RESUMO
The interaction between regulatory T (Treg) cells and self-reactive T cells is a crucial mechanism for maintaining immune tolerance. In this study, we investigated the cross-activation of Treg cells by self-antigens and its impact on self-reactive CD8+ T cell responses, with a focus on the P53 signaling pathway. We discovered that major histocompatibility complex (MHC) I-restricted self-peptides not only activated CD8+ T cells but also induced the delayed proliferation of Treg cells. Following HLA-A*0201-restricted Melan-A-specific (pMelan) CD8+ T cells, we observed the direct expansion of Treg cells and concurrent suppression of pMelan+CD8+ T cell proliferation upon stimulation with Melan-A peptide. Transcriptome analysis revealed no significant alterations in specific signaling pathways in pMelan+CD8+ T cells that were co-cultured with activated Treg cells. However, there was a noticeable upregulation of genes involved in P53 accumulation, a critical regulator of cell survival and apoptosis. Consistent with such observation, the blockade of P53 induced a continuous proliferation of pMelan+CD8+ T cells. The concurrent stimulation of Treg cells through self-reactive TCRs by self-antigens provides insights into the immune system's ability to control activated self-reactive CD8+ T cells as part of peripheral tolerance, highlighting the intricate interplay between Treg cells and CD8+ T cells and implicating therapeutic interventions in autoimmune diseases and cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos , Linfócitos T Reguladores , Antígeno MART-1/metabolismo , Autoantígenos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Antígenos CD8/metabolismoRESUMO
In adoptive T cell therapy (ACT), the transfer of tumor-specific T cells is paralleled by the conditioning regimen to increase therapeutic efficacy. Pre-conditioning depletes immune-suppressive cells and post-conditioning increases homeostatic signals to improve the persistence of administered T cells. Identifying the favorable immunological factors involved in a conditioning regimen is important to design effective strategies in ACT. Here, by using an ACT model of murine melanoma, we evaluate the effect of the total body irradiation (TBI) and interleukin-2 (IL-2) treatment combination. The use of a Rag1 knock-out strain, which lacks endogenous T cells, enables the identification of factors in a way that focuses more on transferred T cells. We demonstrate that the TBI/IL-2 combination has no additive effect in ACT, although each conditioning improves the therapeutic outcome. While the combination increases the frequency of transferred T cells in lymphoid and tumor tissues, the activation intensity of the cells is reduced compared to that of the sole TBI treatment. Notably, we show that in the presence of TBI, the IL-2 treatment reduces the frequency of intra-tumoral dendritic cells, which are crucial for T cell activation. The current study provides insights into the immunological events involved in the TBI/IL-2 combination in ACT.
Assuntos
Imunoterapia Adotiva , Interleucina-2 , Melanoma , Animais , Camundongos , Terapia Baseada em Transplante de Células e Tecidos , Interleucina-2/farmacologia , Melanoma/terapia , Camundongos Knockout , Irradiação Corporal Total , Linfócitos TRESUMO
We previously found the SLC3A2-NRG1 (S-N) fusion gene in a lung adenocarcinoma specimen without known driver mutations and validated this in 59 invasive mucinous adenocarcinoma (IMA) samples. Interestingly, KRAS mutation coexisted (62.5%) in 10 out of 16 NRG1 fusions. In this study, we examined the role of mutant KRAS in regulating the S-N fusion protein in KRAS mutant (H358) and wild-type (Calu-3) cells. KRAS mutation-mediated increase in MEK1/2 and ERK1/2 activity enhanced disintegrin and metalloproteinase (ADAM)17 activity, which increased the shedding of NRG1 from the S-N fusion protein. The cleavage of NRG1 also increased the phosphorylation of ERBB2-ERBB3 heterocomplex receptors and their downstream signalling pathways, including PI3K/Akt/mTOR, even under activated KRAS mutation signalling. The concurrence of S-N fusion and KRAS mutation synergistically increased cell proliferation, colony formation, tumour growth, and the cells' resistance to EGFR kinase inhibitors more than KRAS mutation alone. Targeted inhibition of MEK1/2, and ADAM17 significantly induced apoptosis singly and when combined with each mutation singly or with chemotherapy in both the concurrent KRAS mutant and S-N fusion xenograft and lung orthotopic models. Taken together, this is the first study to report that KRAS mutation increased NRG1 cleavage from the S-N fusion protein through ADAM17, thereby enhancing the Ras/Raf/MEK/ERK and ERBB/PI3K/Akt/mTOR pathways. Moreover, the coexistence of KRAS mutant and S-N fusion in lung tumours renders them vulnerable to MEK1/2 and/or ADAM17 inhibitors, at least in part, due to their dependency on the strong positive loop between KRAS mutation and S-N fusion.
Assuntos
Proteína ADAM17/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Neuregulina-1/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , Neuregulina-1/metabolismo , TransfecçãoRESUMO
Adoptive T cell therapy (ACT) requires lymphodepletion preconditioning to eliminate immune-suppressive elements and enable efficient engraftment of adoptively transferred tumor-reactive T cells. As anti-CD4 monoclonal antibody depletes CD4+ immune-suppressive cells, the combination of anti-CD4 treatment and ACT has synergistic potential in cancer therapy. Here, we demonstrate a post-ACT conditioning regimen that involves transient anti-CD4 treatment (CD4post). Using murine melanoma, the combined effect of cyclophosphamide preconditioning (CTXpre), CD4post, and ex vivo primed tumor-reactive CD8+ T-cell infusion is presented. CTXpre/CD4post increases tumor suppression and host survival by accelerating the proliferation and differentiation of ex vivo primed CD8+ T cells and endogenous CD8+ T cells. Endogenous CD8+ T cells enhance effector profile and tumor-reactivity, indicating skewing of the TCR repertoire. Notably, enrichment of polyfunctional IL-18Rαhi CD8+ T cell subset is the key event in CTXpre/CD4post-induced tumor suppression. Mechanistically, the anti-tumor effect of IL-18Rαhi subset is mediated by IL-18 signaling and TCR-MHC I interaction. This study highlights the clinical relevance of CD4post in ACT and provides insights regarding the immunological nature of anti-CD4 treatment, which enhances anti-tumor response of CD8+ T cells.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos Alquilantes/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Ciclofosfamida/farmacologia , Subunidade alfa de Receptor de Interleucina-18/genética , Melanoma Experimental/terapia , Neoplasias Cutâneas/terapia , Linfócitos T Citotóxicos/imunologia , Transferência Adotiva , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Imunoterapia Adotiva/métodos , Interleucina-18/genética , Interleucina-18/imunologia , Subunidade alfa de Receptor de Interleucina-18/agonistas , Subunidade alfa de Receptor de Interleucina-18/imunologia , Ativação Linfocitária , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/mortalidade , Camundongos , Camundongos Endogâmicos C57BL , Receptores CCR4/genética , Receptores CCR4/imunologia , Receptores CCR8/genética , Receptores CCR8/imunologia , Receptores Histamínicos H4/genética , Receptores Histamínicos H4/imunologia , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Análise de Sobrevida , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/transplante , Carga Tumoral/efeitos dos fármacosRESUMO
Adoptive cell therapy (ACT) using tumor-reactive T cells is a promising form of immunotherapy to specifically target cancer. However, the survival and functional maintenance of adoptively transferred T cells remains a challenge, ultimately limiting their efficacy. Here, we evaluated the use of recombinant IL7-Fc in ACT. In a lymphopenic murine melanoma model, IL7-Fc treatment led to the enhanced inhibition of tumor growth with an increased number of adoptively transferred CD8+ T cells in tumor tissue and tumor-draining lymph nodes. Additionally, IL7-Fc further enhanced anti-tumor responses that were induced by recombinant human IL2 in the same mouse model. In contrast, in an immunocompetent murine melanoma model, IL7-Fc dampened the anti-tumor immunity. Further, IL7-Fc decreased the proliferation of adoptively transferred and immune-activated tumor-reactive CD8+ T cells in immunocompetent mice by inducing the massive expansion of endogenous T cells, thereby limiting the space for adoptively transferred T cells. Our data suggest that IL7-Fc is principally beneficial for enhancing the efficacy of tumor-reactive T-cells in lymphopenic conditions for the ACT.
Assuntos
Fragmentos Fc das Imunoglobulinas/imunologia , Imunoterapia Adotiva/métodos , Interleucina-7/imunologia , Linfopenia/imunologia , Melanoma Experimental/terapia , Proteínas Recombinantes de Fusão/administração & dosagem , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Interleucina-7/genética , Interleucina-7/metabolismo , Contagem de Leucócitos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Linfopenia/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The antitumor capabilities of agonistic anti-4-1BB mAbs have made them an attractive target for tumor immunotherapy. However, the adverse side effects associated with agonist antibodies have hindered their clinical development. Here, we aimed to study the immune-related adverse events of repeated doses and long-term use of agonistic anti-4-1BB mAbs. We show that chronic activation of 4-1BB signals induced the accumulation of IFN-γ-producing PD-1+CD8+ T cells in the secondary lymphoid organs of tumor-bearing mice by increasing the number of dividing CD8+ T cells, which was beneficial for suppressing tumor growth in the early phase of anti-4-1BB induction. However, repeated exposure to anti-4-1BB mAbs led to granuloma development in tumor-draining lymph nodes (TDLNs) of mice due to recruitment and accumulation of macrophages via the CD8+ T cell-IFN-γ axis. This was accompanied by excessive lymph node swelling, which impaired the sequential activation of CD8+ T cells. Our data provide insights into the immune-related adverse events of long-term agonist 4-1BB antibody dosing, which should be considered during the clinical development of immunomodulating therapy.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Animais , Granuloma/patologia , Linfonodos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
Hypoxia-inducible factors (HIFs) induced by reduced O2 availability activate the transcription of target genes encoding proteins that play important roles in communication between cancer and stromal cells. Cancer cells were incubated under hypoxic conditions: H1299, A549 (NSCLC); Hep3B, HepG2 (HCC); HCT116, CT26 (Colon cancer); MCF-7, MDAMB231 (Breast cancer); MKN1, MKN5 (Gastric cancer); U87MG, SHSY5Y (Brain cancer); and SKOV3, SNU840 (Ovary cancer). All cells expressed HIF-1α and HIF-2α mRNA and proteins. However, cell proliferation of NSCLC, breast, gastric, and brain cancer cells under hypoxia was more dependent on HIF-1α except for HCC cells where it was more dependent on HIF-2α. Among HIF-1α dependent cells H1299 was the most affected in terms of cell proliferation by HIF-1α knockdown. To examine which cytokines are secreted in NSCLC cells by HIF-1α to communicate with stromal cells, we performed a cytokine-profiling array with H1299. We screened the top 14 cytokines which were dependent on the HIF-1α expression pattern. Among them, midkine (MDK) expression was affected the most in response to HIF-1α. MDK is a heparin-binding growth factor that promotes angiogenesis and carcinogenesis. Indeed, MDK significantly increased HUVEV endothelial cell migration and neo- vascularization in chick chorioallantoic membrane assay (CAM) assay via paracrine signaling. In addition, MDK secreted from NSCLC cells interacted with Notch2 which activated the Notch signaling pathway and induced EMT, upregulated NF-κB, and increased cancer promotion. However, in response to MDK knock down, siRNA or the MDK inhibitor, iMDK treatment not only decreased MDK-induced migration and angiogenesis of endothelial cells but also abrogated the progression and metastasis of NSCLC cells in in vitro and in vivo orthotopic and spontaneous lung metastasis models. Consequently, iMDK treatment significantly increased mice survival rates compared with the control or MDK expression group. MDK plays a very important role in the progression and metastasis of NSCLC cells. Moreover, the MDK targeting strategy provides a potential therapeutic target for the treatment of MDK-expressing lung cancers.
RESUMO
Antibody applications in cancer immunotherapy involve diverse strategies, some of which redirect T cell-mediated immunity via engineered antibodies. Affinity is a trait that is crucial for these strategies, as optimal affinity reduces unwanted side effects while retaining therapeutic function. Antibody-antigen pairs possessing a broad affinity range are required to define optimal affinity and to investigate the affinity-associated functional profiles of T cell-engaging strategies such as bispecific antibodies and chimeric antigen receptor-engineered T cells. Here, we demonstrate the unique binding characteristic of the developed antibody clone MVR, which exhibits robust binding to B-lymphoid cell lines. Intriguingly, MVR specifically recognizes the highly polymorphic human leukocyte antigen (HLA)-DR complex and exhibits varying affinities that are dependent upon the HLA-DRB1 allele type. Remarkably, MVR binds to the conformational epitope that consists of two hypervariable regions. As an application of MVR, we demonstrate an MVR-engineered chimeric antigen receptor (CAR) that elicits affinity-dependent function in response to a panel of target cell lines that express different HLA-DRB1 alleles. This tool evaluates the effect of affinity on cytotoxic killing, polyfunctionality, and activation-induced cell death of CAR-engineered T cells. Collectively, MVR exhibits huge potential for the evaluation of the affinity-associated profile of T cells that are redirected by engineered antibodies.
RESUMO
RELT (tumor necrosis factor receptor superfamily member 19-like, TNFRSF19L) is a TNFR superfamily member that is primarily expressed in immune cells and lymphoid tissues, but whose immunological function is not well-defined. Here, we show that RELT is expressed by naive T cells and DCs, and their activation or maturation decreases RELT expression. Using RELT knockout (RELT-/- ) mice, we demonstrate that RELT deficiency selectively promotes the homeostatic proliferation of CD4+ T cells but not CD8+ T cells, and enhances anti-tumor CD8+ T-cell responses. We also demonstrate, using an adoptive transfer model in which RELT is knocked-out in either the transferred transgenic CD8+ T cells or the recipient melanoma-bearing mice, that RELT on multiple immune cells limits the hyper-response of tumor-specific CD8+ T cells. Hyper-responsiveness of RELT-deficient T cells was induced by promoting their proliferation. Taken together, our findings suggest that RELT acts as a negative regulator that controls the early phase of T-cell activation probably by promoting T-cell apoptosis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica/imunologia , Ativação Linfocitária , Receptores do Fator de Necrose Tumoral/genética , Transferência Adotiva , Animais , Apoptose , Linfócitos T CD8-Positivos/imunologia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Chimeric antigen receptor (CAR) T cell therapy is an effective method for treating specific cancers. CARs are normally designed to recognize antigens, which are highly expressed on malignant cells but not on T cells. However, when T cells are engineered with CARs that recognize antigens expressed on the T cell surface, CAR T cells exhibit effector function on other T cells, which results in fratricide, or killing of neighboring T cells. Here, using human leukocyte antigen-DR (HLA-DR)-targeted CAR T cells, we show that weak affinity between CAR and HLA-DR reduces fratricide and induces sustained CAR downregulation, which consequently tunes the avidity of CAR T cells, leading to desensitization. We further demonstrate that desensitized CAR T cells selectively kill Epstein-Barr virus-transformed B cells with enhanced HLA-DR expression, while sparing normal B cells. Our study supports an avidity-tuning strategy that permits sensing of antigen levels by CAR T cells.
Assuntos
Linfócitos B/citologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/fisiologia , Animais , Anticorpos Monoclonais/genética , Linfócitos B/virologia , Morte Celular , Feminino , Antígenos HLA-DR/metabolismo , Cadeias HLA-DRB1/metabolismo , Herpesvirus Humano 4/patogenicidade , Humanos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfócitos T/citologiaRESUMO
Chimeric antigen receptor (CAR) T cells have outstanding therapeutic potential for treating blood cancers. The prospects for this technology have accelerated basic research, clinical translation and Big Pharma's investment in the field of T-cell therapeutics. This interest has led to the discovery of key factors that affect CAR T-cell efficacy and play pivotal roles in T-cell immunology. Herein, we introduce advances in adoptive immunotherapy and the birth of CAR T cells, and review CAR T-cell studies that focus on three important features: CAR constructs, target antigens and T-cell phenotypes. At last, we highlight novel strategies that overcome the tumor microenvironment and circumvent CAR T-cell side effects, and consider the future direction of CAR T-cell development.
Assuntos
Imunoterapia Adotiva , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/química , Receptores de Antígenos Quiméricos/genética , Linfócitos T/citologia , Microambiente Tumoral/imunologiaRESUMO
4-1BB (CD137) is a strong enhancer of the proliferation of CD8+ T cells. Since these cells require increased production of energy and biomass to support their proliferation, we hypothesized that 4-1BB signaling activated glucose and fatty acid metabolism. We found that treatment with agonistic anti-4-1BB mAb promoted the proliferation of CD8+ T cells in vitro, increasing their size and granularity. Studies with a glycolysis inhibitor and a fatty acid oxidation inhibitor revealed that CD8+ T cell proliferation required both glucose and fatty acid metabolism. Anti-4-1BB treatment increased glucose transporter 1 expression and activated the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK)-acetyl-CoA carboxylase (ACC) signaling pathway, which may be responsible for activating the metabolism of glucose and fatty acids. We also examined whether blocking glucose or fatty acid metabolism affected cell cycle progression and the anti-apoptotic effect of 4-1BB signaling. The increase of anti-apoptotic factors and cyclins in response to anti-4-1BB treatment was completely prevented by treating CD8+ T cells with the fatty acid oxidation inhibitor, etomoxir, but not with the glycolysis inhibitor, 2-deoxy-D-glucose. We conclude that anti-4-1BB treatment activates glucose and fatty acid metabolism thus supporting the increased demand for energy and biomass, and that fatty acid metabolism plays a crucial role in enhancing the cell cycle progression of anti-CD3-activated CD8+ T cells in vitro and the anti-apoptotic effects of 4-1BB signaling on these cells.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Transdução de Sinais , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Acetil-CoA Carboxilase/metabolismo , Animais , Apoptose , Cálcio/metabolismo , Ciclo Celular , Proliferação de Células , Separação Celular , Tamanho Celular , Metabolismo dos Lipídeos , Camundongos Endogâmicos C57BL , OxirreduçãoRESUMO
VSIG4/CRIg (V-set and immunoglobulin domain containing 4) is a transmembrane receptor of the immunoglobulin superfamily that is expressed specifically on macrophages and mature dendritic cells. VSIG4 signaling accelerates phagocytosis of C3-opsonized bacteria, thereby efficiently clearing pathogens within macrophages. We found that VSIG4 signaling triggered by C3-opsonized Listeria (opLM) or by agonistic anti-VSIG4 monoclonal antibody (mAb) induced macrophages to form autophagosomes. VSIG4-induced autophagosomes were selectively colocalized with the intracellular LM while starvation-induced autophagosomes were not. Consistent with these results, the frequency of autophagosomes induced by infection with opLM was lower in VSIG4-deficient bone marrow-derived macrophages (BMDMs) than in WT BMDMs. Furthermore, when VSIG4 molecules were overexpressed in HeLa cells, which are non-macrophage cells, VSIG4 triggering led to efficient uptake of LM, autophagosome formation, and killing of the infected LM. These findings suggest that VSIG4 signaling not only promotes rapid phagocytosis and killing of C3-opsonized intracellular bacteria, as previously reported, but also induces autophagosome formation, eliminating the LM that have escaped from phagosomes. We conclude that VSIG4 signaling provides an anti-immune evasion mechanism that prevents the outgrowth of intracellular bacteria in macrophages.
Assuntos
Autofagia , Infecções Bacterianas/imunologia , Macrófagos/metabolismo , Receptores de Complemento/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Autofagossomos/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Células da Medula Óssea/citologia , Linhagem Celular , Células HeLa , Humanos , Imunoglobulina G/imunologia , Listeria/metabolismo , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/imunologia , Fagossomos/metabolismo , Transdução de SinaisRESUMO
HIV-1 escape from CTL is predictable based on the Human Leukocyte Antigen (HLA) class I alleles expressed by the host. As such, HIV-1 sequences circulating in a population of hosts will harbor escape mutations specific to the HLA alleles of that population. In theory, this should increase the frequency of escape mutation transmission to persons expressing the restricting HLA allele, thereby compromising host immunity to the incoming HIV-1 strain. However, the clinical impact of infection with HIV-1 containing immune escape mutations has not conclusively been demonstrated. Japan's population features limited HLA diversity which is driving population-level HIV adaptation: for example, >60% of Japanese express HLA-A*24:02 and its associated Nef-Y135F escape mutation represents the population consensus. As such, Japan is an ideal population in which to examine this phenomenon. Here, we combine genetic and immunological analyses to identify A*24:02-positive individuals likely to have been infected with Y135F-containing HIV-1. Over a ~5 year follow-up, these individuals exhibited significantly lower CD4 counts compared to individuals inferred to have been infected with wild-type HIV-1. Our results support a significant negative clinical impact of pathogen adaptation to host pressures at the population level.
Assuntos
Adaptação Biológica , Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Adolescente , Adulto , Idoso , Alelos , Apresentação de Antígeno , Contagem de Linfócito CD4 , Progressão da Doença , Feminino , Genótipo , Infecções por HIV/imunologia , HIV-1/classificação , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Filogenia , Estudos Retrospectivos , Carga Viral , Adulto Jovem , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologiaRESUMO
BACKGROUND: Human Leukocyte Antigen (HLA) class I restricted Cytotoxic T Lymphocytes (CTLs) exert substantial evolutionary pressure on HIV-1, as evidenced by the reproducible selection of HLA-restricted immune escape mutations in the viral genome. An escape mutation from tyrosine to phenylalanine at the 135th amino acid (Y135F) of the HIV-1 nef gene is frequently observed in patients with HLA-A*24:02, an HLA Class I allele expressed in ~70% of Japanese persons. The selection of CTL escape mutations could theoretically result in the de novo creation of novel epitopes, however, the extent to which such dynamic "CTL epitope switching" occurs in HIV-1 remains incompletely known. RESULTS: Two overlapping epitopes in HIV-1 nef, Nef126-10 and Nef134-10, elicit the most frequent CTL responses restricted by HLA-A*24:02. Thirty-five of 46 (76%) HLA-A*24:02-positive patients harbored the Y135F mutation in their plasma HIV-1 RNA. Nef codon 135 plays a crucial role in both epitopes, as it represents the C-terminal anchor for Nef126-10 and the N-terminal anchor for Nef134-10. While the majority of patients with 135F exhibited CTL responses to Nef126-10, none harboring the "wild-type" (global HIV-1 subtype B consensus) Y135 did so, suggesting that Nef126-10 is not efficiently presented in persons harboring Y135. Consistent with this, peptide binding and limiting dilution experiments confirmed F, but not Y, as a suitable C-terminal anchor for HLA-A*24:02. Moreover, experiments utilizing antigen specific CTL clones to recognize endogenously-expressed peptides with or without Y135F indicated that this mutation disrupted the antigen expression of Nef134-10. Critically, the selection of Y135F also launched the expression of Nef126-10, indicating that the latter epitope is created as a result of escape within the former. CONCLUSIONS: Our data represent the first example of the de novo creation of a novel overlapping CTL epitope as a direct result of HLA-driven immune escape in a neighboring epitope. The robust targeting of Nef126-10 following transmission (or in vivo selection) of HIV-1 containing Y135F may explain in part the previously reported stable plasma viral loads over time in the Japanese population, despite the high prevalence of both HLA-A*24:02 and Nef-Y135F in circulating HIV-1 sequences.
Assuntos
Epitopos de Linfócito T/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Linfócitos T Citotóxicos/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Linhagem Celular , Epitopos de Linfócito T/genética , Células HEK293 , HIV-1/genética , Antígeno HLA-A24/genética , Antígeno HLA-A24/imunologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Mutação , RNA Viral/genética , RNA Viral/imunologia , Carga Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
We investigated the crystal structure of an HLA-A*2402-restricted CTL epitope in the HIV-1 nef gene (Nef134-10) before (pHLA) or after TCR docking. The wild type epitope and two escape mutants were included in the study. Y135F was an early-appearing major mutation, while F139L was a late-appearing mutation which was selected in the patients without Y135F. F139 was an eminent feature of the Nef134-10 epitope. Wild type-specific TCR was less fit to F139L mutant suggesting that F139L is an escape from the CTL against the wild type epitope. Although Y135F mutation disrupted the hydrogen bond to HLA-A*2402 His70, newly formed hydrogen bond between T138 and His70 kept the conformation of the epitope in the reconstituted pMHC. TCR from Y135F- or dually-specific CTL had unique mode of binding to the mutant epitope. Y135F has been reported as a processing mutant but CTL carrying structurally adequate TCR can be found in the patients.
Assuntos
Epitopos de Linfócito T/imunologia , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Antígenos/química , Antígenos/genética , Antígenos/imunologia , Cromo/metabolismo , Feminino , Infecções por HIV/genética , Antígeno HLA-A24/química , Antígeno HLA-A24/genética , Antígeno HLA-A24/imunologia , Humanos , Masculino , Modelos Moleculares , Mutação , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Ligação Proteica/imunologia , Conformação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologiaRESUMO
In this report, we describe a novel method for directly preparing enzyme-labeled antibodies harvested from IgM-producing hybridoma cells. We constructed expression vectors for antibody light (L) chain-enzyme fusion proteins by linking either the genes for the murine lambda L chain or its constant region (C(L)) with one of two proteins, either the secreted placental alkaline phosphatase or Gaussia luciferase (Gluc). When the vectors were transfected into anti-NP (4-hydroxy-3-nitrophacetyl) IgM-producing myeloma cells, secretion of the IgM-enzyme complex from the gene-transfected cells was confirmed by a direct enzyme-linked immunosorbent assay with an immobilized antigen. Furthermore, when human hybridoma HF10B4, a cell line that produces anti-human lung cancer IgM, was transfected with the vector containing L-Gluc, a significantly stronger signal was obtained for the human lung carcinoma SBC-1 cells than for cervical HeLa cells. Because successful production of an active IgM-enzyme complex containing a heterologous L chain-enzyme fusion was observed, the L-chain fusion method will be a generally applicable method for preparing various IgM-enzyme complexes.