Graft Source

Integration of humoral and cellular HLA-specific immune responses in cord blood allograft rejection

Article metrics

Abstract

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.

Introduction

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

Patient

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.

Table 1 HLA typing of the patient and donor

Human samples

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.

Colony-forming assay

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.

Results

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.

Figure 1
figure1

Inhibition of colony-forming unit for granulocyte-macrophage (CFU-GM) colonies by DSAB*54:01. (a) Detection of anti-HLA Abs. Patient's serum at the time of graft rejection was screened for class I and class II HLA Abs with LABScreen PRA (One Lambda, Canoga Park, CA, USA). A sample with positive screening was further evaluated to determine HLA specificities by using a LABScreen Single Antigen kit (One Lambda). Anti-HLA Ab positivity was defined as the presence of Abs with mean fluorescence intensity (MFI)>1000. The results shown are representative of a single Ag assay. Abs against HLA class II Ags were not detected. (b, c) The effect of CDC activities of DSAB*54:01 was evaluated by colony-forming assay. Unrelated HLA-B*54:01-positive BMMNC (1 × 104) were incubated with the patient serum for 1.5 h at 37 °C. After washing, the cells were incubated with (b) rabbit complement (One Lambda) or (c) pooled human serum as a source of complement for 1 h at 37 °C, and then they were seeded in MethoCult H4034 (Stem Cell Technologies, Vancouver, Canada). CFU-GM colonies were counted on day 14 of incubation. (d) The effect of Ab-dependent cellular cytotoxicity (ADCC) activities of DSAB*54:01 was evaluated by colony-forming assay. Unrelated HLA-B*54:01-positive BMMNC (1 × 104) were incubated with the patient serum and natural killer (NK) cells (3 × 104) for 6 h at 37 °C, and they were seeded in MethoCult. NK cells were isolated from the patient PBMC by using an NK Cell Isolation kit (Miltenyi Biotech). CFU-GM colonies were counted on day 14 of incubation. The ratios of CFU-GM number relative to control (BMMNC alone) are presented. Data are representative of at least two independent experiments with the means and s.e. of triplicate determinations. DSA+ stands for DSAB*54:01-positive serum (patient serum), and DSA− stands for anti-HLA Ab-negative serum (volunteer donor serum). There are significant differences in CFU-GM colony formation between ‘NK cells+DSA-positive serum’ and ‘no NK cells (control)’, ‘NK cells only’ and ‘NK cells+DSA-negative serum’ groups (*P<0.01: unpaired Student’s t-test). Normal distribution of the data was confirmed using the Kolmogorov–Smirnov test. An F-test was used to test for equal variance.

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+CD4CD8+ 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.

Figure 2
figure2

HLA specificity of CTL clones and inhibition of colony-forming unit for granulocyte-macrophage (CFU-GM) colonies by the CTL clones. (a) B-LCLs (2 × 103 per well) from the donor and patient were used as target cells for the CTL clones. Cytotoxicity was determined by a chromium release assay. Percent-specific lysis was calculated as ((experimental c.p.m.–spontaneous c.p.m.)/(maximum c.p.m.–spontaneous c.p.m.)) × 100. Specific lysis is shown as the mean of triplicate cultures at various E:T ratios. (b) Clonotype distribution plots of TCR-β sequences from patient PBMC obtained at the time of graft rejection. Total RNA was extracted from patient PBMC and TRB gene products were amplified through 5’ RACE PCR. Amplified products were sequenced with a Roche 454 sequencer. Sequence data were submitted online to IMGT/HighV-QUEST (http://www.imgt.org) and CDR3-β amino-acid sequences were determined. The number of reads was 38 166. Each dot represents a distinct CDR3-β amino-acid sequence. The CTL#1 was the third most frequent clonotype, and the CTL#2 was the eighth most frequent clonotype (Supplementary Table S2). (c, e) B-LCL (5 × 103) of the donor was pre-cultured with either anti-HLA class I Ab (100 μg/mL; W6/32, BioLegend, San Diego, CA, USA) or class II Ab (200 μg/mL; TU39, BD Bioscience, San Jose, CA, USA), and stimulated IFN-γ production by the CTL#1 (c) or CTL#2 (e). (d, f) COS cells transfected with various HLA cDNA constructs were co-cultured with the CTL#1 (d) or CTL#2 (f). HLA-B*54:01, Cw*01:02 and DRB1*15:02 were mismatched donor Ags. IFN-γ production was measured in the supernatant by ELISA using a plate reader equipped with a 650 nm filter. (g, h) The effect of CTLs for engraftment were evaluated by colony-forming assays. Unrelated HLA-B*54:01-positive (g) and DRB1*15:02-positive (h) BMMNCs (1 × 104) were co-cultured with the CTL#1 and #2, respectively, at various E:T ratios for 16 h at 37 °C, and CFU-GM colonies were counted. CTL#2 and CTL#1 were used as control CTL for (g) and (h), respectively. The ratios of the CFU-GM number relative to control (BMMNC alone) are presented. Data are representative of at least two independent experiments with the means and s.e. of triplicate determinations. Significant differences in the inhibition of CFU-GM compared with the control group are shown (*P<0.05, **P<0.01 and ***P<0.001: unpaired Student’s t-test). No routine authentication of cell line or routine mycoplasma testing was performed.

Table 2 Characteristics and clonotypes of isolated CTL clones

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.

Figure 3
figure3

Presence of DSAB*54:01 and CTL#1 before transplantation. (a) Detection of anti-HLA Abs. Cryopreserved pre-transplant patient serum was screened for class I and class II HLA Abs with LABScreen PRA. A sample with positive screening was further evaluated to determine HLA specificities by using a LABScreen Single Antigen kit. Anti-HLA Ab positivity was defined as the presence of Abs with mean mean fluorescence intensity (MFI)>1000. The results shown are representative of a single Ag assay. Abs against HLA class II Ags were not detected. (b, c) Detection of lymphocytes with the CTL#1 (b) or CTL#2 (c) clone-specific TCR in pre-transplant patient PBMC. Semi-nested PCR was performed on cDNA with primer sets specific for the uniquely rearranged TCR Vβ and CDR3 of the CTL clones. cDNA from the CTL clones was used as a positive control. cDNAs prepared from unrelated healthy volunteers were used as negative controls. PCR to β-actin was used as an internal control in each assay. Data are representative of at least two independent experiments. (d) Clonotype distribution plots of TCR-β sequences from patient PBMC obtained at pre-transplant. The number of reads was 41 267. Each dot represents a distinct CDR3-β amino-acid sequence. The CTL#1 was the 1197th most frequent clonotype, and the CTL#2 was undetectable (Supplementary Table S4).

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.

Figure 4
figure4

The existence of HLA-specific T cells in patients with anti-HLA Abs. (ac) Results of ELISPOT assay for three patients (patients #2, #3 and #4) with anti-HLA Abs are shown. Responder CD8+ T cells were plated in 96-well MultiScreen-IP filter plates (Millipore, Billerica, MA, USA) coated with anti-human IFN-γ Ab (1-D1K, Mabtech, Nacka, Sweden) and tested against stimulator cells. HLA class I-deficient 721.221 cells transduced with various HLA class I cDNAs were used as stimulator cells. The plates were incubated for 16 h at 37 °C without interleukin-2, washed and incubated with biotinylated anti-human IFN-γ Ab (7-B6-1, Mabtech) for 2 h at room temperature. After addition of streptavidin (Fitzgerald, Acton, MA, USA), the plates were developed with a 3-amino-9-ethylcarbozol substrate kit (Vector Laboratories, Burlingame, CA, USA). The resulting spots were automatically counted by using an ELIPHOTO Counter (Minerva Tech, Tokyo, Japan). The mean number of spots was calculated after subtraction of the number of spots obtained with medium alone. Each bar represents the mean and s.e. of spots in triplicate wells. Data are representative of at least three independent experiments. (a) Patient #2 with HLA-A*24:02 was positive for anti-HLA-A*02:01 Ab (mean fluorescence intensity (MFI), 1600) and negative for anti-HLA-B*51:01 Ab. (b) Patient #3 with HLA-A2 was positive for anti-HLA-B*51:01 Ab (MFI, 13 233) and negative for anti-HLA-B*07:02 Ab. (c) Patient #4 with HLA-A2 was positive for anti-HLA-B*07:02 Ab (MFI, 1391) and negative for anti-HLA-A*24:02 Ab. Significant differences in the numbers of IFN-γ-producing T cells against Ab-positive HLA compared with those against autologous HLA and Ab-negative HLA are shown (*P<0.05: unpaired Student’s t-test). (df) Results of ELISPOT assay for three patients without anti-HLA Abs (HLA Ab-negative patients #9, #10, and #11) are shown. (d) Ab-negative patient #9 had HLA-A*24:02 and B*07:02. (e) Ab-negative patient #10 had HLA-B*07:02. (f) Ab-negative patient #11 had HLA-A*24:02.

Discussion

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.

References

  1. 1

    Olsson R, Remberger M, Schaffer M, Berggren DM, Svahn BM, Mattsson J et al. Graft failure in the modern era of allogeneic hematopoietic SCT. Bone Marrow Transplant 2013; 48: 537–543.

  2. 2

    Rocha V, Labopin M, Sanz G, Arcese W, Schwerdtfeger R, Bosi A et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 2004; 351: 2276–2285.

  3. 3

    Laughlin MJ, Eapen M, Rubinstein P, Wagner JE, Zhang MJ, Champlin RE et al. Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 2004; 351: 2265–2275.

  4. 4

    Bierer BE, Emerson SG, Antin J, Maziarz R, Rappeport JM, Smith BR et al. Regulation of cytotoxic T lymphocyte-mediated graft rejection following bone marrow transplantation. Transplantation 1988; 46: 835–839.

  5. 5

    Kernan NA, Flomenberg N, Dupont B, O’Reilly RJ . Graft rejection in recipients of T-cell-depleted HLA-nonidentical marrow transplants for leukemia. Identification of host-derived antidonor allocytotoxic T lymphocytes. Transplantation 1987; 43: 842–847.

  6. 6

    Mattsson J, Ringden O, Storb R . Graft failure after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2008; 14 (Supplement 1): 165–170.

  7. 7

    Storb R . B cells versus T cells as primary barrier to hematopoietic engraftment in allosensitized recipients. Blood 2009; 113: 1205.

  8. 8

    Voogt PJ, Fibbe WE, Marijt WA, Goulmy E, Veenhof WF, Hamilton M et al. Rejection of bone-marrow graft by recipient-derived cytotoxic T lymphocytes against minor histocompatibility antigens. Lancet 1990; 335: 131–134.

  9. 9

    Fleischhauer K, Kernan NA, O’Reilly RJ, Dupont B, Yang SY . Bone marrow-allograft rejection by T lymphocytes recognizing a single amino acid difference in HLA-B44. N Engl J Med 1990; 323: 1818–1822.

  10. 10

    Fleischhauer K, Zino E, Mazzi B, Sironi E, Servida P, Zappone E et al. Peripheral blood stem cell allograft rejection mediated by CD4(+) T lymphocytes recognizing a single mismatch at HLA-DP beta 1*0901. Blood 2001; 98: 1122–1126.

  11. 11

    Narimatsu H, Murata M, Terakura S, Sugimoto K, Naoe T . Potential role of a mismatched HLA-specific CTL clone developed pre-transplant in graft rejection following cord blood transplantation. Biol Blood Marrow Transplant 2008; 14: 397–402.

  12. 12

    Pei J, Akatsuka Y, Anasetti C, Lin MT, Petersdorf EW, Hansen JA et al. Generation of HLA-C-specific cytotoxic T cells in association with marrow graft rejection: analysis of alloimmunity by T-cell cloning and testing of T-cell-receptor rearrangements. Biol Blood Marrow Transplant 2001; 7: 378–383.

  13. 13

    Eng HS, Bennett G, Chang SH, Dent H, McDonald SP, Bardy P et al. Donor human leukocyte antigen specific antibodies predict development and define prognosis in transplant glomerulopathy. Hum Immunol 2011; 72: 386–391.

  14. 14

    Reinsmoen NL, Nelson K, Zeevi A . Anti-HLA antibody analysis and crossmatching in heart and lung transplantation. Transpl Immunol 2004; 13: 63–71.

  15. 15

    Terasaki PI . A personal perspective: 100-year history of the humoral theory of transplantation. Transplantation 2012; 93: 751–756.

  16. 16

    Arias M, Rush DN, Wiebe C, Gibson IW, Blydt-Hansen TD, Nickerson PW et al. Antibody-mediated rejection: analyzing the risk, proposing solutions. Transplantation 2014; 98 (Suppl 3): S3–S21.

  17. 17

    Ciurea SO, Thall PF, Wang X, Wang SA, Hu Y, Cano P et al. Donor-specific anti-HLA Abs and graft failure in matched unrelated donor hematopoietic stem cell transplantation. Blood 2011; 118: 5957–5964.

  18. 18

    Costa LJ, Moussa O, Bray RA, Stuart RK . Overcoming HLA-DPB1 donor specific antibody-mediated haematopoietic graft failure. Br J Haematol 2010; 151: 94–96.

  19. 19

    Cutler C, Kim HT, Sun L, Sese D, Glotzbecker B, Armand P et al. Donor-specific anti-HLA antibodies predict outcome in double umbilical cord blood transplantation. Blood 2011; 118: 6691–6697.

  20. 20

    Ruggeri A, Rocha V, Masson E, Labopin M, Cunha R, Absi L et al. Impact of donor-specific anti-HLA antibodies on graft failure and survival after reduced intensity conditioning-unrelated cord blood transplantation: a Eurocord, Societe Francophone d’Histocompatibilite et d’Immunogenetique (SFHI) and Societe Francaise de Greffe de Moelle et de Therapie Cellulaire (SFGM-TC) analysis. Haematologica 2013; 98: 1154–1160.

  21. 21

    Spellman S, Bray R, Rosen-Bronson S, Haagenson M, Klein J, Flesch S et al. The detection of donor-directed, HLA-specific alloantibodies in recipients of unrelated hematopoietic cell transplantation is predictive of graft failure. Blood 2010; 115: 2704–2708.

  22. 22

    Takanashi M, Atsuta Y, Fujiwara K, Kodo H, Kai S, Sato H et al. The impact of anti-HLA antibodies on unrelated cord blood transplantations. Blood 2010; 116: 2839–2846.

  23. 23

    Brunstein CG, Noreen H, DeFor TE, Maurer D, Miller JS, Wagner JE . Anti-HLA antibodies in double umbilical cord blood transplantation. Biol Blood Marrow Transplant 2011; 17: 1704–1708.

  24. 24

    Detrait M, Dubois V, Sobh M, Morisset S, Tedone N, Labussiere H et al. Impact of anti-HLA antibodies on allogeneic hematopoietic stem cell transplantation outcomes after reduced-intensity conditioning regimens. Exp Hematol 2012; 40: 792–799.

  25. 25

    Ciurea SO, de Lima M, Cano P, Korbling M, Giralt S, Shpall EJ et al. High risk of graft failure in patients with anti-HLA antibodies undergoing haploidentical stem-cell transplantation. Transplantation 2009; 88: 1019–1024.

  26. 26

    Ottinger HD, Rebmann V, Pfeiffer KA, Beelen DW, Kremens B, Runde V et al. Positive serum crossmatch as predictor for graft failure in HLA-mismatched allogeneic blood stem cell transplantation. Transplantation 2002; 73: 1280–1285.

  27. 27

    Focosi D, Zucca A, Scatena F . The role of anti-HLA antibodies in hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2011; 17: 1585–1588.

  28. 28

    Nordlander A, Uhlin M, Ringden O, Kumlien G, Hauzenberger D, Mattsson J . Immune modulation to prevent antibody-mediated rejection after allogeneic hematopoietic stem cell transplantation. Transpl Immunol 2011; 25: 153–158.

  29. 29

    Yoshihara S, Taniguchi K, Ogawa H, Saji H . The role of HLA antibodies in allogeneic SCT: is the ‘type-and-screen’ strategy necessary not only for blood type but also for HLA? Bone Marrow Transplant 2012; 47: 1499–1506.

  30. 30

    Yoshihara S, Maruya E, Taniguchi K, Kaida K, Kato R, Inoue T et al. Risk and prevention of graft failure in patients with preexisting donor-specific HLA antibodies undergoing unmanipulated haploidentical SCT. Bone Marrow Transplant 2012; 47: 508–515.

  31. 31

    Barge AJ, Johnson G, Witherspoon R, Torok-Storb B . Antibody-mediated marrow failure after allogeneic bone marrow transplantation. Blood 1989; 74: 1477–1480.

  32. 32

    Fang JP, Xu LH, Yang XG, Wu YF, Weng WJ, Xu HG . Panel reactive antibody in thalassemic serum inhibits proliferation and differentiation of cord blood CD34+ cells in vitro. Pediatr Hematol Oncol 2009; 26: 338–344.

  33. 33

    Murata M, Warren EH, Riddell SR . A human minor histocompatibility antigen resulting from differential expression due to a gene deletion. J Exp Med 2003; 197: 1279–1289.

  34. 34

    Sugimoto K, Murata M, Terakura S, Naoe T . CTL clones isolated from an HLA-Cw-mismatched bone marrow transplant recipient with acute graft-versus-host disease. J Immunol 2009; 183: 5991–5998.

  35. 35

    Kato T, Terakura S, Murata M, Sugimoto K, Murase M, Iriyama C et al. Escape of leukemia blasts from HLA-specific CTL pressure in a recipient of HLA one locus-mismatched bone marrow transplantation. Cell Immunol 2012; 276: 75–82.

  36. 36

    Puisieux I, Even J, Pannetier C, Jotereau F, Favrot M, Kourilsky P . Oligoclonality of tumor-infiltrating lymphocytes from human melanomas. J Immunol 1994; 153: 2807–2818.

  37. 37

    van Heijst JW, Ceberio I, Lipuma LB, Samilo DW, Wasilewski GD, Gonzales AM et al. Quantitative assessment of T cell repertoire recovery after hematopoietic stem cell transplantation. Nat Med 2013; 19: 372–377.

  38. 38

    Li S, Lefranc MP, Miles JJ, Alamyar E, Giudicelli V, Duroux P et al. IMGT/HighV QUEST paradigm for T cell receptor IMGT clonotype diversity and next generation repertoire immunoprofiling. Nat Commun 2013; 4: 2333.

  39. 39

    Barrett AJ, Faille A, Saal F, Balitrand N, Gluckman E . Marrow graft rejection and inhibition of growth in culture by serum in aplastic anaemia. J Clin Pathol 1978; 31: 1244–1248.

  40. 40

    Brand A, Doxiadis IN, Roelen DL . On the role of HLA antibodies in hematopoietic stem cell transplantation. Tissue Antigens 2013; 81: 1–11.

  41. 41

    Parker DC . T cell-dependent B cell activation. Annu Rev Immunol 1993; 11: 331–360.

  42. 42

    Conlon TM, Saeb-Parsy K, Cole JL, Motallebzadeh R, Qureshi MS, Rehakova S et al. Germinal center alloantibody responses are mediated exclusively by indirect-pathway CD4 T follicular helper cells. J Immunol 2012; 188: 2643–2652.

  43. 43

    Ott PA, Berner BR, Herzog BA, Guerkov R, Yonkers NL, Durinovic-Bello I et al. CD28 costimulation enhances the sensitivity of the ELISPOT assay for detection of antigen-specific memory effector CD4 and CD8 cell populations in human diseases. J Immunol Methods 2004; 285: 223–235.

  44. 44

    Koyama M, Hashimoto D, Nagafuji K, Eto T, Ohno Y, Aoyama K et al. Expansion of donor-reactive host T cells in primary graft failure after allogeneic hematopoietic SCT following reduced-intensity conditioning. Bone Marrow Transplant 2014; 49: 110–115.

  45. 45

    Ottmann OG, Nocka KH, Moore MA, Pelus LM . Differential expression of class II MHC antigens in subpopulations of human hematopoietic progenitor cells. Leukemia 1988; 2: 677–686.

  46. 46

    Shiratori S, Ito M, Yoneoka M, Hayasaka K, Hayase E, Iwasaki J et al. Successful engraftment in HLA-mismatched bone marrow transplantation despite the persistence of high-level donor-specific anti-HLA-DR antibody. Transplantation 2013; 96: e34–e44.

  47. 47

    Donohue J, Homge M, Kernan NA . Characterization of cells emerging at the time of graft failure after bone marrow transplantation from an unrelated marrow donor. Blood 1993; 82: 1023–1029.

  48. 48

    Taylor PA, Ehrhardt MJ, Roforth MM, Swedin JM, Panoskaltsis-Mortari A, Serody JS et al. Preformed antibody, not primed T cells, is the initial and major barrier to bone marrow engraftment in allosensitized recipients. Blood 2007; 109: 1307–1315.

  49. 49

    Xu H, Chilton PM, Tanner MK, Huang Y, Schanie CL, Dy-Liacco M et al. Humoral immunity is the dominant barrier for allogeneic bone marrow engraftment in sensitized recipients. Blood 2006; 108: 3611–3619.

Download references

Acknowledgements

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.

Author contributions

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.

Author information

Correspondence to M Murata.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on Bone Marrow Transplantation website

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading