Biology of Blood and Marrow Transplantation
Volume 14, Issue 4 , Pages 397-402, April 2008

Potential Role of a Mismatched HLA-Specific CTL Clone Developed Pre-Transplant in Graft Rejection following Cord Blood Transplantation

Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan

Received 5 November 2007; accepted 1 January 2008. published online 15 February 2008.

Article Outline

Abstract 

Graft rejection is a serious complication in cord blood transplantation (CBT), but little is known about the mechanism of rejection. To investigate the potential role of T lymphocytes in graft rejection, we isolated a CD8+ cytotoxic T lymphocyte (CTL) clone of recipient origin from blood obtained from a patient with graft rejection after CBT from an HLA-mismatched unrelated donor. The isolated CTL clone specifically recognized an HLA-B∗1501 molecule as an alloantigen, which was expressed in donor cells but not in recipient cells. The results of a microchimerism analysis specific for HLA-B∗1501 and a polymerase chain reaction assay specific for the T cell receptor on DNA from pretransplant peripheral blood mononuclear cells revealed that the patient was exposed to HLA-B∗1501 prior to CBT, and that the CTL clone was in the patient's blood prior to transplantation. The present study demonstrates a potential role for pretransplant CTL in graft rejection following CBT.

Key Words: Graft failure, HLA class I antigen, T lymphocyte, Microchimerism, Reduced-intensity stem cell transplantation

 

Back to Article Outline

Introduction 

Although cord blood transplantation (CBT) from unrelated donors is an attractive treatment for patients with hematologic disease 1, 2, 3, 4, 5, 6, graft rejection is a serious complication and is associated with a high rate of mortality 3, 4, 7, 8. The mechanisms of graft rejection have been extensively studied in bone marrow transplantation (BMT). One mechanism proposed by the studies in human and animal models is an immunologic response in which the host-derived T lymphocytes recognize donor-specific antigens 9, 10, 11, 12, 13, 14. For CBT, however, the role of the host-derived T lymphocytes in graft rejection has never been demonstrated in humans.

Here, we demonstrate a potential role of the host-derived cytotoxic T lymphocytes (CTLs) for graft rejection after CBT.

Back to Article Outline

Materials and Methods 

Study Patient 

A 59-year-old woman with acute myelomonocytic leukemia received CBT from an unrelated male donor. The details of the CBT procedure and the clinical course of the patient were reported previously [15]. Briefly, the preparative regimen consisted of 125 mg/m2 fludarabine and 180 mg/m2 melphalan, and 2.9 × 107/kg nuclear cells were infused. White blood cell (WBC) and neutrophil counts increased to 100/μL and 60/μL, respectively, with 65% of donor chimerism on day 16, but both counts subsequently decreased to less than the detection limit of the autohemocytometer on day 26. Graft rejection was diagnosed based on severe marrow hypoplasia and a complete loss of donor chimerism in bone marrow cells on day 28. A second CBT from an unrelated female donor was conducted on day 35. The infused nuclear cell dose was 2.9 × 107/kg. DNA typing of the HLA-A, -B, and -DRB1 loci demonstrated that the recipient was A∗1101/A∗2402, B∗4404/B∗5603, and DRB1∗1201/DRB1∗1302, the first CBT donor was A∗1101/A∗2402, B∗1501/B∗5603, and DRB1∗0901/DRB1∗1201, and the second CBT donor was A∗2402/A∗3303, B∗4403/B∗5101, and DRB1∗1201/DRB1∗1302. The patient had HLA antibodies, including those against HLA-A33, and broad HLA-DR antigens prior to transplantation, whereas HLA antibody against HLA-B∗1501 was not detected. The patient has one 19-year-old daughter. DNA typing of the HLA-A, -B, and -DRB1 loci demonstrated that her daughter was A∗1101/A∗2601, B∗1501/B∗5603, and DRB1∗0405/DRB1∗1201.

Cell Culture 

CTL clones were isolated from a blood sample as described previously [16]. Briefly, peripheral blood mononuclear cells (PBMCs) obtained from the patient on day 20 were cultured in interleukin-2-containing media without stimulator cells for 14 days, and T lymphocyte clones were isolated by limiting dilution.

Transfection and CTL Stimulation Assays 

The patient's B-LCL were transfected by electroporation with the pEAK10 plasmid (Edge BioSystems, Gaithersburg, MD) encoding HLA-B∗1501 cDNA, selected for 3 days with 0.8 μg/mL of puromycin, and assayed as a target for N19D8 CTL. Cytotoxicity was determined using a chromium release assay [16].

Interferon-γ release assays were conducted as previously described [16]. Briefly, COS cells were transfected with a plasmid encoding HLA-B∗1501, B∗5603 (negative control), or B∗4403 (negative control) cDNA using the FuGENE transfection reagent (Roche, Indianapolis, IN). COS transfectants were cocultured with N19D8 CTL, and interferon-γ production was measured in the supernatant after 24 hours using an enzyme-linked immunosorbent assay (Endogen, Pierce, Rockford, IL).

Polymerase Chain Reaction (PCR) Specific for the T Cell Receptor 

The T cell receptor Vβ repertoire was determined by flow cytometry using an IOTest Beta Mark Kit (Beckman Coulter, Fullerton, CA). The nucleotide sequences of the CTL clone's uniquely rearranged T cell receptor Vβ chain gene were determined by direct DNA sequencing of the amplified PCR product [17]. To determine the presence of the N19D8 clone-specific T cell receptor rearrangement, nested PCR was performed on genomic DNA extracted from a CTL clone N19D8 and PBMCs using a T cell receptor Vβ17-specific primer set for the first PCR: 5′-TTTCAGAAAGGAGATATAGCT-3′ (sense) and 5′-TTCTGATGGCTCAAACAC-3′ (antisense) followed by a second primer set specific for the N19D8 clone T cell receptor: 5′-GGAGATATAGCTGAAGGGTA-3′ (sense) and 5′-CCCCGCAAAGCTCTCA-3′ (antisense). PCR products were sequenced and confirmed to be identical in sequence to the N19D8-specific T cell receptor rearrangement. The PCR was performed with thermalcycler (Model 9600; Perkin-Elmer, Boston, MA) for 35 cycles under the following conditions: denaturation at 95°C for 1 minute, primer annealing at 55°C for 1 minute, and primer extension at 72°C for 1 minute in the first step, and denaturation at 95°C for 1 minute, primer annealing at 67°C for 15 seconds, and primer extension at 72°C for 1 minute in the second step. Each reaction contained 0.4 mL of Advantage 2 Polymerase Mix (Clontech Laboratories Inc., Palo Alto, CA).

Microchimerism Analysis 

The presence of the microchimerism was determined using a nested PCR approach on genomic DNA extracted from a fingernail sample and PBMCs as previously described [18]. Briefly, nested PCR was performed on genomic DNA using an HLA-B-specific primer set for the first PCR: 5′-GGCGGGGGCGCAGGACCTGA-3′ and 5′-GGCGGGGGCGCAGGACCCGG-3′ (1:1 mixture; sense) and 5′-GAGGCCATCCCCGGCGACCTAT-3′ (antisense) followed by a second primer set specific for HLA-B∗1501: 5′-ACCGGGAGACACAGATCTC-3′ (sense) and 5′-CTTGCCGTCGTAGGCGG-3′ (antisense). The touch-down procedure [19] was performed as first-step PCR under the following conditions: (1) denaturation at 96°C for 20 seconds and primer annealing at 72°C for 2 minutes for 5 cycles, (2) denaturation at 96°C for 20 seconds and primer annealing at 70°C for 2 minutes for 5 cycles, (3) denaturation at 96°C for 20 seconds and primer annealing at 68°C for 2 mintues for 4 cycles, and (4) denaturation at 96°C for 20 seconds and primer annealing at 72°C for 2.15 minutes for 15 cycles. The second-step PCR was performed for 28 cycles under the following conditions: denaturation at 94°C for 1 minute, primer annealing at 62°C for 1 minte, and primer extension at 72°C for 1 minute. FastStart Taq DNA Polymerase (Roche) and AmpliTaq Gold PCR Master Mix (Applied Byosistems, Foster City, CA) were used in the first- and the second-step PCR, respectively.

Back to Article Outline

Results 

Isolation of CTL Clone N19D8 

Two CTL clones were isolated from the peripheral blood of the patient just after the onset of graft rejection. One clone lysed B-LCL from the patient but failed to lyse B-LCL from the donor of the first CBT. The other clone, designated N19D8, lysed B-LCL from the donor but not from the patient (Figure 1A). Thus, we further investigated the N19D8 clone because this clone had the possibility of being involved in immunologic graft rejection. Flow cytometry analysis and sex chromosome fluorescein in situ hybridization revealed that the N19D8 clone was a CD3+CD4−CD8+ T cell and originated from the patient (data not shown).

  • View full-size image.
  • Figure 1 

    The N19D8 CTL clone recognizes HLA-B∗1501. (A) Transfection of HLA-B∗1501 cDNA into the B-LCL from the patient restored recognition by N19D8 CTL. The patient's B-LCL were transfected with a plasmid encoding HLA-B∗1501 cDNA and assayed as a target for N19D8 CTL. Cytotoxicity was determined using a chromium release assay. Lysis of the first-donor B-LCL (closed squares), second-donor B-LCL (open squares), patient B-LCL (triangles), and HLA-B∗1501-transfected patient B-LCL (circles) are shown as the mean of triplicate cultures at various E:T ratios. (B) HLA-B∗1501 cDNA stimulated interferon-γ production by N19D8 CTL. COS cells were transfected with a plasmid encoding HLA-B∗1501, B∗5603 (negative control), or B∗4403 (negative control) cDNA. COS transfectants were cocultured with N19D8 CTL, and interferon-γ production was measured in the supernatant, an enzyme-linked immunosorbent assay. Data is shown as the mean of triplicate determinations.

CTL Clone N19D8 Recognizes the HLA-B∗1501 Molecule 

In a cytotoxicity assay using a panel of B-LCL derived from unrelated individuals, N19D8 CTL lysed all of 4 B-LCL lines from unrelated individuals that shared HLA-B∗1501 but failed to lyse B-LCL from the donor of the second CBT and 7 unrelated individuals without B∗1501 (Table 1). These results indicated that N19D8 CTL recognized the HLA-B∗1501 molecule as an alloantigen, which was only expressed in donor cells, or a minor histocompatibility antigen presented by HLA-B∗1501 molecule.

Table 1. Cytotoxicity Assays for N19D8 CTL against B-LCL
Characteristics of B-LCL
B-LCLHLA-AHLA-BSexLysis by CTL (%)∗
Recipient B-LCLA∗1101A∗2402B∗4403B∗5603Female0
First-donor B-LCLA∗1101A∗2402B∗1501B∗5603Male36
Second-donor B-LCLA∗2402A∗3303B∗4403B∗5101Female0
L11A∗0201A∗1101B∗1501 Female34
L17A∗2402A∗3303B∗1501B∗4403Male35
L79A∗2402A∗3303B∗1501B∗4403NA42
JMDA∗0201A∗2901B∗1501B∗4403NA44
L16A∗1101A∗2402B∗5401B∗5101Male0
L19A∗2402A∗3303B∗0702B∗4403Male0
L25A∗1101A∗2402B∗5401B∗5201Female0
L59A∗2603A∗3303B∗3501B∗4403Male0
L73A∗0201A∗2402B∗0702B∗6701Female0
L74A∗1101A∗3303B∗4002B∗5101Female0
RARA∗2901A∗3101B∗2705B∗4403NA0

NA indicates not available.

∗E:T ratio was 10:1.

To determine if the lack of recognition by N19D8 CTL was solely because of the absence of HLA-B∗1501 gene expression, the patient's B-LCL were transfected with HLA-B∗1501 cDNA and used as a target in cytotoxicity assays. The B∗1501-transfected patient B-LCL was lysed almost as well as the first CBT donor's B-LCL (Figure 1A). Furthermore, COS cells transfected with HLA-B∗1501 cDNA alone stimulated interferon-γ production by N19D8 CTL (Figure 1B). Thus, we concluded that the N19D8 CTL clone recognizes mismatched HLA-B∗1501 as an alloantigen.

The Presence of the N19D8 CTL Clone and HLA-B∗1501 Microchimerism Prior to Transplantation 

We next determined whether the N19D8 clone developed prior to transplantation using nested PCR assays specific for the CTL clone's uniquely rearranged T cell receptor Vβ17 chain gene. PCR products were detected by amplification of DNA from pretransplant as well as posttransplant PBMCs (Figure 2), demonstrating that the N19D8 CTL clone developed in the patient prior to the first CBT. We further tested pretransplant PBMCs for chimeric cells with the HLA-B∗1501 gene using PCR. Indeed, the HLA-B∗1501 microchimerism was detected by amplification of DNA from the pretransplant patient PBMCs using standard PCR as well as nested PCR (Figure 3), indicating that the patient was exposed to HLA-B∗1501 prior to CBT. The HLA-A∗2601 microchimerism was also detected from the pretransplant patient PBMCs. From the patient PBMCs 3 months after transplantation, HLA-A∗2601 and HLA-B∗1501 microchimerisms were detected (data not shown).

  • View full-size image.
  • Figure 2 

    Presence of the N19D8 clone in pretransplant peripheral blood from the patient. Detection of lymphocytes with the N19D8 clone-specific T cell receptor in pretransplant PBMCs. Nested PCR was performed on genomic DNA using a T cell receptor Vβ17-specific primer set for the first PCR. Genomic DNA was prepared from PBMCs obtained from the patient 1 month before the first CBT (pre PBMCs) and 3 or 9 months after the second CBT (post PBMCs). Genomic DNA from the N19D8 clone was used as a positive control. Genomic DNA prepared from PBMCs obtained from unrelated volunteers were used as negative controls. PCR to detect β-globin was used as an internal control in each assay.

  • View full-size image.
  • Figure 3 

    Presence of the N19D8 clone in pretransplant peripheral blood from the patient. The presence of HLA-B∗1501 microchimerism in pretransplant PBMCs. Lanes 1 and 6 are standard PCRs specific for HLA-B∗1501 on genomic DNA from pretransplant PBMCs from the patient and B∗1501-positive PBMCs from the patient's daughter (positive control), respectively. Lanes 2-3, 4-5, and 7-8 are nested PCR specific for HLA-B∗1501 on diluted (in parentheses) first PCR products from a posttransplant fingernail sample from the patient (negative control), pretransplant PBMCs from the patient, and PBMCs from the patient's daughter (positive control), respectively. PCR to detect complement factor 4 was used as an internal control in each assay.

Back to Article Outline

Discussion 

Although direct verification would be difficult, it is reasonable to conclude that the N19D8 clone was involved in the graft rejection based on the following clinical and laboratory findings. First, the patient rejected the first cord blood graft with HLA-B∗1501, whereas the second graft without B∗1501 was successfully engrafted. This clinical course is consistent with allo-activity of the N19D8 clone. Second, CD3+CD4−CD8+ T lymphocytes accounted for the majority of the lymphocytes in the patient's blood just after the onset of graft rejection (84.8%, data not shown). Third, no clones that recognize the other mismatched HLA antigens or minor histocompatibility antigens were isolated by the limiting dilution cloning from the patient just after the onset of graft rejection; N19D8 was the only clone with cytotoxic activity against the first donor cells. Last, the patient developed graft loss with a transient lymphocyte increase after reaching transient mix chimerism. It is difficult to differentiate immunologic graft rejection from other causes of graft failure in CBT [7]; however, this clinical course is consistent with those of patients who developed immunologic graft rejection following myeloablative or nonmyeloablative BMT 14, 20. Taken together, the results described above support the involvement of immunologic mechanisms such as cytotoxicity of T lymphocyte in the graft rejection.

The following routes of exposure of the patient to HLA-B∗1501 can be considered. One possibility is that the patient was exposed to HLA-B∗1501 during her pregnancy; her 19-year-old daughter has HLA-B∗1501, presumably as an inherited paternal allele. It is known that fetal hematopoietic chimerism can persist for decades, maintaining a paternal-specific CTL developed during pregnancy over a long period of time 21, 22. Another possibility is exposure via blood transfusions. The patient had previously received blood transfusions from random donors, and development of HLA-B∗1501-specific CTL as a consequence of contamination by HLA-B∗1501-positive WBCs in the transfusion products cannot be completely ruled out. Future studies are needed to determine which of these events was related to graft rejection. It is to be noted that the presence of an HLA-B∗1501 microchimerism was demonstrated even after the onset of the graft rejection. Although the mechanism is uncertain, the N19D8 CTL clone might not be able to completely eliminate B∗1501-positive cells. Further studies focusing on this basic mechanism are warranted.

Interestingly, the patient had the HLA antibodies including those against the second donor HLA, HLA-A∗3303, whereas the patient did not have the HLA antibody against HLA-B∗1501. This suggested that HLA-antibodies did not have clinical impact on the graft rejection in this patient. However, it is inconsistent with the previous studies in solid organ transplantations or bone marrow transplantations 23, 24. This inconsistency indicates the clinical impact of HLA antibodies is controversial in CBT. Future studies would allow a proper interpretation.

In conclusion, the results of the present study demonstrate a potential role for pretransplant CTL in graft rejection following CBT. Further studies focusing on mismatched HLA-specific CTLs should help to establish an optimal strategy for overcoming graft rejection in CBT.

Back to Article Outline

Acknowledgments 

We are grateful to Chika Wakamatsu, Yuka Nomura, and Ryohei Tanizaki for technical assistance. We are also grateful to Dr. Etsuko Maruya and Dr. Hiroh Saji (HLA Laboratory, NPO, Kyoto, Japan) for detecting microchimerism in this study. This work was supported by grants-in-aid from the Ministry of Health, Labor and Welfare of Japan (to M.M.) and the National Institute of Biomedical Innovation (to T.N.).

Back to Article Outline

References 

  1. Wagner JE, Barker JN, DeFor TE, et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood. 2002;100:1611–1618
  2. Barker JN, Weisdorf DJ, DeFor TE, Blazar BR, Miller JS, Wagner JE. Rapid and complete donor chimerism in adult recipients of unrelated donor umbilical cord blood transplantation after reduced-intensity conditioning. Blood. 2003;102:1915–1919
  3. Laughlin MJ, Eapen M, Rubinstein P, 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. Rocha V, Labopin M, Sanz G, 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
  5. Takahashi S, Ooi J, Tomonari A, et al. Comparative single-institute analysis of cord blood transplantation from unrelated donors with bone marrow or peripheral blood stem-cell transplants from related donors in adult patients with hematologic malignancies after myeloablative conditioning regimen. Blood. 2007;109:1322–1330
  6. Narimatsu H, Terakura S, Matsuo K, et al. Short-term methotrexate could reduce early immune reactions and improve outcomes in umbilical cord blood transplantation for adults. Bone Marrow Transplant. 2007;39:31–39
  7. Narimatsu H, Kami M, Miyakoshi S, et al. Graft failure following reduced-intensity cord blood transplantation for adult patients. Br J Haematol. 2006;132:36–41
  8. Shimada K, Narimatsu H, Morishita Y, Kohno A, Saito S, Kato Y. Severe regimen-related toxicity of second transplantation for graft failure following reduced-intensity cord blood transplantation in an adult patient. Bone Marrow Transplant. 2006;37:787–788
  9. Vogt MH, Goulmy E, Kloosterboer FM, et al. UTY gene codes for an HLA-B60-restricted human male-specific minor histocompatibility antigen involved in stem cell graft rejection: characterization of the critical polymorphic amino acid residues for T-cell recognition. Blood. 2000;96:3126–3132
  10. Voogt PJ, Fibbe WE, Marijt WA, et al. Rejection of bone-marrow graft by recipient-derived cytotoxic T lymphocytes against minor histocompatibility antigens. Lancet. 1990;335:131–134
  11. Martin PJ. Prevention of allogeneic marrow graft rejection by donor T cells that do not recognize recipient alloantigens: potential role of a veto mechanism. Blood. 1996;88:962–969
  12. Bunjes D, Heit W, Arnold R, et al. Evidence for the involvement of host-derived OKT8-positive T cells in the rejection of T-depleted, HLA-identical bone marrow grafts. Transplantation. 1987;43:501–505
  13. Vogt MH, de Paus RA, Voogt PJ, Willemze R, Falkenburg JH. DFFRY codes for a new human male-specific minor transplantation antigen involved in bone marrow graft rejection. Blood. 2000;95:1100–1105
  14. Kraus AB, Shaffer J, Toh HC, et al. Early host CD8 T-cell recovery and sensitized anti-donor interleukin-2-producing and cytotoxic T-cell responses associated with marrow graft rejection following nonmyeloablative allogeneic bone marrow transplantation. Exp Hematol. 2003;31:609–621
  15. Mizutani E, Narimatsu H, Murata M, Tomita A, Kiyoi H, Naoe T. Successful second cord blood transplantation using fludarabine and cyclophosphamide as a preparative regimen for graft rejection following reduced-intensity cord blood transplantation. Bone Marrow Transplant. 2007;40:85–87
  16. 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
  17. Akatsuka Y, Martin EG, Madonik A, Barsoukov AA, Hansen JA. Rapid screening of T-cell receptor (TCR) variable gene usage by multiplex PCR: application for assessment of clonal composition. Tissue Antigens. 1999;53:122–134
  18. Tokita K, Terasaki P, Maruya E, Saji H. Tumour regression following stem cell infusion from daughter to microchimeric mother. Lancet. 2001;358:2047–2048
  19. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. “Touchdown” PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991;19:4008
  20. Georges G, Storb R. Failure of sustained engraftment: clinical manifestations and treatment. In:  Atkinson K editors. Clinical Bone Marrow and Blood Stem Cell Transplantation. Cambridge, UK: Cambridge University Press; 2000;p. 1088–1188
  21. Kotlan B, Fulop V, Padanyi A, et al. High anti-paternal cytotoxic T-lymphocyte precursor frequencies in women with unexplained recurrent spontaneous abortions. Hum Reprod. 2001;16:1278–1285
  22. Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA. 1996;93:705–708
  23. Barge AJ, Johnson G, Witherspoon R, Torok-Storb B. Antibody-mediated marrow failure after allogeneic bone marrow transplantation. Blood. 1989;74:1477–1480
  24. Terasaki PI. Humoral theory of transplantation. Am J Transplant. 2003;3:665–673

PII: S1083-8791(08)00004-9

doi:10.1016/j.bbmt.2008.01.001

Biology of Blood and Marrow Transplantation
Volume 14, Issue 4 , Pages 397-402, April 2008

Article Tools

* Image Usage & Resolution