RESUMEN

The adoptive transfer of tumor antigen-specific T cells recently achieved clinical efficacy for a fraction of melanoma patients refractory to other therapies. Unfortunately, the application of this strategy to the remaining melanoma and most other cancer patients is hampered by the difficulty to generate high-affinity tumor-reactive T cells. Two strategies are currently developed to extend the feasibility of this therapeutic approach: clinical grade tool production for MHC-peptide multimer-driven sorting of antigen-specific T cells from the endogenous peripheral T cell repertoire and de novo engineering of the missing repertoire by genetic transfer of cloned specific T cell receptor (TCR) into T cells. The expected multiplication of adoptive transfer treatments, by these strategies, and their careful evaluation should enable the cure of a number of otherwise compromised cancer patients and to gain insight into the characteristics of transferred T cells best fitted to eradicate tumor cells, in terms of antigen specificities, phenotype, and functions. In particular, identification of tumor-rejection antigens by this approach would improve the design and efficacy of all immunotherapeutic approaches.

Asunto(s)

Antígenos de Neoplasias/inmunología , Inmunoterapia Adoptiva , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo , Humanos , Ingeniería de Proteínas , Mejoramiento de la Calidad , Linfocitos T/trasplante , Resultado del Tratamiento

RESUMEN

The tumor suppressor gene von Hippel-Lindau (VHL) is involved in the development of sporadic clear-cell renal cell carcinoma (RCC). VHL interferes with angiogenesis and also controls cell adhesion and invasion. Therapies that target VHL-controlled genes are currently being evaluated in RCC patients. RCC is a immunogenic tumor and treatment with interleukin-2 (IL2) or interferon (IFN)-α results in regression in some patients. We used two renal tumor cell lines (RCC6 and RCC4) carrying VHL loss-of-function mutations to investigate the role of mutant VHL in susceptibility to natural killer (NK) cell-mediated lysis. The RCC6 and RCC4 cell lines were transfected with the wild-type gene to restore the function of VHL. The presence of the gene in RCC cells downregulated hypoxia-inducible factor (HIF)-1α and subsequently decreased vascular endothelial growth factor (VEGF) production. Relative to control transfectants and parental cells, pVHL-transfected cell lines activated resting and IL2-activated NK cells less strongly, as assessed by IFNγ secretion, NK degranulation and cell lysis. NKG2A, a human leukocyte antigen (HLA)-I-specific inhibitory NK receptor, controls the lysis of tumor targets. We show that HLA-I expression in RCC-pVHL cells is stronger than that in parental and controls cells, although the expression of activating receptor NK ligands remains unchanged. Blocking NKG2A/HLA-I interactions substantially increased lysis of RCC-pVHL, but had little effect on the lysis of VHL-mutated RCC cell lines. In addition, in response to IFNα, the exponential growth of RCC-pVHL was inhibited more than that of RCC-pE cells, indicating that VHL mutations may be involved in IFNα resistance. These results indicate that a decreased expression of HLA-I molecules in mutated VHL renal tumor cells sensitizes them to NK-mediated lysis. These results suggest that combined immunotherapy with anti-angiogenic drugs may be beneficial for patients with mutated VHL.

Asunto(s)

Citotoxicidad Inmunológica/genética , Células Asesinas Naturales/metabolismo , Mutación , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Western Blotting , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Hipoxia de la Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citotoxicidad Inmunológica/inmunología , Prueba de Complementación Genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interferón-alfa/farmacología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Células Asesinas Naturales/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Interferencia de ARN , Transfección , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo

RESUMEN

BACKGROUND/AIM: Proinflammatory cytokines are key factors in the pathogenesis of Crohn's disease (CD). Activation of nuclear factor kappa B (NFkappaB), which is involved in their gene transcription, is increased in the intestinal mucosa of CD patients. As butyrate enemas may be beneficial in treating colonic inflammation, we investigated if butyrate promotes this effect by acting on proinflammatory cytokine expression. METHODS: Intestinal biopsy specimens, isolated lamina propria cells (LPMC), and peripheral blood mononuclear cells (PBMC) were cultured with or without butyrate for assessment of secretion of tumour necrosis factor (TNF) and mRNA levels. NFkappaB p65 activation was determined by immunofluorescence and gene reporter experiments. Levels of NFkappaB inhibitory protein (IkappaBalpha) were analysed by western blotting. The in vivo efficacy of butyrate was assessed in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis. RESULTS: Butyrate decreased TNF production and proinflammatory cytokine mRNA expression by intestinal biopsies and LPMC from CD patients. Butyrate abolished lipopolysaccharide (LPS) induced expression of cytokines by PBMC and transmigration of NFkappaB from the cytoplasm to the nucleus. LPS induced NFkappaB transcriptional activity was decreased by butyrate while IkappaBalpha levels were stable. Butyrate treatment also improved TNBS induced colitis. CONCLUSIONS: Butyrate decreases proinflammatory cytokine expression via inhibition of NFkappaB activation and IkappaBalpha degradation. These anti-inflammatory properties provide a rationale for assessing butyrate in the treatment of CD.

Asunto(s)

Butiratos/uso terapéutico , Enfermedad de Crohn/tratamiento farmacológico , Fármacos Gastrointestinales/uso terapéutico , FN-kappa B/efectos de los fármacos , Adolescente , Adulto , Anciano , Animales , Western Blotting , Células Cultivadas , Enfermedad de Crohn/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación de Medicamentos , Femenino , Humanos , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Estudios Prospectivos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

To design an efficient procedure to expand high avidity melanoma reactive T cells and to perform immunotherapies, we compared conditions of peripheral blood lymphocyte (PBL) stimulation by Melan-A/MART-1 peptides. Avidity of induced CTLs was evaluated by measuring their lysis and cytokine secretion to peptide-pulsed transporter-associated protein-deficient cells and to melanoma cells. In side-by-side experiments, we show that melanoma cells, either allogeneic or autologous, induced the growth of high avidity Melan-A-reactive CTLs from all donors, whereas essentially low avidity T cells were induced by peptide-pulsed PBLs. We also show that at least two cytokines, interleukin-6 and interleukin-2, were required to promote the growth of high avidity CTLs. Once sorted by tetramer labeling or cloning, the specificity and reactivity to tumor cells of peptide-specific T cells induced by allogeneic melanoma cells were confirmed. We then describe a relatively simple and efficient procedure that allowed us to obtain systematically high amounts (in the range of billion) of high avidity Melan-A/ MART-1-specific T cells from the PBLs of HLA-A2 melanoma patients and healthy donors in 3 months. Because this antigen is expressed by most melanoma tumors, this procedure should be useful for checking the efficiency of adoptive immunotherapy of melanoma tumors and using functionally well-defined Melan-A/MART-1-specific CTLs in a large group of patients.

Asunto(s)

Linfocitos/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Antígenos de Neoplasias , División Celular/efectos de los fármacos , División Celular/inmunología , Células Clonales , Citotoxicidad Inmunológica , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Linfocitos/citología , Linfocitos/efectos de los fármacos , Antígeno MART-1 , Melanoma/patología , Proteínas de Neoplasias/farmacología , Oligopéptidos/inmunología , Oligopéptidos/farmacología , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/inmunología

RESUMEN

Many mechanisms have been proposed to explain why immune responses against human tumor antigens are generally ineffective. For example, tumor cells have been shown to develop active immune evasion mechanisms. Another possibility is that tumor antigens are unable to optimally stimulate tumor-specific T cells. In this study we have used HLA-A2/Melan-A peptide tetramers to directly isolate antigen-specific CD8(+) T cells from tumor-infiltrated lymph nodes. This allowed us to quantify the activation requirements of a representative polyclonal yet monospecific tumor-reactive T cell population. The results obtained from quantitative assays of intracellular Ca(2+) mobilization, TCR down-regulation, cytokine production and induction of effector cell differentiation indicate that the naturally produced Melan-A peptides are weak agonists and are clearly suboptimal for T cell activation. In contrast, optimal T cell activation was obtained by stimulation with recently defined peptide analogues. These findings provide a molecular basis for the low immunogenicity of tumor cells and suggest that patient immunization with full agonist peptide analogues may be essential for stimulation and maintenance of anti-tumor T cell responses in vivo.

Asunto(s)

Antígenos de Neoplasias/inmunología , Activación de Linfocitos , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T/inmunología , Calcio/metabolismo , Línea Celular , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Humanos , Antígeno MART-1 , Receptores de Antígenos de Linfocitos T/análisis

RESUMEN

HLA-A*0201 melanoma patients often develop a CTL response to an immunodominant peptide derived from the melanocyte lineage-specific protein Melan-A/MART-1. We have shown previously that the antigenic peptide most often involved is the decapeptide Melan-A(26-35) (EAAGIGILTV). We also observed some clonal diversity in the fine specificity of Melan-A-specific CTL. To substantiate this observation, we have now tested a series of Melan-A(26-35) variant peptides containing single alanine substitutions for binding to HLA-A*0201 and recognition by polyclonal and monoclonal Melan-A-specific CTL. Substitution of several residues with alanine reduced peptide binding activity by > 10-fold. In contrast, substitution of E26 with alanine (AAAGIGILTV) resulted in a 5-fold higher binding activity as well as in stronger stability of the corresponding HLA-A*0201/peptide complexes. Interestingly, the peptide variant AAAGIGILTV was recognized more efficiently than the natural decapeptide by short term cultured, tumor-infiltrated lymph node cell cultures and a number of Melan-A-specific CTL clones derived from different individuals. Moreover, this analysis revealed that the fine specificity of the CTL response to the Melan-A immunodominant epitope is quite diverse at the clonal level. At least three distinct patterns of fine specificity were identified. This diversity appears to reflect the diversity of the TCR repertoire available for this Ag, since similar results were obtained with a panel of Melan-A-specific CTL clones derived from a single melanoma patient. These findings have important implications for the formulation of Melan-A peptide-based vaccines as well as for the monitoring of Melan-A-specific CTL responses in melanoma patients.

Asunto(s)

Antígenos de Neoplasias/inmunología , Antígeno HLA-A2/inmunología , Epítopos Inmunodominantes/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Alelos , Sustitución de Aminoácidos , Antígenos de Neoplasias/química , Línea Celular Transformada , Células Cultivadas , Reordenamiento Génico de Linfocito T , Antígeno HLA-A2/genética , Humanos , Epítopos Inmunodominantes/química , Linfocitos Infiltrantes de Tumor/inmunología , Antígeno MART-1 , Proteínas de Neoplasias/química , Fragmentos de Péptidos/química , Unión Proteica , Células Tumorales Cultivadas

RESUMEN

Subsets of T and natural killer (NK) lymphocytes express the CD94-NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94-NKG2A heterodimer inhibits both antigen-driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma-specific human T cell clones. Similarly, CD16-mediated NK cell cytotoxicity is extinguished by cross-linking of the CD94-NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine-based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP-1 and SHP-2, but not with the polyinositol phosphatase SHIP Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho-ITIM interact directly with the SH2 domains of SHP-1 and SHP-2 with a high affinity. Engagement of the CD94-NKG2A heterodimer therefore appears as a protein-tyrosine phosphatase-based strategy that negatively regulates both antigen-induced T cell response and antibody-induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.

Asunto(s)

Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos CD/farmacología , Antígenos CD/fisiología , Células Asesinas Naturales/efectos de los fármacos , Lectinas Tipo C , Activación de Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Glicoproteínas de Membrana/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Linfocitos T Citotóxicos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antígenos CD/química , Dimerización , Humanos , Inmunosupresores/farmacología , Péptidos y Proteínas de Señalización Intracelular , Células Asesinas Naturales/inmunología , Cinética , Linfocitos Infiltrantes de Tumor , Sustancias Macromoleculares , Melanoma/inmunología , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Subfamília D de Receptores Similares a Lectina de las Células NK , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/química , Ratas , Receptores de IgG/fisiología , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

The Melan-A/MART-1 gene, which is expressed by normal melanocytes as well as by most fresh melanoma samples and melanoma cell lines, codes for Ags recognized by tumor-reactive CTL. HLA-A*0201-restricted Melan-A-specific CTL recognize primarily the Melan-A(27-35) (AAGIGILTV) and the Melan-A(26-35) (EAAGIGILTV) peptides. The sequences of these two peptides are not necessarily optimal as far as binding to HLA-A*0201 is concerned, since both lack one of the dominant anchor amino acid residues (leucine or methionine) at position 2. In this study we introduced single amino acid substitutions in either one of the two natural peptide sequences with the aim of improving peptide binding to HLA-A*0201 and/or recognition by specific CTL. Surprisingly, analogues of the Melan-A(27-35) peptide, which bound more efficiently than the natural nonapeptide to HLA-A*0201, were poorly recognized by tumor-reactive CTL. In contrast, among the Melan-A(26-35) peptide analogues tested, the peptide ELAGIGILTV was not only able to display stable binding to HLA-A2.1 but was also recognized more efficiently than the natural peptide by two short-term cultured tumor-infiltrated lymph node cell cultures as well as by five of five tumor-reactive CTL clones. Moreover, in vitro generation of tumor-reactive CTL by stimulation of PBMC from HLA-A*0201 melanoma patients with this particular peptide analogue was much more efficient than that observed with either one of the two natural peptides. These results suggest that the Melan-A(26-35) peptide analogue ELAGIGILTV may be more immunogenic than the natural peptides in HLA-A*0201 melanoma patients and should thus be considered as a candidate for future peptide-based vaccine trials.

Asunto(s)

Antígenos de Neoplasias/inmunología , Citotoxicidad Inmunológica , Epítopos Inmunodominantes/inmunología , Activación de Linfocitos , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Línea Celular Transformada , Células Cultivadas , Células Clonales , Citotoxicidad Inmunológica/efectos de los fármacos , Epítopos de Linfocito T/inmunología , Antígenos HLA-A/inmunología , Antígenos HLA-A/metabolismo , Humanos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Antígeno MART-1 , Melanoma/inmunología , Melanoma/patología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

To better understand how Ag density influences the various biologic responses of CTL, we analyzed lysis and, at the single cell level, cytokine production by a panel of melanoma-specific CTL clones. Titration experiments done with peptide-pulsed TAP-deficient T2 cells indicated that: 1) Ag density affects both the fraction of responding cells and the amount of cytokine secreted by each cell. 2) Different responses have a relative Ag requirement that may vary between clones. Lysis and secretion of IFN-gamma, and for most clones' secretion of TNF-alpha, required lower Ag densities, by one or two logs, than IL-2 and granulocyte-macrophage CSF secretion. 3) In a significant fraction of IFN-gamma-secreting cells, IL-2 production is not induced. 4) A large fraction of cloned cells is refractory to lymphokine gene activation for about 2 wk after previous stimulation. Together these data indicate that CTL biologic responses are controlled by variable Ag thresholds and by additional parameters affecting activation requirements of each cell. A similar heterogeneity of cytokine responses was observed to Ag endogenously presented by melanoma cells. As a consequence, most melanoma lines, including those with the highest Ag expression, could trigger only low CTL fractions to secrete IL-2 and, also for most clones, granulocyte-macrophage CSF. This may be an important component of the inefficiency of specific CTL in cancer patients.

Asunto(s)

Antígenos de Neoplasias/inmunología , Citotoxicidad Inmunológica , Melanoma/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Células COS , Citocinas/inmunología , Citometría de Flujo , Humanos , Melanoma/patología , Células Tumorales Cultivadas

RESUMEN

The Melan-A/MART-1 gene product is frequently recognized by tumor-specific HLA-A2-restricted CTL. An immunodominant nonapeptide has been localized to the region spanning residues 27-35. However, the decapeptide including residues 26-35 (the nonapeptide extended NH2 terminally by one residue) appeared to be recognized as efficiently as the nonapeptide. In this study, we show that the optimal length immunodominant peptide appears to correspond to the decapeptide 26-35, as assessed by quantitative analyses of both 4 polyclonal and 13 monoclonal populations of specific CTL. Functional assays of peptide binding to HLA-A2 indicate that the decapeptide is significantly a more efficient binder than the nonapeptide. Moreover, analogues of the decapeptide including substitutions at a secondary HLA-A2 peptide anchor further improve decapeptide binding. Finally, we show that the functional (9 CTL clones analyzed) and structural TCR repertoire (7 CTL clones) of a group of specific CTL clones is rather diverse. The findings reported here may have important implications for future peptide-based melanoma vaccination trials as well as for the monitoring of specific CTL responses in vivo.

Asunto(s)

Antígenos de Neoplasias/inmunología , Antígeno HLA-A2/inmunología , Epítopos Inmunodominantes/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno , Antígenos de Neoplasias/genética , Secuencia de Bases , Células Clonales/inmunología , Reordenamiento Génico de Linfocito T , Humanos , Epítopos Inmunodominantes/genética , Antígeno MART-1 , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Mutación Puntual , Unión Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Especificidad por Sustrato

RESUMEN

Recent experimental data have shown that mice could be immunized efficiently, in particular against cancer, by the injection of antigen-loaded dendritic cells (DC) or macrophages (MPH). In the present work, these two antigen-presenting cells (APC) were prepared in humans from circulating mononuclear cells (MNC). MPH were obtained from MNC that were cultured in hydrophobic plastic bags and purified by elutriation. DC were from the culture of adherent elutriation-purified monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The two APC were prepared in parallel from the same donors and their phenotype and antigen-presenting capacity were compared. DC differed from MPH by a higher expression of HLA-DR and CD23 and a lower expression of CD14, CD64 and of adhesion molecules. DC and MPH were comparably effective in (a) enhancing the mitotic response of autologous lymphocytes to immobilized anti-CD3 (accessory function); (b) presenting melanoma peptides to specific cytotoxic T lymphocyte (CTL) clones; and (c) stimulating the generation of CTL directed against a myxovirus influenza peptide. However, DC were more effective than MPH in inducing the mitotic response of allogeneic peripheral blood leucocytes (PBL), possibly because of their higher expression of HLA class II molecules. In conclusion, DC and MPH prepared from blood MNC did not differ substantially in their ability to activate HLA class I-restricted T-cell responses by exogenous peptide presentation.

Asunto(s)

Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Macrófagos/inmunología , Péptidos/inmunología , Técnicas de Cultivo de Célula , Diferenciación Celular/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Inmunofenotipificación , Interleucina-4/inmunología , Activación de Linfocitos , Melanoma/inmunología , Monocitos/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología

RESUMEN

Coculture of melanoma cells and T cell clones derived from tumor-infiltrating lymphocytes (TIL) generally results in lysis of the antigen-bearing tumor cells but to inefficient proliferation and IL-2 secretion by responder T cells. This suboptimal activation is classically explained by an inability of tumor cells to provide costimulatory signals. Here we analyzed the responses to synthetic peptides of HLA-A2.1-restricted CTL clones specific for melanoma antigens MART-1 and NA17-A. We showed that peptide concentrations ranging from 1 pM to 10 nM efficiently sensitized the peptide transporter-deficient T2 cells to lysis. T2 cells pulsed with melanoma peptides also induced TIL proliferation and detectable secretion of IL-2, IFN-gamma and GM-CSF, but only for peptide concentrations 10- to 10,000-fold higher than those required for lysis. Hence this suggests that partial triggering of TIL clones by melanoma cells could be due to expression of appropriate MHC-peptide complexes at subthreshold levels. In support of this, we showed that melanoma cells, unable to trigger IL-2 secretion, developed this ability when incubated with the appropriate peptide. These results indicate that the level of antigens expressed on melanoma tumors critically affects TIL activation status and thus, the efficiency of specific immune reactions mediated by these cells.

Asunto(s)

Citotoxicidad Inmunológica , Antígenos HLA/inmunología , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Antígenos de Neoplasias/inmunología , Células Clonales , Técnicas de Cocultivo , Citocinas/biosíntesis , Antígenos HLA/metabolismo , Antígeno HLA-A2/inmunología , Humanos , Interleucina-2/biosíntesis , Antígeno MART-1 , Complejo Mayor de Histocompatibilidad , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Células Tumorales Cultivadas

RESUMEN

CD4+ T cell clones derived from lymphocytes infiltrating four human melanomas specifically recognized melanoma-derived tumor epitopes as shown by secretion of tumor necrosis factor (TNF) in vitro upon interaction with autologous melanoma cells, whereas they did not recognize HLA class II-expressing autologous lymphoblasts or HLA class II mismatched allogeneic melanoma cells. Specificity was further established by demonstrating that TNF responses to tumor cells were inhibited by HLA-DR or HLA-DQ monoclonal antibodies. Most of these clones cross-reacted with allogeneic melanoma cells expressing a potentially restricting HLA allele or a structurally similar one. These data show that shared epitopes of human melanoma cells presented on HLA class II molecules are frequently recognized by autologous CD4+ T lymphocytes.

Asunto(s)

Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos HLA-D/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Alelos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Linfocitos T CD4-Positivos/metabolismo , Bovinos , Células Clonales/inmunología , Células Clonales/metabolismo , Genes MHC Clase II , Antígenos HLA-D/genética , Humanos , Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfotoxina-alfa/metabolismo

RESUMEN

B7-1 expression, induced by transfection in poorly immunogenic murine tumours, was shown to elicit a T cell-mediated rejection of these tumours and further active immunity against the non-transfected tumour. We therefore asked to what level similarly induced expression of B7 on human melanoma cells would affect the antigen-dependent responses of tumour-specific T cell clones in vitro. Data presented show that B7-1 expression by melanoma lines: (i) significantly induced, or improved, an IL-2-dependent proliferative response of such clones to the antigen; (ii) increased the amount of IL-2 produced by two clones in response to the parental non-transfected tumour cells; and (iii) increased the TNF responses of all the CD4+ clones. However, despite these clear co-stimulatory effects on antigen-induced responses of all T cell clones, which demonstrated an effective interaction of the B7-1 transfected molecule with one or the other of its counter-receptors expressed on T cell clones, B7 co-stimulation did not correct the defect of IL-2 secretion exhibited by many of these clones in response to in vitro antigen presentation by melanoma cells. We further show that defective IL-2 secretion in response to melanoma antigens was not due to a T cell clone refractoriness induced by the culture, since one of these clones could be induced to secrete IL-2 by an antigen-expressing melanoma line, upon increased lymphocyte function associated antigen-3 expression induced by gene transfection. Together these data suggest that defective IL-2 secretion by many tumour-infiltrating lymphocytes clones in response to antigen presentation by melanoma cells in vitro is not exclusively due to the inability of these cells to provide an appropriate co-stimulation through the B7-1 molecule.

Asunto(s)

Antígeno B7-1/fisiología , Interleucina-2/metabolismo , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígeno B7-1/genética , Antígenos CD28/inmunología , Antígenos CD58/genética , Antígenos CD58/fisiología , Línea Celular , Células Clonales , Fibrosarcoma , Humanos , Melanoma/patología , Ratones , Proteínas de Neoplasias/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Subgrupos de Linfocitos T/metabolismo , Transfección

RESUMEN

Human melanomas are infiltrated by tumor-reactive T lymphocytes. However, the ability of these cells to elicit a specific anti-tumor response in vivo remains to be established. Because lymphokine production is critical for T cell functions, we have analyzed the capacity of melanoma-specific tumor-infiltrating lymphocyte (TIL) clones to produce major lymphokines: interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), as well as tumor necrosis factor (TNF), in response to direct antigen presentation by autologous and allogeneic tumor cells. We report here that, upon stimulation by autologous melanoma cells, all TIL clones secreted TNF but only a few of them produced significant amounts of IL-2, IL-4 or IFN-gamma. Nonetheless, all these clones consistently produced two or three of these last lymphokines upon stimulation with phorbol myristate acetate and calcium ionophore, as well as IL-2 upon CD3 stimulation, showing the existence of three lymphokine profiles among them: Th1, Th0 and a profile characterized by IL-2 and IL-4, but not IFN-gamma secretion. Stimulation of TIL clones by allogeneic melanoma lines sharing the appropriate HLA-peptide complexes revealed that defective IL-2 production seemed to be a constant feature for some clones, while it was, for other clones, dependent on the antigen-presenting tumor cells. For this last type of clone, we further showed that defective IL-2 induction resulted from an LFA-3 defect of some melanoma cells or from distinct yet undefined defects of other melanoma lines. Our data suggest that defective lymphokine secretion may be an essential component of the in vivo failure of melanoma-reactive TIL to control tumor development. Interestingly both CD4+ and CD8+ TIL clones from one patient were fully activated by the autologous melanoma cells in vitro, supporting a potential role of such TIL in spontaneous or induced tumor rejection.

Asunto(s)

Citocinas/biosíntesis , Melanoma/inmunología , Subgrupos de Linfocitos T/inmunología , Anticuerpos Monoclonales , Antígenos CD/fisiología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD58 , Antígenos CD8/inmunología , Humanos , Interleucina-2/biosíntesis , Ionóforos/farmacología , Linfocitos Infiltrantes de Tumor/inmunología , Glicoproteínas de Membrana/fisiología , Linfocitos T Citotóxicos/inmunología , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas

RESUMEN

To try to understand the functional significance of human melanoma-infiltrating lymphocytes (TIL), a clonal analysis of the specificity, T cell receptor (TcR) diversity and activation requirements of these lymphocytes isolated from four different tumors was carried out. Supporting the presence of in vivo primed tumor-specific T lymphocytes in these four tumors, a high frequency of the Cd8+ and CD4+ clones, obtained from the TIL cultured for a few days with recombinant interleukin (rIL)-2 and autologous tumor cells, exhibited a restricted lysis or proliferation in response to the autologous tumor cell line. In contrast, no tumor-specific clone was obtained from freshly extracted TIL, suggesting that the frequency of tumor-specific effectors remained low in these tumors. Only the CD8+ clones lysed the autologous tumor cells and their activity was major histocompatibility complex MHC class I restricted. Significant expansion of CD4+ and CD8+ tumor-specific clones required regular restimulation by autologous melanoma cells but also the addition of exogenous IL-2 and of Epstein-Barr virus-transformed B feeder cells. Five different tumor-specific clones, three CD8+ and two CD4+ clones were identified in a single tumor on the basis of their TcR gene configuration. Together, these data suggest that a spontaneous and diverse immune response, mediated by tumor-specific CD4+ as well as CD8+ T lymphocytes, arises in most MHC-bearing human melanomas but that antigen-MHC complex presentation by tumor cells does not, at least in vitro, allow a significant proliferation of these lymphocytes.

Asunto(s)

Antígenos CD4/análisis , Antígenos CD8/análisis , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Receptores de Antígenos de Linfocitos T/análisis , Células Clonales , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interleucina-2/farmacología , Células Tumorales Cultivadas

RESUMEN

We have sought to determine how much amino acid diversity is tolerable at position 69 of the Ak alpha chain, a position previously implicated as a peptide contact site. Slot-machine mutagenesis was used to create a set of 11 mutant Ak alpha cDNAs, each specifying a different amino acid at position 69. These cDNAs were individually expressed in L cells together with a wild-type Ak beta cDNA to produce a panel of mutant antigen-presenting cell lines. The ability of each member of this panel to present a hen egg lysozyme and a bovine ribonuclease peptide to various T hybridomas was assessed. We found that a surprising degree of amino acid diversity is tolerable at Ak alpha position 69: even charged (Glu, Arg) or bulky (Trp, Tyr) residues can be accommodated without abrogating cell-surface expression of Ak, peptide binding to it, or T cell recognition of it. We discuss the implications of these findings for models of T cell recognition of the class II molecule/antigen duplex.

Asunto(s)

Antígenos de Histocompatibilidad Clase II/química , Animales , Células Presentadoras de Antígenos/química , Secuencia de Bases , Sitios de Unión , Línea Celular , Antígenos de Histocompatibilidad Clase II/genética , Hibridomas/inmunología , Células L/inmunología , Ratones , Datos de Secuencia Molecular , Mutagénesis , Receptores de Antígenos de Linfocitos T/fisiología , Relación Estructura-Actividad , Linfocitos T/inmunología

RESUMEN

We discovered by chance that the R28 T cell hybridoma has dual specificity. It responds to a peptide derived from ribonuclease presented by cells displaying Ak molecules and it reacts, in the absence of added antigen, to cells expressing Ak complexes with a single amino acid substitution at position 69 of the alpha chain. Modelling and functional studies suggest that residue 69 is a peptide contact residue, prompting the hypothesis that R28's alloreactivity is a cross-reactive response to an unknown peptide bound in the 'groove' of the mutant Ak complex. In this report, we employ a competition assay to confirm that this alloresponse involves a groove-binding peptide, demonstrate that this peptide derives from or depends on fetal calf serum and exploit a panel of antigen-presenting cell lines--each displaying an Ak complex with a different position 69 substitution--to establish that the alloresponse is not just a heteroclitic response to ribonuclease, itself. We speculate that much of the alloreactivity against murine class II molecules that is revealed in vitro may prove to be directed at bovine serum-derived peptides, suggesting that in this context, alloreactivity is a misnomer.

Asunto(s)

Células Presentadoras de Antígenos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Clonación Molecular , Análisis Mutacional de ADN , Hibridomas , Inmunidad Celular , Complejo Mayor de Histocompatibilidad , Datos de Secuencia Molecular , Péptidos/inmunología , Ribonucleasas/inmunología , Relación Estructura-Actividad , Transfección

RESUMEN

A CD8+ clone, identified by its T-cell receptor gamma- and beta-gene configuration, was shown to preferentially develop, in the bulk culture of melanoma tumor-infiltrating lymphocytes with recombinant interleukin 2 after 1 month. Thirteen CD8+ clones were obtained by limiting dilution culture of tumor-infiltrating lymphocytes from 43-days old culture. Four of these clones, analyzed for T-cell receptor rearrangements, exhibited exactly the same T-cell receptor gene pattern as tumor-infiltrating lymphocytes from the bulk culture, showing, therefore, that all the CD8+ clones were subclones. All the 13 CD8+ subclones were strongly cytotoxic for autologous melanoma cells but did not kill K562. A more complete cytotoxicity analysis showed that the clones did not kill autologous fibroblasts or Con A blasts or allogeneic tumor targets. Furthermore, specific killing was inhibited by monoclonal antibodies against CD3, CD8, T-cell receptors alpha beta, and class I major histocompatibility complex antigens indicating that effector-to-target cell recognition was mediated through the T-cell receptor in a major histocompatibility complex-restricted fashion. These data showed that human melanoma-specific cytotoxic T lymphocytes can be obtained from melanoma TIL and that a single cytotoxic T lymphocyte clone can be expanded to more than 10(10) cells without a loss of autotumor specificity.

Asunto(s)

Melanoma/inmunología , Linfocitos T Citotóxicos/inmunología , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T/fisiología , Complejo CD3 , Antígenos CD8 , Células Clonales , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase I/fisiología , Humanos , Melanoma/secundario , Receptores de Antígenos de Linfocitos T/fisiología , Células Tumorales Cultivadas

RESUMEN

In vitro culture of metastatic melanoma fragments with 150 units of recombinant interleukin 2 resulted in the successive expansion of CD4+ and then CD8+ tumor-infiltrating lymphocytes (TIL) throughout a 2-month period. TIL cultured for 43 days and consisting of 95% CD8+ and 10% CD4+ T lymphocytes were cloned by limiting dilution (LD). Thirteen CD8+ and thirty-one CD4+ clones were obtained, indicating that the frequency of clonogenic CD8+ proliferative T lymphocytes was much lower than that of their CD4+ homologues. When LD was performed in the presence of autologous melanoma cells the frequency of CD8+ clones was increased by factor 4. The DNA from TIL of day 43 bulk culture and of six CD8+ clones was hybridized with T cell receptor (TcR) beta and gamma probes. Identical configuration of the nonfunctional gamma and functional beta TcR genes was found in "bulk culture" and cloned TIL. The CD8+ clones therefore derived from a clonal population of CD8+ cells which had expanded in vitro before the LD. All the CD8+ clones tested were strongly cytotoxic for autologous melanoma cells but did not kill autologous fibroblasts or concanavalin A blasts, or any of the 10 allogeneic tumor targets tested, including 5 melanomas, 2 breast cell lines, 1 neuroblastoma, K-562 and the Epstein-Barr virus-transformed cell line used as a feeder. Furthermore, specific killing was inhibited by monoclonal antibodies against CD3, CD8, TcR alpha/beta and against class I major histocompatibility complex antigens indicating that these cytotoxic T lymphocyte clones recognized autologous tumor cells through the TcR, in an HLA class I-restricted manner. These data show that it is feasible to obtain tumor-specific cytotoxic T lymphocytes from melanoma TIL with a simple culture technique and that a single clone could be expanded to more than 10(10) cells which should allow testing of immunotherapeutic potential of these cells by adoptive transfer into melanoma patients.

Asunto(s)

Citotoxicidad Inmunológica , Reordenamiento Génico de Linfocito T , Melanoma/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Anticuerpos Monoclonales/inmunología , Linfocitos T CD4-Positivos/inmunología , División Celular , Células Cultivadas , Células Clonales , Humanos