Anti-PD-1 therapy triggers Tfh cell-dependent IL-4 release to boost CD8 T cell responses in tumor-draining lymph nodes.

Anti-PD-1 therapy targets intratumoral CD8+ T cells to promote clinical responses in cancer patients. Recent evidence suggests an additional activity in the periphery, but the underlying mechanism is unclear. Here, we show that anti-PD-1 mAb enhances CD8+ T cell responses in tumor-draining lymph nodes by stimulating cytokine production in follicular helper T cells (Tfh). In two different models, anti-PD-1 mAb increased the activation and proliferation of tumor-specific T cells in lymph nodes. Surprisingly, anti-PD-1 mAb did not primarily target CD8+ T cells but instead stimulated IL-4 production by Tfh cells, the major population bound by anti-PD-1 mAb. Blocking IL-4 or inhibiting the Tfh master transcription factor BCL6 abrogated anti-PD-1 mAb activity in lymph nodes while injection of IL-4 complexes was sufficient to recapitulate anti-PD-1 mAb activity. A similar mechanism was observed in a vaccine model. Finally, nivolumab also boosted human Tfh cells in humanized mice. We propose that Tfh cells and IL-4 play a key role in the peripheral activity of anti-PD-1 mAb.

Tumor-intrinsic sensitivity to the pro-apoptotic effects of IFN-γ is a major determinant of CD4+ CAR T-cell antitumor activity.

CD4+ T cells and CD4+ chimeric antigen receptor (CAR) T cells display highly variable antitumor activity in preclinical models and in patients; however, the mechanisms dictating how and when CD4+ T cells promote tumor regression are incompletely understood. With the help of functional intravital imaging, we report that interferon (IFN)-γ production but not perforin-mediated cytotoxicity was the dominant mechanism for tumor elimination by anti-CD19 CD4+ CAR T cells. Mechanistically, mouse or human CD4+ CAR T-cell-derived IFN-γ diffused extensively to act on tumor cells at distance selectively killing tumors sensitive to cytokine-induced apoptosis, including antigen-negative variants. In anti-CD19 CAR T-cell-treated patients exhibiting elevated CAR CD4:CD8 ratios, strong induction of serum IFN-γ was associated with increased survival. We propose that the sensitivity of tumor cells to the pro-apoptotic activity of IFN-γ is a major determinant of CD4+ CAR T-cell efficacy and may be considered to guide the use of CD4+ T cells during immunotherapy.

IL2/Anti-IL2 Complex Combined with CTLA-4, But Not PD-1, Blockade Rescues Antitumor NK Cell Function by Regulatory T-cell Modulation.

High-dose IL2 immunotherapy can induce long-lasting cancer regression but is toxic and insufficiently efficacious. Improvements are obtained with IL2/anti-IL2 complexes (IL2Cx), which redirect IL2 action to CD8+ T and natural killer (NK) cells. Here, we evaluated the efficacy of combining IL2Cx with blockade of inhibitory immune pathways. In an autochthonous lung adenocarcinoma model, we show that the IL2Cx/anti-PD-1 combination increases CD8+ T-cell infiltration of the lung and controls tumor growth. In the B16-OVA model, which is resistant to checkpoint inhibition, combination of IL2Cx with PD-1 or CTLA-4 pathway blockade reverses that resistance. Both combinations work by reinvigorating exhausted intratumoral CD8+ T cells and by increasing the breadth of tumor-specific T-cell responses. However, only the IL2Cx/anti-CTLA-4 combination is able to rescue NK cell antitumor function by modulating intratumoral regulatory T cells. Overall, association of IL2Cx with PD-1 or CTLA-4 pathway blockade acts by different cellular mechanisms, paving the way for the rational design of combinatorial antitumor therapies.

A common transcriptomic program acquired in the thymus defines tissue residency of MAIT and NKT subsets.

Mucosal-associated invariant T (MAIT) cells are abundant T cells with unique specificity for microbial metabolites. MAIT conservation along evolution indicates important functions, but their low frequency in mice has hampered their detailed characterization. Here, we performed the first transcriptomic analysis of murine MAIT cells. MAIT1 (RORγtneg) and MAIT17 (RORγt+) subsets were markedly distinct from mainstream T cells, but quasi-identical to NKT1 and NKT17 subsets. The expression of similar programs was further supported by strong correlations of MAIT and NKT frequencies in various organs. In both mice and humans, MAIT subsets expressed gene signatures associated with tissue residency. Accordingly, parabiosis experiments demonstrated that MAIT and NKT cells are resident in the spleen, liver, and lungs, with LFA1/ICAM1 interactions controlling MAIT1 and NKT1 retention in spleen and liver. The transcriptional program associated with tissue residency was already expressed in thymus, as confirmed by adoptive transfer experiments. Altogether, shared thymic differentiation processes generate "preset" NKT and MAIT subsets with defined effector functions, associated with specific positioning into tissues.

Induction of anergic or regulatory tumor-specific CD4+ T cells in the tumor-draining lymph node.

CD4+ T cell antitumor responses have mostly been studied in transplanted tumors expressing secreted model antigens (Ags), while most mutated proteins in human cancers are not secreted. The fate of Ag-specific CD4+ T cells recognizing a cytoplasmic Ag in mice bearing autochthonous tumors is still unclear. Here we show, using a genetically engineered lung adenocarcinoma mouse model, that naive tumor-specific CD4+ T cells are activated and proliferate in the tumor-draining lymph node (TdLN) but do not differentiate into effectors or accumulate in tumors. Instead, these CD4+ T cells are driven toward anergy or peripherally-induced Treg (pTreg) differentiation, from the early stage of tumor development. This bias toward immune suppression is restricted to the TdLN, and is maintained by Tregs enriched in the tumor Ag-specific cell population. Thus, tumors may enforce a dominant inhibition of the anti-tumor CD4 response in the TdLN by recapitulating peripheral self-tolerance mechanisms.

Rationale for treating primary Sjögren's syndrome patients with an anti-CD6 monoclonal antibody (Itolizumab).

CD6 is a cell surface receptor expressed on the majority of T cells and a subset of B cells. When expressed, CD6 contributes to lymphocyte activation through its extracellular domain 1, while adhesion and cellular migration are related to the extracellular scavenger receptor cysteine-rich domain (SRCR-D)-3 of CD6. Itolizumab, clone T1h, is a newly developed humanized IgG1 monoclonal antibody that targets CD6 SRCR-D1 and blocks immune activation. Itolizumab has been proposed to be effective in autoimmune diseases such as rheumatoid arthritis, Sjögren's syndrome and multiple sclerosis. In Sjögren's syndrome, the utilization of itolizumab as therapeutic option is reinforced by our recent observation that ALCAM, the CD6 ligand, is overexpressed and that CD6-positive T and B cells are detected within salivary glands from Sjögren's syndrome patients. In this study, itolizumab-positive target cells were characterized within both peripheral blood and salivary glands in order to provide rational for anti-CD6 treatment in Sjögren's syndrome.

Non-classical binding of a polyreactive α-type anti-idiotypic antibody to B cells.

Detailed information on the immunological relevance of α-type anti-idiotypic antibodies is lacking after more than 30 years since Jerne postulated his Idiotypic Network Theory. The B7Y33 mutant is a mouse-human chimeric version of the B7 MAb, a polyreactive α-type anti-idiotypic antibody, generated against an anti-GM2 ganglioside IgM Ab1 antibody. It retained the unusual self-binding activity and multispecificity of the parental murine antibody, being able to recognize several anti-ganglioside IgM antibodies as well as non-immunoglobulin antigens. Previous work with the murine B7 MAb suggested that this antibody might have immunoregulatory properties, and therefore we investigated the possible interaction of B7Y33 with immune cells. We found that B7Y33 binds to human and murine B lymphocytes. Inhibition assays using flow cytometry indicated that this antibody is capable of binding the Fc γ receptor II (FcγRII). The recognition of FcγRII-expressing K562, Raji and Daudi human cell lines, together with the capability of inhibiting the binding of an anti-human FcγRII antibody to these cells, suggest that B7Y33 interacts with both the FcγRIIa and FcγRIIb isoforms. We evaluated the contribution to the binding of different surface-exposed residues at the top of the heavy chain variable region (VH) CDR loops through the construction of mutants with substitutions in the three conventional VH CDRs (HCDRs) and the "HCDR4", located in the framework 3 (HFR3). In addition, we assessed the involvement of the Fc region by performing key mutations in the CH2 domain. Furthermore, chimeric hybrid molecules were obtained by combining the B7Y33 heavy chain with unrelated light chains. Our results indicate that the multispecificity and self-binding properties of B7Y33 are not linked to its recognition of B lineage cells, and that this phenomenon occurs in a non-classical way with the participation of both the variable and constant regions of the antibody. Two possible models for this interaction are proposed, with B7Y33 binding to two FcγRIIb molecules through the Fc and Fv regions, or simultaneously to FcγRIIb and another unknown antigen on B cells. The FcγRIIb has recently received great attention as an attractive target for therapies directed to B lymphocytes. The recognition of peripheral B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) patients by B7Y33 suggests its potential application for the treatment of B cell malignancies.

Aberrant expression of CD6 on B-cell subsets from patients with Sjögren's syndrome.

CD6 is one of a pair of related genes encoding CD5-associated receptors on all T cells and a subset of B cells. The current availability of "T1h", a humanized anti-CD6 monoclonal antibody for B cell-mediated autoimmune disorders revives analysis of the B-cell subset expression of CD6, particularly in primary Sjögren's syndrome (SS). Refined phenotype of B-lymphocytes peripheral blood (PB), bone marrow and tonsils revealed that the overlap between the expression of CD6 is less close to that of CD5 than currently acknowledged. In contrast to CD5, CD6 is absent on transitional B cells, while present on mature and memory B cells. Interestingly, the PB proportion of CD6(+) B cells is decreased in patients with primary SS, as opposed to those with rheumatoid arthritis. The reduction in primary SS does not result from the shedding of CD6 from the membrane of B cells, but from the lowering of memory B lymphocytes. It may result from the ability of CD6 to make transmigration of CD27(+) memory B cells into the salivary glands (SGs) easier. Consistent with this view is our finding that CD166 (one of the ligands for CD6) is highly expressed on epithelial cells of patients' SGs. This study is relevant in that the humanized T1h anti-CD6 becomes an alternative to anti-CD20 for treatment of primary SS.

Recombinant nucleocapsid-like particles from dengue-2 virus induce protective CD4+ and CD8+ cells against viral encephalitis in mice.

Virus-like particles are a highly effective type of subunit vaccine that mimics the overall structure of virus particles without containing infectious genetic material. In this work, a particulate form of the recombinant capsid protein from dengue-2 was evaluated in mice to determine the level of protection against viral challenge and to measure the antigen-induced cell-mediated immunity (CMI). The nucleocapsid-like particles (NLPs) adjuvanted with alum did not induce antiviral antibodies. However, splenocytes from the immunized animals secreted high levels of IFN-gamma upon virus stimulation, and a significant protection rate was achieved after challenge with lethal dengue-2 virus. Finally, both IFN-gamma secretion and protection against viral encephalitis were demonstrated to be dependent on CD4(+) and CD8(+) cells. This study provides new evidences regarding the protective role of the CMI in the mouse model without the induction of neutralizing antibodies. Further studies in non-human primates or humanized mice should be carried out to elucidate the usefulness of the NLPs as a potential vaccine candidate against dengue disease.

Towards the definition of a chimpanzee and human conserved CD6 domain 1 epitope recognized by T1 monoclonal antibody.

Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.

Biological activity in vitro of anti-epidermal growth factor receptor monoclonal antibodies with different affinities.

Epidermal growth factor receptor (EGFR) is frequently overexpressed in epithelial tumors and is associated with a poor prognosis. An increasing interest in developing anti-EGFR therapies has resulted in the evaluation of monoclonal antibodies with the capacity to bind to the EGFR, inhibiting EGFR-dependent cellular transformation. A differential toxicity and therapeutic effect in vivo are associated with the affinity and isotype of the molecule. In this study, we examined the biological activities of three monoclonal antibodies (MAbs) -- Ior egf/r3 (mouse IgG2a, 10(-9) M), Nimotuzumab (humanized IgG1, 10(-9) M), and Cetuximab (human/mouse chimeric IgG1, 10(-10) M) -- considering inhibition of cell proliferation, apoptosis, and complement-mediated cell death in squamous cell carcinoma A431 in vitro. All the antibodies bound to the EGFR on these cells, inhibiting the receptor phosphorylation, as measured by flow cytometry, inmunocytochemistry, and Western blot. Exposure to the different antibodies inhibited cell proliferation in culture in a range from 50 to 80% compared to controls. Furthermore, similar capabilities to induce either complement-mediated cytotoxicity (ranging between 70 and 90%) or a two-fold increase in the rate of apoptotic cells were found when tumor cells were exposed to the antibodies. These results suggest that the affinity between specific anti-EGFR antibodies and its receptor could affect, but not determine their biological activity at least in those cell lines that exhibit high sensitivity to withheld EGFR. Our findings also confirm previous evidences that blocking EGFR in A431 cells by means of antibodies significantly changes tumor cell biology by promoting apoptosis while decreasing tumor cell proliferation.

Immune responses in breast cancer patients immunized with an anti-idiotype antibody mimicking NeuGc-containing gangliosides.

A phase I clinical trial was conducted in patients with stage III/IV breast cancer who were treated with the anti-idiotype mAb 1E10 specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with antigens expressed on human melanoma and breast carcinoma cells. Patients were treated with 1 or 2 mg of aluminum hydroxide-precipitated 1E10 mAb every other week for six injections. Two patients at each dose were reimmunized 7-9 months after completing the induction phase. In hyperimmune sera from eight of the nine patients who received at least four doses of anti-Id vaccine preparations, strong specific responses were observed both against 1E10 mAb and NeuGc-GM3 ganglioside (Ab3 Id+Ag+). Nonclassical Ab1' antibodies (Id-Ag+) were also elicited by 1E10 mAb vaccine treatment. There were no differences between the two levels of dose tested in relation to toxicity and immunogenicity. No evidence of serious or unexpected effects was observed.