PD-1 and Tim-3 Regulate the Expansion of Tumor Antigen-Specific CD8+ T Cells Induced by Melanoma Vaccines

Although melanoma vaccines stimulate tumor antigen (TA)-specific CD8+ T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8+ T cells with regard to the inhibitory T cell co-receptors PD-1 and Tim-3 in metastatic melanoma patients who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant (IFA), CpG oligodeoxynucleotide (CpG) and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid TA-specific CD8+ T-cell responses detected ex vivo, however, TA-specific CD8+ T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8+ T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8+ T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8+ T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T cell responses and increase the likelihood of clinical responses in advanced melanoma patients

CD4CD8αα Lymphocytes, A Novel Human Regulatory T Cell Subset Induced by Colonic Bacteria and Deficient in Patients with Inflammatory Bowel Disease

It has become evident that bacteria in our gut affect health and disease, but less is known about how they do this. Recent studies in mice showed that gut Clostridium bacteria and their metabolites can activate regulatory T cells (Treg) that in turn mediate tolerance to signals that would ordinarily cause inflammation. In this study we identify a subset of human T lymphocytes, designated CD4CD8αα T cells that are present in the surface lining of the colon and in the blood. We demonstrate Treg activity and show these cells to be activated by microbiota; we identify F. prausnitzii, a core Clostridium strain of the human gut microbiota, as a major inducer of these Treg cells. Interestingly, there are fewer F. prausnitzii in individuals suffering from inflammatory bowel disease (IBD), and accordingly the CD4CD8αα T cells are decreased in the blood and gut of patients with IBD. We argue that CD4CD8αα colonic Treg probably help control or prevent IBD. These data open the road to new diagnostic and therapeutic strategies for the management of IBD and provide new tools to address the impact of the intestinal microbiota on the human immune system

TIGIT in cancer immunotherapy

Tumors evade immune-mediated recognition through multiple mechanisms of immune escape. On chronic tumor antigen exposure, T cells become dysfunctional/exhausted and upregulate various checkpoint inhibitory receptors (IRs) that limit T cells’ survival and function. During the last decade, immunotherapies targeting IRs such as programmed cell death receptor 1 (PD-1) and anticytotoxic T lymphocyte-associated antigen 4 (CTLA-4) have provided ample evidence of clinical benefits in many solid tumors. Beyond CTLA-4 and PD-1, multiple other IRs are also targeted with immune checkpoint blockade in the clinic. Specifically, T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is a promising new target for cancer immunotherapy. TIGIT is upregulated by immune cells, including activated T cells, natural killer cells, and regulatory T cells. TIGIT binds to two ligands, CD155 (PVR) and CD112 (PVRL2, nectin-2), that are expressed by tumor cells and antigen-presenting cells in the tumor microenvironment. There is now ample evidence that the TIGIT pathway regulates T cell-mediated and natural killer cell-mediated tumor recognition in vivo and in vitro. Dual PD-1/TIGIT blockade potently increases tumor antigen-specific CD8+ T cell expansion and function in vitro and promotes tumor rejection in mouse tumor models. These findings support development of ongoing clinical trials with dual PD-1/TIGIT blockade in patients with cancer

PD-1 and Tim-3 regulate the expansion of tumor antigen-specific CD8⁺ T cells induced by melanoma vaccines.

Although melanoma vaccines stimulate tumor antigen-specific CD8(+) T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8(+) T cells with regard to the inhibitory T-cell coreceptors PD-1 and Tim-3 in patients with metastatic melanoma who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant, CpG oligodeoxynucleotide (CpG), and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid tumor antigen-specific CD8(+) T-cell responses detected ex vivo, however, tumor antigen-specific CD8(+) T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8(+) T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8(+) T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8(+) T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T-cell responses and increase the likelihood of clinical responses in patients with advanced melanoma

DP8α LPL specifically respond to the gut commensal bacterium <i>F. prausnitzii</i>.

<p>Flow cytometry analysis of the proliferative response (VPD dilution and FlowJo analysis) of DP8α LPL (a) or CD4 LPL lines (b) after 3 d of co-culture with allogeneic monocytes alone or loaded overnight with F in the presence or absence of an anti-MHC class-II antibody or of an irrelevant antibody (IgG), or with monocytes loaded with B, L, or E: representative cytometry data and mean percentage of VPD low cells (<i>n</i> = 6: two independent experiments performed with three DP8α LPL lines); **<i>p</i><0.01 (paired <i>t</i>-test). (c) Flow cytometry analysis of the IFN-γ and IL-10 responses of DP8α LPL lines (<i>n</i> = 4) after 6 h of stimulation by monocytes, loaded overnight by F (1∶5) in the presence or absence of anti MHC class-II antibody or of an irrelevant antibody, or not loaded, or loaded with B, L, or E: representative response of the C139 DP8α LPL line and the percentages of cells secreting IL-10 or IFN- γ or both in independent experiments (two to seven experiments performed with four DP8α LPL lines: C114, black circles; C139, white circles; C192, white squares; and C140, black squares; ***<i>p</i><0.001 (paired <i>t</i>-test). (d) Flow cytometry analysis of the proliferative response of freshly dissociated LPL from three donors stimulated by allogeneic monocytes (black symbols) and of sorted DP8α and CD4 LPL from one donor (grey symbols) stimulated by autologous monocytes. Responses were analyzed after 5 d of co-culture with monocytes alone or monocytes loaded overnight with F or E: mean percentage of F specific divided cells (with deduction of the divided cells to monocytes alone) (calculated on FlowJo software) and representative cytometry data of the proliferative response of the freshly sorted DP8α LPL population.</p

Specificity of DP8α-LPL lines and-PBL lines for <i>F. prausnitzii</i>.

<p>DP8α LPL lines (<i>n</i> = 3, C114, C139, C140) and one PBL line (DTC4) were stimulated for 6 h by monocytes, loaded overnight by different Clostridium species (1∶5).</p>a<p>Percent IL-10 or IFN-γ positive cells.</p

Presence of F-reactive DP8α T lymphocytes in the blood of healthy individuals.

<p>(a) Flow cytometry analysis of the frequencies of DP8α T lymphocytes in the blood of 18 donors: representative dot plot of PBMC co-labelled with anti-CD3, anti-CD4, and anti-CD8α antibodies, representative histogram of CD8β expression by gated CD8, CD4, and DP8α PBMC, and the percentages of DP8α lymphocytes among the CD3 and the CD3CD4 PBMC. (b) Proliferative responses of DP8α peripheral blood T cells to F in the presence or absence of an anti-MHC class-II antibody or an irrelevant antibody, and to B, L, and E. PBMC were cultured for 5 d with the antibody and/or indicated bacteria at a bacterium∶PBMC ratio 1∶1: representative FlowJo analysis of the VPD dilution and percentage of F-specific divided DP8α T cells in the PBMC from several donors (<i>n</i> = 18); ***<i>p</i><0.001 and **<i>p</i><0.01 (paired <i>t</i>-test). (c) Percent VPD dilution in paired DP8α and CD4 T cells among PBMC co-cultured with F for 5 d; ***<i>p</i><0.001 (paired <i>t</i>-test). (d) CTLA4 and LAG3 expression in proliferative DP8α and CD4 PBL after 5 d of co-culture with allogeneic monocytes loaded overnight with F (<i>n</i> = 2). (e) Flow cytometry analysis of the proliferative response of freshly isolated DP8α or CD4 PBL after 5 d of co-culture with autologous monocytes alone or loaded overnight with F or E: representative cytometry data of a freshly sorted DP8α PBL population and mean percentage of F-specific divided cells (calculated on FlowJo software) (experiments performed with five freshly isolated DP8α PBL (black circles) and two CD4 PBL (white circles).</p