Histocompatibility Leukocyte Antigen-A2402-restricted Cytotoxic T Lymphocytes Recognizing Adenocarcinoma in Tumor-infiltrating Lymphocytes of Patients with Colon Cancer

To cast light on T cell-mediated specific immunity at the tumor site of colon cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) from colon cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted cytotoxicity against adenocarcinoma. IL 2-activated TIL from all four HLA-A24 patients examined lysed HLA-A2402+ adenocarcinomas, but not HLA-A2402 tumors. Those of two of the four cases also lysed HLA-A2402⁺ squamous cell carcinomas. CD8⁺ cytotoxic T lymphocyte (CTL) clones recognizing HLA-A2402⁺ adenocarcinomas were established from one CTL line. This CTL line produced IFN-γ upon recognition of an HLA-A2402^- adenocarcinoma transfected with HLA-A2402 cDNA. These results suggest the presence of HLA-A2402-restricted CTL recognizing adenocarcinoma at the tumor site of colon cancer. Furthermore, HLA-A31-restricted CTL activity was found in IL-2-activated TIL from one of two HLA-A31⁺ patients, suggesting the existence of HLA-class I-restricted CTL involving an allele other than A24     

A newly identified MAGE-3-derived epitope recognized by HLA-A24-restricted cytotoxic T lymphocytes

Five MAGE-3-derived peptides carrying an HLA-A24-binding motif were synthesized. Binding capacity of these peptides was analyzed by an HLA-class-I stabilization assay. Two of the 5 peptides bound to HLA-A*2402 molecule with high affinity, and 3 peptides with low affinity. Peripheral-blood mononuclear cells (PBMC) depleted of CD4+T cells were stimulated with the peptides to determine whether these peptides would induce cytotoxic T lymphocytes (CTL) from PBMCs obtained from 7 healthy HLA-A*2402+ donors. Peptide M3-p97 (TFPDLESEF; corresponding to amino-acid residues 97-105 of MAGE-3), with high binding capacity to the HLA-A*2402 molecule, elicited the peptide-specific and HLA-A24-restricted CD8+CTL lines in 2 of the 7 donors, while none of the 4 other peptides induced CTL specific for the corresponding peptide in any of the donors. CTL lines induced by stimulation with peptide M3-p97 exhibited cytolytic activities against HLA-A*2402 transfectant cell lines (C1R-A*2402) in the presence of peptide M3-p97, but not in unloaded or irrelevant peptide-pulsed C1R-A*2402 cells. The CTL lines and a cloned CD8+CTL isolated from one of the bulk populations by limiting dilution could lyse MAGE-3+/HLA-A*2402+ squamous-cell-carcinoma(SCC) lines but neither MAGE-3-/HLA-A*2402+ nor MAGE-3+/HLA-A*2402- SCC lines, indicating that M3-p97 can be naturally processed and presented on the tumor-cell surface in association with HLA-A*2402 molecules. Combined with the 4 currently reported CTL epitopes derived from MAGE-3 and presented by HLA-A1, HLA-A2, HLA-A24 or HLA-B44, identification of this CTL epitope presented by the HLA-A*2402 molecule will extend the application of MAGE-3-derived peptides for immunotherapy for cancer patients.     

MHC-dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas

Tumor-infiltrating lymphocytes (TIL) were grown from 23 urothelial carcinomas. Phenotyping analysis showed that the TIL cultures were mainly CD3⁺. Although CD4⁺ and CD8⁺ T-cell sub-sets were grown in culture, CD4⁺ T-cell sub-sets predominated over CD8⁺ T cells. Immunohistochemical studies performed on 5 tumor specimens confirmed this observation, and indicated that CD4⁺ T cells surrounded the tumor islets, whereas CD8⁺ T lymphocytes were localized among the tumor cells. Five short-term carcinoma cell lines established from these urothelial tumors were used as target cells in cytolysis assays in order to investigate the functional anti-tumor activity of autologous TIL. TIL from 4/5 tumors were lytic and 3 TIL lines displayed MHC-class-I-dependent cytotoxicity directed against autologous tumor cells. CD4⁺ T-cell-depletion experiments performed on TIL line 07 confirmed that CD8⁺ MHC-class-I-dependent CTL were the predominant effectors. Finally, experiments performed on 6 allogeneic urothelial-cancer cell lines matched for HLA-class-I molecules showed that TIL07 exhibited selective lytic activity toward tumor 07. These data indicate that CD8⁺ MHC-class-I-dependent CTL present in urothelial carcinomas are functional and may participate in the anti-tumor immune response. Int. J. Cancer 71:585-594, 1997. © 1997 Wiley-Liss, Inc.     

Histocompatibility Leukocyte Antigen-A2-restricted and Tumor-specific Cytotoxic T Lymphocytes from Tumor-infiltrating Lymphocytes of a Patient with Testicular Embryonal Cancer

T lymphocytes play an important role in tumor rejection. To understand T cell-mediated specific immunity at the tumor site of testicular embryonal cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) of a patient with testicular embryonal cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted and tumor-specific cytotoxicity. We established a CD3⁺CD4^-CD8⁺ cytotoxic T lymphocyte (CTL) line from the IL-2-activated TIL of a 37-year-old patient with testicular embryonal cancer. A 6 h ⁵¹Cr-release assay was performed to measure the cytotoxicity of the CTL. The CD3⁺CD4^-CD8⁺ CTL line showed cytotoxicity against HLA-A2⁺ tumor cells, including freshly isolated autologous tumor cells, adenocarcinoma cell lines from various organs (lung, breast, pancreas, colon and kidney) and squamous cell carcinomas (esophagus and oral cavity). No other cell lines examined, including an autologous tumor cell line and HLA-A2" tumor cell lines, were lysed by this CTL line. These results suggest the existence of HLA-A2-restricted and tumor-specific CTL at the tumor site of testicular embryonal cancer.     

Identification of GP100-derived,melanoma-specific cytotoxic T-lymphocyte epitopes restricted by HLA-A3 supertype molecules by primary in vitro immunization with peptide-pulsed dendritic cells

The human melanocyte lineage-specific antigen gp100 contains several epitopes recognized by cytotoxic T lymphocytes (CTL). However, most of the epitopes reported to date are HLA-A2.1-restricted. Despite the high frequency of HLA-A2.1 in melanoma patients, effective population coverage requires the identification of epitopes restricted by other frequent HLA alleles. Herein, HLA-A3 binding, gp100-derived synthetic peptides were tested for their capacity to elicit anti-melanoma CTL in vitro using CD8⁺ T cells from healthy donors as responders and peptide-pulsed autologous dendritic cells as antigen-presenting cells. Of 7 peptides tested, 2 (gp100[9₈₇] and gp100[10₈₆] ) induced CTLs that killed melanoma cell lines expressing HLA-A3 and gp100. Additional MHC-binding studies to various HLA molecules belonging to the HLA-A3 superfamily (HLA-A*1101, -A*3101, -A*3301 and -A*6801) were performed to determine whether these CTL epitopes could further increase potential population coverage. Further experiments indicated that the peptide gp100\[9₈₇\], which bound to HLA-A11 with high affinity, was capable of inducing specific CTLs that killed melanoma cells expressing gp100 and HLA-A11 molecules. Our results indicate that the gp100\[9₈₇\] peptide corresponds to a CTL epitope which may be restricted by either the HLA-A3 or HLA-A11 allele, emphasizing its utility for the design and development of epitope-based therapies for melanoma. Int. J. Cancer, 78:518–524, 1998. © 1998 Wiley-Liss, Inc.     

Development of HLA-A2402/K(b) transgenic mice

HLA-transgenic mice have been developed to facilitate studies of HLA-restricted cytotoxic responses, e.g., for the identification of immunodominant HLA-restricted CTL epitopes and the optimization of peptide or DNA vaccine constructs for human use. We have developed HLA-A2402/K(b)-transgenic mice expressing chimeric human (alpha1 and alpha2 domains of HLA-A2402) and mouse (alpha3, transmembrane and cytoplasmic domains of H-2K(b)) class I molecules. Immunization of these HLA-A2402/K(b)-transgenic mice with various known HLA-A24-restricted immunodominant cancer CTL epitope peptides derived from gp100, MAGE-1, MAGE-3, Her2/neu, CEA and TERT induced HLA-A24-restricted, peptide-specific CTLs. Using these transgenic mice, we identified a novel HLA-A24-restricted CTL epitope, PSA(152-160), encoded by human prostate-specific antigen. Staining with HLA tetramers showed that the cytotoxic activity induced by immunizing with PSA(152-160) in HLA-A2402/K(b) transgenic mice was HLA-A2402-restricted and CD8-dependent. Therefore, PSA(152-160) might be a candidate peptide for vaccination of HLA-A24(+) patients with prostate cancer. Our results suggest that HLA-A2402/K(b) transgenic mice might be useful in the search for HLA-A24-restricted CTL epitopes functioning as human cancer antigens and for the development of peptide-based cancer immunotherapy.     

Peptide-specific ctl in tumor-infiltrating lymphocytes from metastatic melanomas expressing mart-1/melan-a,gp100 and tyrosinase genes: A study in an unselected group of hla-a2.1-positive patients

Peptide specificity of cultured tumor-infiltrating lymphocytes (TIL) was systematically investigated in a group of HLA-A2.1⁺ metastatic melanoma patients consecutively referred to our department for surgical treatment. Seven samples from 6 patients were studied. All surgical specimens showed evidence of gp100, MART-1/Melan-A and Tyrosinose gene expression as detectable by reverse PCR (rPCR). Cultured TIL from 2 patients displayed cytotoxic activity against autologous or HLA-matched EBV-transformed cells previously pulsed with MART-1/Melan-A_27-35 peptide. In contrast, no CTL activity against gp 100_280-288 or tyrosinase_1-9 peptides could be observed. TIL were then repeatedly stimulated in vitro with the same peptides. After 6 restimulation courses at weekly intervals, specific recognition of gp100_280-288 and MART-1/Melan-A_27-35 peptides was detectable in 3 and 5 TIL populations, respectively. In one case Tyrosinase_1-9-specific CTL could be demonstrated. Two TIL populations from metastases resected from a melanoma patient at 6 months' distance showed a different peptide specificity pattern, and no specific CTL could be generated from simultaneously sampled peripheral blood mononuclear cells (PBMC). All peptide-specific CTL populations also displayed significant cytotoxic activity against HLA-A2.1 matched melanoma cell lines expressing the antigens under investigation. Our data indicate that CTL specific for MART-Melan-A_27-35, gp100_280-288 or Tyrosinase_1-9 peptides could be expanded with varying frequency from TIL derived from 4 out of 6 HLA-A2.1⁺ patients whose tumors expressed the genes encoding these tumor-associated antigens (TAA). © 1995 Wiley-Liss, Inc.     

Targeting of human p53-overexpressing tumor cells by an HLA A*0201-restricted murine T-cell receptor expressed in Jurkat T cells

A potent anti-human (hu) p53 CD8+ CTL response develops in HLA A*0201 transgenic (Tg) mice after immunization with peptides corresponding to HLA A*0201 motifs from hu p53. Mice immunized with the hu P53(149-157) peptide develop a CTL response that is of moderately high affinity and is capable of recognizing hu tumor cells expressing mutated p53. In this report, the mRNAs encoding the predominantly expressed T-cell receptor (TCR) sequences were molecularly cloned from a murine (mu) CTL clone derived from immunized Tg mice, which recognized endogenously processed hu p53 restricted by HLA A*0201. The separate A and B chain TCR cDNAs were transfected in the corresponding TCR A- and B- Jurkat-CD3- mutant T-cell lines, and each rescued CD3 surface expression. Both TCR chains were simultaneously introduced into Jurkat-CD3+ cells, and the transfected Jurkat cells recognized hu T2 cells sensitized with the p53(149-157) CTL epitope but not T2 cells sensitized with a nonspecific CTL epitope. Breast, pancreatic, and sarcoma tumor cell lines, which overexpress endogenous mutated p53, were recognized in the presence of anti-CD28 costimulation, only if they also expressed HLA A*0201. Normal hu fibroblasts established from skin cultures were not recognized. These results represent the first time that a p53-specific TCR capable of recognizing hu cancer cells was heterologously expressed in a naive recipient cell, converting that cell to one recognizing hu tumor cells with mutated p53. This TCR represents a candidate molecule for a genetic strategy in combating hu cancer by an adoptive immunotherapy approach, which uses the strong xenorecognition of hu p53 in mice.     

Determination of cellularly processed HLA-A2402-restricted novel CTL epitopes derived from two cancer germ line genes, MAGE-A4 and SAGE.

PURPOSE: For identification of CTL epitopes useful for cancer vaccines, it is crucial to determine whether cognate epitopes are presented on the cell surface of target cancer cells through natural processing of endogenous proteins. For this purpose, we tried to use the cellular machinery of both mice and human to define naturally processed CTL epitopes derived from two "cancer germ line" genes, MAGE-A4 and SAGE. EXPERIMENTAL DESIGN: We vaccinated newly produced HLA-A2402 transgenic mice with DNA plasmids encoding target antigens. Following screening of synthesized peptides by splenic CD8(+) T cells of vaccinated mice, we selected candidate epitopes bound to HLA-A2402. We then examined whether human CD8(+) T cells sensitized with autologous CD4(+) PHA blasts transduced by mRNA for the cognate antigens could react with these selected peptides in an HLA-A2402-restricted manner. RESULTS: After DNA vaccination, murine CD8(+) T cells recognizing MAGE-A4(143-151) or SAGE(715-723) in an HLA-A2402-restricted manner became detectable. Human CTLs specific for these two peptides were generated after sensitization of HLA-A2402-positive CD8(+) T cells with autologous CD4(+) PHA blasts transduced with respective mRNA. CTL clones were cytotoxic toward tumor cell lines expressing HLA-A2402 and cognate genes. Taken together, these CTL epitopes defined in HLA-A24 transgenic mice are also processed and expressed with HLA-A2402 in human cells. The presence of SAGE(715-723)-specific precursors was observed in HLA-A2402-positive healthy individuals. CONCLUSIONS: Two novel HLA-A2402-restricted CTL epitopes, MAGE-A4(143-151) and SAGE(715-723), were identified. Our approach assisted by cellular machinery of both mice and human could be widely applicable to identify naturally processed CTL epitopes.     

Identification of SPARC as a candidate target antigen for immunotherapy of various cancers

To establish efficient anticancer immunotherary, it is important to identify tumor‐associated antigens (TAAs) directing the immune system to attack cancer. A genome‐wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA‐A24 (A*2402)‐restricted and SPARC‐derived CTL epitopes. We previously identified H‐2K^d‐restricted and SPARC‐derived CTL epitope peptides in BALB/c mice, of which H‐2K^d‐binding peptide motif is comparable with that of HLA‐A24 binding peptides. By using these peptides, we tried to induce HLA‐A24 (A*2402)‐restricted and SPARC‐reactive human CTLs and demonstrated an antitumor immune response. The SPARC‐A24‐1_143–151 (DYIGPCKYI) and SPARC‐A24‐4_225–234 (MYIFPVHWQF) peptides‐reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA‐A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA‐A24 (A*2402). Furthermore, the adoptive transfer of the SPARC‐specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA‐A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.     

Identification of novel Lck‐derived T helper epitope long peptides applicable for HLA‐A2+ cancer patients as cancer vaccine

The present study attempted to identify T helper epitope long peptides capable of inducing cytotoxic T lymphocytes (CTL) from Lck antigen (p56^Lck), the src family tyrosine kinase, which is known to be aberrantly expressed in metastatic cancers cells, in order to develop a long peptide‐based cancer vaccine for HLA‐A2⁺ cancer patients. Based on the biding motif to the HLA‐DR and HLA‐A2 alleles, 94 peptides were prepared from the Lck antigen. These peptides were screened for their reactivity to immunoglobulin G (IgG) from plasma of cancer patients, followed by testing of their ability to induce both CD4⁺ and CD8⁺ T lymphocytes showing not only peptide‐specific IFN‐γ production but cytotoxicity against HLA‐A2⁺ cancer cells from peripheral blood mononuclear cells (PBMC) of HLA‐A2⁺ cancer patients. Among 94 peptides tested, the three T helper epitope long peptides and their inner CTL epitope short peptides with HLA‐A2 binding motifs were frequently recognized by IgG of cancer patients, and efficiently induced both CD4⁺ IFN‐γ⁺ and CD8⁺ IFN‐γ⁺ T lymphocytes. Patients' PBMC stimulated with these long peptides showed cytotoxicity against HLA‐A2⁺ Lck⁺ cancer cells in HLA‐class I and HLA‐class II dependent manners. These three peptides might be useful for long peptide‐based vaccines for HLA‐A2⁺cancer patients with Lck⁺ tumor cells.     

Immunological evaluation of peptide vaccination for cancer patients with the HLA‐A26 allele

To develop a peptide vaccine for cancer patients with the HLA‐A26 allele, which is a minor population worldwide, we investigated the immunological responses of HLA‐A26⁺/A26⁺ cancer patients to four different CTL epitope peptides under personalized peptide vaccine regimens. In personalized peptide vaccine regimens, two to four peptides showing positive peptide‐specific IgG responses in pre‐vaccination plasma were selected from the four peptide candidates applicable for HLA‐A26⁺/A26⁺ cancer patients and administered s.c. Peptide‐specific CTL and IgG responses along with cytokine levels were measured before and after vaccination. Cell surface markers in PBMCs and plasma cytokine levels were also measured. In this study, 21 advanced cancer patients, including seven lung, three breast, two pancreas, and two colon cancer patients, were enrolled. Their HLA‐A26 genotypes were HLA‐A26:01 (n = 24), HLA‐A26:03 (n = 10), and HLA‐A26:02 (n = 8). One, 14, and 6 patients received two, three, and four peptides, respectively. Grade 1 or 2 skin reactions at the injection sites were observed in the majority of patients, but no severe adverse events related to the vaccination were observed. Peptide‐specific CTL responses were augmented in 39% or 22% of patients after one or two cycles of vaccination, respectively. Notably, peptide‐specific IgG were augmented in 63% or 100% of patients after one or two cycles of vaccination, respectively. Personalized peptide vaccines with these four CTL epitope peptides could be feasible for HLA‐A26⁺ advanced cancer patients because of their safety and higher rates of immunological responses.     

Identification of SART3-derived peptides capable of inducing HLA-A2-restricted and tumor-specific CTLs in cancer patients with different HLA-A2 subtypes

We recently identified the SART3 antigen encoding shared tumor epitopes recognized by HLA-A2402-restricted and tumor-specific CTLs. Our study investigated whether the SART3 antigen encodes peptides recognized by the HLA-A2-restricted CTLs. The HLA-A2-restricted and tumor-specific CTL line recognized COS-7 cells co-transfected with the SART3 gene and either HLA-A0201, -A0206 or -A0207 cDNA but not those co-transfected with the SART3 gene and HLA-A2402 or -A2601 cDNA. The 2 SART3 peptides at positions 302 to 310 and 309 to 317 possessed the ability to induce HLA-A2-restricted and tumor-specific CTLs from peripheral blood mononuclear cells of cancer patients with various histological types and different HLA-A2 subtypes. Therefore, these 2 peptides could be useful for specific immunotherapy of a relatively large number of HLA-A2(+) cancer patients.     

Usage of T-cell receptor Vβ chain genes in fresh and cultured tumor-infiltrating lymphocytes from human melanoma

Tumor-infiltrating lymphocytes (TIL) freshly obtained from human malignant melanomas as well as the same TIL grown in the presence of interleukin 2 (IL2) were studied for gene expression of the T-cell receptor (TCR) variable β regions (Vβ). To perform the TCR-Vβ analysis, total RNA was isolated from TIL and reverse-transcribed into cDNA, which was then amplified by PCR using 22 different 5′ primers specifically recognizing the sequences of 20 Vβ gene families and a 3′ primer annealing to the constant region of the β chain. The TCR-α constant region (Cα) gene was co-amplified as a standard for the calculation of the percentage of each TCR-Vβ gene expressed. The frequency of individual Vβ regions expressed on TIL was computed from the ratio of cpm Vβ to cpm Ca for each Vβ region in relation to the total of all 22 ratios. With fresh TIL obtained from 8 different melanomas, oligoclonal distribution of Vβ genes expressed on TIL was observed, in comparison with a broader and unrestricted distribution seen with peripheral-blood T cells of 8 normal individuals. The oligoclonal patterns of Vβ-gene expression in fresh melanoma TIL were distinct in every tumor. Several of the Vβ-genes usually expressed in normal PBL were not expressed in fresh TIL. In melanoma TIL cultured in the presence of IL2 and IL4 and in the absence of autologous tumor (AuTu) or antigen-presenting cells for 23 to 65 days, selection of T-cell lines expressing a restricted number of Vβ genes occurred. Although in 4/5 TIL cultures this selection involved the Vβ7 gene, no relationship could be established between Vβ gene expression in fresh TIL and that in T-cell lines outgrowing in long-term cultures. Selection in culture of CD3⁺ CD8⁺ T-cell lines with Vβ-gene expression restricted to I or 2 Vβ families did not correlate with the presence or level of AuTu cytotoxicity mediated by these T cells. The results indicate that in TIL cultures random selection of T-cell lines with reactivity not relevant to AuTu may account for poor expression or loss of AuTu cytotoxicity by most TIL cultured long-term in the presence of cytokines and in the absence of specific antigenic stimulation.     

The shared tumor associated antigen cyclin‐A2 is recognized by high‐avidity T‐cells

Cyclin‐A2, a key cell cycle regulator, has been shown to be overexpressed in various types of malignancies with little expression in normal tissue. Such tumor‐associated genes potentially are useful targets for cancer immunotherapy. However, high‐avidity cyclin‐specific T cells are considered to be thymically deleted. We identified at least one nonameric HLA‐A*0201 binding cyclin‐A2 epitope by a reverse immunology approach. Using a highly efficient T‐cell expansion system that is based on CD40‐activated B (CD40‐B) cells as sole antigen‐presenting cells we successfully generated cyclin‐A2 specific CTL from HLA‐A*0201⁺ donors. Interestingly, high‐avidity cyclin‐A2 specific CTL lines, which recognized peptide‐pulsed and antigen expressing target cells, were indeed generated by stimulation with CD40‐B cells when pulsed with low concentrations of peptide, whereas CD40‐B cells pulsed at saturating concentrations could only induce low‐avidity CTL, which recognized peptide‐pulsed target cells only. One high‐avidity CTL line was subcloned and CTL clones, whose peptide concentration required for half‐maximal lysis were less than 1 nM, could lyse cyclin‐A2 expressing tumor cells. Taken together, cyclin A2 is an attractive candidate for immune intervention in a significant number of cancer patients and high‐avidity T cells can be readily generated using CD40‐B cells as antigen‐presenting cells. © 2009 UICC     

Patient‐individualized CD8+ cytolytic T‐cell therapy effectively combats minimal residual leukemia in immunodeficient mice

Adoptive transfer of donor‐derived cytolytic T‐lymphocytes (CTL) has evolved as a promising strategy to improve graft‐versus‐leukemia (GvL) effects in allogeneic hematopoietic stem‐cell transplantation. However, durable clinical responses are often hampered by limited capability of transferred T cells to establish effective and sustained antitumor immunity in vivo. We therefore analyzed GvL responses of acute myeloid leukemia (AML)‐reactive CD8⁺ CTL with central and effector memory phenotype in a new allogeneic donor‐patient specific humanized mouse model. CTL lines and clones obtained upon stimulation of naive CD45RA⁺ donor CD8⁺ T cells with either single HLA antigen‐mismatched or HLA‐matched primary AML blasts, respectively, elicited strong leukemia reactivity during cytokine‐optimized short to intermediate (i.e., 2–8 weeks) culture periods. Single doses of CTL were intravenously infused into NOD/scidIL2Rcg^null mice when engraftment with patient AML reached bone marrow infiltration of 1–5%, clinically defining minimal residual disease status. This treatment resulted in complete regression of HLA‐mismatched and strong reduction of HLA‐matched AML infiltration, respectively. Most importantly, mice receiving AML‐reactive CTL showed significantly prolonged survival. Transferred CTL were detectable in murine bone marrow and spleen and demonstrated sustained AML‐reactivity ex vivo. Moreover, injections with human IL‐15 clearly promoted CTL persistence. In summary, we show that naive donor‐derived CD8⁺ CTL effectively combat patient AML blasts in immunodeficient mice. The donor‐patient specific humanized mouse model appears suitable to evaluate therapeutic efficacy of AML‐reactive CTL before adoptive transfer into patients. It may further help to identify powerful leukemia rejection antigens and T‐cell receptors for redirecting immunity to leukemias even in a patient‐individualized manner.     

Identification of immunodominant HLA-B7-restricted CD8<Superscript>+</Superscript> cytotoxic T cell epitopes derived from mammaglobin-A expressed on human breast cancers

Mammaglobin-A (MGBA), a 10-kD protein, is over expressed in 80% of primary and metastatic human breast cancers. Breast cancer patients demonstrate high frequencies of CD8⁺ cytotoxic T lymphocytes (CTL) specific to MGBA. Defining CD8⁺ CTL responses to HLA class I-restricted MGBA-derived epitopes assumes significance in the context of our ongoing efforts to clinically translate vaccine strategies targeting MGBA for prevention and/or treatment of human breast cancers. In this study, we define the CD8⁺ CTL response to MGBA-derived candidate epitopes presented in the context of HLA-B7, which has a frequency of 17.7% in Caucasian and 15.5% in African American populations. We identified seven MGBA-derived candidate epitopes with high predicted binding scores for HLA-B7 using a computer algorithm. Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-B7 indicated that MGBA B7.3 (VSKTEYKEL), B7.6 (KLLMVLMLA), B7.7 (NPQVSKTEY), and B7.1 (YAGSGCPLL) have the highest HLA-B7 binding affinities. Further, two CD8⁺ CTL cell lines generated in vitro against T2.B7 cells individually loaded with MGBA-derived candidate epitopes showed significant cytotoxic activity against MGBA B7.1, B7.3, B7.6, and B7.7. In addition, the same CD8⁺ CTL lines lysed the HLA-B7⁺/MGBA⁺ human breast cancer cell line DU-4475 but had no significant cytotoxicity against HLA-B7⁻ or MGBA⁻ breast cancer cell lines. Cold-target inhibition studies strongly suggest that MGBA B7.3 is an immunodominant epitope. In summary, our results define HLA-B7-restriced, MGBA-derived, CD8⁺ CTL epitopes with all of the necessary features for developing novel vaccine strategies against HLA-B7 expressing breast cancer patients.