RESUMO
In our opinion the most urgent needs to improve patient outcomes are: 1) a deeper ability to measure cancer immunobiology, and 2) increased availability of agents that, coupled with predictive biomarkers, will be used to tailor anti-cancer immunity. Tailoring effective immunotherapy will entail combinations of immunotherapeutics that augment priming of anti-cancer immunity, boost expansion of effector and memory cells of the T, B and NK lineage, amplify innate immunity and relieve checkpoint inhibition. Alternatives to inducing adaptive immunity to cancer include synthetic immunology that incorporate bi-specifics that target T cells to cancer or adoptive immunotherapy with gene-modified immune cells.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Terapia Baseada em Transplante de Células e Tecidos/métodos , Imunoterapia/métodos , Neoplasias/terapia , Terapia Combinada , Humanos , Neoplasias/imunologiaRESUMO
BACKGROUND: One of today's greatest hurdles for cancer immunotherapy is the absence of information regarding which tumor antigens are already recognized by patients receiving immunotherapies, and whether those therapies then boost or generate an immune response against tumor proteins. For CD8+ T cells in particular, patient-specific immune recognition and responses at the level of individual tumor antigens are rarely characterized. Because of this, some immunologists have turned to serum antibodies as an alternative measure of antigen-specific anti-tumor immunity. In this work, we sought to simultaneously interrogate serum IgG and CD8+ T cell recognition of individual tumor antigens to determine whether antigen-specific serum IgG antibodies provide a window into the behavior of antigen-specific CD8+ T cell responses. Using antibody-based assays to evaluate immune response repertoires and focus T cell antigen exploration could afford substantial advantages for discovering and monitoring the anti-cancer immune responses of patients enrolled on clinical trials. METHODS: We vaccinated female BALB/c mice with a novel combination of an autophagosome-enriched vaccine derived from 4T1 mammary carcinoma along with poly-I:C adjuvant, then screened serum for IgG binding to arrays of 15mer peptides containing known mutation sites in 4T1. Simultaneously, we primed CD8+ T cell cultures from these same animals with 8-11mer peptides derived from these antigens. These primed T cells were then stimulated to measure recognition of the peptides or live 4T1 cells by IFNγ release. RESULTS: Vaccinated animals demonstrate increases in antigen-specific CD8+ T cell recognition of 4T1 tumor cells and peptides. For proteins confirmed in 4T1 cells and vaccine by mass spectrometry, there is a correlation between this increased CD8+ T cell IFNγ release and serum IgG binding to individual peptide antigens. CONCLUSIONS: These results suggest it is possible to observe some features of a patient's antigen-specific T cell repertoire via an antibody surrogate, which has implications for tumor antigen discovery and clinical monitoring of antigen-specific anti-tumor immunity.
Assuntos
Antígenos de Neoplasias/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer , Imunoglobulina G/sangue , Neoplasias Mamárias Experimentais/imunologia , Peptídeos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Neoplasias Mamárias Experimentais/terapia , Camundongos Endogâmicos BALB C , Poli I-C/farmacologia , VacinaçãoRESUMO
Purpose: Antibodies specific for inhibitory checkpoints PD-1 and CTLA-4 have shown impressive results against solid tumors. This has fueled interest in novel immunotherapy combinations to affect patients who remain refractory to checkpoint blockade monotherapy. However, how to optimally combine checkpoint blockade with agents targeting T-cell costimulatory receptors, such as OX40, remains a critical question.Experimental Design: We utilized an anti-PD-1-refractory, orthotopically transplanted MMTV-PyMT mammary cancer model to investigate the antitumor effect of an agonist anti-OX40 antibody combined with anti-PD-1. As PD-1 naturally aids in immune contraction after T-cell activation, we treated mice with concurrent combination treatment versus sequentially administering anti-OX40 followed by anti-PD-1.Results: The concurrent addition of anti-PD-1 significantly attenuated the therapeutic effect of anti-OX40 alone. Combination-treated mice had considerable increases in type I and type II serum cytokines and significantly augmented expression of inhibitory receptors or exhaustion markers CTLA-4 and TIM-3 on T cells. Combination treatment increased intratumoral CD4+ T-cell proliferation at day 13, but at day 19, both CD4+ and CD8+ T-cell proliferation was significantly reduced compared with untreated mice. In two tumor models, sequential combination of anti-OX40 followed by anti-PD-1 (but not the reverse order) resulted in significant increases in therapeutic efficacy. Against MMTV-PyMT tumors, sequential combination was dependent on both CD4+ and CD8+ T cells and completely regressed tumors in approximately 30% of treated animals.Conclusions: These results highlight the importance of timing for optimized therapeutic effect with combination immunotherapies and suggest the testing of sequencing in combination immunotherapy clinical trials. Clin Cancer Res; 23(20); 6165-77. ©2017 AACRSee related commentary by Colombo, p. 5999.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Imunoterapia , Terapia de Alvo Molecular , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores OX40/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Imunofenotipagem , Imunoterapia/métodos , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores OX40/metabolismo , Taxa de Sobrevida , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Fatores de Tempo , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stroke induction in immunologically competent mice not only produces local ischemia and brain damage, but also induces early inflammatory changes in brain and peripheral immune responses. Although immune elements clearly are activated after brain vascular occlusion, the relative contribution of T and B lymphocytes to the developing lesion has not been quantified. We evaluated effects 22 h after middle cerebral artery occlusion (90 mins) on histologic injury and peripheral immune activation in severe combined immunodeficient (SCID) mice lacking T and B cells. Cortical and total infarct volumes were strikingly reduced in male SCID mice (n=14, 33+/-4% of contralateral cortex, n=10, 52+/-3% of contralateral hemisphere) versus immunologically intact C57BL/6 mice (wild type, n=9, 57+/-5% of contralateral cortex, 57+/-4% of contralateral hemisphere) (P<0.01). Striatal infarction was not altered (77+/-7% of contralateral striatum in SCID, 84+/-7% in wild type), suggesting that the core of the evolving ischemic lesion was not impacted by lack of T and B cells. As expected, inflammatory factors from immune cells in ischemic SCID brains were essentially absent, with the exception of interleukin-1beta increase in both SCID and wild type tissue. Spleen cell numbers were low in SCID mice, but were further reduced 22 h after stroke, with substantial reduction in most inflammatory factors except for increased expression of interferon-gamma and macrophage inflammatory protein (MIP)-2. These data quantify the damaging effect of T and B lymphocytes on early, evolving ischemic brain injury, and further implicate interleukin-1beta in brain and interferon-gamma and MIP-2 in spleen as inflammatory factors produced by cells other than T and B cells.
Assuntos
Linfócitos B/imunologia , Linfócitos B/fisiologia , Inflamação/imunologia , Inflamação/patologia , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/patologia , Linfócitos T/imunologia , Linfócitos T/fisiologia , Animais , Encéfalo/patologia , Química Encefálica/fisiologia , Isquemia Encefálica/patologia , Contagem de Células , Infarto Cerebral/patologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Citometria de Fluxo , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/imunologiaRESUMO
Inflammation results in CNS damage in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. It is uncertain how much repair of injured myelin and axons can occur following highly selective anti-inflammatory therapy in EAE and MS. In this study, SJL/J mice with established EAE were treated successfully with an antigen-specific recombinant T cell receptor ligand (RTL), RTL401, a mouse I-A(s)/PLP-139-151 construct, after the peak of EAE. To define the mechanisms by which late application of RTL401 inhibits EAE, we evaluated mice at different time points to assess the levels of neuroinflammation and myelin and axon damage in their spinal cords. Our results showed that RTL401 administered after the peak of acute EAE induced a marked reduction in inflammation in the CNS, associated with a significant reduction of demyelination, axonal loss and ongoing damage. Electron microscopy showed that RTL-treated mice had reduced pathology compared with mice treated with vehicle and mice at the peak of disease, as demonstrated by a decrease in continued degeneration, increase in remyelinating axons and the presence of an increased number of small, presumably regenerative axonal sprouts. These findings indicate that RTL therapy targeting encephalitogenic T cells may promote CNS neuroregenerative processes.
Assuntos
Axônios , Encefalomielite Autoimune Experimental/fisiopatologia , Encefalomielite Autoimune Experimental/terapia , Imunoterapia , Bainha de Mielina , Regeneração Nervosa/efeitos dos fármacos , Proteínas Recombinantes de Fusão/uso terapêutico , Medula Espinal/ultraestrutura , Animais , Axônios/ultraestrutura , Esquema de Medicação , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Epitopos , Feminino , Ligantes , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica , Bainha de Mielina/ultraestrutura , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/uso terapêuticoRESUMO
Alpha B-crystallin (alphaB) is a small heat shock protein that is strongly up-regulated in multiple sclerosis (MS) brain tissue, and can induce strong T cell responses. Assessing a potential encephalitogenic function for alphaB protein in MS and experimental autoimmune encephalomyelitis (EAE) has been challenging due to its ubiquitous expression that likely maintains central and peripheral tolerance to this protein in mice. To address this issue, we obtained alphaB-knockout (alphaB-KO) mice in H-2b background that lack immune tolerance to alphaB protein, and thus are capable of developing alphaB-specific T cells that could be tested for encephalitogenic activity after transfer into alphaB-expressing wild type (WT) mice. We found that T cell lines from spleens of alphaB protein-immunized alphaB-KO mice proliferated strongly to alphaB protein itself, and the majority of T cells were CD4+ and capable of secreting pro-inflammatory Th1 cytokines upon restimulation. However, transfer of such alphaB-reactive T cells back into WT recipients was not sufficient to induce EAE, compared to the transfer of mouse MOG-35-55 peptide-reactive T cells from the same donors that induced severe EAE in recipients. Moreover, alphaB-specific T cells failed to augment severity of actively induced EAE in WT mice that were expressing high levels of alphaB message in the CNS at the time of transfer. These results suggest that alphaB-specific T cells are immunocompetent but not encephalitogenic in 129SvEv mice, and that immune tolerance may not be the main factor that limits the encephalitogenic potential of alphaB.
Assuntos
Encefalomielite Autoimune Experimental/etiologia , Linfócitos T/imunologia , Cadeia B de alfa-Cristalina/imunologia , Sequência de Aminoácidos , Animais , Encefalomielite Autoimune Experimental/imunologia , Glicoproteínas/imunologia , Linfonodos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/imunologia , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/fisiologiaRESUMO
Induction of stroke not only produces local ischemia and brain damage, but also has profound effects on peripheral immune responses. In the current study, we evaluated effects on spleen and blood cells 4 days after stroke induction. Surprisingly, there was a less inflammatory cytokine profile in the middle cerebral artery occlusion-affected right brain hemisphere at 96 h compared with earlier time points. Moreover, our results demonstrate that stroke leads to splenic atrophy characterized by a reduction in organ size, a drastic loss of splenocyte numbers, and induction of annexin V+ and TUNEL+ cells within the spleen that are in the late stages of apoptosis. The consequence of this process was to reduce T cell proliferation responses and secretion of inflammatory cytokines, resulting in a state of profound immunosuppression. These changes produced a drastic reduction in B cell numbers in spleen and blood, and a novel increase in CD4+FoxP3+ regulatory T cells. Moreover, we detected a striking increase in the percentage of nonapoptotic CD11b+ VLA-4-negative macrophages/monocytes in blood. Immunosuppression in response to brain injury may account for the reduction of inflammatory factors in the stroke-affected brain, but also potentially could curtail protective immune responses in the periphery. These findings provide new evidence to support the contention that damage to the brain caused by cerebral ischemia provides a powerful negative signal to the peripheral immune system that ultimately induces a drastic state of immunosuppression caused by cell death as well as an increased presence of CD4+FoxP3+ regulatory T cells.
Assuntos
Infarto da Artéria Cerebral Média/imunologia , Macrófagos/patologia , Baço/imunologia , Baço/patologia , Linfócitos T Reguladores/patologia , Animais , Atrofia , Encéfalo/imunologia , Encéfalo/metabolismo , Isquemia Encefálica/sangue , Isquemia Encefálica/imunologia , Isquemia Encefálica/patologia , Antígeno CD11b/biossíntese , Antígeno CD11b/sangue , Morte Celular/imunologia , Quimiocinas/biossíntese , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Fatores de Transcrição Forkhead/biossíntese , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/patologia , Contagem de Leucócitos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timo/imunologia , Timo/patologia , Regulação para Cima/imunologiaRESUMO
Clinical experimental stroke induces injurious local brain inflammation. However, effects on the peripheral immune system have not been well characterized. We quantified mRNA and protein levels for cytokines, chemokines, and chemokine receptors (CCR) in brain, spinal cord, peripheral lymphoid organs (spleen, lymph node, blood, and cultured mononuclear cells from these sources), and blood plasma after reversible middle cerebral artery occlusion (MCAO) or sham treatment in male C57BL/6 mice. Middle cerebral artery occlusion induced a complex, but organ specific, pattern of inflammatory factors in the periphery. At both 6 and 22 h after MCAO, activated spleen cells from stroke-injured mice secreted significantly enhanced levels of TNF-alpha, IFN-gamma, IL-6, MCP-1, and IL-2. Unstimulated splenocytes expressed increased chemokines and CCR, including MIP-2 and CCR2, CCR7 and CCR8 at 6 h; and MIP-2, IP-10, and CCR1 and CCR2 at 22 h. Also at 22 h, T cells from blood and lymph nodes secreted increased levels of inflammatory cytokines after activation. As expected, there were striking proinflammatory changes in postischemic brain. In contrast, spinal cord displayed suppression of all mediators, suggesting a compensatory response to intracranial events. These data show for the first time that focal cerebral ischemia results in dynamic and widespread activation of inflammatory cytokines, chemokines, and CCR in the peripheral immune system.