RESUMO
BACKGROUND: Cell transplantation-based treatments for neurological disease are promising, yet graft rejection remains a major barrier to successful regenerative therapies. Our group and others have shown that long-lasting tolerance of transplanted stem cells can be achieved in the brain with systemic application of monoclonal antibodies blocking co-stimulation signaling. However, it is unknown if subsequent injury and the blood-brain barrier breach could expose the transplanted cells to systemic immune system spurring fulminant rejection and fatal encephalitis. Therefore, we investigated whether delayed traumatic brain injury (TBI) could trigger graft rejection. METHODS: Glial-restricted precursor cells (GRPs) were intracerebroventricularly transplanted in immunocompetent neonatal mice and co-stimulation blockade (CoB) was applied 0, 2, 4, and 6 days post-grafting. Bioluminescence imaging (BLI) was performed to monitor the grafted cell survival. Mice were subjected to TBI 12 weeks post-transplantation. MRI and open-field test were performed to assess the brain damage and behavioral change, respectively. The animals were decapitated at week 16 post-transplantation, and the brains were harvested. The survival and distribution of grafted cells were verified from brain sections. Hematoxylin and eosin staining (HE) was performed to observe TBI-induced brain legion, and neuroinflammation was evaluated immunohistochemically. RESULTS: BLI showed that grafted GRPs were rejected within 4 weeks after transplantation without CoB, while CoB administration resulted in long-term survival of allografts. BLI signal had a steep rise following TBI and subsequently declined but remained higher than the preinjury level. Open-field test showed TBI-induced anxiety for all animals but neither CoB nor GRP transplantation intensified the symptom. HE and MRI demonstrated a reduction in TBI-induced lesion volume in GRP-transplanted mice compared with non-transplanted mice. Brain sections further validated the survival of grafted GRPs and showed more GRPs surrounding the injured tissue. Furthermore, the brains of post-TBI shiverer mice had increased activation of microglia and astrocytes compared to post-TBI wildtype mice, but infiltration of CD45+ leukocytes remained low. CONCLUSIONS: CoB induces sustained immunological tolerance towards allografted cerebral GRPs which is not disrupted following TBI, and unexpectedly TBI may enhance GRPs engraftment and contribute to post-injury brain tissue repair.
Assuntos
Lesões Encefálicas Traumáticas , Rejeição de Enxerto/imunologia , Tolerância Imunológica/imunologia , Células-Tronco Neurais/transplante , Transplante de Células-Tronco/métodos , Aloenxertos , Animais , Anticorpos Monoclonais/farmacologia , Antígeno B7-1/antagonistas & inibidores , Antígeno B7-2/antagonistas & inibidores , Antígenos CD28/antagonistas & inibidores , Antígenos CD40/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/transplanteRESUMO
Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.
Assuntos
Antígenos CD28/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Citocinas/biossíntese , Feminino , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Transdução de Sinais , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immune checkpoint immunotherapy is a pillar of human oncology treatment with potential for non-human species. The first checkpoint immunotherapy approved for human cancers targeted the CTLA4 protein. CTLA4 can inhibit T cell activation by capturing and internalizing CD80 and CD86 from antigen presenting cells, a process called trans-endocytosis. Similarly, CD28 can capture CD80 and CD86 via trogocytosis and retain the captured ligands on the surface of the CD28-expressing cells. The wild Tasmanian devil (Sarcophilus harrisii) population has declined by 77% due to transmissible cancers that evade immune defenses despite genetic mismatches between the host and tumors. We used a live cell-based assay to demonstrate that devil CTLA4 and CD28 can capture CD80 and CD86. Mutation of evolutionarily conserved motifs in CTLA4 altered functional interactions with CD80 and CD86 in accordance with patterns observed in other species. These results suggest that checkpoint immunotherapies can be translated to evolutionarily divergent species.
Assuntos
Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD28/metabolismo , Antígeno CTLA-4/metabolismo , Marsupiais/imunologia , Motivos de Aminoácidos/genética , Animais , Antígenos CD28/antagonistas & inibidores , Células CHO , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/genética , Células Cultivadas , Clonagem Molecular , Cricetulus , Espécies em Perigo de Extinção , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Microscopia Intravital , Marsupiais/metabolismo , Mutação , TrogocitoseRESUMO
Immune checkpoint inhibitors (ICI) aim to restore the immune system anti-tumor function by blocking two inhibitory axes: CTLA-4/CD28 and PD1/PDL1. ICI is established as a treatment option for multiple cancers, but their remarkable clinical impact is observed only in a fraction of patients. Together with their adverse effects and high cost, it's imperative to identify patients who are likely to benefit from this type of treatment. Genomic features represent promising candidates as predictive biomarkers of response to ICI, with agnostic FDA-approvals of an anti-PD1 drug for tumors with microsatellite instability and tumors with a high mutational burden. Other genomic markers are also emerging to help refine patient selection. In this review, we discuss recent progress in genomic biomarkers development and its challenges, with a focus on alterations in the neoantigen burden, immune, and oncogenic pathways.
Assuntos
Biomarcadores Tumorais/genética , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/efeitos adversos , Neoplasias/terapia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/imunologia , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/genética , Antígenos CD28/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Genômica/tendências , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologiaRESUMO
An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1 Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.
Assuntos
Antígenos CD28/antagonistas & inibidores , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Citocinas/biossíntese , Feminino , Humanos , Imunoterapia Adotiva , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fatores de Transcrição NFATC/genética , Células NIH 3T3 , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptor de Morte Celular Programada 1/genética , Receptores de Antígenos Quiméricos/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Therapeutic targeting of immune checkpoints has garnered significant attention in the area of cancer immunotherapy, in which efforts have focused in particular on cytotoxic T lymphocyte antigen 4 (CTLA4) and PD1, both of which are members of the CD28 family. In autoimmunity, these same pathways can be targeted to opposite effect: to curb the over-exuberant immune response. The CTLA4 checkpoint serves as an exemplar, whereby CTLA4 activity is blocked by antibodies in cancer immunotherapy and augmented by the provision of soluble CTLA4 in autoimmunity. Here, we review the targeting of co-stimulatory molecules in autoimmune diseases, focusing in particular on agents directed at members of the CD28 or tumour necrosis factor receptor families. We present the state of the art in co-stimulatory blockade approaches, including rational combinations of immune inhibitory agents, and discuss the future opportunities and challenges in this field.
Assuntos
Doenças Autoimunes/terapia , Antígenos CD28/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Imunoterapia/métodos , Proteína Coestimuladora de Linfócitos T Induzíveis/antagonistas & inibidores , Receptores OX40/antagonistas & inibidores , Doenças Autoimunes/imunologia , Humanos , Transdução de SinaisRESUMO
Autologous chimeric antigen receptor engineered T-cell therapies are beginning to dramatically change the outlook for patients with several hematological malignancies. Yet methods to activate and expand these cells are limited, often pose challenges to automation, and have biological limitations impacting the output of the injectable dose. This study describes the development of a novel, highly flexible, soluble DNA-based T-cell activation and expansion platform which alleviates the limitations of current technologies and provides rapid T-cell activation and expansion.
Assuntos
DNA/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Anticorpos Monoclonais/farmacologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/imunologia , Complexo CD3/antagonistas & inibidores , Complexo CD3/imunologia , Proliferação de Células , DNA/genética , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Lentivirus/genética , Ativação Linfocitária/efeitos dos fármacos , Cultura Primária de Células/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/efeitos dos fármacos , Transdução GenéticaRESUMO
Hypersonins A-D (1-4), four 1,2-seco-homoadamantane type polycyclic polyprenylated acylphloroglucinols (PPAPs) possessing a new bicyclo[4.3.1]decane-3-methoxycarbonyl architecture, were obtained from Hypericum wilsonii. The structures of hypersonins A-D were identified by spectroscopic data, electronic circular dichroism comparison, and X-ray crystallographic data. Hypersonins A-D are the first seco-homoadamantane-type PPAPs with cleavage at the C-1-C-2 bond. Hypersonin A (1) showed moderate inhibitory activity to anti-CD3/anti-CD28 monoclonal antibody-induced proliferation of murine splenocytes, with an IC50 value of 8.3 ± 0.2 µM.
Assuntos
Hypericum/química , Compostos Policíclicos/farmacologia , Animais , Anticorpos Bloqueadores , Antineoplásicos Fitogênicos/química , Antígenos CD28/antagonistas & inibidores , Complexo CD3/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Compostos Policíclicos/química , Baço/citologia , Difração de Raios XRESUMO
Both conventional and regulatory CD4+ T-cells rely on costimulatory signals mediated by cell surface receptors including CD28 for full activation. We showed previously that stimulation of CD4+ Foxp3+ regulatory T-cells by superagonistic anti-CD28 monoclonal antibodies (mAb) improves myocardial healing after experimental myocardial infarction (MI). However, the effect of ligand binding blocking anti-CD28 monoclonal antibodies has not yet been tested in this context. We hypothesize that ligand blocking anti-CD28 mAb treatment might favorably impact on healing after MI by limiting the activation of conventional CD4+ T-cells. Therefore, we studied the therapeutic effect of the recently characterized mAb E18 which blocks ligand binding to CD28 in a mouse permanent coronary ligation model. E18 or an irrelevant control mAb was applied once on day two after myocardial infarction to wildtype mice. Echocardiography was performed on day 7 after MI. E18 treatment improved the survival and reduced the incidence of left ventricular ruptures after experimental myocardial infarction. Accordingly, although we found no difference in infarct size, there was significantly less left ventricular dilation after E18 treatment in surviving animals as determined by echocardiography at day 7 after MI. In sham operated control mice neither antibody had an impact on body weight, survival, and echocardiographic parameters. Mechanistically, compared to control immunoglobulin, E18 treatment reduced the number of CD4+ T-cells and monocytes/macrophages within the infarct and periinfarct zone on day 5. This was accompanied by an upregulation of arginase which is a marker for alternatively differentiated macrophages. The data indicate that CD28-dependent costimulation of CD4+ T-cells impairs myocardial healing and anti-CD28 antibody treatment constitutes a potentially clinically translatable approach to improve the outcome early after MI.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD28/antagonistas & inibidores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Infarto do Miocárdio/tratamento farmacológico , Animais , Anticorpos Monoclonais/uso terapêutico , Arginase/imunologia , Arginase/metabolismo , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Ecocardiografia , Humanos , Ligantes , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Miocárdio/imunologia , Miocárdio/patologiaAssuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/terapia , Ferritinas/sangue , Imunoglobulinas Intravenosas/uso terapêutico , Interleucina-1/antagonistas & inibidores , Interleucina-6/antagonistas & inibidores , Pneumonia Viral/diagnóstico , Pneumonia Viral/terapia , Vacinas Virais/imunologia , Idoso de 80 Anos ou mais , Antígenos CD/sangue , Antígenos de Diferenciação Mielomonocítica/sangue , Síndrome Antifosfolipídica , Betacoronavirus/imunologia , Biomarcadores/sangue , Antígenos CD28/antagonistas & inibidores , COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Humanos , Imunização Passiva , Imunoglobulinas Intravenosas/administração & dosagem , Síndrome de Ativação Macrofágica , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/imunologia , Receptores de Superfície Celular/sangue , Insuficiência Respiratória/sangue , Insuficiência Respiratória/complicações , SARS-CoV-2 , Choque Séptico , Vacinas Virais/efeitos adversos , Soroterapia para COVID-19RESUMO
Iron uptake via the transferrin receptor (CD71) is a pivotal mechanism for T cell proliferation. Yet, it is incompletely understood if targeting of CD71 also affects the differentiation and functional polarization of primary human T cells. In this study, we demonstrate that inhibition of iron ingestion with blocking mAbs against CD71 induces nonproliferating T cells, which release high amounts of IL-2. Targeting of CD71 with blocking or nonblocking mAbs did not alter major signaling pathways and the activation of the transcription factors NF-κB, NFAT, or AP-1 as analyzed in Jurkat T cells. Growth arrest in iron-deficient (Fe-def) T cells was prevented upon addition of exogenous iron in the form of ferric ammonium citrate but was not reversible by exogenous IL-2. Surprisingly, protein synthesis was found to be intact in Fe-def T cells as demonstrated by comparable levels of CD69 upregulation and cytokine production with iron-sufficient T cells upon stimulation with CD3 plus CD28 mAbs. Indeed, high amounts of IL-2 were detectable in the supernatant of Fe-def T cells, which was accompanied with a reduced cell surface expression of IL-2R. When we used such Fe-def T cells in allogeneic MLRs, we observed that these cells acquired an accessory cell function and stimulated the proliferation of bystander T cells by providing IL-2. Thus, the results of our study demonstrate that iron deprivation causes nonproliferating, altruistic T cells that can help and stimulate other immune cells by providing cytokines such as IL-2.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Proliferação de Células/efeitos dos fármacos , Deficiências de Ferro , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Antígenos CD/imunologia , Doadores de Sangue , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/imunologia , Complexo CD3/antagonistas & inibidores , Complexo CD3/imunologia , Feminino , Compostos Férricos/farmacologia , Sangue Fetal/citologia , Humanos , Interleucina-2/metabolismo , Células Jurkat , Camundongos , Compostos de Amônio Quaternário/farmacologia , Receptores da Transferrina/antagonistas & inibidores , Receptores da Transferrina/imunologiaRESUMO
CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti-tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti-tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumour response. Here, we show that ex vivo administration of a function-blocking anti-CD5 MAb to primary mouse CTLs of both tumour-naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti-CD3/anti-CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN-γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function-blocking MAb treatment enhanced the capacity of CD8+ T cells to kill 4T1-mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumour immunity.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Antígenos CD28/imunologia , Antígenos CD5/imunologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Antineoplásicos Imunológicos , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/genética , Antígenos CD5/antagonistas & inibidores , Antígenos CD5/genética , Extratos Celulares/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Proteína Ligante Fas/genética , Proteína Ligante Fas/imunologia , Feminino , Regulação da Expressão Gênica , Interferon gama/genética , Interferon gama/imunologia , Ativação Linfocitária/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Transdução de Sinais , Linfócitos T Citotóxicos/imunologia , Receptor fas/genética , Receptor fas/imunologiaRESUMO
BACKGROUND: Pre-clinical and clinical studies have shown that the infusion of CAR T cells with a naive-like (TN) and central memory (TCM) phenotype is associated with prolonged in vivo T cell persistence and superior anti-tumor effects. To optimize the maintenance of such populations during the in vitro preparation process, we explored the impact of T cell exposure to both traditional [fetal bovine serum (FBS), human AB serum (ABS)] and non-traditional [human platelet lysate (HPL) - a xeno-free protein supplement primarily used for the production of clinical grade mesenchymal stromal / stem cells (MSCs)] serum supplements. METHODS: Second generation chimeric antigen receptor with CD28 and CD3ζ endodomain targeting prostate stem cell antigen (PSCA) (P28z) or CD19 (1928z) were constructed and used for this study. After retroviral transduction, CAR T cells were divided into 3 conditions containing either FBS, ABS or HPL and expanded for 7 days. To evaluate the effect of different sera on CAR T cell function, we performed a series of in vitro and in vivo experiments. RESULTS: HPL-exposed CAR T cells exhibited the less differentiated T cell phenotype and gene signature, which displayed inferior short-term killing abilities (compared to their FBS- or ABS-cultured counterparts) but superior proliferative and anti-tumor effects in long-term in vitro coculture experiments. Importantly, in mouse xenograft model, HPL-exposed CAR T cells outperformed their ABS or FBS counterparts against both subcutaneous tumor (P28z T cells against Capan-1PSCA) and systemic tumor (1928z T cells against NALM6). We further observed maintenance of less differentiated T cell phenotype in HPL-exposed 1928z T cells generated from patient's PBMCs with superior anti-tumor effect in long-term in vitro coculture experiments. CONCLUSIONS: Our study highlights the importance of serum choice in the generation of CAR T cells for clinical use.
Assuntos
Plaquetas/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Sobrevivência Celular , Técnicas de Cocultura , Citocinas/metabolismo , Modelos Animais de Doenças , Edição de Genes , Engenharia Genética , Humanos , Memória Imunológica , Imunofenotipagem , Imunoterapia Adotiva , Células-Tronco Mesenquimais/metabolismo , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores de Antígenos Quiméricos/genética , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Abatacepte/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo/terapia , Recidiva Local de Neoplasia/terapia , Terapia de Salvação , Linfócitos T/transplante , Idoso , Antígeno B7-2/antagonistas & inibidores , Antígenos CD28/antagonistas & inibidores , Terapia Combinada , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas , Humanos , Transfusão de Linfócitos , Masculino , Melfalan/administração & dosagem , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Recidiva Local de Neoplasia/patologia , Prognóstico , Doadores de Tecidos , Condicionamento Pré-Transplante , Transplante Haploidêntico , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados , Irradiação Corporal TotalRESUMO
PURPOSE: Targeting nonspecific, tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CAR-T) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However, decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here, we demonstrate the prime importance of the type of intracellular signaling on the function of low-affinity CAR-T cells. EXPERIMENTAL DESIGN: We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. RESULTS: We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (K d < 1.9 × 10-6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity, preservation of a central memory phenotype, and significantly improved in vivo antitumor function, while preserving their ability to discriminate target antigen density. CONCLUSIONS: A combinatorial costimulatory design allows the use of very low-affinity binding domains (K d < 1 µmol/L) for the construction of safe but also optimally effective CAR-T cells. Thus, very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs.
Assuntos
Antígenos CD28/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Modelos Animais de Doenças , Ordem dos Genes , Engenharia Genética , Vetores Genéticos/genética , Humanos , Memória Imunológica , Imunoterapia Adotiva , Ativação Linfocitária/imunologia , Camundongos , Mieloma Múltiplo/etiologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Fenótipo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Retroviridae/genética , Transdução de Sinais , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite the development of several targeted therapies for rheumatoid arthritis (RA), there is still no reliable drug-specific predictor to assist rheumatologists in selecting the most effective targeted therapy for each patient. Recently, a gene signature caused by impaired induction of PTPN22 in anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) was observed in healthy at-risk individuals. However, the downstream target genes of PTPN22 and the molecular mechanisms regulating its expression are still poorly understood. Here we report that the PTPN22 gene signature is also present in PBMC from patients with active RA and can be reversed after effective treatment. The expression of PTPN22 correlates with that of more than 1000 genes in Th cells of anti-CD3 stimulated PBMC of healthy donors and is inhibited by TNFα or CD28 signals, but not IL-6, through distinct mechanisms. In addition, the impaired induction of PTPN22 in PBMC of patients with active RA can be normalized in vitro by several targeted therapies. More importantly, the in vitro normalization of PTPN22 expression correlates with clinical response to the targeted therapies in a longitudinal RA cohort. Thus, in vitro normalization of PTPN22 expression by targeted therapies can potentially be used to predict clinical response.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Regulação da Expressão Gênica , Terapia de Alvo Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Adulto , Idoso , Artrite Reumatoide/diagnóstico , Biomarcadores , Antígenos CD28/antagonistas & inibidores , Feminino , Humanos , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Cytokine release syndrome (CRS) is a serious and potentially life-threatening complication typically associated with biological drug products. Pre-clinical testing in vitro and in vivo studies using non-human primates had failed to reliably predict CRS. To determine if bone marrow-thymus-liver (BLT) humanized mice with a fully engrafted human immune system or a CD34-humanized mouse model could predict CRS, we tested an anti-CD28 monoclonal antibody (mAb) similar to TGN1412. This TGN1412 analogue (TGN1412A) was initially tested in vitro and found to produce significant dose-dependent increases in cytokine production. For in vivo studies, adalimumab, an anti-tumor necrosis factor-alpha antibody known not to cause CRS, served as a negative control. We evaluated immune cell activation and cytokine expression in three independent experiments. In BLT humanized mice, significant increases in levels of human cytokines were identified in animals treated with anti-CD28 mAb. As expected, CD28+ cell detection was strongly reduced in the anti-CD28 treated group. Increased T cell activation was also observed. The control group did not show reductions in CD28+ T-cells and did not experience increased cytokine levels. Responses by CD34-humanized mice showed no significant differences between adalimumab and anti-CD28 treatment at doses used to test BLT-humanized mice. These results suggest that the TGN1412A produces similar results in vitro to the original TGN1412 monoclonal antibody. The BLT immune humanized mice but not the CD34 humanized mice produce both robust and specific cytokine responses and may represent a pre-clinical model to identify CRS.
Assuntos
Anticorpos Monoclonais Humanizados/toxicidade , Antígenos CD28/antagonistas & inibidores , Síndrome da Liberação de Citocina/etiologia , Citocinas/sangue , Linfócitos T/efeitos dos fármacos , Animais , Antígenos CD34/imunologia , Antígenos CD28/sangue , Antígenos CD28/imunologia , Células Cultivadas , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/imunologia , Citocinas/imunologia , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Transplante de Fígado , Camundongos Endogâmicos NOD , Camundongos SCID , Medição de Risco , Linfócitos T/imunologia , Linfócitos T/metabolismo , Timo/embriologia , Timo/transplanteRESUMO
CTLA-4Ig (belatacept) blocks the CD80/CD86 ligands for both CD28 and CTLA-4; thus, in addition to the intended effect of blocking CD28-mediated costimulation, belatacept also has the unintended effect of blocking CTLA-4-mediated coinhibition. Recently, anti-CD28 domain antibodies (dAb) that selectively target CD28 while leaving CTLA-4 intact were shown to more effectively inhibit alloimmune responses and prolong graft survival. However, the impact of selective CD28 blockade on protective immunity has not been extensively investigated. Here, we sought to compare the impact of CTLA-4Ig vs anti-CD28dAb on CD8+ T cell immunity to a transplant-relevant pathogen, a murine homolog of Epstein-Barr virus. Mice were infected with murine gammaherpesvirus-68 (MHV) and treated with vehicle, CTLA-4Ig, or anti-CD28dAb. Although anti-CD28dAb resulted in a decrease in virus-specific CD8+ T cell numbers as compared to CTLA-4Ig, cytolytic function and the expression of markers of high-quality effectors were not different from CTLA-4Ig treated animals. Importantly, MHV-68 viral load was not different between the treatment groups. These results suggest that preserved CTLA-4 coinhibition limits MHV-specific CD8+ T cell accumulation, but the population that remains retains cytolytic function and migratory capacity and is not inferior in its ability to control viral burden relative to T cell responses in CTLA-4Ig-treated animals.
Assuntos
Abatacepte/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos CD28/antagonistas & inibidores , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunossupressores/farmacologia , Ativação Linfocitária/imunologia , Animais , Antígenos CD28/imunologia , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
INTRODUCTION: Since the approval of belatacept in 2011 for use in the setting of de novo kidney transplantation, this CD80/86 - CD28 co-stimulation blocker has been shown to be a valuable treatment option for maintenance immunosuppression. Areas covered: In this setting, belatacept has been associated with superior glomerular filtration rate as compared to calcineurin inhibitor-based treatments because of the absence of nephrotoxicity. Additionally, belatacept avoids the cardiovascular side effects (e.g. hypertension and dyslipidemia) caused by a CNI-based-regimen. Nevertheless, belatacept-treated recipients have a higher rate of acute rejections and a higher risk of lymphoproliferative disorders. Expert opinion: Data suggest a benefit from early conversion vs. late conversion of belatacept in a conversion setting following CNI-related toxicity. Randomized studies are currently comparing belatacept to tacrolimus, instead of cyclosporine, as was done in the Belatacept Evaluation of Nephroprotection and Efficacy as a First-line Immunosuppression Trial (BENEFIT). The benefits and limitations of belatacept seem to be the same when tacrolimus is used instead of cyclosporine. Finally, we also report in this review on the immunological data available so far that explain belatacept's limitations and the higher rate of acute rejection. The goal is to find the optimal immunosuppressive strategy to improve efficacy and safety at post-transplantation.
Assuntos
Abatacepte/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Transplante de Rim , Rim/metabolismo , Animais , Antígeno B7-1/antagonistas & inibidores , Antígenos CD28/antagonistas & inibidores , Ensaios Clínicos como Assunto , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
The low affinity Fcγ receptor, FcγRIIA, harbors a common missense mutation, rs1801274 (G>A, Arg131His) that modifies binding affinity to human IgG2 and mouse IgG1 antibodies and is associated with increased risk of autoimmune disease. Despite the important role of the Arg131His variant, little is understood about heterozygous genotype effects on global gene expression and cytokine production during an FcγR-dependent response. To address this gap in knowledge, we treated human whole-blood samples from 130 individuals with mouse IgG1 anti-CD3 and anti-CD28 antibodies and characterized the genome-wide gene expression profiles and cytokine production among individuals stratified by rs1801274 genotype. Our analysis revealed that the levels of four cytokines (IFNγ, IL-12, IL-2, TNFα) and global gene expression patterns differed between all three genotype classes. Surprisingly, the heterozygotes showed suboptimal T cell activation compared to cells from individuals homozygous for the higher-affinity FcγRIIA allele (GG; Arg/Arg). The results of this study demonstrate that IgG response varies among all rs1801274 genotype classes and results in profound differences in both cytokine responses and gene expression patterns in blood leukocytes. Because even heterozygotes showed dampened global responses, our data may provide insight into the heterogeneity of outcomes in cytokine release assays and immunotherapy efficacy.