CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia

Wilms tumor gene product WT1 and proteinase 3 are overexpressed antigens in acute myeloid leukemia (AML), against which cytotoxic T lymphocytes can be elicited in vitro and in murine models. We performed this study to investigate whether WT1- and proteinase 3-specific CD8 T cells spontaneously occur in AML patients. T cells recognizing HLA-A2.1-binding epitopes from WT1 or proteinase 3 could be detected ex vivo in 5 of 15 HLA-A2-positive AML patients by interferon-gamma (IFN-gamma) ELISPOT assay and flow cytometry for intracellular IFN-gamma and in 3 additional patients by flow cytometry only. T cells producing IFN-gamma in response to proteinase 3 were further characterized in one patient by 4-color flow cytometry, identifying them as CD3(+)CD8(+)CD45RA(+) CCR7(-) T cells, resembling cytotoxic effector T cells. In line with this phenotype, most of the WT1- and proteinase-reactive T cells were granzyme B(+). These results provide for the first time evidence for spontaneous T-cell reactivity against defined antigens in AML patients. These data therefore support the immunogenicity of WT1 and proteinase 3 in acute leukemia patients and the potential usefulness of these antigens for leukemia vaccines.     

Allorestricted cytotoxic T cells specific for human CD45 show potent antileukemic activity

Recent advances have made haploidentical transplantation for leukemia feasible, but the rigorous T-cell depletion used contributes to the high relapse rates observed. We have attempted to improve the graft-versus-leukemia (GVL) effect by generating allorestricted cytotoxic T lymphocytes (CTLs) directed against human CD45. Such CTLs should recognize patient hematopoietic cells including leukemia, enhancing donor cell engraftment and improving the GVL effect, but they should not recognize host nonhematopoietic tissues or donor cells from the graft. Using the T2 binding assay, 4 CD45-derived peptides were found to bind HLA-A2 molecules. These peptides were used to generate cytotoxic T-cell lines from HLA-A2(-) donors by sequential stimulation with peptide-pulsed HLA-A2(+) stimulators, and the lines obtained were screened for peptide-specific cytotoxicity. Using one of these peptides (P1218), it was possible to generate peptide-specific, allorestricted CTLs in 3 of 7 responders. P1218-specific CTL lines show potent cytotoxicity against hematopoietic cell lines coexpressing HLA-A2 and CD45 but not CD45 loss variants. Studies with stable transfectants of 293 cells demonstrated recognition by P1218-specific CTLs of endogenously expressed CD45. Likewise P1218-specific CTLs recognized peripheral blood mononuclear cells (PBMCs) from HLA-A2(+) patients with chronic myeloid leukemia (CML) and leukemic blasts in HLA-A2(+) patients with acute myeloid leukemia (AML), but they were unable to lyse HLA-A2(+) fibroblasts or HLA-A2(-) normal PBMCs. Coculture of CD34(+) PBMCs and bone marrow mononuclear cells (BMMCs) with P1218-specific CTL significantly inhibited colony-forming unit-granulocyte macrophage (CFU-GM) formation in HLA-A2(+) healthy controls and CML patients but resulted in no significant inhibition in HLA-A2(-) healthy controls. These studies demonstrate that P1218-specific cytotoxic T lymphocytes (CTLs) have potent activity against leukemic progenitors and suggest that adoptive immunotherapy with allorestricted CTLs directed against CD45 epitopes may be useful in restoring the GVL effect after HLA-A2-mismatched haploidentical transplantation. Further, because P1218-specific CTLs also recognize healthy HLA-A2(+) progenitors, such CTLs could also contribute to host myeloablation and enhance donor cell engraftment.     

Acute myeloid leukemia (AML)-reactive cytotoxic T lymphocyte clones rapidly expanded from CD8(+) CD62L((high)+) T cells of healthy donors prevent AML engraftment in NOD/SCID IL2Rgamma(null) mice

OBJECTIVE: Current in vitro techniques for isolating leukemia-reactive cytotoxic T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield responder populations with unknown biological significance. This study aimed at the development of a more reliable approach, allowing generation and expansion of acute myeloid leukemia (AML)-reactive CTLs using primary in vitro stimulation. MATERIALS AND METHODS: We established allogeneic mini-mixed lymphocyte-leukemia cultures (mini-MLLCs) by stimulating donor CD8(+) T cells with human leukocyte antigen (HLA) class I-matched AML blasts in microtiter plates. Before culture, CD8(+) T cells were separated into CD62L((high)+) and CD62L((low)+/neg) subsets enriched for naive/central memory and effector memory cells, respectively. RESULTS: In eight different related and unrelated donor/AML pairs, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs expressed T-cell receptors of single Vbeta-chain families, indicating their clonal origin. The majority of CTL clones were obtained from mini-MLLCs initiated with CD62L((high)+) cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10(8)-10(10) cells within 6 to 8 weeks. Three of four representative CTL clones were capable of completely preventing engraftment of human primary AML blasts in nonobese diabetic/severe combined immune deficient IL2Rgamma(null) mice. CONCLUSION: The mini-MLLC approach allows the efficient in vitro expansion of AML-reactive CTL clones from CD8(+)CD62L((high)+) precursors of healthy donors. These CTLs can inhibit leukemia engraftment in immunodeficient mice, suggesting their potential biological relevance.     

Clinical grade expansion of CD45RA, CD45RO, and CD62L-positive T-cell lines from HLA-compatible donors: high cytotoxic potential against AML and ALL cells

OBJECTIVE: Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. MATERIALS AND METHODS: Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span, granulocyte macrophage colony-stimulating factor, interleukin-4, and calcium ionophore. All B-precursor ALL (N22) and AML (N13), but not T-cell ALL (N3), differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). RESULTS: Upon in vitro culture, donor-derived CTL acquired a memory T phenotype, showing concomitant high CD45RA, CD45RO, CD62L expression. CD8(+) cells, but not CD4(+) cells, were granzyme, perforine, and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%, 30:1 E:T ratio), but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%, 1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. CONCLUSIONS: Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation.     

Dendritic cells fused with core binding factor-beta positive acute myeloid leukaemia blast cells induce activation of cytotoxic lymphocytes

Several reports have described various strategies of dendritic cell (DC) vaccination to induce specific T-cell responses in patients with acute myeloid leukaemia (AML). About 50-60% of AML cases blasts have chromosomal abnormalities, such as inv(16) or t(8,21), which could encode for leukaemia-specific antigenic peptide sequences, possibly presented in the context of self-major histocompatibility complex (MHC) molecules. As the co-culture of AML blasts with T lymphocytes seldom resulted in T-cell stimulation, we fused AML blasts with autologous DC to enhance this effect. The fusion cells expressed MHC class I and II, CD40, B7-1, B7-2, CD209 and several adhesion molecules. In a mixed lymphocyte hybrid reaction, the fusion cells induced the proliferation of autologous T cells. Moreover, in the special case of fusion cells established from AML blasts with the chromosomal abnormality inv(16), the autologous T lymphocytes could be primed to induce cytotoxicity against up to 70% autologous AML blasts in a effector:target ratio of 20:1. Blocking assays demonstrated that the lysis was chiefly mediated by CD8(+), CCR7(-) T lymphocytes, which could be further expanded in the form of effector memory CD8(+) T cells by repeated co-cultures with the autologous fusion cells.     

Ex vivo characterization of polyclonal memory CD8+ T-cell responses to PRAME-specific peptides in patients with acute lymphoblastic leukemia and acute and chronic myeloid leukemia

Preferentially expressed antigen of melanoma (PRAME) is aberrantly expressed in hematologic malignancies and may be a useful target for immunotherapy in leukemia. To determine whether PRAME is naturally immunogenic, we studied CD8(+) T-cell responses to 4 HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and healthy donors. CD8(+) T cells recognizing PRAME peptides could be detected ex vivo in 4 of 10 ALL, 6 of 10 AML, 3 of 10 CML patients, and 3 of 10 donors by HLA-A2 tetramer analysis and flow cytometry for intracellular interferon-gamma. The frequency of PRAME-specific CD8(+) T cells was greater in patients with AML, CML, and ALL than healthy controls. All peptides were immunogenic in patients, while responses were only detected to PRA300 in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to greater than or equal to 2 PRAME epitopes compared with low PRAME expression levels (4/7 vs 0/23, P = .001), suggesting a PRAME-driven T-cell response. PRAME-specific T cells were readily expanded in short-term cultures in donors and patients. These results provide evidence for spontaneous T cell reactivity against multiple epitopes of PRAME in ALL, AML, and CML. The potential for developing PRAME as a target for immunotherapy in leukemia deserves further exploration.     

Expression and induction of costimulatory and adhesion molecules on acute myeloid leukemic cells: implications for adoptive immunotherapy

OBJECTIVE: Previously, we observed an increased recognition of malignant cells by cytotoxic T lymphocytes (CTL) when the target cells were cultured in vitro for 24 hours. In this study, we analyzed the expression of costimulatory and adhesion molecules on acute myeloid leukemia (AML) cells and determined whether 24-hour culture of the cells was associated with upregulation of these molecules. We analyzed whether this incubation period improved recognition of AML cells by CTL. MATERIALS AND METHODS: Expression of costimulatory and adhesion molecules on leukemic blasts of 34 patients comprising each AML FAB subclassification were analyzed directly and after 24 hours of culture, and the recognition of these AML cells by an HLA-A2 restricted CTL clone was determined. Blocking studies were performed with antibodies against CD54, CD58, and CD11a. RESULTS: Immunophenotyping showed a low expression of CD80 and CD40 and a variable CD86 expression on most AML cells. CD54 expression was generally low, CD58 expression was high, and CD11a expression was variable, with a higher expression in AML M0, M1, M4, and M5. Twenty-four hours of culture resulted in a significant upregulation of CD40, CD54, and CD58. Impaired recognition of AML cells by the HLA-A2 restricted CTL clone was enhanced 100-200% by 24 hours of preincubation of the leukemic cells. Blocking studies showed the importance of multiple adhesion molecules on the AML cells. CONCLUSION: Low expression of multiple costimulatory and adhesion molecules on AML could be upregulated by 24 hours of culture, which was associated with increased recognition of the AML blasts by CTL. Blocking multiple adhesion molecules completely abolished CTL recognition, showing the importance of the combination of these molecules for T-cell interaction with AML.     

Characterization of immunologic properties of a second HLA-A2 epitope from a granule protease in CML patients and HLA-A2 transgenic mice

The serine proteases, neutrophil elastase (HNE) and proteinase 3 (PR3), are aberrantly expressed in human myeloid leukemias. T-cell responses to these proteins have been correlated with remission in patients with chronic myeloid leukemia (CML). Human PR3/HNE-specific CD8(+) T cells predominantly recognize a nonameric HLA-A2-restricted T-cell epitope called PR1 which is conserved in both Ags. However, CML patients have CD8(+) T cells in peripheral blood recognizing an additional HLA-A2 epitope termed PR2. To assess immunologic properties of these Ags, novel recombinant vaccinia viruses (rVV) expressing PR3 and HNE were evaluated in HLA-A2 transgenic (Tg) mice (HHDII). Immunization of HHDII mice with rVV-PR3 elicited a robust PR3-specific CD8(+) T-cell response dominated by recognition of PR2, with minimal recognition of the PR1 epitope. This result was unexpected, because the PR2 peptide has been reported to bind poorly to HLA. To account for these findings, we proposed that HHDII mice negatively selected PR1-specific T cells because of the presence of this epitope within murine PR3 and HNE, leading to immunodominance of PR2-specific responses. PR2-specific splenocytes are cytotoxic to targets expressing naturally processed PR3, though PR1-specific splenocytes are not. We conclude that PR2 represents a functional T-cell epitope recognized in mice and human leukemia patients. These studies are registered at www.clinicaltrials.gov as NCT00716911.     

Leukemia blast-induced T-cell anergy demonstrated by leukemia-derived dendritic cells in acute myelogenous leukemia

OBJECTIVE: To elucidate the mechanism of immunologic escape of leukemia cells and establish an effective anti-leukemia immunotherapy, we attempted to generate dendritic cells from leukemia cells in patients with acute myelogenous leukemia (AML). Using these leukemia-derived dendritic cells, we investigated leukemia cell-associated T-cell anergy. MATERIALS AND METHODS: Leukemia cells of 30 patients with AML were cultured with granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha. Cultured leukemia cells were evaluated for antigen-presenting ability by mixed leukocyte culture (MLC). Normal lymphocytes, which were cocultured with leukemia blasts in the first MLC, were cultured with leukemia-derived dendritic cells in the second MLC. RESULTS: In cultures of leukemia cells from 21 of 30 patients examined, cells with stellate morphology and cell fractions with CD1a(+) and/or CD83(+) were present. Autologous MLC using lymphocytes obtained in remission phase as responders as well as allogeneic MLC demonstrated antigen-presenting ability in leukemia-derived dendritic cells. Leukemia cells of FAB-M0, M1, M2, M3, or M6 morphology/phenotype gave rise to dendritic cells as well as leukemia cells of M5. The leukemic origin of dendritic cells was suggested by in situ hybridization. By coculture with CD80(-) leukemia blasts, the response of normal lymphocytes to leukemia-derived dendritic cells cultured from the same individual as that of leukemia blasts was markedly reduced, compared with the lymphocytes cultured with leukemia blasts from a different individual as leukemia blasts. CONCLUSIONS: Escape of leukemia cells from anti-leukemia immunity may be associated with T-cell anergy caused by leukemia blasts. The results of the present study suggest that leukemia-derived dendritic cells can be applied efficiently in anti-leukemia immunotherapy.     

An anti-PR1/HLA-A2 T-cell receptor-like antibody mediates complement-dependent cytotoxicity against acute myeloid leukemia progenitor cells

PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)-like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [K(D)] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin(-)CD34(+)CD38(-) leukemia stem cells (LSCs) but not normal leukocytes (P < .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P < .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML.     

Increased HTLV type 1 tax specific CD8+ cells in HTLV type 1-asociated myelopathy/tropical spastic paraparesis: correlation with HTLV type 1 proviral load

Less than 1% of individuals infected with the human T lymphotropic virus type 1 (HTLV-1) develop an inflammatory neurological disorder, termed HTLV-1-associated myelopathy/tropical spastic (HAM/TSP), while the vast majority of those infected remain asymptomatic HTLV-1 carriers (ACs). The fundamental viroimmunological differences between these groups are not well understood. To address this issue, we have investigated HTLV-1-specific T cell responses and measured the proviral load in these groups. Frequencies of HTLV-1-specific CD8(+) cells were demonstrated to be significantly higher in HAM/TSP patients than in ACs by using intracellular cytokine staining and soluble divalent HLA-A2/Ig fusion protein loaded with HTLV-1 Tax 11-19 peptide. It is consistent with the observed increase in HTLV-1-specific cytotoxic T lymphocytes in HAM/TSP patients. These CD8(+) cells produced interferon (IFN)-gamma in recognition of HTLV-1 antigens bound to HLAs on the infected CD4(+) cells. Using phenotypic markers indicative for T cell differentiation, memory and/or effector HTLV-1 Tax-specific CD8(+) cells were found to be increased in HLA-A2 HAM/TSP patients. HTLV-1 proviral load was elevated in HAM/TSP patients when compared to ACs. In addition, the proviral load in HAM/TSP patients correlated with the frequency of HTLV-1-specific IFN-gamma(+)CD8(+) cells or Tax-HLA-A2/Ig(+)CD8(+) cells, especially with the effector cells. In contrast, the proviral load inversely correlated with memory cells. These results suggest that HTLV-1 antigens may continuously stimulate HTLV-1-specific CD8(+) cells and differentiate them from memory cells into effector cells in vivo. These differentiated HTLV-1-specific CD8(+) cells may play a role in the pathogenesis of HAM/TSP.     

Use of B cell-bound HLA-A2 class I monomers to generate high-avidity, allo-restricted CTLs against the leukemia-associated protein Wilms tumor antigen

Recent studies have detected Wilms tumor antigen (WT1)-specific cytotoxic T lymphocytes (CTLs) in patients with acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) and demonstrated that most of these CTLs were low avidity. Although HLA-mismatched donors can mount high-avidity CTLs against HLA-A2-presented peptides of WT1, a dominant anti-alloimmune response usually obscures detection of peptide-specific CTLs. Here we explored the feasibility of using recombinant HLA-A2 monomers containing single peptide epitopes as immunogens to generate peptide-specific CTLs from allogeneic donors. We demonstrate that the coating of HLA-A2(-) B lymphocytes with A2/peptide monomers provides a strong stimulus for autologous peptide-specific CTLs. After 3 to 5 rounds of stimulation a population of CD8(+) T cells binding A2/peptide tetramers is easily detectable by fluorescence-activated cell sorting analysis. Furthermore, sorted A2/WT1 tetramer-positive CTLs display strong cytotoxic activity against leukemia cells expressing WT1 endogenously but not against WT1(-) human tumor cells. Thus, HLA/peptide monomers may be useful to isolate peptide-specific donor lymphocytes for treatment of patients with leukemia after HLA-mismatched transplantation.     

T-cell receptor usage by melanoma-specific clonal and highly oligoclonal tumor-infiltrating lymphocyte lines.

Tumor-infiltrating lymphocytes (TIL) obtained from human melanomas can specifically lyse autologous tumor in vitro and mediate tumor regression in vivo. To develop more effective therapeutic reagents and to further understand the T-cell response to tumors, the diversity of T-cell receptors (TCRs) involved in melanoma antigen recognition has been studied. The TCR variable (V) genes, joining (J) segments, and N diversity regions used by five clonal lines and one highly oligoclonal, melanoma-specific, CD8+ TIL line were examined utilizing PCR amplification with V gene subfamily-specific primers and anchor PCR. The TIL lysed multiple allogeneic melanomas expressing matched surface major histocompatibility complex class I molecules. TCR analysis confirmed the clonal nature of the TIL lines; however, the TCR repertoire was diverse. Even among the three HLA-A2 restricted TIL (TIL 1200, TIL F2-2, and TIL-5), no common V gene usage was found. Comparison of the third complementarity-determining regions of the TCRs from the HLA-A2 restricted TIL revealed no homology. Results presented here identify T-cell clonotypes that recognize epitopes on highly prevalent, shared melanoma tumor-associated antigens presented in the context of HLA-B55, HLA-A1, and HLA-A2. These T cells and the antigens they recognize represent important components for the design of new immunotherapies for patients with advanced melanoma.     

Vaccination with autologous tumor-loaded dendritic cells induces clinical and immunologic responses in indolent B-cell lymphoma patients with relapsed and measurable disease: a pilot study

Eighteen relapsed patients with measurable indolent non-Hodgkin lymphoma (NHL) were vaccinated with dendritic cells (DCs) loaded with killed autologous tumor cells. Six patients had objective clinical responses including 3 continuous complete responses (CRs) and 3 partial responses (PRs), with a median follow up of 50.5 months. Eight patients had stable disease, whereas 4 had progressive disease. Clinical responses were significantly associated with a reduction in CD4(+)CD25(+)FOXP3(+) regulatory T cells, an increase in CD3(-)CD56(dim)CD16(+) natural killer (NK) cells, and maturation of lymphocytes to the effector memory stage in either postvaccination peripheral blood or tumor specimen samples. In partial responding patients, vaccination significantly boosted the IFN-gamma-producing T-cell response to autologous tumor challenge. In one HLA-A*0201(+) patient who achieved CR, IL-4 release by circulating T cells in response to tumor-specific IgH-encoded peptides was also documented. Immunohistochemical analysis of tumor biopsies using biotin-conjugated autologous serum samples revealed a tumor-restricted humoral response only in the postvaccination serum from responding patients. Collectively these results demonstrate that vaccination with tumor-loaded DCs may induce both T- and B-cell responses and produces clinical benefits in indolent NHL patients with measurable disease. This study is registered with the Istituto Superiore di Sanità: http://www.iss.it with protocol number 7578-PRE 21-801.     

Early lymphocyte recovery after intensive timed sequential chemotherapy for acute myelogenous leukemia: peripheral oligoclonal expansion of regulatory T cells

Few published studies characterize early lymphocyte recovery after intensive chemotherapy for acute myelogenous leukemia (AML). To test the hypothesis that lymphocyte recovery mirrors ontogeny, we characterized early lymphocyte recovery in 20 consecutive patients undergoing induction timed sequential chemotherapy for newly diagnosed AML. Recovering T lymphocytes were predominantly CD4(+) and included a greatly expanded population of CD3(+)CD4(+)CD25(+)Foxp3(+) T cells. Recovering CD3(+)CD4(+)CD25(+)Foxp3(+) T cells were phenotypically activated regulatory T cells and showed suppressive activity on cytokine production in a mixed lymphocyte reaction. Despite an initial burst of thymopoiesis, most recovering regulatory T cells were peripherally derived. Furthermore, regulatory T cells showed marked oligoclonal skewing, suggesting that their peripheral expansion was antigen-driven. Overall, lymphocyte recovery after chemotherapy differs from ontogeny, specifically identifying a peripherally expanded oligoclonal population of activated regulatory T lymphocytes. These differences suggest a stereotyped immunologic recovery shared by patients with newly diagnosed AML after induction timed sequential chemotherapy. Further insight into this oligoclonal regulatory T-cell population will be fundamental toward developing effective immunomodulatory techniques to improve survival for patients with AML.     

Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region

Donor-derived T lymphocytes directed against minor histocompatibility antigens (mHags) exclusively expressed on cells of the hematopoietic lineages can eliminate hematologic malignancies. Transfer of T-cell receptors (TCRs) directed against these mHags into T lymphocytes may provide a strategy to generate antileukemic T cells. To investigate the feasibility of this strategy the TCR usage of mHag HA-2-specific T-cell clones was characterized. Thirteen different types of HA-2-specific T-cell clones were detected, expressing TCRs with diversity in TCR alpha- and beta-chain usage, however, containing in the TCR alpha chain a single conserved gene segment J alpha 42, indicating that J alpha 42 is involved in HA-2-specific recognition. We transferred various HA-2 TCRs into T lymphocytes from HLA-A2-positive HA-2-negative individuals resulting in T cells with redirected cytolytic activity against HA-2-expressing target cells. Transfer of chimeric TCRs demonstrated that the HA-2 specificity is not only determined by the J alpha 42 region but also by the N-region of the alpha chain and the CDR3 region of the beta chain. Finally, when HA-2 TCRs were transferred into T cells from HLA-A2-negative donors, the HA-2 TCR-modified T cells exerted potent antileukemic reactivity without signs of anti-HLA-A2 alloreactivity. These results indicate that HA-2 TCR transfer may be used as an alternative strategy to generate HA-2-specific T cells to treat hematologic malignancies of HLA-A2-positive, HA-2-expressing patients that received transplants from HLA-A2-matched or -mismatched donors.     

Identification and characterization of epitopes of the receptor for hyaluronic acid-mediated motility (RHAMM/CD168) recognized by CD8+ T cells of HLA-A2-positive patients with acute myeloid leukemia

The receptor for hyaluronic acid-mediated motility (RHAMM/CD168) has been described as a leukemia-associated antigen. To define T-cell epitopes of RHAMM/CD168 toward specific immunotherapies for acute myeloid leukemia (AML), 10 potential HLA-A2-binding RHAMM/CD168 peptides (R1 to R10) were synthesized based on computer algorithms and screened by enzyme-linked immunospot (ELISPOT) analysis using CD8+ T cells isolated from peripheral blood (PB) of patients with AML and healthy donors. We found that CD8+ cells from 7 of 13 (54%) patients with AML presensitized with peptides R3 (ILSLELMKL) or R5 (SLEENIVIL) specifically recognized T2 cells pulsed with R3 (39%) or R5 (15%) peptide. In contrast, only 4 of 21 (19%) healthy volunteers had CD8+ cells reactive with R3- or R5-pulsed T2 cells after presensitization. The presence of R3 peptide-specific effector T cells in the peripheral blood of patients with AML could be confirmed by staining as HLA-A2/R3 peptide tetramer+ CCR7-CD45RA+ cells. In chromium-51 release assays, peptide-primed CD8+ T cells from patients with AML were able to lyse RHAMM/CD168 peptide-pulsed T2 cells, AML blasts, and dendritic cells generated thereof (AML DCs). Transfection of COS7 cells with RHAMM/CD168 cDNA revealed that peptides R3 and R5 are naturally processed epitopes of RHAMM/CD168 that are presented in an HLA-A2-restricted manner. In summary, RHAMM/CD168 is a promising target for immunotherapies in patients with AML, and we have therefore initiated a clinical vaccination trial with R3 peptide. Because RHAMM/CD168 is also expressed in various other hematologic malignancies and solid tumors, vaccines targeting this antigen may have even wider application.     

Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4+ T cells and is quickly expressed on the cell surface in an antigen-specific manner

CD40 ligand (CD40L) is an essential effector cytokine for macrophage activation, dendritic cell licensing, and T-cell-dependent antibody responses. Although CD40L is known to be made de novo following antigen recognition, several reports have described surface mobilization of preformed, intracellular CD40L in certain CD4(+) effector T cells. Here we show that rapid surface expression of preformed CD40L following antigen recognition is a general property of both effector and memory CD4(+) T cells, including in vitro and in vivo activated T-cell-receptor transgenic T cells, memory phenotype CD4(+) T cells from pathogen-free naive mice, and polyclonal virus-specific effector and memory T cells. Intracellular CD40L is stored in secretory lysosomes, and colocalizes more strongly with Fas ligand than with CTLA-4, two other molecules that are delivered to the cell surface following antigen recognition. Stimulated surface expression of preformed CD40L is found in memory CD4(+) T cells from CD40-deficient mice, indicating that it does not depend on CD40-induced internalization for delivery to the secretory compartment. We suggest that delivery of preformed CD40L to antigen-presenting cells (APCs) could enable antigen-specific activation of APCs in transient interactions that are too brief to permit de novo synthesis of CD40L.     

Significant mobilization of both conventional and regulatory T cells with AMD3100

In this study, we used the rhesus macaque model to determine the impact that AMD3100 has on lymphocyte mobilization, both alone and in combination with G-CSF. Our results indicate that, unlike G-CSF, AMD3100 substantially mobilizes both B and T lymphocytes into the peripheral blood. This led to significant increases in the peripheral blood content of both effector and regulatory T-cell populations, which translated into greater accumulation of these cells in the resulting leukapheresis products. Notably, CD4(+)/CD25(high)/CD127(low)/FoxP3(+) Tregs were efficiently mobilized with AMD3100-containing regimens, with as much as a 4.0-fold enrichment in the leukapheresis product compared with G-CSF alone. CD8(+) T cells were mobilized to a greater extent than CD4(+) T cells, with accumulation of 3.7 ± 0.4-fold more total CD8+ T cells and 6.2 ± 0.4-fold more CD8(+) effector memory T cells in the leukapheresis product compared with G-CSF alone. Given that effector memory T-cell subpopulations may mediate less GVHD compared with other effector T-cell populations and that Tregs are protective against GVHD, our results indicate that AMD3100 may mobilize a GVHD-protective T-cell repertoire, which would be of benefit in allogeneic hematopoietic stem cell transplantation.     

HLA class I-restricted lysis of leukemia cells by a CD8(+) cytotoxic T-lymphocyte clone specific for WT1 peptide

The Wilms tumor (WT1) gene has been reported to be preferentially expressed in acute leukemia cells, regardless of leukemia subtype and chronic myelogenous leukemia cells in blast crisis, but not in normal cells. This finding suggests strongly that WT1 protein is a potential target of immunotherapy for human leukemia. In this study, we established a CD8(+) cytotoxic T-lymphocyte (CTL) clone directed against a WT1-derived peptide and examined its immunologic actions on leukemia cells. A CD8(+) CTL clone, designated TAK-1, which lysed autologous cells loaded with a WT1-derived 9-mer peptide consisting of the HLA-A24 (HLA-A*2402)-binding motifs was established by stimulating CD8(+) T lymphocytes from a healthy individual repeatedly with WT1 peptide-pulsed autologous dendritic cells. TAK-1 was cytotoxic to HLA-A24-positive leukemia cells expressing WT1, but not to HLA-A24-positive lymphoma cells that did not express WT1, HLA-A24-negative leukemia cells, or HLA-A24-positive normal cells. Treating leukemia cells with an antisense oligonucleotide complementary to the WT1 gene resulted in reduced TAK-1-mediated cytotoxicity, suggesting that target antigen of TAK-1 on leukemia cells is the naturally processed WT1 peptide in the context of HLA-A24. TAK-1 did not inhibit colony formation by normal bone marrow cells of HLA-A24-positive individuals. Because WT1 is overexpressed ubiquitously in various types of leukemia cells, but not in normal cells, immunotherapy using WT1 peptide-specific CTL clones should be an efficacious treatment for human leukemia. (Blood. 2000;95:286-293)