RESUMO
Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno CTLA-4/antagonistas & inibidores , Imunoterapia/efeitos adversos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Inibidores do Fator de Necrose Tumoral/farmacologia , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Colite/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Sulfato de Dextrana/farmacologia , Feminino , Doença Enxerto-Hospedeiro , Hepatite/tratamento farmacológico , Humanos , Ipilimumab/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nivolumabe/efeitos adversos , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Invariant natural killer T (iNKT) cells are a T-cell subset with potent immunomodulatory properties. Experimental evidence in mice and observational studies in humans indicate that iNKT cells have antitumor potential as well as the ability to suppress acute and chronic graft-versus-host-disease (GVHD). Murine iNKT cells differentiate during thymic development into iNKT1, iNKT2, and iNKT17 sublineages, which differ transcriptomically and epigenomically and have subset-specific developmental requirements. Whether distinct iNKT sublineages also differ in their antitumor effect and their ability to suppress GVHD is currently unknown. In this work, we generated highly purified murine iNKT sublineages, characterized their transcriptomic and epigenomic landscape, and assessed specific functions. We show that iNKT2 and iNKT17, but not iNKT1, cells efficiently suppress T-cell activation in vitro and mitigate murine acute GVHD in vivo. Conversely, we show that iNKT1 cells display the highest antitumor activity against murine B-cell lymphoma cells both in vitro and in vivo. Thus, we report for the first time that iNKT sublineages have distinct and different functions, with iNKT1 cells having the highest antitumor activity and iNKT2 and iNKT17 cells having immune-regulatory properties. These results have important implications for the translation of iNKT cell therapies to the clinic for cancer immunotherapy as well as for the prevention and treatment of GVHD.
Assuntos
Doença Enxerto-Hospedeiro , Efeito Enxerto vs Tumor/imunologia , Ativação Linfocitária , Linfoma de Células B , Células T Matadoras Naturais/imunologia , Neoplasias Experimentais , Animais , Epigenômica , Feminino , Perfilação da Expressão Gênica , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Masculino , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapiaRESUMO
In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.
Assuntos
Quimiotaxia/imunologia , Armadilhas Extracelulares/imunologia , Interleucina-8/imunologia , Neutrófilos/imunologia , Acetilcisteína/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais/imunologiaRESUMO
The Radiation and Dust Sensor is one of six sensors of the Mars Environmental Dynamics Analyzer onboard the Perseverance rover from the Mars 2020 NASA mission. Its primary goal is to characterize the airbone dust in the Mars atmosphere, inferring its concentration, shape and optical properties. Thanks to its geometry, the sensor will be capable of studying dust-lifting processes with a high temporal resolution and high spatial coverage. Thanks to its multiwavelength design, it will characterize the solar spectrum from Mars' surface. The present work describes the sensor design from the scientific and technical requirements, the qualification processes to demonstrate its endurance on Mars' surface, the calibration activities to demonstrate its performance, and its validation campaign in a representative Mars analog. As a result of this process, we obtained a very compact sensor, fully digital, with a mass below 1 kg and exceptional power consumption and data budget features.
Assuntos
Poeira , Meio Ambiente Extraterreno , AtmosferaRESUMO
BACKGROUND: Anti-programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) monoclonal antibodies (mAbs) show remarkable clinical anti-tumour efficacy. However, rational combinations are needed to extend the clinical benefit to primary resistant tumours. The design of such combinations requires the identification of the kinetics of critical immune cell populations in the tumour microenvironment. METHODS: In this study, we compared the kinetics of immune cells in the tumour microenvironment upon treatment with immunotherapy combinations with different anti-tumour efficacies in the non-inflamed tumour model TC-1/A9. Tumour-bearing C57BL/6J mice were treated with all possible combinations of a human papillomavirus (HPV) E7 long peptide, polyinosinic-polycytidylic acid (PIC) and anti-PD-1 mAb. Tumour growth and kinetics of the relevant immune cell populations were assessed over time. The involvement of key immune cells was confirmed by depletion with mAbs and immunophenotyping with multiparametric flow cytometry. RESULTS: The maximum anti-tumour efficacy was achieved after intratumoural administration of HPV E7 long peptide and PIC combined with the systemic administration of anti-PD-1 mAb. The intratumoural immune cell kinetics of this combination was characterised by a biphasic immune response. An initial upsurge of proinflammatory myeloid cells led to a further rise in effector CD8+ T lymphocytes at day 8. Depletion of either myeloid cells or CD8+ T lymphocytes diminished the anti-tumour efficacy of the combination. CONCLUSIONS: The anti-tumour efficacy of a successful immunotherapy combination in a non-inflamed tumour model relies on an early inflammatory process that remodels the myeloid cell compartment.
Assuntos
Anticorpos Monoclonais/farmacologia , Células Mieloides/imunologia , Neoplasias/imunologia , Fragmentos de Peptídeos/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor 3 Toll-Like/metabolismo , Animais , Proliferação de Células , Combinação de Medicamentos , Feminino , Humanos , Ligantes , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Mieloides/patologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Células Tumorais Cultivadas , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Natural killer (NK) cells exist as subsets based on expression of inhibitory receptors that recognize major histocompatibility complex I (MHCI) molecules. NK cell subsets bearing MHCI binding receptors for self-MHCI have been termed as "licensed" and exhibit a higher ability to respond to stimuli. In the context of bone marrow transplantation (BMT), host licensed-NK (L-NK) cells have also been demonstrated to be responsible for the acute rejection of allogeneic and MHCI-deficient BM cells (BMCs) in mice after lethal irradiation. However, the role of recipient unlicensed-NK (U-NK) cells has not been well established with regard to allogeneic BMC resistance. After NK cell stimulation, the prior depletion of host L-NK cells resulted in a marked increase of donor engraftment compared with the untreated group. Surprisingly, this increased donor engraftment was reduced after total host NK cell depletion, indicating that U-NK cells can actually promote donor allogeneic BMC engraftment. Furthermore, direct coculture of U-NK cells with allogeneic but not syngeneic BMCs resulted in increased colony-forming unit cell growth in vitro, which was at least partially mediated by granulocyte macrophage colony-stimulating factor (GM-CSF) production. These data demonstrate that host NK cell subsets exert markedly different roles in allogeneic BMC engraftment where host L- and U-NK cells reject or promote donor allogeneic BMC engraftment, respectively.
Assuntos
Transplante de Medula Óssea , Células Matadoras Naturais/imunologia , Animais , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Fator Estimulador de Colônias de Macrófagos/biossíntese , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transplante HomólogoRESUMO
CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft-versus-host disease (GVHD) in murine models of allogeneic hematopoietic stem cell transplantation. We recently demonstrated that a single treatment of the agonistic antibody to DR3 (death receptor 3, αDR3) to donor mice resulted in the expansion of donor-derived Treg and prevented acute GVHD, although the precise role of DR3 signaling in GVHD has not been elucidated. In this study, we comprehensively analyzed the immunophenotype of Treg after DR3 signal activation, demonstrating that DR3-activated Treg (DR3-Treg) had an activated/mature phenotype. Furthermore, the CD25+Foxp3+ subpopulation in DR3-Treg showed stronger suppressive effects in vivo. Prophylactic treatment of αDR3 to recipient mice expanded recipient-derived Treg and reduced the severity of GVHD, whereas DR3 activation in mice with ongoing GVHD further promoted donor T-cell activation/proliferation. These data suggest that the function of DR3 signaling was highly dependent on the activation status of the T cells. In conclusion, our data demonstrated that DR3 signaling affects the function of Treg and T-cell activation after alloantigen exposure in a time-dependent manner. These observations provide important information for future clinical testing using human DR3 signal modulation and highlight the critical effect of the state of T-cell activation on clinical outcomes after activation of DR3.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/imunologia , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Doença Aguda , Animais , Perfilação da Expressão Gênica , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Isoantígenos/imunologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/imunologia , Transplante HomólogoRESUMO
CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been shown to effectively prevent graft-versus-host disease (GVHD) when adoptively transferred in murine models of hematopoietic cell transplantation and in phase 1/2 clinical trials. Critical limitations to Treg clinical application are the paucity of cells and limited knowledge of the mechanisms of in vivo function. We hypothesized that inflammatory conditions in GVHD modify Treg characteristics and activity. We found that peripheral blood of recipient animals during acute GVHD (aGVHD) induces Treg activation and enhances their function. The serum contains high levels of tumor necrosis factor-α (TNF-α) that selectively activates Tregs without impacting CD4(+)FoxP3(-) T cells. TNF-α priming induces Treg in vivo proliferation, whereas it limits the ability of CD4 and CD8 conventional T cells (Tcons) to proliferate and induce GVHD. TNF-α-primed Tregs prolong animal survival as compared with unprimed Tregs when used at an unfavorable Treg:Tcon ratio, demonstrating enhanced in vivo efficacy of TNF-α-primed Tregs. Because TNF-α is produced by several immune cells during inflammation, our work elucidates aspects of the physiologic mechanisms of Treg function. Furthermore, TNF-α priming of Tregs provides a new tool to optimize Treg cellular therapies for GVHD prevention and treatment.
Assuntos
Transferência Adotiva/métodos , Antígenos CD4/imunologia , Fatores de Transcrição Forkhead/imunologia , Doença Enxerto-Hospedeiro/terapia , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Antígenos CD4/análise , Células Cultivadas , Fatores de Transcrição Forkhead/análise , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/transplante , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Adoptive transfer of freshly isolated natural occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) prevents graft-versus-host disease (GVHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor-derived Treg have been mainly used, as they share the same MHC with CD4(+) and CD8(+) conventional T cells (Tcon) that are primarily responsible for GVHD. Third party-derived Treg are a promising alternative for cellular therapy, as they can be prepared in advance, screened for pathogens and activity, and banked. We explored MHC disparities between Treg and Tcon in HCT to evaluate the impact of different Treg populations in GVHD prevention and survival. Third-party Treg and donor Treg are equally suppressive in ex vivo assays, whereas both donor and third-party but not host Treg protect from GVHD in allogeneic HCT, with donor Treg being the most effective. In an MHC minor mismatched transplantation model (C57BL/6 â BALB/b), donor and third-party Treg were equally effective in controlling GVHD. Furthermore, using an in vivo Treg depletion mouse model, we found that Treg exert their main suppressive activity in the first 2 d after transplantation. Third-party Treg survive for a shorter period of time after adoptive transfer, but despite the shorter survival, they control Tcon proliferation in the early phases of HCT. These studies provide relevant insights on the mechanisms of Treg-mediated protection from GVHD and support for the use of third-party Treg in clinical trials.
Assuntos
Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Biomarcadores/sangue , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Fatores de Transcrição Forkhead/sangue , Expressão Gênica , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Teste de Histocompatibilidade , Subunidade alfa de Receptor de Interleucina-2/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/transplante , Doadores de Tecidos , Transplante Homólogo , Irradiação Corporal TotalRESUMO
Dysregulated donor T cells lead to destruction of host tissues resulting in graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). We investigated the impact of highly purified (>95%) donor CD4(+) invariant natural killer T (iNKT) cells on GVHD in a murine model of allogeneic HCT. We found that low doses of adoptively transferred donor CD4(+) iNKT cells protect from GVHD morbidity and mortality through an expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). These Tregs express high levels of the Ikaros transcription factor Helios and expand from the Treg pool of the donor graft. Furthermore, CD4(+) iNKT cells preserve T-cell-mediated graft-versus-tumor effects. Our studies reveal new aspects of the cellular interplay between iNKT cells and Tregs in the context of tolerance induction after allogeneic HCT and set the stage for clinical translation.
Assuntos
Transferência Adotiva , Linfócitos T CD4-Positivos/fisiologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Células T Matadoras Naturais/fisiologia , Linfócitos T Reguladores/imunologia , Animais , Contagem de Linfócito CD4 , Células Cultivadas , Feminino , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/mortalidade , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Because of increasing interest in the removal of immunosuppressive pathways in cancer, the combination of IL-2 with Abs to neutralize TGF-ß, a potent immunosuppressive cytokine, was assessed. Combination immunotherapy resulted in significantly greater antitumor effects. These were correlated with significant increases in the numbers and functionality of NK cells, NK cell progenitors, and activated CD8 T cells, resulting in the observed antitumor effects. Combination immunotherapy also was accompanied by lesser toxicities than was IL-2 therapy alone. Additionally, we observed a dual competition between NK cells and activated CD8 T cells such that, after immunotherapy, the depletion of either effector population resulted in the increased total expansion of the other population and compensatory antitumor effects. This study demonstrates the efficacy of this combination immunotherapeutic regimen as a promising cancer therapy and illustrates the existence of potent competitive regulatory pathways between NK cells and CD8 T cells in response to systemic activation.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Interleucina-2/uso terapêutico , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Proteína Ligante Fas/imunologia , Feminino , Imunoterapia/métodos , Células Matadoras Naturais/transplante , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília C de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Linfócitos T Reguladores/imunologia , Receptor fas/imunologiaRESUMO
Natural killer (NK) cells show differential functionality based on their capability of binding to self-MHC consistent with licensing. Here we show in vivo confirmation of the physiologic effects of licensing with differential effects of NK subsets on anti-murine cytomegalovirus (anti-MCMV) responses after syngeneic hematopoietic stem cell transplantation (HSCT) or regulatory T-cell (Treg) depletion. After HSCT, depletion of licensed NK cells led to far greater viral loads in target organs early after infection compared with nondepleted and unlicensed depleted mice. There was a preferential expansion of licensed, C-type lectin-like activating receptor Ly49H+ NK cells with increased IFNγ production after infection in nondepleted mice post-HSCT and after Treg depletion. Adoptive transfer of licensed NK subsets into immunodeficient hosts provided significantly greater MCMV resistance compared with transfer of total NK populations or unlicensed subsets. In non-HSCT mice, only concurrent depletion of Tregs or TGF-ß neutralization resulted in detection of NK licensing effects. This suggests that licensed NK cells are the initial and rapidly responding population of NK cells to MCMV infection, but are highly regulated by Tregs and TGF-ß.
Assuntos
Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Proliferação de Células , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Interferon gama/biossíntese , Depleção Linfocítica , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Carga Viral/imunologiaRESUMO
Natural killer (NK) cells express inhibitory receptors with varied binding affinities to specific major histocompatibility complex class I (MHC-I) haplotypes. NK cells can be classified as licensed or unlicensed based on their ability or inability to bind MHC-I, respectively. The role of donor vs host MHC on their development after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not known. Following reciprocal MHC-disparate allogeneic transplants and during de novo NK-cell recovery, depletion of the licensed and not unlicensed population of NK cells as determined by the licensing patterns of donor MHC-I haplotypes, resulted in significantly increased susceptibility to murine cytomegalovirus (MCMV) infection. A corresponding expansion of the licensed Ly49H(+) NK cells occurred with greater interferon γ production by these cells than unlicensed NK cells in the context of donor MHC-I. Thus, NK licensing behavior to MCMV corresponds to the donor, and not recipient, MHC haplotype after allo-HSCT in mice.
Assuntos
Infecções por Citomegalovirus/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Muromegalovirus/imunologia , Animais , Células da Medula Óssea/citologia , Feminino , Haplótipos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Transplante HomólogoRESUMO
NK subsets have activating and inhibitory receptors that bind MHC-I. Ly49A is a mouse inhibitory receptor that binds with high affinity to H2(d) in both a cis- and trans-manner. Ly49A cis-associations limit trans-interactions with H2(d)-expressing targets as well as mAb binding. We demonstrate that cis-interactions affect mAb effector functions. In vivo administration of anti-Ly49A depleted NK cells in H2(b) but not H2(d) mice. Despite lack of depletion, in vivo treatment with anti-Ly49A reduced NK killing capabilities and inhibited activation, partially due to its agonistic effect. These data explain the previously described in vivo effects on bone marrow allograft rejection observed with anti-Ly49A treatment in H2(d)-haplotype mice. However, prior treatment of mice with poly(I:C) or mouse CMV infection resulted in increased Ly49A expression and Ly49A(+) NK cell depletion in H2(d) mice. These data indicate that, although Ly49 mAbs can exert similar in vivo effects in mice with different MHC haplotypes, these effects are mediated via different mechanisms of action correlating with Ly49A expression levels and can be altered within the same strain contingent on stimuli. This illustrates the marked diversity of mAb effector functions due to the regulation of the level of expression of target Ags and responses by stimulatory incidents such as infection.
Assuntos
Células da Medula Óssea/imunologia , Transplante de Medula Óssea/imunologia , Células Matadoras Naturais/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Anticorpos Monoclonais , Citomegalovirus/imunologia , Feminino , Rejeição de Enxerto/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Poli I-C/farmacologiaRESUMO
Natural killer (NK) cells can mediate the rejection of bone marrow allografts and exist as subsets based on expression of inhibitory/activating receptors that can bind MHC. In vitro data have shown that NK subsets bearing Ly49 receptors for self-MHC class I have intrinsically higher effector function, supporting the hypothesis that NK cells undergo a host MHC-dependent functional education. These subsets also play a role in bone marrow cell (BMC) allograft rejection. Thus far, little in vivo evidence for this preferential licensing across mouse strains with different MHC haplotypes has been shown. We assessed the intrinsic response potential of the different Ly49(+) subsets in BMC rejection by using ß2-microglobulin deficient (ß2m(-/-)) mice as donors. Using congenic and allogeneic mice as recipients and depleting the different Ly49 subsets, we found that NK subsets bearing Ly49s, which bind "self-MHC" were found to be the dominant subset responsible for ß2m(-/-) BMC rejection. This provides in vivo evidence for host MHC class I-dependent functional education. Interestingly, all H2(d) strain mice regardless of background were able to resist significantly greater amounts of ß2m(-/-), but not wild-type BMC than H2(b) mice, providing evidence that the rheostat hypothesis regarding Ly49 affinities for MHC and NK-cell function impacts BMC rejection capability.
Assuntos
Transplante de Medula Óssea/imunologia , Rejeição de Enxerto/imunologia , Células Matadoras Naturais/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Feminino , Antígenos H-2/imunologia , Antígenos H-2/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Ligação Proteica/imunologia , Transplante Homólogo , Microglobulina beta-2/genética , Microglobulina beta-2/imunologia , Microglobulina beta-2/metabolismoRESUMO
BACKGROUND: Lymphocytic choriomeningitis virus (LCMV) belongs to the Arenavirus family known for inducing strong cytotoxic T-cell responses in both mice and humans. LCMV has been engineered for the development of cancer immunotherapies, currently undergoing evaluation in phase I/II clinical trials. Initial findings have demonstrated safety and an exceptional ability to activate and expand tumor-specific T lymphocytes. Combination strategies to maximize the antitumor effectiveness of LCMV-based immunotherapies are being explored. METHODS: We assessed the antitumor therapeutic effects of intratumoral administration of polyinosinic:polycytidylic acid (poly(I:C)) and systemic vaccination using an LCMV-vector expressing non-oncogenic versions of the E6 and E7 antigens of human papillomavirus 16 (artLCMV-E7E6) in a bilateral model engrafting TC-1/A9 cells. This cell line, derived from the parental TC-1, exhibits low MHC class I expression and is highly immune-resistant. The mechanisms underlying the combination's efficacy were investigated through bulk RNA-seq, flow cytometry analyses of the tumor microenvironment, selective depletions using antibodies and clodronate liposomes, Batf3 deficient mice, and in vivo bioluminescence experiments. Finally, we assessed the antitumor effectiveness of the combination of artLCMV-E7E6 with BO-112, a GMP-grade poly(I:C) formulated in polyethyleneimine, currently under evaluation in clinical trials. RESULTS: Intratumoral injection of poly(I:C) enhanced the antitumor efficacy of artLCMV-E7E6 in both injected and non-injected tumor lesions. The combined treatment resulted in a significant delay in tumor growth and often complete eradication of several tumor lesions, leading to significantly improved survival compared with monotherapies. While intratumoral administration of poly(I:C) did not impact LCMV vector biodistribution or transgene expression, it significantly modified leucocyte infiltrates within the tumor microenvironment and amplified systemic efficacy through proinflammatory cytokines/chemokines such as CCL3, CCL5, CXCL10, TNF, IFNα, and IL12p70. Upregulation of MHC on tumor cells and a reconfiguration of the gene expression programs related to tumor vasculature, leucocyte migration, and the activation profile of tumor-infiltrating CD8+ T lymphocytes were observed. Indeed, the antitumor effect relied on the functions of CD8+ T lymphocytes and macrophages. The synergistic efficacy of the combination was further confirmed when BO-112 was included. CONCLUSION: Intratumoral injection of poly(I:C) sensitizes MHClow tumors to the antitumor effects of artLCMV-E7E6, resulting in a potent therapeutic synergy.
Assuntos
Vírus da Coriomeningite Linfocítica , Neoplasias , Poli I-C , Animais , Humanos , Camundongos , Injeções Intralesionais , Distribuição Tecidual , Imunoterapia/métodos , Adjuvantes Imunológicos , Microambiente TumoralRESUMO
Natural killer (NK) cell subsets can be defined by the differential expression of inhibitory receptors for MHC class I molecules. Early after congenic HSCT, we found that Ly49G2(high) single-positive NK cells repopulated, displayed an activated phenotype, and were highly cytolytic. Over time, this subset was replaced with NK cells with a normal pattern of Ly49 expression. Treatment of mice with IL-2 also resulted in the rapid expansion of these Ly49G2(high) single-positive NK cells. Only the Ly49g (Klra7) Pro1 transcript was highly induced in both HSCT- and IL-2-treated recipients. MHC-independent expansion of the Ly49G2(+) subset was also observed after Listeria monocytogenes or mouse cytomegalovirus infection. Our data indicate that during reconstitution after HSCT and various activation stimuli, Ly49G2(+) NK cells represent the "first-responder" NK cells, which occur independently of NK-cell licensing via Ly49-MHC interactions. These data suggest that the inhibitory Ly49G2 receptor represents an activation marker on mouse NK cells under various conditions.
Assuntos
Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , Receptores Imunológicos/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citomegalovirus/imunologia , Feminino , Regulação da Expressão Gênica , Interleucina-2/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Listeria monocytogenes/imunologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos , Neoplasias/imunologia , Receptores Imunológicos/genética , Proteínas Recombinantes/metabolismo , Transcrição GênicaRESUMO
NK-cell reactivity against cancer is conceivably suppressed in the tumor microenvironment by the interaction of the inhibitory receptor NKG2A with the non-classical MHC-I molecules HLA-E in humans or Qa-1b in mice. We found that intratumoral delivery of NK cells attains significant therapeutic effects only if co-injected with anti-NKG2A and anti-Qa-1b blocking monoclonal antibodies against solid mouse tumor models. Such therapeutic activity was contingent on endogenous CD8 T cells and type-1 conventional dendritic cells (cDC1). Moreover, the anti-tumor effects were enhanced upon combination with systemic anti-PD-1 mAb treatment and achieved partial abscopal efficacy against distant non-injected tumors. In xenografted mice bearing HLA-E-expressing human cancer cells, intratumoral co-injection of activated allogeneic human NK cells and clinical-grade anti-NKG2A mAb (monalizumab) synergistically achieved therapeutic effects. In conclusion, these studies provide evidence for the clinical potential of intratumoral NK cell-based immunotherapies that exert their anti-tumor efficacy as a result of eliciting endogenous T-cell responses.
Assuntos
Anticorpos Monoclonais , Neoplasias , Camundongos , Humanos , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos de Histocompatibilidade Classe I , Células Matadoras Naturais , Linfócitos T CD8-Positivos , Microambiente TumoralRESUMO
Previous studies have shown that local delivery of tumor antigen-specific CD8+ T lymphocytes engineered to transiently express single-chain IL-12 mRNA is highly efficacious. Peritoneal dissemination of cancer is a frequent and often fatal patient condition usually diagnosed when the tumor burden is too large and hence uncontrollable with current treatment options. In this study, we have modeled intracavitary adoptive T cell therapy with OVA-specific OT-I T cells electroporated with IL-12 mRNA to treat B16-OVA and PANC02-OVA tumor spread in the peritoneal cavity. Tumor localization in the omentum and the effects of local T-cell encounter with the tumor antigens were monitored, the gene expression profile evaluated, and the phenotypic reprogramming of several immune subsets was characterized. Intraperitoneal administration of T cells promoted homing to the omentum more effectively than intravenous administration. Transient IL-12 expression was responsible for a favorable reprogramming of the tumor immune microenvironment, longer persistence of transferred T lymphocytes in vivo, and the development of immunity to endogenous antigens following primary tumor eradication. The efficacy of the strategy was at least in part recapitulated with the adoptive transfer of lower affinity transgenic TCR-bearing PMEL-1 T lymphocytes to treat the aggressive intraperitoneally disseminated B16-F10 tumor. Locoregional adoptive transfer of transiently IL-12-armored T cells appears to offer promising therapeutic advantages in terms of anti-tumor efficacy to treat peritoneal carcinomatosis.