Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

After antigenic challenge, naive T lymphocytes enter a program of proliferation and differentiation during the course of which they acquire effector functions and may ultimately become memory cells. In humans, the pathways of effector and memory T-cell differentiation remain poorly defined. Here we describe the properties of 2 CD8 ؉ T-lymphocyte subsets, RA ؉ CCR7 ؊ 27 ؉ 28 ؉ and RA ؉ CCR7 ؊ 27 ؉ 28 ؊ , in human peripheral blood. These cells display phenotypic and functional features that are intermediate between naive and effector T cells. Like naive T lymphocytes, both subsets show relatively long telomeres. However, unlike the naive population, these T cells exhibit reduced levels of T-cell receptor excision circles (TRECs), indicating they have undergone additional rounds of in vivo cell division. Furthermore, we show that they also share effector-type properties. At equivalent in vivo replicative history, the 2 subsets express high levels of Fas/CD95 and CD11a, as well as increasing levels of effector mediators such as granzyme B, perforin, interferon ␥, and tumor necrosis factor ␣. Both display partial ex vivo cytolytic activity and can be found among cytomegalovirus-specific cytolytic T cells. Taken together, our data point to the presence of T cells with intermediate effector-like functions and suggest that these subsets consist of T lymphocytes that are evolving toward a more differentiated effector or effector-memory stage.

The low frequency of self-peptide–specific T cells in the human preimmune repertoire has so far precluded their direct evaluation. Here, we report an unexpected high frequency of T cells specific for the self-antigen Melan-A/MART-1 in CD8 single–positive thymocytes from human histocompatibility leukocyte antigen-A2 healthy individuals, which is maintained in the peripheral blood of newborns and adults. Postthymic replicative history of Melan-A/MART-1–specific CD8 T cells was independently assessed by quantifying T cell receptor excision circles and telomere length ex vivo. We provide direct evidence that the large T cell pool specific for the self-antigen Melan-A/MART-1 is mostly generated by thymic output of a high number of precursors. This represents the only known naive self-peptide–specific T cell repertoire directly accessible in humans.

Some cancer patients mount spontaneous T- and B-cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T-cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure-function properties of the melanocyte/melanoma tumor antigen Melan-A/MART-1, the assessment of the T-cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine-induced CTL responses to this antigen in patients with metastatic melanoma.

Using optimal culture conditions in which the transforming growth factor 11 (TGF-j1) inhibitory loop has been interrupted by antiuense TGF-131 oligonudeotides or antf-TGF-1 serum, we have compared the proliferative capacities and the abUities ofthe CD34+ CD38-ceil populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38-fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38-cells from a typical umbilical cord blood sample produce equivalent numbers of colonyformdngunits (CFU)-gnulocyte/erythrocyte/macrophage/ megakaryocyt, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult tansplantation. In addition, the colonies resulting from the umbical cord blood samples were signiflcantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparatons, as esimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is pardaly compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbiical cord blood is a suitable source of cells for adult transplantation.Small amounts of near-term or neonatal mouse blood has been demonstrated to have the potential to completely reconstitute the hematopoietic system of adult mice (1). This finding has prompted extensive analysis of human umbilical cord blood as a potential source for cells for hematopoietic reconstitution in man (1-4). Various nonmalignant (3, 5) and malignant (7,8) hematopoietic diseases and malignant nonhematopoietic diseases (9, 10) have been successfully treated in children by using umbilical cord blood transplantation. Umbilical cord blood is easily obtainable and therefore could be used to develop banks of HLA-typed cells for general use in adult transplantation ifthe engraftment potential were high enough. Here we report methods for evaluating the relative potentials for engraftment and their use in comparing umbilical cord blood and bone marrow as cellular sources for transplantation.In the past, engraftability of bone marrow samples has largely been analyzed through estimation of the content of colony-forming unit (CFU)-anulocyte/macrophage (GM) (11,13 Purfication of CD34+ Cells. Mononuclear cells were obtained as described (20,23). Hematopoietic progenitors expressing the CD34 antigen were purified using the Applied Immune Sciences (Santa Clara, CA) procedure [soybean agglutinin (SBA) and CD34 CELLector flasks] (20, 24). These cells were then recovered, concentrated, and used for fluorescence-activated cell sorting (FACS), phenotypic anal-

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Avidity of Ag recognition by tumor-specific T cells is one of the main parameters that determines the potency of a tumor rejection Ag. In this study we show that the relative efficiency of staining of tumor Ag-specific T lymphocytes with the corresponding fluorescent MHC class I/peptide multimeric complexes can considerably vary with staining conditions and does not necessarily correlate with avidity of Ag recognition. Instead, we found a clear correlation between avidity of Ag recognition and the stability of MHC class I/peptide multimeric complexes interaction with TCR as measured in dissociation kinetic experiments. These findings are relevant for both identification and isolation of tumor-reactive CTL.

The membrane receptor 2B4 is a CD2 family member that is involved in lymphocyte activation. A fraction of human CD8+ αβ T cells up-regulate 2B4 in vivo, and here we demonstrate that this correlates with the acquisition of effector cell properties such as granzyme B and perforin expression, rapid IFN-γ production, and down-regulation of the lymph node homing chemokine receptor CCR7. In PBLs from healthy donors, cytomegalovirus-specific effector T cells were 2B4 positive, whereas naive melanoma Ag (Melan-A/melanoma Ag recognized by T cells-1)-specific T cells were 2B4 negative. In melanoma patients, Melan-A-specific T cells up-regulated 2B4 in parallel with in vivo differentiation. This occurred in PBLs after vaccination with Melan-A peptides and in tumor-infiltrated lymph nodes, likely through disease-associated activation of Melan-A-specific T cells. Thus, 2B4 expression correlates with CD8+ T cell differentiation in vivo.