Soluble CTLA-4 attenuates T cell activation and modulates anti-tumor immunity.

CTLA-4 is a crucial immune checkpoint receptor involved in the maintenance of immune homeostasis, tolerance, and tumor control. Antibodies targeting CTLA-4 have been promising treatments for numerous cancers, but the mechanistic basis of their anti-tumoral immune-boosting effects is poorly understood. Although the ctla4 gene also encodes an alternatively spliced soluble variant (sCTLA-4), preclinical/clinical evaluation of anti-CTLA-4-based immunotherapies have not considered the contribution of this isoform. Here, we explore the functional properties of sCTLA-4 and evaluate the efficacy of isoform-specific anti-sCTLA-4 antibody targeting in a murine cancer model. We show that expression of sCTLA-4 by tumor cells suppresses CD8+ T cells in vitro and accelerates growth and experimental metastasis of murine tumors in vivo. These effects were accompanied by modification of the immune infiltrate, notably restraining CD8+ T cells in a non-cytotoxic state. sCTLA-4 blockade with isoform-specific antibody reversed this restraint, enhancing intratumoral CD8+ T cell activation and cytolytic potential, correlating with therapeutic efficacy and tumor control. This previously unappreciated role of sCTLA-4 suggests that the biology and function of multi-gene products of immune checkpoint receptors need to be fully elucidated for improved mechanistic understanding of cancer immunotherapies.

Hiding in Plain Sight: Soluble Immunomodulatory Receptors.

Alternatively spliced natural soluble isoforms of immunomodulatory receptors [cytotoxic T lymphocyte antigen-4 (CTLA-4), 4-1BB, and programmed death-1 (PD-1)/PD-L1] have been overlooked in favor of their cell-surface-bound counterparts that have generated blockbuster antibodies for the treatment of cancer. We propose that the soluble variants of these receptors contribute to immune regulation and offer potential as targets for immunotherapy.

The soluble isoform of CTLA-4 as a regulator of T-cell responses.

CTLA-4 is a crucial immune regulator that mediates both negative costimulation signals to T cells, and regulatory T (Treg)-cell extrinsic control of effector responses. Here we present evidence supporting a novel mechanism for this extrinsic suppression, executed by the alternatively spliced soluble CTLA-4 isoform (sCTLA-4). Analyses of human T cells in vitro show that sCTLA-4 secretion can be increased during responses, and has potent inhibitory properties, since isoform-specific blockade of its activity significantly increased Ag-driven proliferation and cytokine (IFN-γ, IL-17) secretion. Treg cells were demonstrated to be a prominent source of sCTLA-4, which contributed to suppression in vitro when their numbers were limiting. The soluble isoform was also produced by, and inhibited, murine T cells responding to Ag in vitro, and blockade of its activity in vivo protected against metastatic spread of melanoma in mice. We conclude that sCTLA-4 is an important immune regulator, responsible for at least some of the inhibitory effects previously ascribed to the membrane-bound isoform. These results suggest that the immune system exploits the different CTLA-4 isoforms for either intrinsic or extrinsic regulation of T-cell activity.

On the Road to Immunotherapy-Prospects for Treating Head and Neck Cancers With Checkpoint Inhibitor Antibodies.

Head and neck cancers (HNC) represent a heterogeneous cluster of aggressive malignancies that account for 3% of all cancer cases in the UK. HNC is increasing in frequency particularly in the developing world, which is related to changes in risk factors. Unfortunately, the mortality rate is high, which is chiefly attributed to late diagnosis at stages where traditional treatments fail. Cancer immunotherapy has achieved great successes in anti-tumor therapy. Checkpoint inhibitor (CI) antibodies enhance anti-tumor activity by blocking inhibitory receptors to drive tumor-specific T and NK cell effector responses. Since their introduction in 2011, CI antibodies have been approved for many cancer types including HNC. Here, we examine the development of CI therapies and look forward to future developments for treatment of HNC with CI therapies.

Deciphering the Role of Regulatory CD4 T Cells in Oral and Oropharyngeal Cancer: A Systematic Review.

Background: Recruiting regulatory CD4 T cells (Tregs) into the tumor microenvironment is an important tumor escape mechanism. Diminishing these suppressive cells is therefore one of the targets of cancer immunotherapy. Selective depletion of Tregs has proven successful in enhancing anti-tumor immunity and therapeutic efficacy in multiple tumor types. However, the role of Tregs in oral/oropharyngeal cancers is unclear with conflicting evidence regarding the effect of these suppressive cells on tumor prognosis. In this study, we sought to review the role of Tregs in oral/oropharyngeal cancer with the aim of deciphering the controversy regarding their effect on tumor progression and prognosis. Methods: A systematic review of the literature pertaining to the role of Tregs in oral/oropharyngeal cancer was performed using Scopus, Embase, and PubMed. Forty-five records were deemed eligible and data describing methodology of Treg detection, tumor type, and association with prognosis were extracted. Results: Of the 45 eligible manuscripts accepted for this systematic review, thirty-nine studies reported data from human subjects while the remaining studies focused on animal models. Sixteen studies were carried out using peripheral blood samples, while samples from the tumor site were analyzed in 18 studies and 11 studies assessed both blood and tumor samples. The transcriptional factor, Foxp3, was the most commonly used marker for Treg identification (38/45). The findings of 25 studies suggested that an increase in Tregs in the tumor microenvironment and/or peripheral blood was associated with poorer prognosis. These conclusions were attributed to the suppression of immune responses and the consequent tumor progression. Conversely, nine studies showed an increase in Tregs in peripheral blood and/or tumor microenvironment was related to a favorable prognosis, particularly in the presence of human papilloma virus (HPV), the status of which was only assessed in 11 studies. Conclusions: This review underlines the importance of host immunity in the behavior of oral/oropharyngeal cancer. Furthermore, we report an apparent lack of clarity regarding the true role Tregs play in oral/oropharyngeal cancer progression which could be attributed to inconsistent detection techniques of Tregs. Our results therefore highlight the need for clearer methodologies and more robust phenotyping when defining Tregs.

Controlling autoimmunity--Lessons from the study of red blood cells as model antigens.

The characterization of human and animal red blood cell (RBC) autoantigens in autoimmune hemolytic anemia (AIHA) has provided an opportunity study the control of specific autoimmune responses of unequivocal pathogenic relevance. The results reveal that censorship of the autoimmune helper T (Th) cell repertoire by deletion and anergy is very incomplete in healthy individuals, even for widely distributed, abundant self-antigens on RBC. There is strong evidence that autoaggression by surviving Th cells is normally held in check by other mechanisms, including failure to display the epitopes that they recognize, and active immunoregulation. AIHA is one of the first human autoimmune diseases in which regulatory T (Tr) cells that are specific for the major autoantigens have been identified. These Tr cells recognize the dominant naturally processed epitopes, and recent studies suggest that disease develops when other determinants, to which such tolerance is less secure, and which are normally inefficiently presented, are displayed at higher levels. Together, the results raise the possibility that therapy for diseases such as AIHA could be based on switching the balance of the response back towards regulation, in particular by the administration of the dominant peptides recognized by specific Tr cells.

Immunoregulatory soluble CTLA-4 modifies effector T-cell responses in systemic lupus erythematosus.

BACKGROUND: The 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). METHODS: The 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. RESULTS: We 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. CONCLUSION: The 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.

Isolation, polarization, and expansion of CD4⁺ helper T cell lines and clones using magnetic beads.

Autoreactive CD4⁺ helper T cells specific for a range of nucleoprotein-derived autoantigens are an important feature of systemic lupus erythematosus, driving B cell differentiation and autoantibody production and contributing to the inflammatory lesions caused by immune complex deposition. Several peptide epitopes from nucleoprotein antigens have been identified and offer a means selectively to manipulate T cell responses by skewing toward a profile of cytokines that is less pro-inflammatory. Antigen-specific T cell lines and clones can be useful in the study of helper T cell subsets because their life span is prolonged and many individual cells can be generated, allowing particular phenotypes to be studied in detail. Magnetic beads offer a robust and convenient method for the isolation, polarization, and expansion of T cells, which can be adapted for a broad range of applications.

Targeting the alternatively spliced soluble isoform of CTLA-4: prospects for immunotherapy?

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.

Clonal regulatory T cells specific for a red blood cell autoantigen in human autoimmune hemolytic anemia.

Regulatory T (Tr) cells have the potential to treat immune-mediated disease, but cloning such cells for study from patients with autoimmune disease has proven difficult. Here, we describe autoantigen-specific, interleukin-10 (IL-10)-secreting Tr cell clones recovered ex vivo from a patient with autoimmune hemolytic anemia (AIHA) and characterize their phenotype, origin, and regulatory function. These IL-10+ Tr cells recognized a peptide, 72H-86L, derived from the Rh red blood cell autoantigen and shared phenotypic characteristics with both natural and inducible Tr cells. The clones also expressed different Tr markers depending on activation state: high levels of CD25 and LAG-3 when expanding nonspecifically, but FoxP3 after activation by the autoantigen they recognize. Despite a discrete Tr phenotype, these cells stably expressed the T helper 1 (Th1) signature transcription factor T-bet, suggesting they derive from Th1 T cells. Finally, the contribution of CTLA-4 in activating these IL-10+ Tr cells was confirmed by analyzing responses to transgenic B7.1-like molecules that preferentially bind either CD28 or CTLA-4. Overall, these Tr cells have a functional phenotype different from those described in previous studies of human Tr populations, which have not taken account of antigen specificity, and understanding their properties will enable them to be exploited therapeutically in AIHA.

Deletion of the dominant autoantigen in NZB mice with autoimmune hemolytic anemia: effects on autoantibody and T-helper responses.

The mechanisms underlying apparently spontaneous autoimmune diseases, such as autoimmune hemolytic anemia (AIHA) in New Zealand Black (NZB) mice, are unknown. Here, we determine the contribution of the dominant red blood cell (RBC) autoantigen, the anion exchanger protein Band 3, to the development of NZB autoimmune responses. The approach was to prevent Band 3 expression in NZB mice by disrupting the AE1 gene. AE1(-/-) NZB mice produced RBC autoantibodies at the same levels as the wild-type strain, but they differed in recognizing antigens that correspond to glycophorins, rather than Band 3. Splenic T-helper (Th) cells from wild-type NZB mice proliferated strongly against multiple Band 3 peptides, particularly the dominant epitope within aa861-874. This helper response was severely attenuated in AE1(-/-) animals, leaving only weak proliferation to peptide aa861-874. The results demonstrate that the defect in self-tolerance in NZB AIHA is directed to the RBC type, and is not specific for, or dependent on, Band 3. However, the predisposition to RBC autoimmunity may be focused onto Band 3 by weak Th cell cross-reactivity between the helper dominant epitope and an exogenous antigen. The redundancy of the major autoantigen illustrates the requirement for specific therapy to induce dominant forms of tolerance, such as T-cell regulation.

Histone peptide-induced nasal tolerance: suppression of murine lupus.

Induced mucosal tolerance has been shown to be beneficial in preventing or treating a number of murine and human autoimmune disorders. However, this particular form of therapy has not been thoroughly tested in systemic lupus erythematosus. In this study, we investigated the conditions for induction of nasal tolerance using a histone peptide named H471 expressing a dominant T cell epitope in the histone protein H4 of mononucleosome in lupus-prone SNF(1) female mice. We also tested the effect of chronic peptide nasal treatment on the development of autoimmune reactivities in these mice. Results demonstrated that a dose-dependent nasal tolerance to peptide H471 can be achieved before or after peptide sensitization in SNF(1) mice. In addition, tolerance to mononucleosomes was induced by nasal instillation of SNF(1) mice with H471. This was accompanied by an increase in IL-10 and suppression of IFN-gamma production by lymph node cells. Suppression of Th1-type cytokines was also observed in SNF(1) mice that were nasally administered with H471 before intradermal injection with the peptide. Finally, chronic nasal instillation of mice with the H471 peptide not only suppressed the development of autoantibodies, but also altered the severity of glomerulonephritis in lupus-prone SNF(1) mice.

Autoreactivity in collagen-induced arthritis of rats: a potential role for T cell responses to self MHC peptides.

Collagen-induced arthritis (CIA) is a chronic inflammatory arthropathy of rats which follows immunization with bovine type II collagen (bCII). T cell lines generated from arthritic rats have been shown to be self-reactive and proliferate in an autologous MLR, which is MHC-dependent. However, the peptides which drive this autoreactive response remain to be elucidated. T cell lines, generated initially to bCII, were cultured with synthetic peptides representing potential autoreactive self epitopes. C1q-c(50-64) peptide, which demonstrates sequence homology to the bCII(184-198) peptide, failed to stimulate T cell proliferation suggesting that the autologous MLR was not due to antigen cross-reactivity with this self peptide. In contrast, several peptides from the amino-terminal region of the RT1D(u) MHC class II molecule stimulated proliferative responses. These results suggest that immunization with bCII leads to activation of a population of autoreactive T cells which respond in an autologous MLR, and that this response could be due, in part, to T cell reactivity to self MHC peptides.

Interleukin-10-mediated regulatory T-cell responses to epitopes on a human red blood cell autoantigen.

Regulatory T cells have been shown to control animal models of immune-mediated pathology by inhibitory cytokine production, but little is known about such cells in human disease. Here we characterize regulatory T-cell responses specific for a human red blood cell autoantigen in patients with warm-type autoimmune hemolytic anemia. Peripheral blood mononuclear cells from patients with autoimmune hemolytic anemia were found either to proliferate and produce interferon-gamma or to secrete the regulatory cytokine interleukin 10 when stimulated in vitro with a major red blood cell autoantigen, the RhD protein. Flow cytometric analysis confirmed that the majority of the responding cells were of the CD4(+) phenotype. Serial results from individual patients demonstrated that this bias toward proliferative or interleukin-10 responses was unstable over time and could reverse in subsequent samples. Epitope mapping studies identified peptides from the sequence of the autoantigen that preferentially induced interleukin-10 production, rather than proliferation, and demonstrated that many contain naturally processed epitopes. Responses to such peptides suppressed T-cell proliferation against the RhD protein, an inhibition that was mediated largely by interleukin 10 and dependent on cytotonic T lymphocyte-associated antigen (CTLA-4) costimulation. Antigenic peptides with the ability to stimulate specific regulatory cells may represent a new class of therapeutic agents for immune-mediated disease.

Characterization of the dominant autoreactive T-cell epitope in spontaneous autoimmune haemolytic anaemia of the NZB mouse.

NZB mice spontaneously develop autoimmune haemolytic anaemia (AIHA) due to a T helper-dependent autoantibody response against the erythrocyte anion channel protein, Band 3. Here, we characterize the recognition of the Band 3 sequence 861-874, which carries the dominant, I-E(d)-restricted T cell epitope. The ability of N and C-terminal truncated versions of peptide 861-874 to elicit NZB splenic T-cell proliferation indicated that the core epitope spans residues 862-870. Next, a set of alanine substitution analogues was tested to determine which residues functioned either as MHC anchor or TCR contact residues. A combination of proliferation and MHC:peptide binding assays identified residues 862(L), 864(V), 865(L), and 869(K) as I-E(d) anchor residues, and 868(V) as the only TCR contact residue. The ability of the wild-type sequence 861-874 to compete with a high affinity reference peptide for binding to I-E(d) indicates that the escape of pathogenic NZB T cells from purging of the autoreactive repertoire cannot be attributed to ineffective presentation of peptide 861-874 by its restricting element. It will now be possible to design altered peptide ligands of Band 3 861-874, in order to further dissect the mechanisms responsible for the maintenance and loss of T cell tolerance to RBC autoantigens, and to modulate the immune response in AIHA.