CTLA-4 is an inhibitory protein that contributes to immune homeostasis and tolerance, a role that has led to its exploitation as a therapeutic in several clinical settings including cancer and autoimmune disease. Development of CTLA-4 therapies focused largely on the full-length receptor isoform but other CTLA-4 isoforms are also expressed, including a secretable form of CTLA-4 (soluble CTLA-4 [sCTLA-4]). The contribution of sCTLA-4 to immune regulation has been less well studied, primarily because it was identified some years after the original description of CTLA-4. Here, we examine how sCTLA-4 might contribute to immune regulation and ask whether it might be a biomarker to inform current CTLA-4 therapies or represent a novel CTLA-4 target for future therapeutics.

BackgroundThe inhibitory CTLA-4 molecule is a crucial regulator of immune responses and a target for therapeutic intervention in both autoimmunity and cancer. In particular, CTLA-4 is important in controlling antigen-specific immunity, including responses to autoantigens associated with autoimmune disease. Here, we investigate cytokine responses to a range of lupus-associated autoantigens and assess whether the alternatively spliced isoform of CTLA-4, soluble CTLA-4 (sCTLA-4), contributes to immune regulation of autoantigen-specific immunity in systemic lupus erythematosus (SLE).MethodsThe cell culture supernatant production of sCTLA-4 as well as the cytokines IL-10, IFN-γ, and IL-17 from peripheral blood mononuclear cells (PBMC) from lupus patients and age- and sex-matched healthy volunteer donors were measured in response to previously identified histone and small nuclear ribonucleoprotein (snRNP) autoantigen-derived peptides (H391-105, H471-93, and U170K131-151) by ELISA. We also examined the functional contribution of sCTLA-4 to immune regulation in the context of these autoantigenic peptides following blockade of sCTLA-4 with a selective anti-sCTLA-4 monoclonal antibody, JMW-3B3.ResultsWe identified responses to autoantigenic peptides, which revealed qualitative differences in cytokine (IL-10, IL-17, and IFN-γ) profiles between SLE patients and healthy donors. PBMC from healthy donors responded to each of the lupus peptides by secreting IFN-γ and IL-17, but PBMC from SLE patients produced IL-10. Although we did not observe differences in the levels of serum or PBMC culture supernatant sCTLA-4 in either cohort, blockade of sCTLA-4 in PBMC cultures responding to antigen enhanced the cytokine profiles associated with each group.ConclusionThe results show that lupus autoantigen-derived peptides display varied immunogenicity in lupus versus healthy volunteer donors, while sCTLA-4 acts to regulate the T-cell activity independently of response profile.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1075-1) contains supplementary material, which is available to authorized users.

Inhibitory receptors of the human leukocyte immunoglobulin-like receptor family are constitutively expressed on all myeloid cell types and regulate their functional activity. We demonstrate that ligation of the human leukocyte antigen class I-specific receptor LILRB1, during the differentiation of monocytes to dendritic cells in vitro, results in increased expression of the nuclear factor κB inhibitor protein ABIN1 (also known as TNIP1). Similarly increased expression of ABIN1/TNIP1 was observed in the "immunosuppressive" monocyte populations of patients with non-Hodgkin lymphoma ex vivo. Reducing expression of ABIN1/TNIP1 using small interfering ribonucleic acid allows dendritic cells and immunosuppressive monocytes to respond to stimulation by allowing nuclear factor κB translocation to the nucleus (P < 0.001), increasing cell surface expression of antigen presentation and costimulatory molecules (P < 0.01), increasing phagocytic capacity (P < 0.001), secreting proinflammatory cytokines (P < 0.01), and an increasing ability to stimulate T cell responses (P < 0.05). Our study, therefore, identifies an important functional role for ABIN1/TNIP1 in mediating the effects of LILRB1 ligation-induced inhibitory effects on immune responses. Our findings suggest that inhibiting the LILRB1-ABIN1/TNIP1 pathway in antigen-presenting cells could be a therapeutic approach to stimulate antitumor immune responses. Conversely, stimulation of the pathway may also ameliorate autoimmune diseases in which TNIP1 is a susceptibility gene.

Malignant melanoma is an aggressive form of cancer, which can be treated with anti-CTLA-4 and anti-PD-1 checkpoint inhibitor antibodies but while anti-CTLA-4 antibodies have clear benefits for some patients with melanoma, productive responses are difficult to predict and often associated with serious immune related adverse events. Antibodies specific to CTLA-4 bind two major isoforms of CTLA-4 in humans, the receptor isoform and a second naturally secretable, soluble isoform - sCTLA-4. The primary aim here was to examine the effect of selectively blocking the function of sCTLA-4 on in vitro immune responses from volunteer healthy or melanoma patient PBMC samples. Addition of recombinant sCTLA-4 to healthy PBMC samples demonstrated sCTLA-4 to have immunosuppressive capacity comparable to recombinant CTLA4-Ig, partially reversible upon antibody blockade. Further, we identified a mechanistic relationship where melanoma patient TGFβ2 serum levels correlated with sCTLA-4 levels and provided the basis for a novel protocol to enhance sCTLA-4 production and secretion by T cells with TGFβ2. Finally, a comparison of selective antibody blockade of sCTLA-4 demonstrated that both healthy and melanoma patient effector cytokine responses can be significantly increased. Overall, the data support the notion that sCTLA-4 is a contributory factor in cancer immune evasion.

BackgroundImmTAC molecules are bispecific fusion proteins consisting of an affinity-enhanced T cell receptor fused to an anti-CD3 effector that can redirect T cells to target cells. Tebentafusp, a gp100-directed ImmTAC, has demonstrated survival benefit in metastatic uveal melanoma [1]. We previously reported that a tumor microenvironment with a high immunosuppressive CD163+ tumor-associated macrophage (TAM): CD3 T cell ratio was associated with reduced benefit from tebentafusp [2]. Here, we explored whether IL-2 could potentiate T cells to overcome inhibition of ImmTAC-mediated killing by TAM-like M2 macrophages.MethodsTumor biopsies from a Phase 2 trial of metastatic uveal melanoma HLA-A*02:01+ patients treated with tebentafusp (NCT02570308) were used to quantify CD163+ TAMs and CD3+ T cells by immunohistochemistry (N=107) and to measure gene expression by bulk RNAseq (N=70). Pro-inflammatory M1 and TAM-like M2 macrophages were generated in vitro from healthy donors (N=5) and their effect on ImmTAC-mediated T cell activation and tumor killing was assessed against THP-1 tumor cells. T cells were untreated or pre-treated for 4 days with commercially sourced IL-2 or IL-15.ResultsIn vitro, ImmTAC-mediated T cell killing of tumor cells was reduced by 85±5% in the presence of TAM-like M2 but not M1 macrophages. Consistent with this finding, below median CD163:CD3 ratio was associated with greater tumor shrinkage (TS) (odds ratio OR=2.9, p=0.014) and longer overall survival (OS) (hazard ratio HR=0.4, p=0.001) in tebentafusp-treated patients. We next explored in vitro whether the T cell activating cytokines IL-2 and IL-15 could overcome TAM-mediated inhibition of T cell redirection by ImmTAC. At clinically relevant doses, IL-2 but not IL-15 treatment resulted in dose dependent restoration of ImmTAC-mediated T cell killing of tumor cells in the presence of TAM-like M2 macrophages—60% and 83% restoration of killing at 50 and 150 U/ml of IL-2, respectively. Consistent with this observation, increased expression of IL2RB (HR 0.3, p<0.001) and IL2RG (HR 0.4, p=0.002), but not IL2RA (HR 0.8, p=0.5) in tumors was associated with longer OS on tebentafusp.ConclusionsLow CD163+ TAM to CD3 T cell ratio and high IL2RB/G expression in tumors at baseline were associated with longer OS and greater TS in tebentafusp-treated metastatic uveal melanoma patients in a Phase 2 trial. In vitro, TAM-like M2 macrophages suppressed ImmTAC-mediated T cell killing of tumor cells, an effect abrogated by IL-2. These observations provide strong rationale for combining IL-2 biased to IL2RB/G with ImmTAC molecules to enhance benefit in tumors with high levels of TAMs.Trial RegistrationNCT02570308ReferencesPiperno-Neumann S, Hassel JC, Rutkowski P et al. Abstract CT002: Phase 3 randomized trial comparing tebentafusp with investigator’s choice in first line metastatic uveal melanoma. Cancer Res. 2021; 81 (13 Supplement) CT002.Hassel JC, Benlahrech A, Stanhope S, et al. Abstract 1673: Uveal melanoma study patients with low CD163:CD3 ratio in tumor biopsy and low serum IL-6 showed enhanced tumor shrinkage (TS) and overall survival (OS) on tebentafusp. Cancer Res. 2021; 81 (13 Supplement) 1673.Ethics ApprovalThe Oxford A REC approved protocol 13/SC/0226 was used to obtain written consent for all blood donations and was fully approved by the National Research Ethics Committee South Central.