One CD4+TCR and One CD8+TCR Targeting Autochthonous Neoantigens Are Essential and Sufficient for Tumor Eradication.

PURPOSE: To achieve eradication of solid tumors, we examined how many neoantigens need to be targeted with how many T-cell receptors (TCR) by which type of T cells. EXPERIMENTAL DESIGN: Unmanipulated, naturally expressed (autochthonous) neoantigens were targeted with adoptively transferred TCR-engineered autologous T cells (TCR-therapy). TCR-therapy used CD8+ T-cell subsets engineered with TCRs isolated from CD8+ T cells (CD8+TCR-therapy), CD4+ T-cell subsets engineered with TCRs isolated from CD4+ T cells (CD4+TCR-therapy), or combinations of both. The targeted tumors were established for at least 3 weeks and derived from primary autochthonous cancer cell cultures, resembling natural solid tumors and their heterogeneity as found in humans. RESULTS: Relapse was common with CD8+TCR-therapy even when targeting multiple different autochthonous neoantigens on heterogeneous solid tumors. CD8+TCR-therapy was only effective against homogenous tumors artificially derived from a cancer cell clone. In contrast, a combination of CD8+TCR-therapy with CD4+TCR-therapy, each targeting one neoantigen, eradicated large and established solid tumors of natural heterogeneity. CD4+TCR-therapy targeted a mutant neoantigen on tumor stroma while direct cancer cell recognition by CD8+TCR-therapy was essential for cure. In vitro data were consistent with elimination of cancer cells requiring a four-cell cluster composed of TCR-engineered CD4+ and CD8+ T cells together with antigen-presenting cells and cancer cells. CONCLUSIONS: Two cancer-specific TCRs can be essential and sufficient to eradicate heterogeneous solid tumors expressing unmanipulated, autochthonous targets. We demonstrate that simplifications to adoptive TCR-therapy are possible without compromising efficacy.

Single CAR-T cell treatment controls disseminated ovarian cancer in a syngeneic mouse model.

BACKGROUND: Treatment of some blood cancers with T cells that express a chimeric antigen receptor (CAR) against CD19 have shown remarkable results. In contrast, CAR-T cell efficacy against solid tumors has been difficult to achieve. METHODS: To examine the potential of CAR-T cell treatments against ovarian cancers, we used the mouse ovarian cancer cell line ID8 in an intraperitoneal model that exhibits disseminated solid tumors in female C57BL/6J mice. The CAR contained a single-chain Fv from antibody 237 which recognizes a Tn-glycopeptide-antigen expressed by ID8 due to aberrant O-linked glycosylation in the absence of the transferase-dependent chaperone Cosmc. The efficacy of four Tn-dependent CARs with varying affinity to Tn antigen, and each containing CD28/CD3ζ cytoplasmic domains, were compared in vitro and in vivo in this study. RESULTS: In line with many observations about the impact of aberrant O-linked glycosylation, the ID8Cosmc knock-out (ID8Cosmc-KO) exhibited more rapid tumor progression compared with wild-type ID8. Despite the enhanced tumor growth in vivo, 237 CAR and a mutant with 30-fold higher affinity, but not CARs with lower affinity, controlled advanced ID8Cosmc-KO tumors. Tumor regression could be achieved with a single intravenous dose of the CARs, but intraperitoneal administration was even more effective. The CAR-T cells persisted over a period of months, allowing CAR-treated mice to delay tumor growth in a re-challenge setting. The most effective CARs exhibited the highest affinity for antigen. Antitumor effects observed in vivo were associated with increased numbers of T cells and macrophages, and higher levels of cleaved caspase-3, in the tumor microenvironment. Notably, the least therapeutically effective CAR mediated tonic signaling leading to antigen-independent cytokine expression and it had higher levels of the immunosuppressive cytokine interleukin10. CONCLUSION: The findings support the development of affinity-optimized CAR-T cells as a potential treatment for established ovarian cancer, with the most effective CARs mediating a distinct pattern of inflammatory cytokine release in vitro. Importantly, the most potent Tn-dependent CAR-T cells showed no evidence of toxicity in tumor-bearing mice in a syngeneic, immunocompetent system.

Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides.

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors.

Cooperation of genes in HPV16 E6/E7-dependent cervicovaginal carcinogenesis trackable by endoscopy and independent of exogenous estrogens or carcinogens.

Human papillomavirus (HPV) infection is necessary but insufficient for progression of epithelial cells from dysplasia to carcinoma-in situ (CIS) to invasive cancer. The combination of mutant cellular and viral oncogenes that regulate progression of cervical cancer (CC) remains unclear. Using combinations of HPV16 E6/E7 (E+), mutant Kras (mKras) (K+) and/or loss of Pten (P-/-), we generated autochthonous models of CC without exogenous estrogen, carcinogen or promoters. Furthermore, intravaginal instillation of adenoCre virus enabled focal activation of the oncogenes/inactivation of the tumor suppressor gene. In P+/+ mice, E6/E7 alone (P+/+E+K-) failed to cause premalignant changes, while mKras alone (P+/+E-K+) caused persistent mucosal abnormalities in about one-third of mice, but no cancers. To develop cancer, P+/+ mice needed both E6/E7 and mKras expression. Longitudinal endoscopies of P+/+E+K+ mice predicted carcinoma development by detection of mucosal lesions, found on an average of 23 weeks prior to death, unlike longitudinal quantitative PCRs of vaginal lavage samples from the same mice. Endoscopy revealed that individual mice differed widely in the time required for mucosal lesions to appear after adenoCre and in the time required for these lesions to progress to cancer. These cancers developed in the transition zone that extends, unlike in women, from the murine cervix to the distal vagina. The P-/-E+K+ genotype led to precipitous cancer development within a few weeks and E6/E7-independent cancer development occurred in the P-/-E-K+ genotype. In the P-/-E+K- genotype, mice only developed CIS. Thus, distinct combinations of viral and cellular oncogenes are involved in distinct steps in cervical carcinogenesis.

Impact of TCR Diversity on the Development of Transplanted or Chemically Induced Tumors.

Burnet postulated that the diversity of T-cell receptors (TCR) allows T cells to protect against the development of cancers that display antigens with a similar, seemingly endless diversity. To test this hypothesis, we developed a strategy in which a single breeding pair of mice gives rise to four groups of sibling mice. Three of the four groups had a similar number of CD8+ T cells, but TCR diversity was either broad, significantly reduced, or absent when expressing only one type of TCR. The fourth group had no T cells. All mice shared the same housing, and, therefore, their microbial environment was similar. Only slight differences in the intestinal flora were observed under these conditions. An undisturbed broad TCR repertoire was required for the rejection of inoculated cancers displaying the natural antigenic heterogeneity of primary tumors, whereas even one type of TCR was sufficient to protect against artificial cancers stably expressing cognate antigens. The three groups of mice with limited or no TCR repertoire showed an increased risk of developing primary tumors after chemical induction. However, the risk of early death or morbidity in these cohorts of mice was significantly higher than in mice with a diverse TCR repertoire, and it remains unknown whether mice with reduced TCR diversity, who died early without cancer, would have developed tumors with higher, lower, or equal probability after induction. Together, TCR diversity seems crucial to overcome the natural genetic instability of cancers and their antigenic heterogeneity, which impacts the design of cellular therapies.

Multiple cancer-specific antigens are targeted by a chimeric antigen receptor on a single cancer cell.

Human cancer cells were eradicated by adoptive transfer of T cells transduced with a chimeric antigen receptor (CAR) made from an antibody (237Ab) that is highly specific for the murine Tn-glycosylated podoplanin (Tn-PDPN). The objectives were to determine the specificity of these CAR-transduced T (CART) cells and the mechanism for the absence of relapse. We show that although the 237Ab bound only to cell lines expressing murine Tn-PDPN, the 237Ab-derived 237CART cells lysed multiple different human and murine cancers not predicted by the 237Ab binding. Nevertheless, the 237CART cell reactivities remained cancer specific because all recognitions were dependent on the Tn glycosylation that resulted from COSMC mutations that were not present in normal tissues. While Tn was required for the recognition by 237CART, Tn alone was not sufficient for 237CART cell activation. Activation of 237CART cells required peptide backbone recognition but tolerated substitutions of up to 5 of the 7 amino acid residues in the motif recognized by 237Ab. Together, these findings demonstrate what we believe is a new principle whereby simultaneous recognition of multiple independent Tn-glycopeptide antigens on a cancer cell makes tumor escape due to antigen loss unlikely.

Transfer of Allogeneic CD4+ T Cells Rescues CD8+ T Cells in Anti-PD-L1-Resistant Tumors Leading to Tumor Eradication.

Adoptively transferred CD8+ T cells can stabilize the size of solid tumors over long periods of time by exclusively recognizing antigen cross-presented on tumor stroma. However, these tumors eventually escape T-cell-mediated growth control. The aim of this study was to eradicate such persistent cancers. In our model, the SIYRYYGL antigen is expressed by cancer cells that lack the MHC-I molecule Kb needed for direct presentation, but the antigen is picked up and cross-presented by tumor stroma. A single injection of antigen-specific 2C CD8+ T cells caused long-term inhibition of tumor growth, but without further intervention, tumors started to progress after approximately 3 months. Escape was associated with reduced numbers of circulating 2C cells. Tumor-infiltrating 2C cells produced significantly less TNFα and expressed more of the "exhaustion" markers PD-1 and Tim-3 than T cells from lymphoid organs. High-dose local ionizing radiation, depletion of myeloid-derived suppressor cells, infusions of additional 2C cells, and antibodies blocking PD-L1 did not prevent tumor escape. In contrast, adoptive transfer of allogeneic CD4+ T cells restored the numbers of circulating Ag-specific CD8+ T cells and their intratumoral function, resulting in tumor eradication. These CD4+ T cells had no antitumor effects in the absence of CD8+ T cells and recognized the alloantigen cross-presented on tumor stroma. CD4+ T cells might also be effective in cancer patients when PD-1/PD-L1 blockade does not rescue intratumoral CD8+ T-cell function and tumors persist. Cancer Immunol Res; 5(2); 127-36. ©2017 AACR.

Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma.

Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.

Eradication of Large Solid Tumors by Gene Therapy with a T-Cell Receptor Targeting a Single Cancer-Specific Point Mutation.

PURPOSE: Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic nonsynonymous single nucleotide substitutions. We determined whether adoptively transferred T cells can reject large, well-established solid tumors when engineered to express a single type of T-cell receptor (TCR) that is specific for a single AAS. EXPERIMENTAL DESIGN: By exome and RNA sequencing of an UV-induced tumor, we identified an AAS in p68 (mp68), a co-activator of p53. This AAS seemed to be an ideal tumor-specific neoepitope because it is encoded by a trunk mutation in the primary autochthonous cancer and binds with highest affinity to the MHC. A high-avidity mp68-specific TCR was used to genetically engineer T cells as well as to generate TCR-transgenic mice for adoptive therapy. RESULTS: When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intratumor vessels and eradicate large, long-established solid tumors. When the neoepitope was targeted as autochthonous antigen, T cells caused cancer regression followed by escape of antigen-negative variants. Escape could be thwarted by expressing the antigen at increased levels in all cancer cells or by combining T-cell therapy with local irradiation. Therapeutic efficacies of TCR-transduced and TCR-transgenic T cells were similar. CONCLUSIONS: Gene therapy with a single TCR targeting a single AAS can eradicate large established cancer, but a uniform expression and/or sufficient levels of the targeted neoepitope or additional therapy are required to overcome tumor escape. Clin Cancer Res; 22(11); 2734-43. ©2015 AACRSee related commentary by Liu, p. 2602.

Adoptively transferred immune T cells eradicate established tumors despite cancer-induced immune suppression.

Myeloid-derived CD11b(+)Gr1(+) suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are considered a major obstacle for effective adoptive T cell therapy. Myeloid cells suppress naive T cell proliferation ex vivo and can prevent the generation of T cell responses in vivo. We find, however, that adoptively transferred immune T cells eradicate well-established tumors in the presence of MDSCs and TAMs, which are strongly immunosuppressive ex vivo. These MDSCs and TAMs were comparable in numbers and immunosuppressive capacity among different tumor models. Longitudinal microscopy of tumors in vivo revealed that after T cell transfer, tumor vasculature and cancer cells disappeared simultaneously. During T cell-mediated tumor destruction, the tumor stroma contained abundant myeloid cells (mainly TAMs) that retained their suppressive properties. Preimmunized but not naive mice resisted immune suppression caused by an unrelated tumor burden, supporting the idea that in vivo, myeloid immunosuppressive cells can suppress naive but not memory T cell responses.

IL-15 in tumor microenvironment causes rejection of large established tumors by T cells in a noncognate T cell receptor-dependent manner.

A major challenge of cancer immunotherapy is the persistence and outgrowth of subpopulations that lose expression of the target antigen. IL-15 is a potent cytokine that can promote organ-specific autoimmunity when up-regulated on tissue cells. Here we report that T cells eradicated 2-wk-old solid tumors that expressed IL-15, eliminating antigen-negative cells. In contrast, control tumors that lacked IL-15 expression consistently relapsed. Interestingly, even tumors lacking expression of cognate antigen were rejected when expressing IL-15, indicating that rejection after adoptive T-cell transfer was independent of cognate antigen expression. Nevertheless, the T-cell receptor of the transferred T cells influenced the outcome, consistent with the notion that T-cell receptor activation and effector status determine whether IL-15 can confer lymphokine killer activity-like properties to T cells. The effect was limited to the microenvironment of tumors expressing IL-15; there were no noticeable effects on contralateral tumors lacking IL-15. Taken together, these results indicate that expression of IL-15 in the tumor microenvironment may prevent the escape of antigen loss variants and subsequent tumor recurrence by enabling T cells to eliminate cancer cells lacking cognate antigen expression in a locally restricted manner.

Spleen cells from young but not old immunized mice eradicate large established cancers.

PURPOSE: Solid tumors that have grown two weeks or longer in mice and have diameters larger than 1 cm are histologically indistinguishable from autochthonous human cancers. When experimental tumors reach this clinically relevant size, they are usually refractory to most immunotherapies but may be destroyed by adoptive T-cell transfer. However, TCR-transgenic T cells and/or tumor cells overexpressing antigens are frequently used in these experiments. Here we studied the requirements for destroying clinical size, unmanipulated 8101 tumors by adoptive cell therapy. EXPERIMENTAL DESIGN: 8101 arose in an old mouse after chronic exposure to UV light. A cancer line was established, which was never serially transplanted. The immunodominant CD8(+) T cell-recognized antigen of this tumor is caused by a somatic tumor-specific mutation in the RNA helicase p68. 8101 tumors were treated with spleen cells from young naive, or young and old immunized mice to ascertain the characteristics of immune cells that lead to rejection. RESULTS: Here we show that the mutant p68 peptide has an exceptionally high affinity to the presenting MHC class I molecule K(b) and that spleen cells from immunized young syngeneic mice adoptively transferred to Rag(-/-) or cancer-suppressed euthymic mice eradicate 8101 tumors larger than 1 cm in average diameter and established for several weeks. Spleen cells from naive young mice or from old and boosted (reimmunized) mice were ineffective. CONCLUSIONS: Relapse-free destruction of large and long-established tumors expressing a genuine very high-affinity tumor-specific antigen can be achieved by using adoptive transfer of lymphocytes from immunized young individuals.

Densely granulated murine NK cells eradicate large solid tumors.

Natural killer (NK) cells inhibit early stages of tumor formation, recurrence, and metastasis. Here, we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL-15 receptor α on cancer cells was needed to efficiently induce granulated NK cells, and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors.

Progression of cancer from indolent to aggressive despite antigen retention and increased expression of interferon-gamma inducible genes.

Many cancers escape host immunity without losing tumor-specific rejection antigens or MHC class I expression. This study tracks the evolution of one such cancer that developed in a mouse following exposure to ultraviolet light. The primary autochthonous tumor was not highly malignant and was rejected when transplanted into naïve immunocompetent mice. Neoplastic cells isolated from the primary tumor were susceptible to the growth-inhibitory effects of IFNγ in vitro, but expressed very low levels of MHC I antigen and were resistant to tumor-specific T cells unless they were first exposed to IFNγ. Serial passage of the primary tumor cells in vivo led to a highly aggressive variant that caused fast-growing tumors in normal mice. In vitro, the variant tumor cells showed increased resistance to the growth-inhibitory effects of IFNγ but expressed high levels of immunoproteasomes and MHC I molecules and were susceptible to tumor-specific T cells even without prior exposure to IFNγ.

Bystander killing of cancer requires the cooperation of CD4(+) and CD8(+) T cells during the effector phase.

Cancers frequently evade cytotoxic T lymphocyte-mediated destruction through loss or down-regulation of tumor antigens and antigen-presenting major histocompatibility complex molecules. Therefore, we have concentrated our efforts on immunological strategies that destroy nonmalignant stromal cells essential for the survival and growth of cancer cells. In this study, we developed a non-T cell receptor transgenic, immunocompetent tumor model to determine whether tumor-bearing hosts' own immune systems could eliminate cancer cells through stromal targeting and what role CD4(+) T cells play alongside CD8(+) T cells in this process. We found that aggressive cancers could be eradicated by T cell targeting of tumor stroma. However, successful elimination required the cooperation of CD4(+) and CD8(+) T cells not only during the induction phase but also during the effector phase in the tumor microenvironment, implying a new role for CD4(+) T cells that has not been previously described. Our study demonstrates the potential of stromal targeting as a cancer immunotherapy and suggests that successful anticancer strategies must facilitate cooperation between CD4(+) and CD8(+) T cells at the right times and the right places.

Cancer immunotherapy and preclinical studies: why we are not wasting our time with animal experiments.

Experimental research on the immune response to transplanted tumors has led to pioneering discoveries that laid many of the foundations for the current field of immunology. Experimental research in oncology has proven that murine and human tumors have antigens that are truly cancer specific. This article discusses research investigating how can antigens on cancer cells be used to help eradicate cancer.

CD40 ligation restores cytolytic T lymphocyte response and eliminates fibrosarcoma in the peritoneum of mice lacking CD4+ T cells.

Absence of CD4(+) T cell help has been suggested as a mechanism for failed anti-tumor cytotoxic T lymphocytes (CTL) response. We examined the requirement for CD4(+) T cells to eliminate an immunogenic murine fibrosarcoma (6132A) inoculated into the peritoneal cavity. Immunocompetent C3H mice eliminated both single and repeat intraperitoneal (IP) inoculums, and developed high frequency of 6132A-specific interferon-gamma (IFNgamma)-producing CTL in the peritoneal cavity. Adoptive transfer of peritoneal exudate cells (PEC) isolated from control mice, protected SCID mice from challenge with 6132A. In contrast, CD4 depleted mice had diminished ability to eliminate tumor and succumbed to repeat IP challenges. Mice depleted of CD4(+) T cells lacked tumor-specific IFNgamma producing CTL in the peritoneal cavity. Adoptive transfer of PEC from CD4 depleted mice failed to protect SCID mice from 6132A. However, splenocytes isolated from same CD4 depleted mice prevented tumor growth in SCID mice, suggesting that 6132A-specific CTL response was generated, but was not sustained in the peritoneum. Treating CD4 depleted mice with agonist anti-CD40 antibody, starting on days 3 or 8 after initiating tumor challenge, led to persistence of 6132A-specific IFNgamma producing CTL in the peritoneum, and eliminated 6132A tumor. The findings suggest that CTL can be activated in the absence of CD4(+) T cells, but CD4(+) T cells are required for a persistent CTL response at the tumor site. Exogenous stimulation through CD40 can restore tumor-specific CTL activity to the peritoneum and promote tumor clearance in the absence of CD4(+) T cells.

Improved performance of hip DXA using a novel region of interest in the upper part of the femoral neck: in vitro study using bone strength as a standard of reference.

We tested the hypothesis that bone mineral density (BMD) and bone mineral content (BMC) in proximal human femur specimens in the upper neck region of interest (ROI) and femoral neck axis length (FNAL) provide a significantly better prediction of femoral bone strength than standard ROIs in vitro. BMD and BMC were measured in 110 proximal femur specimens using a standard dual-energy X-ray absorptiometry (DXA) scanner. The analysis included a new ROI in the upper neck as well as the standard ROIs. FNAL was obtained from the scan images. The specimens' failure-load was measured in a mechanical loading device, simulating a fall on the greater trochanter. For the standard ROIs, correlations between failure-load and BMD ranged from R2 = 0.64 (shaft ROI) to R2 = 0.70, p < 0.001 (femoral neck). Prediction of strength by BMD did not significantly differ from those of BMC (R2 ranging from 0.65 to 0.75, p < 0.001). In the upper neck ROI, for both BMD and BMC correlations with failure-load were higher (R2 = 0.76 and 0.81, respectively; p < 0.001). A lower, yet still significant, correlation was found between FNAL and bone strength (R2 = 0.23, p < 0.001). Normalization of failure-load with respect to FNAL did not significantly increase the correlations with densitometric measures. This study provides in vitro evidence indicating that among the ROIs of the proximal femur the newly defined upper neck ROI provides the best prediction of bone strength. Only a weak association was observed between failure load and FNAL.

A new murine tumor model for studying HLA-A2-restricted anti-tumor immunity.

HLA-A2/K(b) transgenic mice have been powerful tools for studying HLA-A2-restricted anti-tumor immunity. Two tumor lines were established from an aged HLA-A2/K(b) transgenic mouse that developed spontaneous tumors in the right limb and lung. Histopathologic analysis of the tumor was consistent with an osteosarcoma that had metastasized to the lung. The cells from the primary tumor and the lung metastasis were adapted to culture and are designated Ag201P and Ag201M, respectively. Both Ag201P and Ag201M induced tumors in mice, indicating that the established cell lines are tumorigenic. Both tumor lines expressed HLA-A2/K(b) as assessed by RT-PCR and immunofluorescence analysis. Furthermore, the HLA-A2/K(b) molecules were functional on both tumor lines as demonstrated by their ability to present exogenously loaded HLA-A2-restricted peptides to human HLA-A2-restricted T cells. More importantly, endogenously expressed HLA-A2-restricted epitopes were processed and presented in the context of HLA-A2/K(b) in Ag201P and Ag201M cells to human HLA-A2-restricted T cells. Thus, Ag201P and Ag201M are two new murine tumor lines that express functional HLA-A2/K(b), and represent potentially invaluable tools to study HLA-A2-restricted anti-tumor immunity in mice.