The emerging role of CTLA4 as a cell-extrinsic regulator of T cell responses

The T cell protein cytotoxic T lymphocyte antigen 4 (CTLA4) was identified as a crucial negative regulator of the immune system over 15 years ago, but its mechanisms of action are still under debate. It has long been suggested that CTLA4 transmits an inhibitory signal to the cells that express it. However, not all the available data fit with a cell-intrinsic function for CTLA4, and other studies have suggested that CTLA4 functions in a T cell-extrinsic manner. Here, we discuss the data for and against the T cell-intrinsic and -extrinsic functions of CTLA4.     

Tumour cell‐intrinsic CTLA4 regulates PD‐L1 expression in non‐small cell lung cancer

Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD‐1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non‐small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD‐1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti‐CTLA4 antibody can induce PD‐L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti‐CTLA4 antibody was not able to induce PD‐L1 expression in NSCLC cells. Moreover, anti‐CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell‐intrinsic CTLA4 can regulate PD‐L1 expression and cell proliferation, and that anti‐CTLA4 antibody, by binding to the tumour cell‐intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.     

Immunosuppression in Early Postnatal Days Induces Persistent and Allergen-Specific Immune Tolerance to Asthma in Adult Mice

Bronchial asthma is a chronic airway inflammatory condition with high morbidity, and effective treatments for asthma are limited. Allergen-specific immunotherapy can only induce peripheral immune tolerance and is not sustainable. Exploring new therapeutic strategies is of great clinical importance. Recombinant adenovirus (rAdV) was used as a vector to make cells expressing cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4Ig) a soluble CTLA4 immunoglobulin fusion protein. Dendritic cells (DCs) were modified using the rAdVs together with allergens. Then these modified DCs were transplanted to mice before allergen sensitization. The persistence and specificity of immune tolerance were evaluated in mice challenged with asthma allergens at 3 and 7 months. DCs modified by CTLA4Ig showed increased IL-10 secretion, decreased IL-12 secretion, and T cell stimulation in vitro. Mice treated with these DCs in the early neonatal period developed tolerance against the allergens that were used to induce asthma in the adult stage. Asthma symptoms, lung damage, airway reactivity, and inflammatory response all improved. Humoral immunity indices showed that this therapeutic strategy strongly suppressed mice immune responses and was maintained for as long as 7 months. Furthermore, allergen cross-sensitization and challenge experiments demonstrated that this immune tolerance was allergen-specific. Treatment with CTLA4Ig modified DCs in the early neonatal period, inducing persistent and allergen-specific immune tolerance to asthma in adult mice. Our results suggest that it may be possible to develop a vaccine for asthma.     

Blockade of CD28-B7, but not CD40-CD154, prevents costimulation of allogeneic porcine and xenogeneic human anti-porcine T cell responses

Despite increasing use of swine in transplantation research, the ability to block costimulation of allogeneic T cell responses has not been demonstrated in swine, and the effects of costimulatory blockade on xenogeneic human anti-porcine T cell responses are also not clear. We have compared the in vitro effects of anti-human CD154 mAb and human CTLA4IgG4 on allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses. Both anti-CD154 mAb and CTLA4IgG4 cross-reacted on pig cells. While anti-CD154 mAb and CTLA4IgG4 both inhibited the primary allogeneic pig MLRs, CTLA4IgG4 (7.88 microg/ml) was considerably more inhibitory than anti-CD154 mAb (100 microg/ml) at optimal doses. Anti-CD154 mAb inhibited the production of IFN-gamma by 75%, but did not inhibit IL-10 production, while CTLA4IgG4 completely inhibited the production of both IFN-gamma and IL-10. In secondary allogeneic pig MLRs, CTLA4IgG4, but not anti-CD154 mAb, induced Ag-specific T cell anergy. CTLAIgG4 completely blocked the indirect pathway of allorecognition, while anti-CD154 mAb blocked the indirect response by approximately 50%. The generation of porcine CTLs was inhibited by CTLA4IgG4, but not by anti-CD154 mAb. Human anti-porcine xenogeneic MLRs were blocked by CTLA4IgG4, but only minimally by anti-CD154 mAb. Finally, CTLA4IgG4 prevented secondary xenogeneic human anti-porcine T cell responses. These data indicate that blockade of the B7-CD28 pathway was more effective than blockade of the CD40-CD154 pathway in inhibiting allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses in vitro. These findings have implications for inhibiting cell-mediated immune responses in pig-to-human xenotransplantation.     

Blockade of T cell costimulation by CTLA4-Ig inhibits lung inflammation in murine hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is characterized by an influx of activated T cells in the lungs. The CD28/B7 system provides costimulatory signals essential for complete T cell activation and differentiation. We have previously demonstrated that alveolar macrophages from patients with HP have an up-regulated expression of B7 molecules. In the present study, we investigated the effect of i. p. administration of CTLA4-Ig, a CD28/B7 antagonist, on the lung inflammation of mice inoculated with Saccharoplyspora rectivirgula (SR), a major causative agent of HP. Five groups of C57BL/6 mice were intranasally instilled with SR or saline for 3 consecutive days per wk during 3 wk. CTLA4-Ig was administered starting either after 1 wk of SR challenge or 6 h before the first antigenic exposure and continued during the whole period of sensitization. A control-IgG was given similarly during the 3 wk of SR exposure. The groups included: 1, saline; 2, SR; 3, SR + control-Ig; 4, SR + CTLA4-Ig for the last 2 wk; and 5, SR + CTLA4-Ig for 3 wk. CTLA4-Ig treatment markedly decreased lung inflammation as shown by significantly fewer inflammatory cells in the bronchoalveolar lavage and in lung tissue and reduced SR-specific serum and bronchoalveolar lavage Ig levels. Production of IL-4, IL-10, and IFN-gamma by IL-2-stimulated pulmonary T cells was also decreased by CTLA4-Ig. Administration of CTLA4-Ig did not affect the SR-induced up-regulation of B7-2 expression. These results show that blockade of CD28/B7 interactions by CTLA4-Ig inhibits SR-induced lung inflammation and immune response to SR Ag in mice and may provide a novel approach in the treatment of HP.     

CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment

Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.     

Feline immunodeficiency virus infection phenotypically and functionally activates immunosuppressive CD4+CD25+ T regulatory cells

Disease progression of feline immunodeficiency virus (FIV) infection is characterized by up-regulation of B7.1 and B7.2 costimulatory molecules and their ligand CTLA4 on CD4(+) and CD8(+) T cells. The CD4(+)CTLA4(+)B7(+) phenotype described in FIV(+) cats is reminiscent of CD4(+)CD25(+)CTLA4(+) cells, a phenotype described for immunosuppressive T regulatory (Treg) cells. In the present study, we describe the phenotypic and functional characteristics of CD4(+)CD25(+) T cells in PBMC and lymph nodes (LN) of FIV(+) and control cats. Similar to Treg cells, feline CD4(+)CD25(+) but not CD4(+)CD25(-) T cells directly isolated from LN of FIV(+) cats do not produce IL-2 and fail to proliferate in response to mitogen stimulation. Unstimulated CD4(+)CD25(+) T cells from FIV(+) cats significantly suppress the proliferative response and the IL-2 production of Con A-stimulated autologous CD4(+)CD25(-) T cells compared with unstimulated CD4(+)CD25(+) T cells from FIV(-) cats. Flow-cytometric analysis confirmed the apparent activation phenotype of the CD4(+)CD25(+) cells in LN of chronically FIV(+) cats, because these cells showed significant up-regulation of expression of costimulatory molecules B7.1, B7.2, and CTLA4. These FIV-activated, anergic, immunosuppressive CD25(+)CTLA4(+)B7(+)CD4(+) Treg-like cells may contribute to the progressive loss of T cell immune function that is characteristic of FIV infection.     

A mechanistic study of immune system activation by fusion of antigens with the ligand-binding domain of CTLA4

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4–Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4–Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy.Dhanalakshmi Chinnasamy and Matt Tector contributed equally to this work     

CTLA4-Ig interacts with cultured synovial macrophages from rheumatoid arthritis patients and downregulates cytokine production

Introduction  

Co-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response. Cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig) binding to the B7 molecules on antigen-presenting cells downregulates this activation and represents a recent biological treatment in rheumatoid arthritis (RA). Objectives of the study were to investigate the presence of the B7.2 (CD86) molecule and its masking by CTLA4-Ig on cultures of both RA synovial macrophages (RA SM), and of macrophages differentiated from THP-1 cells (M). In addition, the anti-inflammatory effects of CTLA4-Ig on co-cultures of RA SM and M with activated T cells were tested.     

Control of memory CD4 T cell recall by the CD28/B7 costimulatory pathway

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62L(high)) and effector memory (T(EM); CD62L(low)) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-gamma production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-gamma production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly T(EM) in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on T(EM) responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of T(EM) cells in autoimmunity and chronic disease.     

Construction, and in vitro and in vivo analyses of tetravalent immunoadhesins

Previous observations demonstrated that various immunosuppressive agents and their combination therapies can increase allograft survival rates. However, these treatments may have serious side effects and cannot substantially improve or prolong graft survival in acute graft-versus-host disease (GVHD). To improve the therapeutic potency of divalent immunoadhesins, we have constructed and produced several tetravalent forms of immunoadhesins comprising each of cytotoxic T-lymphocyte-associated antigen-4 (CTLA4), CD2, and lymphocyte activation gene-3 (LAG3). Flow cytometric and T cell proliferation analyses displayed that tetravalent immunoadhesins have a higher binding affinity and more potent efficacy than divalent immunoadhesins. Although all tetravalent immunoadhesins possess better efficacies, tetravalent forms of CTLA4-Ig and LAG3-Ig revealed higher inhibitory effects on T cell proliferation than tetravalent forms of TNFR2-Ig and CD2-Ig. In vitro mixed lymphocytes reaction (MLR) showed that combined treatment with tetravalent CTLA4-Ig and tetravalent LAG3-Ig was highly effective for inhibiting T cell proliferation in both human and murine allogeneic stimulation. In addition, both single tetravalent-form and combination treatments can prevent the lethality of murine acute GVHD. The results of this study demonstrated that co-blockade of the major histocompatibility complex class (MHC)II:T cell receptor (TCR) and CD28:B7 pathways by using tetravalent human LAG3-Ig and CTLA4-Ig synergistically prevented murine acute GVHD.     

Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 +/- 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-gamma, inducible NO synthase, and TGF-beta; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.     

CTLA4 expression is an indicator and regulator of steady-state CD4+ FoxP3+ T cell homeostasis

The presence of FoxP3(+) regulatory T cells (Tregs) is necessary for control of deleterious immune responses in the steady state; however, mechanisms for maintaining the frequency and quality of endogenous Tregs are not well defined. In this study, we used in vivo modulators of the CD28 and CTLA4 pathways administered to intact mice to reveal mechanisms controlling the homeostasis and phenotype of endogenous Tregs. We demonstrate that expression of the negative costimulatory regulator CTLA4 on FoxP3(+) Tregs in vivo is a direct consequence of their rapid, perpetual homeostasis. Up-regulation of CTLA4 expression occurs only on FoxP3(+) Tregs undergoing extensive proliferation and can be abrogated by inhibiting the CD28 pathway, coinciding with a reduction in FoxP3(+) Treg proliferation and frequency. We further demonstrate that CTLA4 negatively regulates steady-state Treg homeostasis, given that inhibiting CTLA4 signaling with an anti-CTLA4 blocking Ab greatly enhances Treg proliferation and overall Treg frequency. Our findings provide new insight into the origin and role of CTLA4 expression on natural FoxP3(+) Tregs and reveal opposing effects of costimulation modulators on the steady-state level and quality of Tregs, with implications regarding their effects on endogenous Tregs in patients receiving immunotherapy.     

Differential requirement for CD80 and CD80/CD86-dependent costimulation in the lung immune response to an influenza virus infection

The CD28 costimulatory pathway is critical to T cell activation. Blockade of the interaction of CD28 with its ligands CD80 and CD86 using CTLA4-Ig has been proposed as a therapy for a number of immune-based disorders. We have used a murine model of influenza virus infection to study the role of CD28-dependent costimulation in the development of antiviral immune responses. In vivo treatment with CTLA4-Ig to block the interaction of CD28 with CD80 and CD86 reduced virus-specific cytotoxicity and IFN-gamma production by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro. It also resulted in decreased numbers of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid, lung, and spleen and lowered virus-specific Ab titers. Mice treated with CTLA4-Ig were able to control and clear the virus infection, but this was delayed compared with controls. Treatment with Y100F-Ig, a mutant form of CTLA4-Ig which selectively binds to CD80 and blocks the CD28-CD80 interaction leaving CD28-CD86 binding intact, did not affect Ab production, spleen cytotoxic precursors, or clearance of virus. However, Y100F-Ig treatment had a clear effect on lung effector cell function. Secretion of IFN-gamma by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro was decreased, and the number of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid and lungs of infected mice was reduced. These results indicate that CD28-dependent costimulation is important in the antiviral immune response to an influenza virus infection. The individual CD28 ligand, CD80, is important for some lung immune responses and cannot always be compensated for by CD86.     

Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo

Flagellin is secreted by many enteric bacteria and, upon reaching the basolateral membrane of the intestinal epithelium, activates Toll-like receptor 5-mediated innate immune signaling pathways. We hypothesized that any flagellin that gets beyond the epithelium might also regulate cells of the adaptive immune system. Here we demonstrate that the clonal expansion of naive DO11.10 CD4 T cells in response to OVA peptide (323-339) was enhanced 3- to 10-fold in the presence of purified bacterial flagellin in vivo. OVA-specific CD4 T cells were also shown to have undergone more cell division in vivo if flagellin was coinjected with OVA. Flagellin administration increased the expression of B7-1 on splenic dendritic cells, and coinjection of CTLA4-Ig, which is known to block B7 function in vivo, completely ablated the adjuvant effect on CD4 T cells. Therefore, a conserved bacterial protein produced by many intestinal microbes can modulate CD4 T cell activation in vivo. Such an adjuvant effect for flagellin has important implications for vaccine development and the generation of CD4 T cell responses to enteric bacteria.     

Inhibition of Phosphoinositide 3-Kinase p110delta Does Not Affect T Cell Driven Development of Type 1 Diabetes Despite Significant Effects on Cytokine Production

Type 1 diabetes is caused by the destruction of insulin producing beta cells by the immune system. The p110δ isoform of PI3K is expressed primarily in cells of haematopoietic origin and the catalytic activity of p110δ is important for the activation of these cells. Targeting of this pathway offers an opportunity to reduce immune cell activity without unwanted side effects. We have explored the effects of a specific p110δ isoform inhibitor, IC87114, on diabetogenic T cells both in vitro and in vivo, and find that although pharmacological inhibition of p110δ has a considerable impact on the production of pro-inflammatory cytokines, it does not delay the onset of diabetes after adoptive transfer of diabetogenic cells. Further, we demonstrate that combination treatment with CTLA4-Ig does not improve the efficacy of treatment, but instead attenuates the protective effects seen with CTLA4-Ig treatment alone. Our results suggest that decreased IL-10 production by Foxp3⁺ CD4⁺ T cells in the presence of IC87114 negates individual anti-inflammatory effects of IC8114 and CTLA4-Ig.     

Antiviral immune responses in CTLA4 transgenic mice.

The role of B7 binding CD28 in the regulation of T- and B-cell responses against viral antigens was assessed in transgenic mice expressing soluble CTLA4-Hgamma1 (CTLA4-Ig tg mice) that blocks B7-CD28 interactions. The results indicate that transgenic soluble CTLA4 does not significantly alter cytotoxic T-cell responses against replicating lymphocytic choriomeningitis virus (LCMV) or vaccinia virus but drastically impairs the induction of cytotoxic T-cell responses against abortively replicating vesicular stomatitis virus (VSV). While the T-independent neutralizing immunoglobulin M (IgM) responses were within normal ranges, the switch to IgG was reduced 4- to 16-fold after immunization with abortively replicating VSV and more than 30-fold after immunization with an inert VSV glycoprotein antigen in transgenic mice. IgG antibody responses to LCMV, as detected by enzyme-linked immunosorbent assay and by neutralizing action, were reduced about 3- to 20-fold and more than 50-fold, respectively. These results suggest that responses in CTLA4-Ig tg mice are mounted according to their independence of T help. While immune responses to nonreplicating or poorly replicating antigens are in general most dependent on T help and B7-CD28 interactions, they are most impaired in CTLA4-Ig tg mice. The results of the present experiments also indicate that highly replicating viruses, because of greater quantities of available antigens and by inducing as-yet-undefined factors and/or cell surface changes, are capable of compensating for the decrease in T help caused by the blocking effects of soluble CTLA4.