Identification of epitope mimics recognized by CTL reactive to the melanoma/melanocyte-derived peptide MART-1(27-35).

CTL reactivity to the epitope MART-1(27-35), of the melanoma (self) antigen MART-1/melan A is frequently observed in tumor-infiltrating lymphocytes and may be readily elicited from the peripheral blood of melanoma patients that express HLA-A*0201. Available data suggest that these observations contrast with those made for other HLA-A*0201-presented melanoma self antigens regarding the regularity of observed CTL responses. Based on preliminary findings, we hypothesized that the CTL response to MART-1 might be augmented in part by T cell encounters with peptides derived from sources other than MART-1, which show sequence similarity to MART-1(27-35). To test this idea, a protein database search for potential MART-1 epitope mimics was done using criteria developed from analyses of effector recognition of singly-substituted peptide analogues of MART-1(27-35). Synthetic peptides were made for a portion of the sequences retrieved; 12/40 peptides tested were able to sensitize target cells for lysis by one or more anti-MART-1 effectors. The peptides recognized correspond to sequences occurring in a variety of proteins of viral, bacterial, and human (self) origin. One peptide derives from glycoprotein C of the common pathogen HSV-1; cells infected with recombinant vaccinia virus encoding native glycoprotein C were lysed by anti-MART-1 effectors. Our results overall indicate that sequences conforming to the A2.1 binding motif and possessing features essential to recognition by anti-MART-1 CTL occur frequently in proteins. These findings further suggest that T cells might encounter a variety of such sequences in vivo, and that epitope mimicry may play a role in modulating the CTL response to MART-1(27-35).

Biochemical identification of a mutated human melanoma antigen recognized by CD4(+) T cells.

CD4(+) T cells play a critical role in generating and maintaining immune responses against pathogens and alloantigens, and evidence suggests an important role for them in antitumor immunity as well. Although major histocompatibility complex class II-restricted human CD4(+) T cells with specific antitumor reactivities have been described, no standard method exists for cloning the recognized tumor-associated antigen (Ag). In this study, biochemical protein purification methods were used in conjunction with novel mass spectrometry sequencing techniques and molecular cloning to isolate a unique melanoma Ag recognized by a CD4(+) tumor-infiltrating lymphocyte (TIL) line. The HLA-DRbeta1*0101-restricted Ag was determined to be a mutated glycolytic enzyme, triosephosphate isomerase (TPI). A C to T mutation identified by cDNA sequencing caused a Thr to Ile conversion in TPI, which could be detected in a tryptic digest of tumor-derived TPI by mass spectrometry. The Thr to Ile conversion created a neoepitope whose T cell stimulatory activity was enhanced at least 5 logs compared with the wild-type peptide. Analysis of T cell recognition of serially truncated peptides suggested that the mutated amino acid residue was a T cell receptor contact. Defining human tumor Ag recognized by T helper cells may provide important clues to designing more effective immunotherapies for cancer.

Identification of a tumor-specific allo-HLA-restricted γδTCR.

γδT cells are key players in cancer immune surveillance because of their ability to recognize malignant transformed cells, which makes them promising therapeutic tools in the treatment of cancer. However, the biological mechanisms of how γδT-cell receptors (TCRs) interact with their ligands are poorly understood. Within this context, we describe the novel allo-HLA-restricted and CD8α-dependent Vγ5Vδ1TCR. In contrast to the previous assumption of the general allo-HLA reactivity of a minor fraction of γδTCRs, we show that classic anti-HLA-directed, γδTCR-mediated reactivity can selectively act on hematological and solid tumor cells, while not harming healthy tissues in vitro and in vivo. We identified the molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 as the essential determinant for recognition and describe the critical role of CD8 as a coreceptor. We conclude that alloreactive γδT-cell repertoires provide therapeutic opportunities, either within the context of haplotransplantation or as individual γδTCRs for genetic engineering of tumor-reactive T cells.

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes.

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.

Minimal determinant expressed by a recombinant vaccinia virus elicits therapeutic antitumor cytolytic T lymphocyte responses.

Anticancer vaccine strategies can now target intracellular antigens that are involved in the process of malignant transformation, such as oncogene products or mutated tumor suppressor genes. Fragments of these antigens, generally 8-10 amino acids in length and complexed with MHC class I molecules, can be recognized by CD8+ T lymphocytes (TCD8+). To explore the possibility of using a genetically encoded, minimally sized fragment of an intracellular antigen as an immunogen, we constructed a recombinant vaccinia virus encoding an 8-residue peptide derived from chicken ovalbumin that is known to associate with the mouse H-2Kb molecule. Compared to standard methods of immunization, recombinant molecule. Compared to standard methods of immunization, recombinant vaccinia virus expressing the minimal determinant as well as full length ovalbumin were the only approaches that elicited specific primary lytic responses in C57BL/6 mice against E.G7OVA, a transfectant of the murine thymoma EL4 containing the ovalbumin gene. Stimulating these effectors in vitro with OVA257-264 peptide induced H-2Kb-restricted TCD8+ that not only lysed but also specifically secreted IFN-gamma in response to an antigen. Furthermore, when transferred adoptively, these anti-OVA257-264 TCD8+ cells significantly reduced the growth of established ovalbumin-transfected tumors in a pulmonary metastasis model system. Synthetic transfected tumors in a pulmonary metastasis model system. Synthetic oligonucleotides encoding minimal antigenic determinants within expression constructs may be a useful approach for treatment of neoplastic disease, thus avoiding the potential hazards of immunizing with full-length cDNAs that are potentially oncogenic.

Characterization of the functional specificity of a cloned T-cell receptor heterodimer recognizing the MART-1 melanoma antigen.

T cells can play a central role in the immune response to cancer, with tumor-specific T-lymphocyte reactivity provided by the T-cell receptor (TCR) alpha and beta chain heterodimer. This study is the first report of the definitive identification and characterization of a functional tumor-associated, antigen-specific TCR by reconstitution in an alternate cell line. Jurkat T cells were transfected with the cDNAs encoding the full-length alpha and beta T-cell receptor chains from the HLA-A2 restricted, melanoma-reactive T-cell clone, clone 5. Expression of the transfected TCR was evaluated by immunofluorescence after down-modulation of the endogenous receptor with Jurkat T-cell receptor beta chain-specific mAb. Jurkat clone 5 TCR+ cells recognized MART-1 peptides presented by T2 cells in a pattern and sensitivity equivalent to native MART-1-reactive T-cells. Recognition of HLA-A2+ melanoma cell lines by the Jurkat clone 5 TCR+ cells, however, did not occur without the addition of exogenous MART-1 peptide. The cloning and expression of functional TCR genes which are capable of specifically recognizing MART-1 antigen provides reagents which could be used for the study of the mechanisms of T-cell/tumor antigen interactions and creates immortalized reagents which can facilitate studies requiring detection of the MART-1 antigen. The tumor reactivity provided by these genes could also have application in novel immunotherapeutic strategies for treating patients with melanoma, including redirection of tumor-infiltrating lymphocyte specificity and bone marrow stem cell therapy.

Identification of MART-1-specific T-cell receptors: T cells utilizing distinct T-cell receptor variable and joining regions recognize the same tumor epitope.

Tumor-specific cytotoxic T lymphocytes (CTLs) can mediate tumor regression in patients with metastatic melanoma and play a central role in the immune response to cancer. The recent identification of shared melanoma antigens has raised the possibility of a limited melanoma-specific T-cell receptor (TCR) repertoire, but subsequent studies have been controversial and difficult to interpret without knowing which tumor-associated antigens (TAAs) are being recognized by specific TCRs. However, the recent cloning of several melanoma TAAs now allows for the identification of the specifically recognized TAA and its epitope. We evaluated the TCR of two clonal CD8+ CTL lines, A42 and 1E2, from two HLA-A2+ patients with metastatic melanoma. Both CTL lines were MART-1 specific, and both demonstrate reactivity to the same epitope when presented in an HLA-A2.1 context. The TCR genes of the two clones were sequenced. All of the productively rearranged A42 TCR beta chain genes were V beta 7/D beta 2.1/J beta 2.7/C beta 2; the TCR alpha chain genes were V alpha 21/J alpha 42/C alpha. The 1E2 TCR beta chain genes were V beta 3/D beta 1.1/J beta 1.1/C beta 1, and TCR alpha chains were V alpha 25/J alpha 54/C alpha. This study is the first report of TCR sequences specific for a melanoma epitope. These TCR clones may be useful for the development of more effective immunotherapies and in studies of the mechanism of T-cell recognition of tumor antigen. They also provide direct evidence that the immune system can provide more than one TCR capable of recognizing a TAA epitope.

MHC class I-restricted recognition of a melanoma antigen by a human CD4+ tumor infiltrating lymphocyte.

It is generally considered that MHC class I-restricted antigens are recognized by CD8+ T cells, whereas MHC class II-restricted antigens are recognized by CD4+ T cells. In the present study, we report an MHC class I-restricted CD4+ T cell isolated from the tumor infiltrating lymphocytes (TILs) of a patient with metastatic melanoma. TIL 1383 I recognized HLA-A2+ melanoma cell lines but not autologous transformed B cells or fibroblasts. The antigen recognized by TIL 1383 I was tyrosinase, and the epitope was the 368-376 peptide. Antibody blocking assays confirmed that TIL 1383 I was MHC class I restricted, and the CD4 and CD8 coreceptors did not contribute significantly to antigen recognition. TIL 1383 I was weakly cytolytic and secreted cytokines in a pattern consistent with it being a Th1 cell. The avidity of TIL 1383 I for peptide pulsed targets is 10-100-fold lower than most melanoma-reactive CD8+ T cell clones. These CD4+ T cells may represent a relatively rare population of T cells that express a T-cell receptor capable of cross-reacting with an MHC class I/peptide complex with sufficient affinity to allow triggering in the absence of the CD4 coreceptor.

Potential use of T cell receptor genes to modify hematopoietic stem cells for the gene therapy of cancer.

The purpose of this review is to illustrate some of the technical and biological hurdles that need to be addressed when developing new gene therapy based clinical trials. Gene transfer approaches can be used to "mark" cells to monitor their persistence in vivo in patients, to protect cells from toxic chemotherapeutic agents, correct a genetic defect within the target cell, or to confer a novel function on the target cell. Selection of the most suitable vector for gene transfer depends upon a number of factors such as the target cell itself and whether gene expression needs to be sustained or transient. The TCR gene transfer approach described here represents one innovative strategy being pursued as a potential therapy for metastatic melanoma. Tumor reactive T cells can be isolated from the tumor infiltrating lymphocytes (TIL) of melanoma patients. A retroviral vector has been constructed containing the T cell receptor (TCR) alpha and beta chain genes from a MART-1-specific T cell clone (TIL 5). Jurkat cells transduced with this virus specifically release cytokine in response to MART-1 peptide pulsed T2 cells, showing that the virus can mediate expression of a functional TCR. HLA-A2 transgenic mice are being used to examine whether transduced bone marrow progenitor cells will differentiate in vivo into mature CD8+ T cells expressing the MART-1-specific TCR. Expression of the human TCR alpha and beta chain genes has been detected by RT-PCR in the peripheral blood of HLA-A2 transgenic mice reconstituted with transduced mouse bone marrow. Expression of the TIL 5 TCR genes in the peripheral blood of these mice was maintained for greater than 40 weeks after bone marrow reconstitution. TIL 5 TCR gene expression was also maintained following transfer of bone marrow from mice previously reconstituted with transduced bone marrow to secondary mouse recipients, suggesting that a pluripotent progenitor or lymphocyte progenitor cell has been transduced.

Loss of T cell-mediated antitumor immunity after construct-specific downregulation of retrovirally encoded T-cell receptor expression in vivo.

Adoptive T-cell therapy is clinically efficacious in the treatment of select cancers. However, it is often difficult to obtain adequate numbers of tumor-specific T cells for therapy. One method for overcoming this limitation is to generate tumor-specific T cells by retrovirally mediated T-cell-receptor (TCR) gene transfer. However, despite instances of therapeutic success, major obstacles remain, including attaining the survival of retrovirally modified T cells in vivo as well as inducing long-term and multi-gene retroviral expression. Using a murine model of adoptively transferred retrovirally modified CD8(+) T cells, where antitumor immunity was dependent on sustained, multigene expression, we found that in vitro assays are poor indicators of in vivo efficacy. Despite persisting for over 9 months in a nonlymphopenic environment, genetically modified T cells exhibited discordant retrovirally mediated gene expression in vivo not readily evident from initial in vitro assays. In particular, one of the two TCR subunit genes necessary for antigen specificity was selectively lost in vivo. As this discordant gene expression was associated with the loss of antitumor immunity, consideration of these findings may provide guidance in the design, evaluation and application of retroviral vectors for use in the treatment of cancer and other human disease.

Mouse Hepa-1 tumor is rejected by H-2Db-restricted CTL despite decreased MHC class I antigen expression.

It has recently been hypothesized that tumor cells with reduced levels of MHC class I antigens are more susceptible to NK-mediated lysis and are rejected by NK cells, whereas tumor cells with normal levels of class I are rejected by tumor-specific CTL. We have tested this hypothesis using a mouse hepatoma system. The Hepa-1 tumor is a spontaneous H-2Kb loss variant that arose from the BW7756 tumor, when BW7756 was adapted to growth in culture. Our studies have shown that despite the loss of H-2Kb antigen, Hepa-1 is not more susceptible to NK lysis than its H-2Kb-transfected variants. These studies also suggested that NK cells were not responsible for rejection of the Hepa-1 tumor. The Hepa-1 tumor, therefore, appears to contradict the hypothesized linkage of MHC levels and NK susceptibility. Because NK cells are not involved in immunity to this tumor, we have sought to identify the effector cell responsible for Hepa-1 rejection. Cytotoxic T lymphocyte assays demonstrate that in vitro, Hepa-1 cells are lysed by Hepa-1-specific H-2Db-restricted CD4-CD8+ T lymphocytes. Footpad assays demonstrate that in vivo, Hepa-1 rejection requires CD4+CD8- and CD4-CD8+ Hepa-1-primed splenocytes. These results indicate that immunity to Hepa-1 is T cell mediated. Hepa-1 is therefore an example of an unusual tumor in that down-regulation of MHC class I antigen expression is associated with increased CTL susceptibility.

H-2Kb antigen expression has no effect on natural killer susceptibility and tumorigenicity of a murine hepatoma.

Recent reports suggested a correlation between decreased expression of tumor cell MHC class I Ag and increased susceptibility to NK cells. These studies led to the hypothesis that tumor cells displaying reduced levels of MHC class I Ag have reduced tumorigenicity in vivo because they are eliminated from the host by endogenous NK cells. The present studies use the murine hepatoma BW7756 and a spontaneous H-2Kb loss variant, Hepa-1, to test this hypothesis. The parental BW7756 tumor is highly malignant in syngeneic C57L/J hosts while Hepa-1 cells do not give rise to tumors, suggesting that the loss of H-2Kb Ag expression correlates with decreased tumorigenicity and NK susceptibility. Hepa-1 cells were therefore transfected with an H-2Kb gene to generate H-2Kb Ag expressing clones. The resulting clones were tested for tumorigenicity. Syngeneic or NK-deficient C57BL/6-beige/beige mice challenged with Hepa-1 or the H-2Kb transfectants rejected the cells, suggesting that reexpression of H-2Kb Ag does not restore tumorigenicity and that NK cells are not involved in Hepa-1 rejection. In vitro H-2Kb Ag-negative and -positive Hepa-1 cells are equally susceptible to tilorone-boosted NK cells, indicating that MHC class I Ag expression also does not affect in vitro NK susceptibility. Tumor challenged athymic nude and sublethally irradiated syngeneic mice develop tumors demonstrating that T cells are probably responsible for rejection of the Hepa-1 tumor, and that H-2Kb Ag expression has no effect on rejection. Inasmuch as the expression of H-2Kb Ag on Hepa-1 cells does not effect tumorigenicity or in vitro NK susceptibility, the previously reported association between reduced MHC class I Ag levels and increased NK susceptibility is not universally applicable.

Transfection and expression of syngeneic H-2 genes does not reduce malignancy of H-2 negative teratocarcinoma cells in the autologous host.

Rejection of the MHC class I negative 402AX teratocarcinoma is accompanied by induction of tumor cell-encoded H-2K and H-2D antigens by the genetically resistant host. To determine whether MHC antigen expression is required for 402AX rejection, we have prepared H-2Db-transfected 402AX cells (402AX/Db). Transfectants express high levels of H-2Db, most of which is not associated with beta 2-microglobulin. MHC syngeneic and allogeneic mice susceptible to 402AX are resistant to 402AX/Db, suggesting that MHC class I antigen expression is required for tumor rejection. Autologous 129 hosts, however, are susceptible to 402AX/Db. 402AX cells transfected with the H-2Kb gene (402AX/Kb) are also lethal in the autologous 129/J host, but rejected by MHC syngeneic and allogeneic mice. Non-129 strain 402AX-susceptible mice pre-immunized with 402AX/Db or simultaneously challenged with 402AX/Db plus 402AX are immune to 402AX. Mice immunized with 402AX/Db produce MHC class I induction factor. 402AX/Db and 402AX cells are lysed equally by natural killer cells, indicating that in 402AX cells the expression of class I antigens is unrelated to NK susceptibility. These studies confirm the requirement for class I expression in 402AX immunity, but demonstrate that in the autologous host immunity requires additional factors beyond class I antigen expression.

Antitumor immunization with a minimal peptide epitope (G9-209-2M) leads to a functionally heterogeneous CTL response.

The goal of experimental clinical protocols using peptide antigen for active vaccination and treatment of patients with metastatic cancer is to induce a vigorous cytotoxic T lymphocyte (CTL) response against the immunizing antigen, and thereby against tumor cells expressing the antigen. However, the magnitude and breadth of human CTL responses induced by peptide immunization, and in particular against antigens expressed by normal tissues as well as tumors, is not well characterized. This issue was examined by characterizing CTL cloids derived from peripheral blood mononuclear cells of three patients who received peptide immunization as treatment for metastatic melanoma. All patients received G9-209-2M peptide, a modified epitope of the gp100 melanoma-associated antigen. The results indicated that the CTL response induced by this peptide antigen was highly heterogeneous both in terms of avidity toward the peptide antigen and recognition of tumor cell lines. Furthermore, avidity of each CTL cloid for the native peptide was highly predictive of tumor reactivity. These results are discussed in terms of their implications for peptide vaccination and adoptive tumor immunotherapy.

Retroviral transduction of a T cell receptor specific for an Epstein-Barr virus-encoded peptide.

The Type II EBV malignancies nasopharyngeal carcinoma and EBV(+) Hodgkin's disease express three subdominant antigens, latency membrane protein (LMP) 1, LMP2, and EBNA-1. While adoptive immunotherapy with T cell lines for Type III EBV malignancy (such as posttransplant lymphoma, PTLD, which expresses the immunodominant EBNA-3 antigens) has been used to prevent and treat PTLD, the generation of class I MHC-restricted CTL suitable for the immunotherapy of Type II EBV malignancy is difficult. This is primarily due to the lack of anti-LMP or EBNA-1 CTL activity in many healthy volunteers. We have engineered, by retroviral transduction of the TCR, CTL that have the potential to recognize subdominant EBV latency antigens. Using the SAMEN retroviral vector we demonstrate the ability to transfer CTL activity from a LMP2 peptide-specific CTL clone to a stimulated PBMC population. TCR-transduced PBMC also secrete IFN-gamma upon coculture with LMP2 targets and maintain expression of the transduced TCR during subsequent mitogenic expansion.

Tumorigenicity and immunogenicity of murine tumor cells expressing an MHC class II molecule with a covalently bound antigenic peptide.

The significance of CD4+ lymphocytes and major histocompatibility complex (MHC) class II-restricted antigens in antitumor immunity has been demonstrated in several animal models as well as in some human tumors. However, because of the lack of known class II-restricted antigens, the participation of CD4+ cells in antitumor responses has not been well characterized. Recent reports showed that class II proteins covalently linked to an antigenic peptide could be constructed and cells expressing these fusion proteins were recognized by specific TH cells. The aim of this study was to determine the effect of the expression of a class II-peptide construct on the tumorigenicity and immunogenicity of transfected murine tumor cells. We have constructed a gene for I-Ed beta chain covalently coupled to the I-Ed-restricted TH cell determinant of sperm whale myoglobin (SWM132-145). This class II fusion protein was recognized by a specific TH cell line on the surface of COS-7 cells or BALB/c sarcoma cells. The sarcoma cells expressing the MHC-peptide complex were rejected by immunocompetent BALB/c mice, and in vivo T-cell subset depletion experiments suggested the importance of CD4+ cells in the rejection. Moreover, splenocytes from mice immunized with tumor cells expressing the I-Ed-SWM complex showed specific peptide recognition in vitro. Such covalent MHC-peptide complexes could prove useful in studies on the role of CD4+ lymphocytes in antitumor immune responses, and also in designing new, more effective vaccine approaches to the immunotherapy of cancer, as class II-restricted tumor-associated antigens are identified for human cancers.

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells.

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.

Quantitation of T-cell receptor frequencies by competitive PCR: generation and evaluation of novel TCR subfamily and clone specific competitors.

T cell receptor (TCR) V gene usage has been used to characterize the immune response to bacteria, viruses, allografts, self antigens, tumor antigens, and superantigens. Sensitive methods to detect changes in the frequency of TCR subfamilies or clonotypes might be useful in evaluating the efficacy of vaccines against infectious agents, immunotherapy treatments for cancer patients, or the status of autoimmune diseases. Two HLA-A2 restricted CTL clones expressing BV17 were isolated from a tumor infiltrating lymphocytes (TIL) culture of a patient with metastatic melanoma. One clone recognized the MART-1(27-35) peptide and the other clone recognized the gp100(209-217) peptide. The frequency of each of these CTL clones in an expanding TIL culture was measured using a novel competitive RT-PCR (cRT-PCR) strategy. cRT-PCR uses a single primer pair to amplify template cDNA simultaneously with a modified DNA competitor molecule. A rapid two-step PCR technique followed by a single cloning step was used to generate a TCR BV17 subfamily specific competitor or competitors specific for the MART-1(27-35) reactive CTL clone (CO-41) and the gp100(209-217) reactive CTL clone (CO-4). Each competitor contained a segment of the TCR BC region that served as an internal reference standard. Using the BV17 competitor we were able to accurately and reproducibly measure cDNA templates at a frequency as low as 1/100,000 using cDNA samples of known TCRBV subfamily composition. This competitor was used to monitor the frequency of BV17 expressing T cells in the TIL and PMBC of a patient with metastatic melanoma. We determined that the frequency of BV17 expressing T cells increased from 4.5% of the culture on day 35 to 60.7% of the culture on day 58. Expansion of the BV17 subfamily was due predominantly to the expansion of the CO-4 clone. This method can be used to meaningfully quantify the precursor frequency of T cell mRNA in prepared samples via TCR subfamily or TCR sequence specific primers.

Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity.

The tumor-associated-Ag MART-1 is expressed by most human melanomas. The genes encoding an alphabeta TCR from a MART-1-specific, HLA-A2-restricted, human T cell clone have been efficiently transferred and expressed in human PBL. These retrovirally transduced PBL cultures were MART-1 peptide reactive, and most cultures recognized HLA-A2+ melanoma lines. Limiting dilution clones were generated from three bulk transduced PBL cultures to investigate the function of individual clones within the transduced cultures. Twenty-nine of 29 CD8+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with MART-1(27-35) peptide, and 23 of 29 specifically secreted IFN-gamma in response to HLA-A2+ melanoma lines. Additionally, 23 of 29 CD8+ clones lysed T2 cells pulsed with the MART-1(27-35) peptide and 15 of 29 lysed the HLA-A2+ melanoma line 888. CD4+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with the MART-1(27-35) peptide. TCR gene transfer to patient PBL can produce CTL with anti-tumor reactivity in vitro and could potentially offer a treatment for patients with metastatic melanoma. This approach could also be applied to the treatment of other tumors and viral infections. Additionally, TCR gene transfer offers unique opportunities to study the fate of adoptively transferred T cells in vivo.

Quantitation of T-cell receptor frequencies by competitive polymerase chain reaction: dynamics of T-cell clonotype frequencies in an expanding tumor-infiltrating lymphocyte culture.

The use of T-cell receptor (TCR) genes as markers for antigen-reactive T cells is dependent on the ability of the TCR genes to rapidly identify antigen-reactive T-cell clonotypes in patient samples. We recently reported a competitive reverse-transcriptase polymerase chain reaction (cRT-PCR) method that can measure the frequency of individual TCRBV subfamilies and clonotypes in mixed lymphocyte populations more accurately than other semiquantitative PCR assays. However, it is impractical to measure changes in the absolute frequency of each TCRBV subfamily to identify those T cells with increasing frequency after antigen stimulation in vivo or in vitro. Therefore, we have modified our cRT-PCR method to more rapidly identify expanding T-cell populations by combining all of the TCRBV subfamily-specific competitors into a single sample to determine the relative abundance of each TCRBV subfamily. Using an expanding TIL 620 culture, we identified four TCRBV (BV2, BV12, BV17, and BV23) subfamilies that expanded over a 23-day period. These subfamilies accounted for 23% of the T cells in the day 35 culture and increased to 57%, 92%, and 80% of the days 44, 51, and 58 cultures respectively. Analysis of DNA sequences demonstrated that the observed expansion was caused primarily by a single clonotype within each subfamily. T cells expressing BV17 and BV23 recognized gp100 and MART-1 respectively. Therefore, this cRT-PCR method can detect expanding T-cell populations based solely on their TCRBV subfamily expression. Furthermore, T-cell expansion in a mixed TIL population was a good predictor of antigen reactivity.