CD8(+) T cells are important effectors, as well as regulators, of organ-specific autoimmunity. Compared with Tc1-type CD8(+) cells, Tc2 cells have impaired anti-viral and anti-tumor effector ...functions, although no data are yet available on their pathogenic role in autoimmunity. Our aim was to explore the role of autoreactive Tc1 and Tc2 cells in autoimmune diabetes. We set up an adoptive transfer model in which the recipients were transgenic mice expressing influenza virus hemagglutinin (HA) specifically in their pancreatic ss islet cells (rat insulin promoter-HA mice) and islet-specific Tc1 and Tc2 cells were generated in vitro from HA-specific CD8(+) cells of TCR transgenic mice (CL4-TCR mice). One million Tc1 cells, differentiated in vitro in the presence of IL-12, transferred diabetes in 100% of nonirradiated adult rat insulin promoter-HA recipients; the 50% diabetogenic dose was 5 x 10(5). Highly polarized Tc2 cells generated in the presence of IL-4, IL-10, and anti-IFN-gamma mAb had a relatively low, but definite, diabetogenic potential. Thus, 5 x 10(6) Tc2 cells caused diabetes in 6 of 18 recipients, while the same dose of naive CD8(+) cells did not cause diabetes. Looking for the cause of the different diabetogenic potential of Tc1 and Tc2 cells, we found that Tc2 cells are at least as cytotoxic as Tc1 cells but their accumulation in the pancreas is slower, a possible consequence of differential chemokine receptor expression. The diabetogenicity of autoreactive Tc2 cells, most likely caused by their cytotoxic activity, precludes their therapeutic use as regulators of autoimmunity.
The two-signal model states that activation of naive T cells requires a signal 1 stimulus through the TCR and a co-stimulatory signal 2. By contrast, signal 1 alone is sufficient for pre-activated T ...cells. Recently, however, it has been shown that under certain conditions T cells can bypass the requirement for co-stimulation. For example, CD28-deficient mice, when immunized with lymphocytic choriomeningitis virus, mount a vigorous cytotoxic T lymphocyte response and clear the virus. As a continuous effort to unravel the mechanisms of T cell activation, we previously reported activation of hybridoma T cells by recombinant single-chain MHC molecules in the absence of antigen-presenting cells. In such reconstitution experiments, since the signals delivered to the T cells are well controlled, the contribution of any known or unknown signals can be ruled out. In the present study, we analyzed the requirements for activation of naive T cells by using splenocytes from TCR transgenic mice as a source of responding cells. We observed that naive CD8+ T cells are fully activated by signal 1 alone, but that co-stimulation lowers their activation threshold. Previously activated T cells are fully responsive, even when the first stimulation was performed in the absence of co-stimulation. They display a low activation threshold and are insensitive to co-stimulation. The physiological relevance of this finding and its consequences for immunotherapy as well as for our understanding of self-tolerance are discussed.
The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell ...lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20 months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10 months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation.
BackgroundThe success of immunotherapy is associated with a remodeling of the entire tumor microenvironment. In this context, a positive cooperation between CD8+ T cells and the activated ...tumor-infiltrating macrophages and monocytes appear necessary for an optimal tumor regression.However, as the mechanisms of such interactions are still unknown, we aimed to uncover the precise identity of the cooperating monocytes/macrophages and CD8+ T cells as well as the nature of their interplay.ModelIn the transplanted PyMT mammary tumor model, we sorted monocytes/macrophages and CD8+ T cells from tumors regressing after STING agonist therapy to perform scRNAseq on these two populations.ResultsWe discovered that the monocyte/macrophage compartment is divided in three main macrophages subsets and two monocyte-like subsets and that the CD8+ T cells comprise a large diversity of phenotypes (effector, naïve, memory-like, regulatory cells). We have followed the dynamic changes of these populations across the stages of tumor regression and noticed that monocyte-like subsets are preferentially enriched upon regression. In parallel, the CD8+ memory subsets were increased during the regression at the expense of regulatory-like populations.PerspectivesWe are currently examining the predicted functions of the different subsets as well as their spatial distribution in the tumor microenvironment, to uncover the mechanisms by which these cells cooperate in tumors treated by immunotherapy.Disclosure Information A. Vermare: None. A. Rouault: None. A. Ventura: None. B. Saint-Pierre: None. W. Zeitouni: None. K. Mulder: None. K. Bailly: None. B. Izac: None. M.V. Guérin: None. C.A. Dutertre: None. N. Bercovici: None.
BackgroundActivation of the STimulator of INterferon Genes (STING) by DMXAA (5,6-dimethylxanthenone-4-acetic acid) can induce a strong production of IFNα/β and the rejection of transplanted primary ...tumors. However, the efficacy of such therapeutic approach for the treatment of spontaneous tumors had still to be evaluated.Material and MethodsWe have tested whether the injection of DMXAA or other STING agonists and TLR4 agonist, could lead to the regression of spontaneous MMTV-PyMT mammary tumors. We also characterized, in time and space, the early signaling events triggered downstream STING and the distribution of infiltrating immune cells in the tumor microenvironment by fluorescence imaging.ResultsWe show that spontaneous MMTV-PyMT mammary tumors are resistant to immunotherapeutic intervention. We demonstrate that TGFβ, abundant in spontaneous tumors, is a key molecule limiting this IFN-induced-tumor regression by DMXAA. Mechanistically, we found that TGFβ blocks the phosphorylation of IRF3 and the ensuing IFNα/β production by tumor infiltrating macrophages. Finally, blocking TGFβ restores the production of IFNα by activated MHCII+ tumor-associated macrophages, and enables tumor regression induced by STING activation.ConclusionsBased on these findings, we propose that the efficacy of many cancer therapies, which are type I IFN-dependent, should be greatly improved by combination with TGFβ blockade.Disclosure InformationN. Bercovici: None. M.V. Guérin: None. F. Regnier: None. J.M. Weiss: None. V. Feuillet: None. L. Vimeux: None. G. Altan-Bonnet: None. E. Donnadieu: None. A. Trautmann: None.
Developing a process to generate dendritic cells (DCs) applicable for multicenter trials would facilitate cancer vaccine development. Moreover, targeting multiple antigens with such a vaccine ...strategy could enhance the efficacy of such a treatment approach. We performed a phase 1/2 clinical trial administering a DC-based vaccine targeting multiple tumor-associated antigens to patients with advanced colorectal cancer (CRC). A qualified manufacturing process was used to generate DC from blood monocytes using granulocyte macrophage colony-stimulating factor and IL-13, and matured for 6 hours with Klebsiella-derived cell wall fraction and interferon-gamma (IFN-gamma). DCs were also loaded with 6 HLA-A*0201 binding peptides derived from carcinoembryonic antigen (CEA), MAGE, and HER2/neu, as well as keyhole limpet hemocyanin protein and pan-DR epitope peptide. Four planned doses of 35x10(6) cells were administered intradermally every 3 weeks. Immune response was assessed by IFN-gamma enzyme-linked immunosorbent spot (ELISPOT). Matured DC possessed an activated phenotype and could prime T cells in vitro. In the trial, 21 HLA-A2+ patients were apheresed, 13 were treated with the vaccine, and 11 patients were evaluable. No significant treatment-related toxicity was reported. T-cell responses to a CEA-derived peptide were detected by ELISPOT in 3 patients. T cells induced to CEA possessed high avidity T-cell receptors. ELISPOT after in vitro restimulation detected responses to multiple peptides in 2 patients. All patients showed progressive disease. This pilot study in advanced CRC patients demonstrates DC-generated granulocyte macrophage colony-stimulating factor and IL-13 matured with Klebsiella-derived cell wall fraction and IFN-gamma can induce immune responses to multiple tumor-associated antigens in patients with advanced CRC.
Although the two-signal model for T cell activation states that a signal-1 through the TCR and a costimulatory signal-2 are required for optimal stimulation, it is now clear that the requirement for ...costimulation can be bypassed under certain conditions. We previously reported that this is the case for naive CD8+ T cells in vitro. In the present study we tested the effect of signal-2 when delivered after signal-1 has been disrupted. Naive CD8+ T cells from TCR transgenic mice were stimulated in vitro by using immobilized recombinant single-chain MHC molecules alone as signal-1. This signal was then stopped after different lengths of time, and anti-CD28 mAb as signal-2 was given either immediately or after a time lag. We found that signal-2 can potentiate a short signal-1 when added sequentially. Moreover, a time lag between the two signals does not abolish this potentiation. If the strength of signal-1, but not its duration, is increased, then the time lag between the delivery of signals 1 and 2 can be lengthen without loss of potentiation. Together, our results indicate that the two signals do not need to be delivered concomitantly to get optimal T cell activation. We suggest that the CD8+ T cells can reach a transient "excited" state after being stimulated with signal-1 alone, characterized by the cell's ability to respond to separate and delayed signal-2.
Dendritic cells (DCs) are much more potent antigen (Ag)-presenting cells than resting B cells for the activation of naive T cells. The mechanisms underlying this difference have been analyzed under ...conditions where ex vivo DCs or B cells presented known numbers of specific Ag-major histocompatibility complex (MHC) complexes to naive CD4(+) T cells from T cell antigen receptor (TCR) transgenic mice. Several hundred Ag-MHC complexes presented by B cells were necessary to elicit the formation of a few T-B conjugates with small contact zones, and the resulting individual T cell Ca2+ responses were all-or-none. In contrast, Ag-specific T cell Ca2+ responses can be triggered by DCs bearing an average of 30 Ag-MHC complexes per cell. Formation of T-DC conjugates is Ag-independent, but in the presence of the Ag, the surface of the contact zone increases and so does the amplitude of the T cell Ca2+ responses. These results suggest that Ag is better recognized by T cells on DCs essentially because T-DC adhesion precedes Ag recognition, whereas T-B adhesion requires Ag recognition. Surprisingly, we also recorded small Ca2+ responses in T cells interacting with unpulsed DCs. Using DCs purified from MHC class II knockout mice, we provide evidence that this signal is mostly due to MHC-TCR interactions. Such an Ag-independent, MHC-triggered calcium response could be a survival signal that DCs but not B cells are able to deliver to naive T cells.
The primary goal of cancer vaccines is to induce CD8+ T cells specific for tumor-associated antigens (TAA) but the characterization of these cells has been difficult because of the low sensitivity of ...ex vivo assays. Here, we focused on TAA-specific CD8+ T-cell responses in melanoma patients after vaccination with autologous dendritic cells loaded with lysates derived from allogeneic tumor-cell lines (Lysate-DC). Out of 40 patients treated, 16 patients developed immune response to tumor-cell lysate and/or CD8+ T cells specific for differentiation and cancer-testis antigens. TAA-specific CD8+ T-cell responses were detected by interferon (IFN)-gamma enzyme-linked immunospot after in vitro sensitization and were, either transient during the treatment period or delayed, that is, observed after completion of all vaccinations. We could not correlate these immune responses to clinical data as none of the patients achieved an overall objective response according to Response Evaluation Criteria in Solid Tumors criteria. Three patients were reported as stable disease and 10 patients presented evidence of antitumor activity. We found that TAA-specific T cells characterized in 4 patients produced perforin ex vivo, but no IFN-gamma in enzyme-linked immunospot. Differential expression of IFN-gamma and perforin was also observed for viral-specific T cells. Altogether, our results show that Lysate-DC therapy elicited tumor-specific CD8+ T cells nonlimited to human leukocyte antigen-A2+ patients, with some T cells secreting perforin ex vivo and IFN-gamma only after restimulation. The differential expression of perforin and IFN-gamma by antitumor and antiviral CD8+ T cells supports that the sole use of IFN-gamma production to monitor T cells overlooks functional T-cell subpopulations triggered by vaccines.
Antigen recognition was analyzed at the single‐cell level by using for the first time T cells which were not altered by in vitro selection, transfection or immortalization. The first consequence of ...antigen recognition by ex vivo naive CD4+ T cells from T cell receptor (TCR)‐transgenic mice is the formation of a “contact zone” with the B cell presenting the antigen. The T cell intracellular calcium (Ca2+ ) response begins after a delay of 30 s on average, following the formation of the contact zone. The T cell response is entirely inhibited by either protein tyrosine kinase or actin polymerization inhibitors but, surprisingly, it is insensitive to inhibitors of phosphoinositide 3‐kinase. Moreover, inhibition of microtubule polymerization and use of Ca2+ ‐free medium do not prevent the beginning of the T cell response, but do reduce the stability of the contact zone and/or the amplitude of the Ca2+ plateau. The critical involvement of the cytoskeleton in antigen recognition on B cells introduces a checkpoint in T cell activation: the initial TCR engagement triggers a Ca2+ response only after an amplification step corresponding to a cytoskeleton‐controlled increase in the number of engaged TCR.
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