Generation of HLA-A2 subtype specific cytotoxic T lymphocytes from cord blood used for cord blood stem cell transplantation

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Abstract

Alloantigen reactive cytotoxic T lymphocytes (CTL) were generated from cord blood (CB) lymphocytes used for cord blood stem cell transplantation (CBSCT). The CTL were cytotoxic against the patient's leukemic cells, as well as the patient's EBV-lymphoblastoid cell line (EBV-LCL), and PHA blasts. The cytotoxicity against patient's EBV-LCL was blocked by anti-HLA-A2 MoAb, and anti-HLA-class I MoAb. The CTL recognized A*0206 positive EBV-LCLs, but not A*0201, A*0204, or A*0207 positive EBV-LCLs, suggesting that this CTL recognizes HLA-A*0206. This case suggests that CB T cells may be competent enough to generate CTL to induce a GVL effect, together with those against A*0206, in patients with CBSCT. Bone Marrow Transplantation (2000) 26, 451–453.

Main

Cord blood (CB) is an alternative source for stem cell transplantation (SCT) and SCT using CB instead of BM has increased recently, yielding promising results.1234 Since it has been reported that CB cells possess a naive phenotype,5 reduced alloreactivity,678910 and weak natural killer (NK) activity,111213 it was speculated that CB cells might lack a graft-versus-leukemia (GVL) effect and result in increased relapse in patients with leukemia. However, increased relapse rates have not been obvious in cord blood stem cell transplantation (CBSCT) for malignant disease so far.1234

Here we reported that CTL specific for an allele of HLA-A2, A*0206 was generated from CB used for CBSCT in a patient with T-ALL, suggesting that CB T cells might be competent enough to generate CTL effective for a GVL effect.

Case report

The patient was an 8-year-old boy with T-ALL. The leukocyte count at diagnosis was 241 200/μl. Chromosomal analysis revealed 46,XY, t(8:14)(q24:q11). Cell surface markers of the leukemic cells were CD2, CD3, CD4dim, CD5+, CD7+, CD8+, and HLA-DR. He achieved CR after induction chemotherapy using Japan Association of Childhood Leukemia Study (JACLS) T-97 protocol with dexamethasone, vincristine, cyclophosphamide, THP-adriamycin, and L-asparaginase. However, he developed central nervous system (CNS) relapse 8 months later. He received craniospinal irradiation, but developed BM relapse 3 months later, following a second CNS relapse. He received CBSCT using a one HLA-antigen mismatched CB identified by serological typing supplied from the Tokai Cord Blood Bank, Nagoya, Japan at the third relapse. The patient's HLA type was HLA-A2 (A*0206), A26 (A*2601); B35, B61 (B*4002); Cw9, Cw10; DR9 (DR*0901), DR4 (DR*0403). The HLA type of the CB was HLA-A2 (A*0201), A26 (A*2601); B35, B61 (B*4002); Cw9, Cw10; DR9 (DR*0901), DR11 (DR*1101). In the Japanese, no allele of HLA-B35 has been found. Therefore, only the HLA-A2 allele was different in HLA-class I between the patient and CB. Numbers of infused CB cells were 1.86 × 107/kg. Conditioning treatment was cytosine arabinoside (3 g/m2 × 2 on days −8, −7, −5, −4) CY (60 mg/kg on days −4 and −3) and TBI 6 Gy on days −2 and −1. GVHD prophylaxis consisted of CsA 5 mg/kg/day starting on day 0 and decreasing to 3 mg/kg/day on day +7 and prednisolone 2 mg/kg/day starting on day +1 and decreasing 1 mg/kg/day on day +7. The WBC exceeded 1.0 × 109/l on day +47, neutrophils exceeded 0.5 × 109/l on day +47 and reticulocytes exceeded 1% on day +55. Stage 2+ skin GVHD developed on day +36 and subsided without any additional specific therapy. He developed hemorrhagic cystitis on day +51 and pneumonia on day +81. He died of renal failure and leukoencephalopathy on day +111.

A bulk CTL was established by stimulating 1 × 105 naive CB lymphocytes frozen in liquid nitrogen after the collection with 1 × 105 of 35 Gy irradiated allele of HLA-A2 mismatched, HLA-class II negative, patient's leukemic cells using 96-well round-bottomed microtiter plate in RPMI 1640 plus 15% pooled human AB serum and 10 mM HEPES by the method published elsewhere.14 On day 6, 1 × 105 viable cells were restimulated with 1 × 106 irradiated leukemic cells, and 2% highly purified IL-2 (Biotest, Dreieich, Germany) in 24-well flat-bottomed culture plate. From day 14, the culture was restimulated and expanded weekly with 1 × 106 irradiated leukemic cells, 100 IU/ml human recombinant IL-2 (Takeda Chemical Industries, Osaka, Japan), and 10% T cell growth factor (TCGF, Biotest). After the third stimulation, the bulk CTL showed strong cytotoxicity against patient's leukemic cells, the leukemic cells pretreated with 10 ng/ml human tumor necrosis factor-α (TNF-α; Boehringer Mannheim, Mannheim, Germany), EBV-LCL of patient's origin, and patient's PHA blasts, but not against donor's EBV-LCL, donor's PHA blasts, or K562 cell line (Table 1). The lysis of patient's EBV-LCL by the bulk CTL was inhibited by HLA-class I MoAb, whereas HLA-class II MoAbs did not (Figure 1). The lysis of the patient's EBV-LCL was also inhibited by anti-HLA-A2 MoAb, MA2.1, suggesting that the bulk CTL recognize the antigenic peptides presented by HLA-A2 molecules on EBV-LCL, leukemic cells, and PHA blasts and kill them. Since the sole mismatched HLA-class I antigen between CB and the patient's leukemic cells detected by high-resolution DNA typing was the allele of HLA-A2, A*0201 vs A*0206, the cytotoxicity of CTL was examined against HLA-A2-positive EBV-LCLs expressing various alleles of HLA-A2 allotypes. The CTL showed clear cytotoxicity against six A*0206 positive EBV-LCLs but not against EBV-LCLs expressing A*0201, A*0204, or A*0207, confirming that the CTL recognizes the allele of HLA-A2, A*0206 (Table 2).

Table 1  Cytotoxicity of CB CTL against cells of various types
Figure 1
figure1

Anti-HLA-A2 MoAb blocks the cytotoxicity of CB CTL against patient's EBV-LCL. MoAbs were added just before the addition of target cells and incubated for 20 min at 37°C. Thereafter, the cytotoxicity against patient's EBV-LCL was studied using a chromium release assay at E/T ratio of 20. MoAbs: anti-HLA class II (Ia-91–5a), anti-HLA class I (wb/32–3a), anti-HLA-A2 (MA2.1). Bars represent the % specific lysis + standard deviation.

Table 2  Cytotoxicity of CB CTL against LCLs expressing various HLA-A2 alleles

Discussion

In the present study, we succeeded in generating CTL detecting the allele of HLA-A2, A*0206 on leukemic cells, EBV-LCL, and PHA blasts by stimulating the CBSCT donor's naive CB lymphocytes with the allele of HLA-A2 mismatched leukemic cells. Our results are, however, not necessarily consistent with the previous reports that (1) CB contained fewer CTL precursors than adult peripheral blood;6 (2) the CB T lymphocytes generated less cytotoxic T cell activity than adult T cells, even after repeated allogeneic stimulation in vitro;10 and (3) CB T cell mitogenesis induced through the CD2 molecule is defective.9 Although it needs to be explained why our results were not consistent with the previous reports, the present study has shown that naive CB T cells were competent enough to generate CTL against a subtype of HLA-A2. In HLA-A*0206, Phe of HLA-A*0201 at position 9 is substituted to Tyr, changing the conformation in the B pocket, so that Val, a slightly smaller hydrophobic amino acid residue than Leu, is preferred at position 2 to anchor the peptide motif in HLA-A*0206.15 Thus, it is conceivable that this conformational change has a significant effect on the binding peptides, resulting in the induction of HLA-A*0206 specific CTL from HLA-A*0201 CB T cells. Since subtype frequency in the Japanese of A*0201, A*0206, A*0207, and A*0210 was 43%, 41%, 14%, and 2%, respectively, the mismatch of A*0201 and A*0206 is the most common one in unrelated SCT in Japan. Therefore, it must be examined whether HLA-A2 allele-specific CTL can be generated in other patients who received HLA-A2 allele mismatched CBSCT.

Since a lower incidence of severe acute GVHD in CBSCT was reported,1234 it is speculated that CB T cells might lack GVL effects. The fact that the CTL against subtype of HLA-A2 was generated in this patient, suggested that the CTL mediating GVL effect can also be generated in this patient after CBSCT. Relapse of leukemia after unrelated CBSCT has not been increased compared to unrelated BMT,1234 supporting the view that GVL effects can be induced even in CBSCT by CTL, although NK cells have been implicated in mediating the GVL effect.11121316 In this reported case, the patient was still in CCR until day +111, although he had received the CBSCT at his third relapse. Grade I acute GVHD was observed in this patient but subsided without further therapy. Further study of patients transplanted with CBSCT may elucidate whether the CB CTL may play a GVL effect in CBSCT and reduce the risk of relapse of leukemia.

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Acknowledgements

We thank Mr Toru Kato for detecting the HLA-A2 allele of EBV-LCLs and Dr Kyogo Ito for providing MA2.1 moAb against HLA-A2. We also thank Mrs Etsuko Maruya for detecting HA-1 allele of LCLs and Mr Dee Lynn Johnson for his advice on this manuscript.

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Correspondence to M Yazaki.

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Keywords

  • cord blood
  • cord blood stem cell transplantation
  • alloreactivity
  • cytotoxic T cells
  • graft-versus-leukemia effect

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