RESUMO
Immunotherapies blocking the PD-1/PD-L1 checkpoint show some efficacy in metastatic breast cancer (mBC) but are often hindered by immunosuppressive mechanisms. Understanding these mechanisms is crucial for personalized treatments, with peripheral blood monitoring representing a practical alternative to repeated biopsies. In the present study, we performed a comprehensive mass cytometry analysis of peripheral blood immune cells in 104 patients with HER2 negative mBC and 20 healthy donors (HD). We found that mBC patients had significantly elevated monocyte levels and reduced levels of CD4+ T cells and plasmacytoid dendritic cells, when compared to HD. Furthermore, mBC patients had more effector T cells and regulatory T cells, increased expression of immune checkpoints and other activation/exhaustion markers, and a shift to a Th2/Th17 phenotype. Furthermore, T-cell phenotypes identified by mass cytometry correlated with functionality as assessed by IFN-γ production. Additional analysis indicated that previous chemotherapy and CDK4/6 inhibition impacted the numbers and phenotype of immune cells. From 63 of the patients, fresh tumor samples were analyzed by flow cytometry. Paired PBMC-tumor analysis showed moderate correlations between peripheral CD4+ T and NK cells with their counterparts in tumors. Further, a CD4+ T cell cluster in PBMCs, that co-expressed multiple checkpoint receptors, was negatively associated with CD4+ T cell tumor infiltration. In conclusion, the identified systemic immune signatures indicate an immune-suppressed environment in mBC patients who had progressed/relapsed on standard treatments, and is consistent with ongoing chronic inflammation. These activated immuno-suppressive mechanisms may be investigated as therapeutic targets, and for use as biomarkers of response or treatment resistance.
RESUMO
We have developed a chimeric antigen receptor (CAR) against the six-transmembrane epithelial antigen of prostate-1 (STEAP1), which is expressed in prostate cancer, Ewing sarcoma, and other malignancies. In the present study, we investigated the effect of substituting costimulatory domains and spacers in this STEAP1 CAR. We cloned four CAR constructs with either CD28 or 4-1BB costimulatory domains, combined with a CD8a-spacer (sp) or a mutated IgG-spacer. The CAR T-cells were evaluated in short- and long-term in vitro T-cell assays, measuring cytokine production, tumor cell killing, and CAR T-cell expansion and phenotype. A xenograft mouse model of prostate cancer was used for in vivo comparison. All four CAR constructs conferred CD4+ and CD8+ T cells with STEAP1-specific functionality. A CD8sp_41BBz construct and an IgGsp_CD28z construct were selected for a more extensive comparison. The IgGsp_CD28z CAR gave stronger cytokine responses and killing in overnight caspase assays. However, the 41BB-containing CAR mediated more killing (IncuCyte) over one week. Upon six repeated stimulations, the CD8sp_41BBz CAR T cells showed superior expansion and lower expression of exhaustion markers (PD1, LAG3, TIGIT, TIM3, and CD25). In vivo, both the CAR T variants had comparable anti-tumor activity, but persisting CAR T-cells in tumors were only detected for the 41BBz variant. In conclusion, the CD8sp_41BBz STEAP1 CAR T cells had superior expansion and survival in vitro and in vivo, compared to the IgGsp_CD28z counterpart, and a less exhausted phenotype upon repeated antigen exposure. Such persistence may be important for clinical efficacy.
Assuntos
Neoplasias da Próstata , Receptores de Antígenos Quiméricos , Animais , Humanos , Masculino , Camundongos , Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos , Citocinas , Modelos Animais de Doenças , Oxirredutases , Próstata , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Receptores de Antígenos Quiméricos/genéticaRESUMO
Adoptive cell therapy with T-cell receptor (TCR)-engineered T cells represents a powerful method to redirect the immune system against tumours. However, although TCR recognition is restricted to a specific peptide-MHC (pMHC) complex, increasing numbers of reports have shown cross-reactivity and off-target effects with severe consequences for the patients. This demands further development of strategies to validate TCR safety prior to clinical use. We reasoned that the desired TCR signalling depends on correct pMHC recognition on the outside and a restricted clustering on the inside of the cell. Since the majority of the adverse events are due to TCR recognition of the wrong target, we tested if blocking the signalling would affect the binding. By over-expressing the c-SRC kinase (CSK), a negative regulator of LCK, in redirected T cells, we showed that peripheral blood T cells inhibited anti-CD3/anti-CD28-induced phosphorylation of ERK, whereas TCR proximal signalling was not affected. Similarly, overexpression of CSK together with a therapeutic TCR prevented pMHC-induced ERK phosphorylation. Downstream effector functions were also almost completely blocked, including pMHC-induced IL-2 release, degranulation and, most importantly, target cell killing. The lack of effector functions contrasted with the unaffected TCR expression, pMHC recognition, and membrane exchange activity (trogocytosis). Therefore, co-expression of CSK with a therapeutic TCR did not compromise target recognition and binding, but rendered T cells incapable of executing their effector functions. Consequently, we named these redirected T cells "dummy T cells" and propose to use them for safety validation of new TCRs prior to therapy.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Quinases da Família src/metabolismo , Proteína Tirosina Quinase CSK , Morte Celular , Células Cultivadas , Humanos , Fosforilação , Ligação Proteica , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Linfócitos T/citologia , Quinases da Família src/genéticaRESUMO
Effector T cells equipped with engineered antigen receptors specific for cancer targets have proven to be very efficient. Two methods have emerged: the Chimeric Antigen Receptors (CARs) and T-cell Receptor (TCR) redirection. Although very potent, CAR recognition is limited to membrane antigens which represent around 1% of the total proteins expressed, whereas TCRs have the advantage of targeting any peptide resulting from cellular protein degradation. However, TCRs depend on heavy signalling machinery only present in T cells which restricts the type of eligible therapeutic cells. Hence, an introduced therapeutic TCR will compete with the endogenous TCR for the signalling proteins and carries the potential risk of mixed dimer formation giving rise to a new TCR with unpredictable specificity. We have fused a soluble TCR construct to a CAR-signalling tail and named the final product TCR-CAR. We here show that, if expressed, the TCR-CAR conserved the specificity and the functionality of the original TCR. In addition, we demonstrate that TCR-CAR redirection was not restricted to T cells. Indeed, after transduction, the NK cell line NK-92 became TCR positive and reacted against pMHC target. This opens therapeutic avenues combing the killing efficiency of NK cells with the diversified target recognition of TCRs.
Assuntos
Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Citometria de Fluxo , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos Quiméricos/química , Proteínas Recombinantes de Fusão , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução GenéticaRESUMO
Dendritic cells (DCs) initiate host immune responses by presenting captured antigens to naive T cells. Hence, DC-binding peptides may be used for antigen targeting to boost naive and memory immune responses. By biopanning peptide phage libraries on human monocyte-derived DCs, we identified novel DC-binding peptides. One of the selected phages, displaying the NW peptide (NWYLPWLGTNDW), bound DCs with high affinity, and its binding was inhibited by the corresponding synthetic peptide. Antigenic peptides or proteins conjugated to the NW peptide bound to DCs and were internalized without negative effects on DC phenotype and function. Ex vivo targeted delivery of CMV-pp65 peptides to DCs via the NW peptide increased T-cell responses in HLA-A2(+)/CMV(+) donors compared to untargeted peptides (P<0.001). Stimulation of CD45RO-depleted peripheral blood mononuclear cells from CMV(-) donors with the NW-pp65 fusion peptides expanded pp65-specific precursor T cells. Moreover, the NW peptide mediated small interfering RNA delivery to DCs, and a significant gene silencing was obtained. Collectively, the data reveal that proteins and nucleic acids can be directed to DCs through the NW peptide, enabling effective uptake and functional effects such as T-cell activation in the context of MHC class I and II molecules.
Assuntos
Antígenos/imunologia , Células Dendríticas/imunologia , Ácidos Nucleicos/imunologia , Oligopeptídeos/imunologia , Sequência de Aminoácidos , Apresentação de Antígeno/imunologia , Antígenos/metabolismo , Ligação Competitiva/imunologia , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Citometria de Fluxo , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Microscopia de Fluorescência , Monócitos/imunologia , Monócitos/metabolismo , Ácidos Nucleicos/metabolismo , Oligopeptídeos/metabolismo , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Ligação Proteica/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/metabolismoRESUMO
We present a 67-year-old non-diabetic male who presented with muscle cramps, paresis, atrophy and fasciculations in the left leg, followed by rapidly progressive muscle stiffness and superimposed spasms which subsequently also affected the right leg and the trunk. GAD65 autoantibodies were elevated in serum and CSF, compatible with systemic and intrathecal synthesis of oligoclonal and high-avidity autoantibodies, and GAD65 specific T cells were clonally expanded in the CSF. The patient did not respond to GABAergic and immunomodulatory treatment or plasma exchange, and died from respiratory failure after 18 months. Autopsy revealed unilateral axonal swelling, chromatolysis and vacuolisation of anterior horn cells of the lower spinal cord, accompanied by microglia proliferation and discrete infiltration of CD8+ cytotoxic T cells. No CD4+ T helper cells, B cells or complement deposition were detected. To our knowledge, this is the first report of stiff person syndrome with lower motor signs restricted to a lower limb, and also the first attempt to characterize the infiltrating T cells. The finding of CD8+ cytotoxic T cells in the absence of B cells in the inflamed area of the spinal cord suggests that the intrathecal synthesis of GAD65 autoantibodies takes place in areas of the CNS not strictly related to the clinically relevant lesions.
Assuntos
Encefalomielite/complicações , Glutamato Descarboxilase/imunologia , Doença dos Neurônios Motores/complicações , Medula Espinal/patologia , Rigidez Muscular Espasmódica/complicações , Linfócitos T Citotóxicos/patologia , Idoso , Autoanticorpos , Encefalomielite/imunologia , Encefalomielite/patologia , Evolução Fatal , Glutamato Descarboxilase/metabolismo , Humanos , Vértebras Lombares , Masculino , Doença dos Neurônios Motores/imunologia , Doença dos Neurônios Motores/patologia , Região Sacrococcígea , Medula Espinal/imunologia , Rigidez Muscular Espasmódica/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Linfócitos T Citotóxicos/metabolismoRESUMO
CD4(+) T cells specific for immunologic non-self determinants on self-IgG, idiotopes (Id), can be raised from cerebrospinal fluid (CSF) and blood of patients with multiple sclerosis (MS). To test if Id-specific CD4(+) T cells have the potential to destroy oligodendrocytes (ODCs), we analyzed their ability to induce apoptosis of human ODC cell lines. Id-specific CD4(+) T cells stimulated with either Id-bearing B cells, Id-peptide presented by other antigen presenting cells, or by anti-CD3/anti-CD28 in the absence of accessory cells induced DNA fragmentation and killed ODCs. Killing required contact between the ODCs and the T cells, it did not depend on the cytokine profile of the T cells, it was independent of other cell types, and was inhibited by a general caspase inhibitor and an anti-Fas antibody. Activated CD4(+) T cells specific for glutamic acid decarboxylase 65 also induced apoptosis, showing that killing does not depend on cognate interaction between T cells and target cells but rather on the activation status of the T cells.
Assuntos
Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Oligodendroglia/citologia , Oligodendroglia/imunologia , Adulto , Anticorpos/imunologia , Caspases/metabolismo , Comunicação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Oligodendroglia/enzimologia , Receptor fas/imunologiaRESUMO
Most patients with stiff person syndrome (SPS) display intrathecal synthesis of oligoclonal and high-avidity IgG against the 65 kDa isoform of glutamic acid decarboxylase (GAD65 IgG), but little is known about the mechanisms driving this immune response. We hypothesized that GAD65-specific T cells accumulating in the central nervous system drive the intrathecal GAD65 IgG production. Accordingly, we were able to clone HLA-DR or DP restricted GAD65-specific T cells from the cerebrospinal fluid (CSF) of all three patients with, but not in one patient without substantial intrathecal production of GAD65 IgG. The CSF T cells recognized four GAD65 epitopes, which were unique to each patient. In two patients, identical or closely related GAD65-specific CSF T cell clones were expanded in vivo. In contrast to the findings in CSF, only one GAD65-specific T cell clone could be raised from the blood of one single patient. Cysteine in amino acid position 474, which is important for enzymatic function of GAD65, was critical for recognition of GAD65(474-484) by HLA-DP restricted CSF T cells. We conclude that GAD65-specific T cells and clonally expanded GAD65-specific B cells coexist intrathecally, where they may collaborate in the synthesis of GAD65 IgG.