RESUMO
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is considered a low-immunogenic (LI) tumor with a "cold" tumor microenvironment and is mostly unresponsive to immune checkpoint blockade therapies. In this study, we decipher the impact of intratumoral heterogeneity of immune determinants on antitumor responses. EXPERIMENTAL DESIGN: We performed spatial proteomic and transcriptomic analyses and multiplex immunofluorescence on multiple tumor regions, including tumor center (TC) and invasive front (IF), from 220 patients with PDAC, classified according to their transcriptomic immune signaling into high-immunogenic PDAC (HI-PDAC, n = 54) and LI PDAC (LI-PDAC, n = 166). Spatial compartments (tumor: pancytokeratin+/CD45- and leukocytes: pancytokeratin-/CD45+) were defined by fluorescence imaging. RESULTS: HI-PDAC exhibited higher densities of cytotoxic T lymphocytes with upregulation of T-cell priming-associated immune determinants, including CD40, ITGAM, glucocorticoid-induced TNF-related receptor, CXCL10, granzyme B, IFNG, and HLA-DR, which were significantly more prominent at the IF than at the TC. In contrast, LI-PDAC exhibited immune-evasive tumor microenvironments with downregulation of immune determinants and a negative gradient from TC to IF. Patients with HI-PDAC had significantly better outcomes but showed more frequently exhausted immune phenotypes. CONCLUSIONS: Our results indicate strategic differences in the regulation of immune determinants, leading to different levels of effectiveness of antitumor responses between HI and LI tumors and dynamic spatial changes, which affect the evolution of immune evasion and patient outcomes. This finding supports the coevolution of tumor and immune cells and may help define therapeutic vulnerabilities to improve antitumor immunity and harness the responsiveness to immune checkpoint inhibitors in patients with PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Microambiente Tumoral , Humanos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Microambiente Tumoral/imunologia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Prognóstico , Biomarcadores Tumorais , Proteômica/métodos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Regulação Neoplásica da Expressão Gênica , Perfilação da Expressão GênicaRESUMO
BACKGROUND & AIMS: Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. METHODS: We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. RESULTS: Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. CONCLUSION: The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.
Assuntos
Carcinoma/patologia , Mucosa Intestinal/patologia , Neoplasias Intestinais/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Animais , Apoptose/genética , Apoptose/imunologia , Carcinogênese/genética , Carcinogênese/imunologia , Carcinogênese/patologia , Carcinoma/diagnóstico , Carcinoma/genética , Modelos Animais de Doenças , Endoscopia , Células Epiteliais/patologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/diagnóstico por imagem , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/genética , Camundongos , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genéticaRESUMO
TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4+ T-helper cell population in the resting (=naïve) and activated (=effector) state, and 3) two commonly used CD4+ Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4+ T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca²+-dependent proliferation of primary CD4+ T-cells indicating that TRPC3 may be involved in Ca²+ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca²+ entry.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação da Expressão Gênica , Canais de Cátion TRPC/genética , Linfócitos T CD4-Positivos/citologia , Cálcio/metabolismo , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Humanos , Células Jurkat , Ativação Linfocitária , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPC/metabolismoRESUMO
Costimulation is a fundamental principle of T-cell activation. In addition to T-cell receptor engagement, the interaction between CD80 and/or CD86 with CD28 and/or cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptors is required to regulate T-cell activation and tolerance. While the importance of costimulation is clearly established, the exact molecular mechanism is unknown. We demonstrate that T-cell proliferation and the ability of CD8(+) T-effector cells to kill were enhanced slightly by CD80 but dramatically by CD86 costimulation. To further analyse the cellular process of costimulation, we developed a single-cell assay to analyse Ca(2+) signals following costimulation with bi-specific antibodies. We found that this stimulation method worked in every human T-cell that was analysed, making it one of the most efficient T-cell activation methods to date for primary human T cells. The enhanced proliferation and killing by costimulation was paralleled by an increase of Ca(2+) influx following CD86 costimulation and it was dependent on CD28/CTLA-4 expression. The enhanced Ca(2+) influx following CD86 costimulation was abrogated by an antibody that interfered with CD28 function. The differences in Ca(2+) influx between CD80 and CD86 costimulation were not dependent on the depletion of Ca(2+) stores but were eliminated by the application of 10 mum 2-aminoethyldiphenyl borate which has recently been shown to enhance stromal interaction molecule 2 (STIM2)-dependent Ca(2+) entry while reducing STIM1-dependent Ca(2+) entry. Our data indicate that differences in the efficiency of costimulation are linked to differences in Ca(2+) entry.
Assuntos
Anticorpos Monoclonais , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteínas Recombinantes de Fusão , Antígeno B7-1/imunologia , Antígeno B7-2/imunologia , Compostos de Boro/farmacologia , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proliferação de Células , Citotoxicidade Imunológica , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Engenharia de Proteínas , Molécula 2 de Interação EstromalRESUMO
Clonal T cell expansion through proliferation is a central process of the adaptive immune response. Apoptosis of activated T cells is required to avoid chronic inflammation. T cell proliferation and apoptosis are often analyzed with stimuli that do not induce formation of a functional immunological synapse. Here we analyze the Ca(2+) dependence of proliferation and apoptosis in primary human CD4(+) T cells following stimulation with anti-CD3/anti-CD28-coated beads, which induce a tight interaction similar to the immunological synapse. We found this focal stimulation to be much more efficient for stimulating IL-2 production and proliferation than non-focal TCR stimuli. Surprising little Ca(2+) entry through Ca(2+) channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca(2+)](i)) elevations of up to 220 nM from a baseline level of around 40 nM were sufficient for maximal proliferation in primary human CD4(+) T cells. We also show that proliferation was very Ca(2+) sensitive in the range 90-120 nM, whereas apoptosis was basically constant for [Ca(2+)](i) levels of 90-120 nM. We conclude that very small changes in [Ca(2+)](i) can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Cálcio/fisiologia , Proliferação de Células , Ativação Linfocitária/imunologia , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Humanos , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismoRESUMO
T helper (Th) cell activation is required for the adaptive immune response. Formation of the immunological synapse (IS) between Th cells and antigen-presenting cells is essential for Th cell activation. IS formation induces the polarization and redistribution of many signaling molecules; however, very little is known about organelle redistribution during IS formation in Th cells. We show that formation of the IS induced cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. Using total internal reflection microscopy, we found that upon stimulation, the distance between the IS and mitochondria was decreased to values<200 nm. Consequently, mitochondria close to the IS took up more Ca2+ than the ones farther away from the IS. The redistribution of mitochondria to the IS was necessary to maintain Ca2+ influx across the plasma membrane and Ca2+-dependent Th cell activation. Our results suggest that mitochondria are part of the signaling complex at the IS and that their localization close to the IS is required for Th cell activation.