RESUMO
Th17 cells have been described as short lived, but this view is at odds with their capacity to trigger protracted damage to normal and transformed tissues. We report that Th17 cells, despite displaying low expression of CD27 and other phenotypic markers of terminal differentiation, efficiently eradicated tumors and caused autoimmunity, were long lived, and maintained a core molecular signature resembling early memory CD8(+) cells with stem cell-like properties. In addition, we found that Th17 cells had high expression of Tcf7, a direct target of the Wnt and ß-catenin signaling axis, and accumulated ß-catenin, a feature observed in stem cells. In vivo, Th17 cells gave rise to Th1-like effector cell progeny and also self-renewed and persisted as IL-17A-secreting cells. Multipotency was required for Th17 cell-mediated tumor eradication because effector cells deficient in IFN-γ or IL-17A had impaired activity. Thus, Th17 cells are not always short lived and are a less-differentiated subset capable of superior persistence and functionality.
Assuntos
Células-Tronco/metabolismo , Células Th17/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Perfilação da Expressão Gênica , Interleucina-17/biossíntese , Camundongos , Camundongos Transgênicos , Neoplasias/imunologia , Células-Tronco/citologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Th17 cells play a critical role in autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Response gene to complement (RGC)-32 is a cell cycle regulator and a downstream target of TGF-ß that mediates its profibrotic activity. In this study, we report that RGC-32 is preferentially upregulated during Th17 cell differentiation. RGC-32-/- mice have normal Th1, Th2, and regulatory T cell differentiation but show defective Th17 differentiation in vitro. The impaired Th17 differentiation is associated with defects in IFN regulatory factor 4, B cell-activating transcription factor, retinoic acid-related orphan receptor γt, and SMAD2 activation. In vivo, RGC-32-/- mice display an attenuated experimental autoimmune encephalomyelitis phenotype accompanied by decreased CNS inflammation and reduced frequency of IL-17- and GM-CSF-producing CD4+ T cells. Collectively, our results identify RGC-32 as a novel regulator of Th17 cell differentiation in vitro and in vivo and suggest that RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases.
Assuntos
Diferenciação Celular/imunologia , Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Células Th17/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/fisiopatologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/deficiência , Proteínas Nucleares/farmacologia , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Immunotherapies that augment antitumor T cells have had recent success for treating patients with cancer. Here we examined whether tumor-specific CD4(+) T cells enhance CD8(+) T-cell adoptive immunotherapy in a lymphopenic environment. Our model employed physiological doses of tyrosinase-related protein 1-specific CD4(+) transgenic T cells-CD4(+) T cells and pmel-CD8(+) T cells that when transferred individually were subtherapeutic; however, when transferred together provided significant (p ≤ 0.001) therapeutic efficacy. Therapeutic efficacy correlated with increased numbers of effector and memory CD8(+) T cells with tumor-specific cytokine expression. When combined with CD4(+) T cells, transfer of total (naïve and effector) or effector CD8(+) T cells were highly effective, suggesting CD4(+) T cells can help mediate therapeutic effects by maintaining function of activated CD8(+) T cells. In addition, CD4(+) T cells had a pronounced effect in the early posttransfer period, as their elimination within the first 3 days significantly (p < 0.001) reduced therapeutic efficacy. The CD8(+) T cells recovered from mice treated with both CD8(+) and CD4(+) T cells had decreased expression of PD-1 and PD-1-blockade enhanced the therapeutic efficacy of pmel-CD8 alone, suggesting that CD4(+) T cells help reduce CD8(+) T-cell exhaustion. These data support combining immunotherapies that elicit both tumor-specific CD4(+) and CD8(+) T cells for treatment of patients with cancer.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/transplante , Vacinas Anticâncer/imunologia , Comunicação Celular , Microambiente Celular , Citotoxicidade Imunológica , Feminino , Memória Imunológica , Imunoterapia Adotiva/tendências , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Oxirredutases/genética , Oxirredutases/metabolismo , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Therapeutic treatment of large established tumors using immunotherapy has yielded few promising results. We investigated whether adoptive transfer of tumor-specific CD8(+) T cells, together with tumor-specific CD4(+) T cells, would mediate regression of large established B16BL6-D5 melanomas in lymphopenic Rag1(-/-) recipients devoid of regulatory T cells. The combined adoptive transfer of subtherapeutic doses of both TRP1-specific TCR transgenic Rag1(-/-) CD4(+) T cells and gp100-specific TCR transgenic Rag1(-/-) CD8(+) T cells into lymphopenic recipients, who received vaccination, led to regression of large (100-400 mm(2)) melanomas. The same treatment strategy was ineffective in lymphoreplete wild-type mice. Twenty-five percent of mice (15/59) had tumors recur (15-180 d postregression). Recurrent tumors were depigmented and had decreased expression of gp100, the epitope targeted by the CD8(+) T cells. Mice with recurrent melanoma had increased CD4(+)Foxp3(+) TRP1-specific T cells compared with mice that did not show evidence of disease. Importantly, splenocytes from mice with recurrent tumor were able to suppress the in vivo therapeutic efficacy of splenocytes from tumor-free mice. These data demonstrate that large established tumors can be treated by a combination of tumor-specific CD8(+) and CD4(+) T cells. Additionally, recurrent tumors exhibited decreased Ag expression, which was accompanied by conversion of the therapeutic tumor-specific CD4(+) T cell population to a Foxp3(+)CD4(+) regulatory T cell population.
Assuntos
Antígenos de Neoplasias/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Linfócitos T Reguladores/imunologia , Regulação para Cima/imunologia , Transferência Adotiva , Animais , Antígenos de Neoplasias/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Feminino , Contagem de Linfócitos , Melanoma Experimental/terapia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Recidiva Local de Neoplasia/terapia , Oxirredutases/biossíntese , Oxirredutases/deficiência , Linfócitos T Reguladores/patologia , Antígeno gp100 de Melanoma/metabolismoRESUMO
Melanocyte differentiation Ags, including tyrosinase-related protein (TRP) 1, are relevant to both autoimmune skin depigmentation (vitiligo) and tumor immunity, because they are expressed by both benign melanocytes and many malignant melanomas. Melanoma patients generate CD4(+) T cells that specifically recognize these proteins. TRP1 contains internal disulfide bonds and is presented by MHC class II molecules. Gamma-IFN-inducible lysosomal thiol reductase (GILT) facilitates the generation of class II-binding peptides by the endocytic reduction of protein disulfide bonds. We show in this study that GILT is required for efficient MHC class II-restricted processing of a TRP1 epitope in vitro and accelerates the onset of vitiligo in TRP1-specific TCR transgenic mice. The presence of GILT confers a small increase in the percentage of autoreactive T cells with an effector memory phenotype that may contribute to earlier disease onset. The onset of vitiligo is associated with a greater increase in the percentage of autoreactive T cells with an effector memory phenotype. Given that many self and tumor Ags have disulfide bonds and are presented on MHC class II, GILT is likely to be important in the pathogenesis of other CD4(+) T cell-mediated autoimmune diseases and for the development of effective cancer immunotherapy.
Assuntos
Anticorpos Antineoplásicos/biossíntese , Antígenos de Neoplasias/imunologia , Doenças Autoimunes/enzimologia , Melanoma Experimental/enzimologia , Melanoma Experimental/imunologia , Glicoproteínas de Membrana/imunologia , Oxirredutases/imunologia , Oxirredutases/fisiologia , Vitiligo/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/genética , Doenças Autoimunes/genética , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Melanoma Experimental/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Oxirredutases/biossíntese , Oxirredutases/deficiência , Oxirredutases/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Vitiligo/enzimologia , Vitiligo/genéticaRESUMO
Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8+ T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4+CD25+ regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8+ T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The gammaC cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of gammaC cytokine-responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8+ T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells.
Assuntos
Transferência Adotiva/métodos , Autoimunidade/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Linfopenia/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Vacinação , Irradiação Corporal TotalRESUMO
Many tumor-associated antigens are derived from nonmutated "self" proteins. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I-restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer.
Assuntos
Autoimunidade , Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Glicoproteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Transferência Adotiva , Animais , Antígenos de Histocompatibilidade Classe I , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Complexo Principal de Histocompatibilidade , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Tolerância a Antígenos Próprios , Taxa de Sobrevida , Vacinação , Antígeno gp100 de MelanomaRESUMO
Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8(+) T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8(+) T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8(+) T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.
Assuntos
Transferência Adotiva , Translocação Bacteriana/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Experimentais/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Irradiação Corporal Total , Animais , Antibacterianos/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/microbiologia , Doenças Autoimunes/patologia , Autoimunidade/efeitos dos fármacos , Autoimunidade/imunologia , Translocação Bacteriana/efeitos da radiação , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Citocinas/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Intestinos/patologia , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Depleção Linfocítica , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/microbiologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Polimixina B/farmacologia , Transdução de Sinais/efeitos dos fármacos , Vitiligo/imunologia , Vitiligo/microbiologia , Vitiligo/patologiaRESUMO
CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-gamma (IFN-gamma) production, whereas depletion of interleukin (IL)-17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer-based immunotherapy of human malignancies.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citotoxicidade Imunológica , Melanoma/imunologia , Animais , Linhagem Celular Tumoral , Interferon Tipo I/imunologia , Interferon gama , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas da Gravidez/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologiaRESUMO
Lymphodepletion with chemotherapeutic agents or total body irradiation (TBI) before adoptive transfer of tumor-specific T cells is a critical advancement in the treatment of patients with melanoma. More than 50% of patients that are refractory to other treatments experience an objective or curative response with this approach. Emerging data indicate that the key mechanisms underlying how TBI augments the functions of adoptively transferred T cells include (a) the depletion of regulatory T cells (T(reg)) and myeloid-derived suppressor cells that limit the function and proliferation of adoptively transferred cells; (b) the removal of immune cells that act as "sinks" for homeostatic cytokines, whose levels increase after lymphodepletion; and (c) the activation of the innate immune system via Toll-like receptor 4 signaling, which is engaged by microbial lipopolysaccharide that translocated across the radiation-injured gut. Here, we review these mechanisms and focus on the effect of Toll-like receptor agonists in adoptive immunotherapy. We also discuss alternate regimens to chemotherapy or TBI, which might be used to safely treat patients with advanced disease and promote tumor regression.
Assuntos
Imunoterapia Adotiva , Neoplasias/terapia , Linfócitos T/transplante , Receptores Toll-Like/agonistas , Animais , Humanos , Intestinos/imunologia , Depleção Linfocítica , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Linfócitos T/imunologia , Receptores Toll-Like/efeitos da radiação , Irradiação Corporal TotalRESUMO
Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of tumor growth compared to either therapy alone. Our studies support a role for 17-AAG and HSP90 inhibition in enhancing cellular immunotherapy for melanoma.
Assuntos
Benzoquinonas/uso terapêutico , Imunoterapia Adotiva , Indóis/uso terapêutico , Lactamas Macrocíclicas/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/terapia , Sulfonamidas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Melanoma Experimental/metabolismo , Camundongos , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , VemurafenibRESUMO
Immunotherapy using adoptive cell transfer is a promising approach that can result in the regression of bulky, invasive cancer in some patients. However, currently available therapies remain less successful than desired. To study the mechanisms of action and possible improvements in cell-transfer therapies, we use a murine model system with analogous components to the treatment of patients. T cell receptor transgenic CD8+ T cells (pmel-1) specifically recognizing the melanocyte differentiation antigen gp100 are adoptively transferred into lympho-depleted mice bearing large, established, 14-day subcutaneous B16 melanoma (0.5-1 cm in diameter) on the day of treatment. Adoptive cell transfer in combination with interleukin interleukin-2 or interleukin-15 cytokine administration and vaccination using an altered form of the target antigen, gp100, can result in the complete and durable regression of large tumor burdens. Complete responders frequently develop autoimmunity with vitiligo at the former tumor site that often spreads to involve the whole coat. These findings have important implications for the design of immunotherapy trials in humans.
Assuntos
Transferência Adotiva , Modelos Animais de Doenças , Imunoterapia , Melanoma/terapia , Metástase Neoplásica/terapia , Animais , Melanoma/imunologia , Camundongos , Metástase Neoplásica/imunologia , Linfócitos T/imunologiaRESUMO
Adoptive transfer of large numbers of tumor-reactive CD8(+) cytotoxic T lymphocytes (CTLs) expanded and differentiated in vitro has shown promising clinical activity against cancer. However, such protocols are complicated by extensive ex vivo manipulations of tumor-reactive cells and have largely focused on CD8(+) CTLs, with much less emphasis on the role and contribution of CD4(+) T cells. Using a mouse model of advanced melanoma, we found that transfer of small numbers of naive tumor-reactive CD4(+) T cells into lymphopenic recipients induces substantial T cell expansion, differentiation, and regression of large established tumors without the need for in vitro manipulation. Surprisingly, CD4(+) T cells developed cytotoxic activity, and tumor rejection was dependent on class II-restricted recognition of tumors by tumor-reactive CD4(+) T cells. Furthermore, blockade of the coinhibitory receptor CTL-associated antigen 4 (CTLA-4) on the transferred CD4(+) T cells resulted in greater expansion of effector T cells, diminished accumulation of tumor-reactive regulatory T cells, and superior antitumor activity capable of inducing regression of spontaneous mouse melanoma. These findings suggest a novel potential therapeutic role for cytotoxic CD4(+) T cells and CTLA-4 blockade in cancer immunotherapy, and demonstrate the potential advantages of differentiating tumor-reactive CD4(+) cells in vivo over current protocols favoring in vitro expansion and differentiation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Leucemia de Células T/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/radioterapia , Melanoma/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Divisão Celular , Modelos Animais de Doenças , Interferon gama/metabolismo , Leucemia de Células T/patologia , Melanoma/patologia , Melanoma Experimental/patologia , Camundongos , Camundongos Transgênicos , Tripsina/genéticaRESUMO
Although FOXP3 is primarily expressed by regulatory CD4 T cells (Treg) in vivo, polyclonal activation of human CD8 T cells can result in the expression of FOXP3 in a fraction of CD8 T cells. However, the cellular lineage and mechanism of FOXP3 induction in CD8 T cells remain unclear. Here, we demonstrate that interleukin-2 (IL-2) induces FOXP3 expression in OKT3-stimulated or antigen-stimulated CD8 T cells, indicating that FOXP3 expression is neither limited to a unique subset of CD8 T cells nor dependent on the mode of T-cell receptor stimulation. In the absence of IL-2, antigen stimulation resulted in T-cell activation and acquisition of effector function without induction of FOXP3, indicating that acquisition of effector function is independent of induction of FOXP3 expression in CD8 T cells. Interestingly, IL-15, but not IL-7 or IL-21, also led to de novo induction of FOXP3 in antigen-specific CD8 T cells, suggesting that signaling by IL-2/IL-15Rbeta chain is pivotal for induction of FOXP3 in human CD8 T cells. These findings indicate that induction of FOXP3 is intrinsic to CD8 T cells that are activated in the presence of IL-2 or IL-15, and in vitro-induced expression of FOXP3 cannot be simply interpreted as an indicator of Treg activity or activation marker.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Antígenos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Memória Imunológica , Interleucina-7/farmacologia , Interleucinas/farmacologia , Ativação Linfocitária , Melanoma/imunologia , Muromonab-CD3/farmacologia , Neoplasias Cutâneas/imunologiaRESUMO
Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) maintains peripheral tolerance by suppressing T-cell activation and proliferation but its precise role in vivo remains unclear. We sought to elucidate the impact of CTLA-4 expression on self/tumor-reactive CD8(+) T cells by using the glycoprotein (gp) 100-specific T-cell receptor (TCR) transgenic mouse, pmel-1. pmel-1 CLTA-4(-/-) mice developed profound, accelerated autoimmune vitiligo. This enhanced autoimmunity was associated with a small but highly activated CD8(+) T-cell population and large numbers of CD4(+) T cells not expressing the transgenic TCR. Adoptive transfer of pmel-1 CLTA-4(-/-) CD8(+) T cells did not mediate superior antitumor immunity in the settings of either large established tumors or tumor challenge, suggesting that the mere absence of CTLA-4-mediated inhibition on CD8(+) T cells did not directly promote enhancement of their effector functions. Removal of CD4(+) T cells by crossing the pmel-1 CLTA-4(-/-) mouse onto a Rag-1(-/-) background resulted in the complete abrogation of CD8(+) T-cell activation and autoimmune manifestations. The effects of CD4(+) CLTA-4(-/-) T cells were dependent on the absence of CTLA-4 on CD8(+) T cells. These results indicated that CD8(+) CLTA-4(-/-) T-cell-mediated autoimmunity and tumor immunity required CD4(+) T cells in which the function was dysregulated by the absence of CTLA-4-mediated negative costimulation.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Neoplasias/imunologia , Animais , Antígenos CD/genética , Antígenos de Diferenciação/genética , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Autoimunidade/genética , Antígeno CTLA-4 , Proliferação de Células , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Ativação Linfocitária/genética , Depleção Linfocítica/métodos , Camundongos , Camundongos Knockout , Neoplasias/genética , Vitiligo/genética , Vitiligo/imunologiaRESUMO
IL-2 is a critical T cell growth factor in vitro, but predominantly mediates tolerance in vivo. IL-2 is mainly produced by CD4(+) Th cells, but the role of Th cell-derived IL-2 in vivo is controversial. We demonstrate that during immunity to a tumor/self-Ag, the predominant role of Th cell-derived IL-2 was to maintain IL-2Ralpha (CD25) on CD4(+) T regulatory cells (T(reg)), which resulted in their maintenance of the T(reg) cell lineage factor, Forkhead/winged helix transcription factor (Foxp3), and tolerance. However, in the absence of T(reg) cells, Th cell-derived IL-2 maintained effector T cells and caused autoimmunity. IL-2R signaling was indispensable for T(reg) cell homeostasis and efficient suppressor function in vivo, but, surprisingly, was not required for their generation, because IL-2(-/-) and CD25(-/-) mice both contained Foxp3(+) T cells in the periphery. IL-2R signaling was also important for CD8(+) T cell immunity, because CD25(-/-) tumor-reactive CD8(+) T cells failed to affect established tumors. Conversely, IL-2R signaling was not required for Th cell function. Lastly, administration of anti-IL-2 plus exogenous IL-15 to tumor-bearing mice enhanced the adoptive immunotherapy of cancer. Therefore, Th cell-derived IL-2 paradoxically controls both tolerance and immunity to a tumor/self-Ag in vivo.
Assuntos
Tolerância Imunológica/imunologia , Interleucina-2/imunologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Autoantígenos/imunologia , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Humanos , Imunoterapia , Interferon gama/metabolismo , Interleucina-2/deficiência , Interleucina-2/genética , Interleucina-2/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Receptores de Interleucina-2/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Timo/imunologia , Timo/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Central memory CD8+ T cells (T(CM)) and effector memory CD8+ T cells (T(EM)) are found in humans and mice; however, their relative contributions to host immunity have only recently been examined in vivo. Further, the ability of T(CM) to treat an established tumor or infection has yet to be evaluated. To address the therapeutic potential of different tumor-reactive CD8+ T cell memory subsets, we used an established model for the in vitro generation of T(CM) and T(EM) by using IL-15 and IL-2, respectively. Adoptively transferred T(CM) exhibited a potent in vivo recall response when combined with tumor-antigen vaccination and exogenous IL-2, leading to the eradication of large established tumors. By contrast, T(EM) were far less effective on a per-cell basis. Microarray analysis revealed that the signature of highly in vivo effective antitumor T cells included the overexpression of genes responsible for trafficking to secondary lymphoid tissues. This gene expression profile correctly predicted the in vitro and in vivo lymphoid-homing attributes of tumor-reactive T cells. Furthermore, we found that homing to secondary lymphoid tissue is required for optimal tumor treatment. Our findings indicated that highly in vivo effective antitumor T cells were those that initially targeted secondary lymphoid tissue, rather than tumor sites, as had previously been postulated. Thus, tumor-reactive CD8+ T cell populations with the phenotypic and functional attributes of T(CM) may be superior to T(EM)/effector T cells for adoptive immunotherapies using concomitant tumor-antigen vaccination.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Regulação Neoplásica da Expressão Gênica , Memória Imunológica/imunologia , Imunoterapia Ativa/métodos , Neoplasias/imunologia , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Interleucina-15/imunologia , Interleucina-2/imunologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Análise em Microsséries , Neoplasias/terapiaRESUMO
CD4(+) T cells control the effector function, memory, and maintenance of CD8(+) T cells. Paradoxically, we found that absence of CD4(+) T cells enhanced adoptive immunotherapy of cancer when using CD8(+) T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4(+)CD25(-) Th cells (Th cells) with tumor/self-reactive CD8(+) T cells and vaccination into CD4(+) T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4(+) T cells that contained a mixture of Th and CD4(+)CD25(+) T regulatory cells (T(reg) cells) or T(reg) cells alone prevented effective adoptive immunotherapy. Maintenance of CD8(+) T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2(-/-) mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring T(reg) cells to be effective.
Assuntos
Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva , Melanoma Experimental/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Tolerância a Antígenos Próprios/imunologia , Linfócitos T Reguladores/transplante , Animais , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Homeostase/imunologia , Imunidade Inata , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Interleucina-2/fisiologia , Interleucina-2/uso terapêutico , Ativação Linfocitária/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/biossíntese , Receptores de Interleucina-2/biossíntese , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante/imunologia , Evasão Tumoral/imunologia , Antígeno gp100 de MelanomaRESUMO
Keratinocyte growth factor (KGF) promotes intestinal epithelial growth. To understand the relevance of intraepithelial lymphocyte (IEL)-derived KGF expression on epithelial growth, we used a mouse model of villus atrophy by the administration of total parenteral nutrition, and a model of villus hypertrophy by the creation of a short bowel syndrome. KGF expression was confined to gammadelta-TCR(+) IELs. IEL-derived KGF expression was highest in the crypts, somewhat less in the lower portion of villi, and markedly lower in the upper portion of villi. Total parenteral nutrition administration was associated with a down-regulation of IEL-derived KGF expression, and short bowel syndrome was associated with an up-regulation of IEL-derived KGF expression. In the absence of gammadelta-TCR(+) IEL, using gammadelta(-/-) mice, intestinal epithelial cell proliferation decreased in control, and in both mucosal atrophy (22% decline) and mucosal hypertrophy (14%) models. These results show that KGF from IELs is an important factor for maintenance of intestinal epithelial cell proliferation and villus growth.
Assuntos
Adjuvantes Imunológicos/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/biossíntese , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Adjuvantes Imunológicos/biossíntese , Animais , Atrofia , Divisão Celular/genética , Divisão Celular/imunologia , Fator 7 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/biossíntese , Hipertrofia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Jejuno/imunologia , Jejuno/metabolismo , Jejuno/patologia , Jejuno/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microdissecção/métodos , Microvilosidades/patologia , Nutrição Parenteral Total , Receptores de Antígenos de Linfócitos T gama-delta/deficiência , Receptores de Antígenos de Linfócitos T gama-delta/genética , Síndrome do Intestino Curto/genética , Síndrome do Intestino Curto/imunologia , Síndrome do Intestino Curto/patologiaRESUMO
OBJECTIVE: To determine the etiology of the loss of epithelial barrier function observed with the administration of total parenteral nutrition (TPN) in a mouse model. SUMMARY BACKGROUND DATA: Removal of enteral nutrition with the administration of TPN is associated with a loss of intestinal epithelial barrier function. The etiology of this barrier loss is not clear. Because intraepithelial lymphocytes (IELs) produce a number of cytokines that may alter epithelial permeability, the authors investigated IEL cytokine expression in a mouse model of TPN. METHODS: Adult C57BL/6 mice received TPN or enteral diet for 7 days. IELs were subsequently harvested and the mRNA expression of cytokines was measured. Epithelial barrier function was assessed in vitro with 51Cr-EDTA in Ussing chambers and was expressed as the permeability coefficient (Papp). RESULTS: IEL mRNA expression of interferon-gamma (IFN-gamma) rose from 0.14 +/- 0.07 in the control (enterally fed) group to 0.44 +/- 0.11 attomoles/microL in the TPN group (P <.05). Transforming growth factor-beta1 declined slightly but not significantly, from 0.75 +/- 0.35 to 0.55 +/- 0.18 attomoles/microL in the control and TPN groups, respectively. Epithelial barrier function declined significantly with TPN compared to controls. To assess the relevance of IFN-gamma changes, permeability in IFN-gamma knockout mice was studied. Barrier function was significantly higher in IFN-gamma knockout mice on TPN compared to C57BL/6 mice that received TPN. CONCLUSIONS: IEL cytokine expression changes significantly with TPN administration. The partial correction with IFN-gamma knockout mice suggests that an upregulation of IFN-gamma expression is one mechanism responsible for the loss of the epithelial barrier associated with TPN.