not shown). These results show that the patient had DSA against HLA-B*54:01 (referred to herein as DSAB*54:01) at graft rejection.
DSA inhibited colony formation of hematopoietic stem cell
To investigate the possibility that DSAB*54:01 inhibited hematopoietic stem cell engraftment, CDC and Ab-dependent cellular cytotoxicity activities were evaluated by colony-forming assays. CDC activities of DSAB*54:01 were assessed by the colony growth of unrelated HLA-B*54:01-positive bone marrow mononuclear cells (BMMNCs) pre-cultured with patient serum and rabbit complement or pooled human serum as a source of complement. Colony-forming units for granulocyte-macrophage were not inhibited by rabbit complement alone, patient serum alone or patient serum with rabbit complement (Figure 1b). Similarly, colony-forming unit for granulocyte-macrophage colony formation was not suppressed by pooled human serum (Figure 1c). Thereby, CDC activities of DSAB*54:01 were not detected by colony-forming assays, although the possibility of a contribution of CDC could not be entirely excluded. DSA-mediated Ab-dependent cellular cytotoxicity activities were assessed by using unrelated HLA-B*54:01-positive BMMNC pre-cultured with patient serum and natural killer cells. The DSAB*54:01 significantly inhibited colony formation when cultured with patient serum and natural killer cells (Figure 1d), whereas colony formation was not inhibited by natural killer cells alone or anti-HLA Ab-negative serum with natural killer cells. These findings suggest that the DSAB*54:01 impaired HLA-B*54:01-positive donor engraftment through at least Ab-dependent cellular cytotoxicity activities in vitro.
Isolation of CTL clones
We next determined whether cellular immunity was also responsible for graft rejection in the patient after CBT. By limiting dilution, two CTL clones, termed CTL#1 and #2, were isolated from the patient PBMC at graft rejection. Both CTL clones had cytotoxicity against B-LCL from the donor but not B-LCL from the patient (Figure 2a). Short tandem repeat analysis showed that both the CTL#1 and #2 were of patient origin (data not shown). Flow cytometric analysis revealed that CTL#1 was CD3+CD4−CD8+ and that CTL#2 was CD3+CD4+CD8− (Table 2). The TCR Vβ repertoire and CDR3 were determined. CTL#1 and #2 used different TRBV genes that shared 75% of their CDR3 sequences (Table 2). Moreover, TCR-β deep sequencing confirmed that the CTL#1 and #2 represented 9.9% and 1.3% of T cells, respectively, at graft rejection (Figure 2b and Supplementary Table S2). These data show that the patient had donor-reactive CTLs at graft rejection and suggest that at least these two independent CTL clones had the possibility of being involved in immunologic graft rejection.
CTL clones recognized the mismatched HLA molecules
To determine whether the CTL clones recognized mismatched donor HLA molecules, an HLA blocking assay was performed with monoclonal Ab specific for HLA class I or class II, and then a CTL stimulation assay was performed using COS cells transfected with mismatched HLA cDNA constructs. The CTL#1 showed HLA class I-restricted recognition of donor B-LCL (Figure 2c), and mismatched HLA class I molecules were studied. IFN-γ production of CTL#1 was significantly increased when stimulated by transfectants expressing HLA-B*54:01 (Figure 2d). These results show that the CTL#1 recognized the mismatched HLA-B*54:01. The CTL#2 showed HLA class II-restricted recognition of donor B-LCL (Figure 2e), and a CTL stimulation assay using a panel of B-LCLs derived from unrelated individuals was performed to study mismatched class II HLA molecules (Supplementary Table S3). Only B-LCLs that shared HLA-DRB1*15:02 (L12, L25, L38, L56, and L147) stimulated IFN-γ production of CTL#2. As HLA class II molecules consist of α and β chains, COS cells transfected with both HLA-DRA1 cDNA obtained from the donor and HLA-DRB1*15:02 were used as stimulators. IFN-γ production of the CTL#2 was significantly increased when stimulated by transfectants expressing HLA-DRB1*15:02 with either HLA-DRA1*01:01 or 01:02 (Figure 2f). Together, these data demonstrate that although the sequences of CTL#1 and #2 were quite similar (Table 2), they recognized the different molecules of mismatched HLA-B*54:01 and DRB1*15:02, respectively, as alloantigens.
CTL clones inhibited colony formation of hematopoietic stem cell
To investigate the possibility that the CTL#1 and #2 inhibited hematopoietic stem cell engraftment, colony-forming assays with either HLA-B*54:01-positive or DRB1*15:02-positive unrelated BMMNC co-cultured with the CTL#1 or #2 were performed. The CTL#1 inhibited colony formation by colony-forming units for granulocyte-macrophage from unrelated BMMNC positive for HLA-B*54:01 (Figure 2g). Similarly, the CTL#2 inhibited colony formation by unrelated BMMNCs positive for HLA-DRB1*15:02 (Figure 2h). These findings suggest that both CTL clones impaired HLA-B*54:01 and DRB1*15:02-positive donor engraftment.
The presence of DSAB*54:01 and CTL#1 before transplantation
We sought to determine whether DSAB*54:01 was present before transplantation. The DSAB*54:01 was detected in cryopreserved pre-transplant patient serum with a higher mean fluorescence intensity as compared with after transplantation (Figures 1a and 3a), which may be explained by absorption of DSAB*54:01 by HLA-B*54:01-positive cord blood and platelet transfusion. Abs against HLA class II Ags were not detected (data not shown). We further determined whether the CTL#1 and #2 developed before transplantation by using semi-nested PCR assays specific for their uniquely rearranged TCR Vβ chains and CDR3s (Table 2). CTL#1-specific PCR products were detected by amplification of cDNA from pre-transplant patient PBMC (Figure 3b), demonstrating that the CTL#1 developed in the patient before CBT. In contrast, no amplification was detected when tested with the CTL#2-specific primer, suggesting that the CTL#2 was not generated before CBT (Figure 3c). In addition, the CTL#1 clonotype was detected and comprised 0.007% of T cells before transplantation, whereas the CTL#2 clonotype was undetectable by using TCR-β deep sequencing (Figure 3d and Supplementary Table S4). Based on these findings, we assume that the patient had the DSAB*54:01 and CTL#1 before CBT, both of which were directed against the same mismatched HLA-B*54:01 molecule of the donor.
Detection of HLA-specific T cells in patients having anti-HLA Abs
While pre-transplant anti-HLA Abs are now routinely screened, it is usually difficult in clinical practice to evaluate HLA-specific T-cell reactivities before transplantation. Based on the results of the patient with cord blood allograft rejection, we hypothesized that the presence of anti-HLA Abs could be an indicator for corresponding HLA-specific T cells. Hence, we further evaluated the existence of HLA-specific CD8+ T cells in seven additional patients having anti-HLA class I Abs by using an IFN-γ ELISPOT assay. These patient characteristics are shown in Supplementary Table S5. CD8+ T cells were isolated from patient blood and tested for corresponding HLA reactivity after stimulation with HLA-transduced 721.221 cells. In patient #2, more IFN-γ-producing CD8+ T cells against Ab-positive HLA-A*02:01 molecule were detected as compared with those against autologous HLA-A*24:02 and irrelevant HLA-B*51:01 molecules (Figure 4a). Similarly, for patient #3 and #4, more IFN-γ-producing CD8+ T cells against Ab-positive HLA molecules were detected as compared with autologous and irrelevant HLA molecules, although the number of spots against the alloantigen corresponding to the Ab was not higher than the number of spots against self or irrelevant Ag in patient #2 (Figures 4b and c). Increased IFN-γ responses to Ab-positive HLA molecules by CD8+ T cells were not detected in the other four patients (data not shown). Taken together, CD8+ T cells specific for corresponding HLA molecules were detected in three of seven patients with anti-HLA class I Abs. Moreover, none of the alloantigens we analyzed were preferentially recognized by CD8+ T cells in three anti-HLA Ab-negative patients (Figures 4d–f). Thus, these data show that the presence of anti-HLA Abs can provide evidence for the existence of corresponding HLA-specific CD8+ T cells.
In this study, we first analyzed the recipient-derived immune responses to mismatched HLA of the donor in a patient with graft rejection after HLA-mismatched CBT. Interestingly, the donor HLA-B*54:01 was targeted by the DSA and CTL, both of which were generated before transplantation. The mean fluorescence intensity for Ab to HLA-B*54:01 was lower after transplantation as compared with before transplantation, and the CTL expanded approximately 1000 times after transplantation. We then evaluated the existence of corresponding HLA-reactive T cells in seven additional patients having anti-HLA Abs and confirmed the existence of donor-reactive CTLs in three patients.
The role of DSAs in allo-SCT has been controversial. Moreover, whether or not DSAs can directly cause graft rejection is unknown.6, 27 Although there were early reports demonstrating that serum from HLA-sensitized patients inhibited bone marrow colony growth in soft agar,39 the effect of DSAs was not fully confirmed because of the low sensitivity methods for anti-HLA Ab testing. With improvement in anti-HLA Ab detection techniques, HLA allele-specific Abs are now distinguishable by highly sensitive bead-based assays.40 The results of this study clearly show that the allele-level DSA had direct cytolytic activity and impaired colony growth by unrelated BMMNC in vitro.
Causes of alloimmunization include pregnancies, blood transfusion and allogeneic transplantation,29 but the mechanism by which alloreactive Abs and T cells are produced remains unclear. Generally, IgG Ab production by B cells is dependent on sufficient help from Ag-specific T cells,41 and this study examined only HLA IgG class Abs, suggesting the potential recognition of the HLA-B*54:01 Ag by patient helper T cells.42 In this study, the patient had the HLA-B*54:01-specific DSA (DSAB*54:01) and CTL (CTL#1) before CBT. Therefore, it could be possible that the DSAB*54:01 and CTL#1 were generated at the same time when the patient was exposed to HLA-B*54:01 Ag (for example, due to blood transfusion) before transplantation.
Naive T cells do not produce IFN-γ within the first 24 h of Ag stimulation, whereas primed memory T cells can produce effector cytokines within several hours after Ag challenge.43 We demonstrated increased IFN-γ responses to corresponding HLA molecules by CD8+ T cells from HLA Ab-positive patients after only a short incubation period, suggesting more corresponding HLA-specific primed precursor T cells as compared with irrelevant HLA-reactive T cells. These findings illustrate that the presence of DSAs not only means a direct deleterious effect on donor cells, but it also reflects the presence of CTLs that target the corresponding HLA molecules. This study did not investigate alloreactive naive T cells, although this subset has been shown to contribute significantly to alloreactivity in allo-SCT. Given the difficulty in detecting HLA-specific CTLs in pre-transplant patient blood, in contrast to the ease of screening for DSAs, our results may suggest that, in addition to reduction of DSAs, conditioning regimens to efficiently suppress patient T cells are required when avoiding donors with HLA Ags corresponding to anti-HLA Abs is difficult.44
Recipient-derived CTLs against mismatched donor HLA or minor histocompatibility Ags have been presumed to be a major cause of graft rejection.6, 7 However, only a few studies have demonstrated the direct association of CTL clones and graft rejection because of the difficulty of isolating CTLs from patient blood after graft rejection.9, 10, 12 We have previously reported that, in CBT, a mismatched class I HLA-specific CTL clone was involved in graft rejection.11 This study demonstrated that HLA-DR Ags were also target molecules for CTLs to mediate graft rejection after CBT. This finding is consistent with early reports that the majority of hematopoietic progenitor cells co-express HLA-DR45, 46 and that CD4+ T cells recognizing mismatched HLA-DR Ag can be associated with graft failure.47
Recently, some animal studies suggest that humoral immunity is the major barrier to engraftment,48, 49 although cellular immunity has been recognized as the primary mechanism of rejection.7 In this study, we could not determine whether humoral or cellular immunity would be a dominant barrier to engraftment. However, based on our study, we could postulate that both humoral and cellular immune responses are responsible at least in part for graft rejection.
In conclusion, the results of this study that used a sample obtained from a case of allograft rejection demonstrate that integrated humoral and cellular immune responses mediate cord blood allograft rejection through targeting mismatched HLA of the donor. Alloreactive DSAs certainly have important roles in the graft rejection of HLA-mismatched allo-SCT, both mediating direct cytotoxicity and reflecting the presence of CTLs. As this study is observational, more sensitive and specific methods to detect HLA-specific memory T cells with more patient samples are needed to clarify whether the presence of DSAs could be an indicator of donor HLA-specific T cells.
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We thank Chika Wakamatsu and Yoko Matsuyama for their technical assistance. This work was supported in part by a grant (H25-Immunology-104 and H26-Immunology-106) from the Ministry of Health, Labor and Welfare, Japan and a Grant-in-Aid for Scientific Research (no. 23591415) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
RH and M Murata designed the study, performed the primary experiments, analyzed the data and wrote the paper. KS and M Murase prepared clinical samples, performed research and analyzed the data. RS, TG, KW, NI, HO and YA performed research. SK and KM prepared clinical samples. ST, HK, T Nishida and T Naoe contributed to the discussion and helped write the paper.
The authors declare no conflict of interest.
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Hanajiri, R., Murata, M., Sugimoto, K. et al. Integration of humoral and cellular HLA-specific immune responses in cord blood allograft rejection. Bone Marrow Transplant 50, 1187–1194 (2015) doi:10.1038/bmt.2015.119
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