Graft-Versus-Tumor Effects

Expansion and activation of minor histocompatibility antigen HY-specific T cells associated with graft-versus-leukemia response

Summary:

The immune system of females is capable of recognizing and reacting against the male-specific minor histocompatibility antigen (mHA), HY. Thus, cytotoxic T-lymphocytes (CTLs) recognizing this antigen may be useful in eradicating leukemic cells of a male patient if they can be generated in vivo or in vitro from a human leukocyte antigen (HLA)-identical female donor. The HLA-A*0201-restricted HY antigen, FIDSYICQV, is a male-specific mHA. Using HLA-A2/HY peptide tetrameric complexes, we reveal a close association between the emergence of HY peptide-specific CD8+ T cells in peripheral blood and molecular remission of relapsed BCR/ABL+ chronic myelogenous leukemia in lymphoid blast crisis in a patient who underwent female-to-male transplantation. Assessment of intracellular cytokine levels identified T cells that produce interferon-γ in response to the HY peptide during the presence of HY tetramer-positive T cells. These results indicate that transplant with allogeneic HY-specific CTLs has therapeutic potential for relapsed leukemia, and that expansion of such T cells may be involved in the development of a graft-versus-leukemia response against lymphoblastic leukemia cells.

Main

Minor histocompatibility antigens (mHAs) are immunogenic peptides derived from polymorphic cellular proteins.1 These peptides bind to human leukocyte antigen (HLA) and are recognized by allogeneic T cells. Following stem cell transplantation (SCT) with HLA-matched donor cells, graft-versus-host disease (GVHD) can arise through disparities in mHAs between the donor and the recipient. Using tetrameric HLA class I–mHA complexes, Mutis et al2 demonstrated that a limited number of mHA-specific T cells expand in peripheral blood (PB) in parallel with increasing GVHD severity. It is believed that donor-derived T cells specific for mHAs play a significant role in the development of graft-versus-leukemia (GVL) effect as well as GVHD after allogeneic SCT. However, there is only limited evidence for the killing of leukemic cells by mHA-specific T cells.

Kern et al3 reported that human cytomegalovirus peptide-specific CD8+ T cells can be detected by flow cytometry in samples whose HLA is known. Kuzushima et al4 showed that the frequency Epstein–Barr virus-specific CD8+ T-cell frequencies are detectable irrespective of HLA typing when PB lymphocytes are incubated with an autologous lymphoblastic cell line (LCL). Both methods are based on multiparameter flow cytometric assays that detect rapid intracellular accumulation of interferon (IFN)γ after in vitro antigen stimulation in the presence of an intracellular transport blocker. By assessing the frequency of IFNγ-producing cells, the presence of functional T cells reactive with target antigens and target cells can be detected.

One male-specific mHA is the HLA-A*0201-restricted peptide, FIDSYICQV, from the male-specific antigen HY.5 Cytotoxic T-lymphocytes (CTLs) recognizing this peptide may be useful in eradicating leukemic cells of a male patient with HLA-A2 if they can be generated in vivo or in vitro from an HLA-identical female donor. By HLA-A2/HY peptide tetramer staining and intracellular IFNγ assessment, we provide the first evidence that the emergence and activation of transferred HY-specific female CTLs contributes to molecular remission of chronic myelogenous leukemia (CML) in a male patient in lymphoid crisis.

To date, a small number of studies describe a clear dominance in T-cell receptor (TCR) variable (V)-gene segment usage in the recognition of certain HLA class I/peptide complexes in humans after SCT.6, 7 We examined PB CD8+ T cells producing intracellular IFNγ of a male patient with CML in lymphoid crisis for the emergence of clonal T-cell proliferation by analyzing the T-cell repertoire as well as in vitro-generated HY-specific CTLs. TCR BV spectratyping showed a similar peak with same size in some BV family between in vitro-generated HY-specific CTLs and in vivo-activated HY-specific CD8+ T cells. These findings suggest the circulation of a functional T-cell clone capable of eradicating lymphoblastic leukemia cells.

Materials and methods

Case report

A patient was a 15-year-old Japanese male (HLA-A*0201-positive) with chronic-phase CML. He had experienced no GVHD after receiving busulfan- and cyclophosphamide- conditioned bone marrow from his HLA-identical sister. At 40 months after bone marrow transfer, lymphoid blast crisis suddenly developed. After treatment with cyclophosphamide, adriamycin, vincristin and prednisolone, the hematologic relapse persisted. At 2 months after chemotherapy, the patient underwent PB stem cell transplantation (PBSCT) from the same donor following conditioning with cytarabine, cyclophosphamide and total body irradiation. An unmanipulated PB stem cell graft including a total of 5.5 × 106 CD34-positive cells/kg was infused. Cyclosporine, which is used to prevent GVHD, was withdrawn on day 21 to induce GVL effect. At 3 weeks after PBSCT, conversion to full donor chimerism was obtained. The patient developed grade II acute GVHD on day 29 after PBSCT, and the disease progressed to extensive chronic GVHD on day 80. At 20 weeks after PBSCT, no BCR/ABL transcripts were detected, and he remained in molecular remission until a relapse at 13 months after PBSCT. The patient died of veno-occlusive disease shortly after the third transplantation with cytarabine- and idarubicin-conditioned bone marrow from the same donor.

Peptide

An HLA-A201-restricted HY peptide was synthesized using a semiautomatic multiple peptide synthesizer based on the reported sequence.5 The purity of the peptide was checked by reverse-phase high-pressure liquid chromatography.

Cell preparation

Cells were obtained from the post transplant patient and his stem cell donor. Peripheral blood mononuclear cells (PBMC) were prepared using density-gradient centrifugation. For the establishment of Epstein–Barr virus (EBV)-transformed LCLs, PBMC from the donor were depleted of T cells using the rosette formation method. A total of 2–3 × 106 non-T cells were incubated in RPMI 1640 (GIBCO, Grand Island, NY, USA) medium containing 10% fetal calf serum (FCS; GIBCO) containing 10% culture medium from an EBV-producing cell line, B95-8, at 37°C for 2 h. The EBV-infected cells were cultured for 3 weeks until transformed LCL cells grew. LCL cells were maintained by changing the medium every 4 to 5 days.

Tetrameric HLA-A2/mHA HY peptide complexes

The generation of HLA-A2/mHA HY tetramers and tetramer labeling of HY-specific T cells was performed as described previously.8

Generation of dendritic cells (DCs) from monocytes

PB monocyte-derived DCs were generated as described previously.9 Briefly, monocytes were isolated by adherence of donor PBMCs to plastic for 2 h. Monocytes were cultured in RPMI 1640 medium supplemented with 10% pooled AB serum, 10 ng/ml recombinant human interleukin-4 (IL-4) and 100 ng/ml recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF) (a gift from Kirin Brewery, Tokyo, Japan). On day 5, 100 U/ml recombinant human tumor necrosis factor (TNF)α was added. On day 8 or 9, the cells were harvested for use as monocyte-derived DCs for antigen presentation. Cultured cells expressed DC-associated antigens, such as CD1a, CD80, CD83, CD86 and HLA class I and class II.

Induction of HY peptide-specific CTLs

DCs were pulsed with an HY peptide for 90 min at 37°C in serum-free RPMI 1640. After washing, 1.0 × 106 peptide- (40 nM) pulsed DCs and 1.0 × 107 donor-derived (autologous) PBMC were cultured together in 24-well culture plates. The culture medium was RPMI 1640 supplemented with 2 mM L-glutamine, minimal essential amino acids, sodium pyruvate and ampicillin (all from GIBCO) plus 10% autologous plasma. The cells were kept at 37°C in a humidified, 5% CO2–air mixture. At days 7, 14 and 21, responder cells were restimulated with peptide-pulsed autologous DCs. From day 21, cultured T cells were suspended in 100 U/ml IL-2- (a gift from Shionogi, Osaka, Japan) containing culture medium and were restimulated weekly with peptide-pulsed autologous monocytes or DCs. T cells were harvested at day 35 and used for the cytotoxicity assay and RNA extraction for T-cell repertoire analysis.

Cytotoxicity (51Cr release) assays

Donor-derived LCL cells and DCs as well as fibroblasts and leukemic cells of the patient were used as target cells in the standard 4-h 51Cr release assay.10 Specific lysis was calculated using the following formula: 100 × (cpm experimental release–cpm spontaneous release)/(cpm maximal release–cpm spontaneous release).

Preparation of target cells

Patient fibroblasts were isolated from a biopsy specimen and cultured in RPMI 1640 plus 10% FCS for 4 weeks. Single-cell suspensions were prepared by trypsinization. Donor-derived LCL cells and DCs and the patient's fibroblasts were suspended in 100 μl of 51Cr solution containing an HY peptide at a concentration of 4 nM. In some experiments, concentrations of the peptide were changed as noted. Bone marrow mononuclear cells containing 98% BCR/ABL+ cells were obtained from the patient just before the second transplantation and were cryopreserved for use as leukemic cells. Thawed leukemic cells were cultured in RPMI 1640 plus 10% pooled AB serum for 24 h before use as a target in the CTL assay.

Blocking of cytotoxicity by monoclonal antibodies (MoAbs)

Polyclonal antibodies (control) or purified MoAbs were added to cultures of HY peptide- (4 nM) pulsed DCs in 96-well plates at a concentration of 10 μg/ml, and CTLs were immediately added to each well. MoAbs were HU-4 (anti-HLA-DR; kindly provided by Dr Akemi Wakisaka, Hokkaido University, Japan) and W6/32 (anti-HLA class I; American Type Culture Collection, Rockville, MD, USA).

Identification and isolation of IFNγ-producing CD8+ T cells by flow cytometry

To detect circulating CD8+ T-lymphocytes that recognize HY peptide, intracellular IFNγ was assessed by flow cytometry as described previously with slight modifications.3, 4 Briefly, donor-derived LCL cells were incubated for an hour with or without HY peptide. PBMCs were taken from the patient 12 weeks after the second transplantation. The CD8+ T-lymphocytes were isolated from PBMCs using magnetic beads coated with an anti-CD8 monoclonal antibody (mAb) according to the manufacturer's instructions (Dynal AS, Oslo, Norway). A total of 106 CD8+ cells were mixed with 106 autologous LCL cells in a culture tube in RPMI 1640 medium and cultured in a humidified 5% CO2 incubator at 37°C for 1 h. Brefeldin A (Sigma, St Louis, MO, USA) was added at a final concentration of 10 μg/ml and the cells were cultured for an additional 5 h. After incubation, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with phosphate-buffered saline, cells were permeabilized with IC Perm (Biosourse International, Camarillo, CA, USA) and stained with PE-labeled anti-CD8 (Coulter, Miami, FL, USA) and FITC-labeled anti-human IFNγ (Biosourse International) MoAbs. Stained cells were analyzed and sorted on a FACScan (Becton Dickinson, San Jose, CA, USA).

RNA extraction and cDNA preparation

Total RNA was extracted from PBMCs or CTLs using a technique described elsewhere,11 and was reverse transcribed into cDNA in a reaction primed with oligo(dT) using SuperScript II reverse transcriptase as recommended by the manufacturer (BRL, Bethesda, MD, USA).

Spectratyping of complementarity-determining region 3 (CDR3)

Conditions for the generation of the CDR3 size spectratyping have been reported previously.6 Briefly, cDNA was polymerase chain reaction (PCR) amplified through 35 cycles (95°C for 1 min, 55°C for 1 min and 72°C for 1 min) with a fluorescent BC primer and primers specific to 24 different BV subfamilies. Analyses of the pseudogenes BV10 and BV19 were excluded from this study.12 A measure of 1 μl of each amplified products was mixed with 2.0 l 100% formamide, heated at 90°C for 3 min and electrophoresed on a 6.75% denaturing polyacrylamide gel. The distribution of CDR3 size within the amplified product of each BV subfamily was analyzed with an automatic sequencer (Applied Biosystems Division, Foster City, CA, USA) equipped with a computer program allowing the determination of the fluorescence intensity of each band. The results are given as peaks corresponding to the intensity of the fluorescence. Expansion of a limited number of T cells was judged when a prominent peak appeared in the CDR3 pattern with or without a reduced number of peaks (five peaks). Given the BV-NDN-BJ sequence of the identical CDR3 size between in HY-specific CD8+ cells in PB and the CTLs, a more specific primer covering CDR3 and different BJ subfamilies13 was designed specifically to amplify cDNA of the BV22+ T-cell clone in both CTLs.

Direct sequencing of PCR products

BV22-BJ PCR products were purified and sequenced as described previously.14

Results

Cytolytic activity of in vitro-generated HY-specific CTLs

Cultured T female donor cells stimulated with autologous HY peptide-pulsed DCs pulsed were able to lyse HY peptide-pulsed autologous DCs and patient fibroblasts, but could not lyse untreated DCs (Figure 1a). Of note, the CTLs lysed nonpeptide pulsed leukemic cells of the patient more efficiently than his fibroblasts. CTLs showed cytotoxicity to HY peptide-loaded autologous DCs, in a peptide concentration-dependent manner (Figure 1b). Cytotoxicity mediated by the CTLs against HY peptide-pulsed autologous DCs was blocked to a similar degree by the addition of MoAb either against anti-class I or anti-CD3, but was not affected by the addition of anti-HLA-DR (Figure 1c). In addition, the CTLs could effectively lyse LCL cells of unrelated males who shared HLA-A*0201. In contrast, apparent cytotoxic activity was neither observed against allogeneic LCL cells that did not possess HLA-A2 nor against LCL cells of unrelated females (Figure 1d).

Figure 1
figure1

Cytotoxic activity of in vitro-generated HY-specific CTLs. (a) Cytotoxicity of cultured T cells stimulated by autologous DCs pulsed with HY peptide. The amount of peptide utilized in (a, b and c) was 4 nM. Target cells: autologous DCs without HY peptide (Δ); autologous DCs pulsed with HY peptide (); fibroblasts of the patient (); leukemic cells of the patient (•). In (b, c, and d), cytotoxicity was determined at an E/T ratio of 10:1. (b) Effect of concentration of HY peptide on cytotoxicity of CTLs. Cytotoxicity of CTLs to autologous DCs loaded with various concentration of HY peptide was tested. (c) Antibody blockade of cytotoxicity against autologous DCs pulsed with HY peptide. Polyclonal antibodies (control), anti-class I MoAb or an anti-class II MoAb were added to cultures for testing blockade of cytotoxicity. (d) Cytotoxicity of HY peptide for 6 h. HY-specific CTLs against LCL cells of unrelated males who share HLA-A2 (A*0201).

Tetramer staining of HY-specific CTLs

The patient in lymphoid crisis with CML relapse developed acute GVHD shortly after the second transplant, which progressed to extensive chronic GVHD. He achieved a molecular remission at 4 months after PBSCT despite the fact that leukemic cells accounted for more than 98% of his bone marrow cells when the conditioning regimen was started. This unusual clinical course appeared to suggest the induction of GVL reactions associated with GVHD.

Other than the HY antigens, there was no disparity in the minor histocompatibility alleles, including HA-1, HA-2, CD31, CD49b and CD62L,15, 16 between the donor and the recipient. Tetramer staining demonstrated the expansion of HY peptide-specific T cells from undetectable prior to PBSCT to 15.9% of the circulating CD8+ T cells 12 weeks after PBSCT (Figure 2a). Thereafter, frequencies of HY tetramer-positive T cells declined and disappeared from the PB, coinciding with molecular relapse. The in vitro-generated HY-specific CTLs were 90% HY tetramer-positive CD8+ T cells (Figure 2b).

Figure 2
figure2

Monitoring and generation of HY-specific CD8+ T cells. (a) Correlation between frequency of circulating HY-tetramer-positive CD8+ T cells and clinical events. (b) HY-tetramer staining of the in vitro-generated HY-specific T cells, showing FITC-conjugated anti-CD8 antibody (x-axis) and PE-conjugated HY-tetramers (y-axis). Appropriate gates were set on vital lymphocytes according to their typical forward- and side-scattering characteristics.

Detection of HY-reactive CD8+ T cells by intracellular cytokine assessment

To demonstrate the HY peptide-driven expansion of CD8+ T cells, we assessed intracellular accumulation of IFNγ in PB CD8+ T cells by flow cytometry. At 12 weeks after PBSCT, 6.8% of PB CD8+ T cells produced intracellular IFNγ in response to HY peptide-pulsed autologous LCL cells (Figure 3a), while IFNγ production was negligible in CD8+ T cells in response to autologous LCL cells without the HY peptide (Figure 3b). These findings indicate that in vivo expansion and activation of HY peptide-reactive T cells occurred after the second transplantation. At that time, the proportion of circulating CD8+ cells positive for HY-tetramer staining was 15.9% as show in Figure 2a. Although inducibility of IFNγ in HY-tetramer-positive CD8+ T cells was not examined, there could be overlapping between CD8+ T cells producing intracellular IFNγ in response to HY peptide and CD8+ T cells stained with HY tetramer, because HY-tetramer staining must detect functional T cells reactive with HY peptide.

Figure 3
figure3

HY-specific IFNγ-producing CD8+ T cells in PB. (a) PB CD8+ T cells were incubated with autologous LCL cells pulsed with after fixation and permeabilization, the cells were stained for CD8 and IFNγ. The frequency of CD8+ T cells producing IFNγ in response to HY peptide is shown as a percentage of total CD8+ cells. (b) IFNγ production was negligible in CD8+ T cells stimulated by autologous LCL cells not pulsed with the HY peptide. (c) CD8+ IFNγ+ T cells detected in Figure 3a were selected by fluorescence-activated cell sorting, and spectratyping was performed. CDR3 sizes of TCR BV subfamilies from CD8+ IFNγ+ T cells are shown together with the data from in vitro-generated HY-specific CTLs.

CDR3 size distribution of TCR BV cDNA of HY-specific T cells

Spectratyping of the TCR BV region was performed on in vitro-generated HY-specific CTLs and PB CD8+ T cells that were stained with intracellular IFNγ MoAb in response to HY peptide stimulation and sorted as shown in Figure 3a. PB HY-reactive CD8+ cells showed prominent skewing within BV4, BV7, BV22 and BV24, and the in vitro-generated HY-specific CTLs showed skewing within BV4, BV7, BV12, BV16 and BV22. The two T-cell populations shared the usage of BV4, BV7 and BV22 (Figure 3c), but only the BV22+ T cells from in vitro-generated HY-specific CTLs and HY-responsive PB CD8+ T cells had a similar peak with the same CDR3 size distribution.

Deduced amino-acid sequence of CDR3 of BV22 cDNA

To determine if HY-specific CTLs circulate in the patient, we subcloned the amplified cDNA of the in vitro-generated BV22+ CTLs and BV22+ T cells that produced intracellular IFNγ in response to HY peptide stimulation as shown in Figure 3a and determined the CDR3 sequence (Table 1). One of three N-D-N sequences of HY-specific CD8+ cells from PB was identical to one of those of the CTLs. These findings indicate that the same cells isolated from the donor are expanding in vivo, and suggest that HY-specific CD8+ T cells in PB of the patient have cytotoxic activity against leukemic cells.

Table 1 Junctional amino-acid sequence of TCR BV22 of HY-specific T cells

Discussion

In mHAs HA-1- and HA-2-matched stem cell transfer between a female donor and a male recipient, we have observed the emergence of HY peptide-specific CTLs that result in the durable remission of relapsed leukemia. The present study utilized tetramer staining to show that HLA-A*0201-restricted HY peptide-specific CD8+ T cells were present in the PB after the development of GVHD and led to the eradication of BCR/ABL transcript-positive leukemic cells. Further, these cells disappeared upon molecular relapse of disease. Assaying for the frequency IFNγ-producing cells by intracellular cytokine staining, we demonstrated the emergence of functional T cells in PB that were reactive with the HY peptide during the presence of HY tetramer+ T cells. The data imply that HY-specific CTLs may have therapeutic potential as adoptive immunotherapy for relapsed leukemia after allogeneic SCT.

In this patient, leukemic relapse had occurred as full-blown disease of CML in lymphoid blast crisis. Although a beneficial effect of donor immunity was expected in the control of leukemic relapse, it would take months to start to work. Thus, we had chosen multiple transplants instead of donor leukocyte infusion (DLI) to reduce leukemic cells sufficiently.

Recently in mHAs HA-1 and/or HA-2 incompatible donor–recipient pairs an association between the emergence of HA-1 or HA-2 tetramer-positive CTLs and the complete disappearance of BCR/ABL+ cells or of myeloma cells was reported.17 Of the three reported patients, one who underwent female-to-male transplantation experienced an increase in HLA-B7-restricted HY-specific T cells as well as an increase in HA-2-specific T cells, but not that of HLA-A2-restricted HY-specific T cells. However, the cell population(s) contributing to the GVL effect were unable to be identified. It is worth noting that this reported case with lymphoid blast crisis of BCR/ABL transcript-positive CML obtained molecular remission after the development of GVHD and the emergence of HY-specific CTLs as seen in the present case.

The in vitro-generated HY peptide-specific CTLs efficiently lysed leukemic cells of the patient, and were also to a lesser extent cytotoxic to the nonhematopoietic cells such as fibroblasts of the patient. Gratwohl et al18 reported that male recipients with CML of female blood or marrow stem cell grafts are at a high risk of GVHD, but benefit from reduced incidence of disease recurrence. These findings provide evidence that HY-specific CTLs may be commonly induced in male patients given a stem cell graft from a female donor, leading to the development of GVL reactions and GVHD. This implies that the availability of a female blood or marrow graft may be beneficial to a leukemic male recipient at high risk of relapse.

In contrast to the ubiquitous expression of HY, HA-1 and HA-2 are exclusively expressed on hematopoietic cells.1 In vitro-generated HA-1- and HA-2-specific CTLs specifically lyse leukemic cells, but not nonhematopoietic cells in a 51Cr release assay.1, 19 Thus, upon HA-1- or HA-2-mismatched SCT and adoptive immunotherapy such as DLI, a low risk of GVHD would be expected. However, HA-1 disparity between a patient and a donor has been associated with the development of GVHD without reducing a rate of relapse.15, 20 Marijt et al17 demonstrated the emergence of HA-1- and HA-2-specific CD8+ T cells in PB of three patients after DLI preceding complete remission of relapsed leukemia. Relapse was associated with the development of GVHD in all three patients. A recent report showed that GVHD does not require alloantigen expression on host epithelium, and its development is primarily mediated by inflammatory cytokines such as TNFα and IL-1.21 This may account for discrepancies between in vitro behavior of HA-1- and HA-2-specific CTLs and clinical observations. Based on these findings, we believe that in mHA-oriented allogeneic immunotherapy the ability of mHAs to induce powerful immune reactions is more important than restriction of mHAs to hematopoietic tissue, and so far it appears that GVHD is an inevitable consequence. In the future, selective blockade of cytokines mediating GVHD21 may be a strategy to preserve GVL, while reducing toxicity of GVHD after mHA-oriented immunotherapy.

TCR BV spectratyping showed a similar peak with same size in a BV22+ family between in vitro-generated HY-specific CTLs and in vivo-activated HY-specific CD8+ T cells, and one shared N-D-N sequence. These findings suggest the expansion of a functional T-cell clone that participates in eradicating lymphoblastic leukemia cells positive for BCR/ABL transcripts, although we were not able to provide direct evidence demonstrating antileukemic activity of HY-specific CD8+ T cells taken from PB of the patient. It would have been beneficial to sort the HY-tetramer-positive cells detectable in PB of the patient, to expand these cells using HY peptide-pulsed LCL cells, and to test their cytotoxicity against the leukemic targets. Restricted TCR BV usage for HA-1-specific CTLs has also been described.7 Spectratyping could be beneficial in monitoring HY-specific CTLs in vivo, because spectratype analysis is more sensitive than tetramer analyses, and can be performed using as little as 500 cells.6

Compared with tetramer staining, a flow cytometric assay assessing intracellular IFNγ levels can be used to screen a large number of allogeneic peptides with a relatively little effort. In allogeneic SCT, this approach should be useful for initial screening of candidates for mHAs derived from polymorphic cellular proteins. Moreover, intracellular IFNγ assessment between CD8+ T cells during the GVL effect or GVHD as a responder and hematopoietic cells or nonhematopoietic cells of a host as a stimulator may enable the detection of undefined mHAs-specific CTLs. As the IFNγ capture assay enables isolation of live T cells stained for surface-associated IFNγ,22 further studies with regard to the function of responding effector T cells could elucidate their putative target antigens.

Another advantage of the flow cytometric cytokine production assay is that it is possible to assess the production of multiple cytokines on an antigen-specific, single-cell basis. It has already been demonstrated by Nazaruk et al23 that a subset of EBV-specific CD8+ T-cell lines produce IL-4 or IL-13 in addition to IFNγ upon stimulation with phorbol myristate acetate and ionomycin. Such a technique could be utilized in determining the cytokine production capabilities of mHA-specific CTLs in PBSCT.

In conclusion, the present data provide evidence that the emergence and activation of HY-specific CD8+ T cells may participate in eradicating lymphoblastic leukemic cells. This implies that in vitro-generated HY-specific CTLs may have therapeutic potential for relapsed leukemia after allogeneic SCT.

References

  1. 1

    Mutis T, Goulmy E . Hematopoietic system-specific antigens as targets for cellular immunotherapy of hematological malignancies. Semin Hematol 2002; 39: 23–31.

  2. 2

    Mutis T, Gillespie G, Schrama E et al. Tetrameric HLA class I-minor histocompatibilty antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease. Nat Med 1999; 5: 839–842.

  3. 3

    Kern F, Surel IP, Brock C et al. T-cell epitope mapping by flow cytometry. Nat Med 1998; 4: 975–978.

  4. 4

    Kuzushima K, Hoshino Y, Fujii K et al. Rapid determination of Epstein–Barr virus-specific CD8+ T-cell frequencies by flow cytometry. Blood 1999; 94: 3094–3100.

  5. 5

    Meadows L, Wang W, den Haan JM et al. The HLA-A*0201-restricted H-Y antigen contains a post translationally modified cysteine that significantly affects T cell recognition. Immunity 1997; 6: 273–281.

  6. 6

    Kondo Y, Shiobara S, Nakao S . Identification of T-cell clones showing expansion associated with graft-versus-leukemia effect on chronic myelogenous leukemia in vivo and in vitro. Exp Hematol 2001; 29: 471–476.

  7. 7

    Verdijk RM, Mutis T, Wilke M et al. Exclusive TCRVbeta chain usage of ex vivo generated minor histocompatibility antigen HA-1 specific cytotoxic T cells: implications for monitoring of immunotherapy of leukemia by TCRBV spectratyping. Hematol J 2002; 3: 271–275.

  8. 8

    Kuzushima K, Hayashi N, Kudoh A et al. Tetramer-assisted identification and characterization of epitopes recognized by HLA A*2402-restricted Epstein–Barr virus-specific CD8+ T cells. Blood 2003; 101: 146–1468.

  9. 9

    Redchenko IV, Rickinson AB . Accessing Epstein–Barr virus-specific T-cell memory with peptide-loaded dendritic cells. J Virol 1999; 73: 334–342.

  10. 10

    Nakao S, Takami A, Takamatsu H et al. Isolation of a T-cell clone showing HLA-DRB1*0405-restricted cytotoxicity for hematopoietic cells in a patient with aplastic anemia. Blood 1997; 89: 3691–3699.

  11. 11

    Chomczynski P, Sacchi N . Single step method of RNA isolation by acid guanidium thiocyanate phenol–chloroform extraction. Anal Biochem 1987; 162: 156–159.

  12. 12

    Currier JR, Deulofeut H, Barron KS, Kehn PJ, Robinson MA . Mitogens, superantigens, and nominal antigens elicit distinctive patterns of TCRB CDR3 diversity. Hum Immunol 1996; 48: 39–51.

  13. 13

    Waase I, Kayser C, Carlson PJ et al. Oligoclonal T cell proliferation in patients with rheumatoid arthritis and their unaffected siblings. Arthritis Rheum 1996; 39: 904–914.

  14. 14

    Casanova JL, Pannetier C, Jaulin C, Kourilsky P . Optimal conditions for directly sequencing double-stranded PCR products with sequenase. Nucleic Acids Res 1990; 18: 4028.

  15. 15

    Goulmy E, Schipper R, Pool J et al. Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versus-host disease after bone marrow transplantation. N Engl J Med 1996; 334: 281–285.

  16. 16

    Maruya E, Saji H, Seki S et al. Evidence that CD31, CD49b, and CD62L are immunodominant minor histocompatibility antigens in HLA identical sibling bone marrow transplants. Blood 1998; 92: 2169–2176.

  17. 17

    Marijt WA, Heemskerk MH, Kloosterboer FM et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia. Proc Natl Acad Sci USA 2003; 100: 2742–2747.

  18. 18

    Gratwohl A, Hermans J, Niederwieser D et al. Female donors influence transplant-related mortality and relapse incidence in male recipients of sibling blood and marrow transplants. Hematol J 2002; 2: 363–370.

  19. 19

    Mutis T, Verdijk R, Schrama E et al. Feasibility of immunotherapy of relapsed leukemia with ex vivo-generated cytotoxic T lymphocytes specific for hematopoietic system-restricted minor histocompatibility antigens. Blood 1999; 93: 2336–2341.

  20. 20

    Gallardo D, Arostegui JI, Balas A et al. Disparity for the minor histocompatibility antigen HA-1 is associated with an increased risk of acute graft-versus-host disease (GvHD) but it does not affect chronic GvHD incidence, disease-free survival or overall survival after allogeneic human leucocyte antigen-identical sibling donor transplantation. Br J Haematol 2001; 114: 931–936.

  21. 21

    Teshima T, Ordemann R, Reddy P et al. Acute graft-versus-host disease does not require alloantigen expression on host epithelium. Nat Med 2002; 8: 575–581.

  22. 22

    Becker C, Pohla H, Frankenberger B et al. Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay. Nat Med 2001; 7: 1159–1162.

  23. 23

    Nazaruk RA, Rochford R, Hobbs MV, Cannon MJ . Functional diversity of the CD8+ T-cell response to Epstein–Barr virus (EBV): implications for the pathogenesis of EBV-associated lymphoproliferative disorders. Blood 1998; 91: 3875–3883.

Download references

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare. We wish to thank Megumi Yoshii for the excellent technical assistance as well as her patience in preparation of the manuscript.

Author information

Correspondence to A Takami.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Takami, A., Sugimori, C., Feng, X. et al. Expansion and activation of minor histocompatibility antigen HY-specific T cells associated with graft-versus-leukemia response. Bone Marrow Transplant 34, 703–709 (2004). https://doi.org/10.1038/sj.bmt.1704583

Download citation

Keywords

  • minor histocompatibility antigen
  • HY
  • cytotoxic T-lymphocytes

Further reading