Interaction Of CD4 Research Articles

Sparing T cells from inhibition

Immunology Programmed cell death 1 (PD-1) is an inhibitory receptor that normally keeps T cell immune responses in check. Immunotherapy targeting PD-1 has proven successful for certain types of cancer, but it remains unclear how PD-1 is regulated. Sugiura et al. found that a costimulatory molecule, CD80, can restrict PD-1 function during the activation of T lymphocytes. Binding of CD80 to the PD-1 ligand PD-L1 in cis on primary activated dendritic cells interfered with the ability of PD-L1 to access PD-1 on T cells, which would otherwise have inhibited T cell activation. Functional insights into PD-L1–CD80 interactions may explain the outcomes of anti–PD-1 and anti–PD-L1 cancer therapy. Science , this issue p. [558][1] [1]: /lookup/doi/10.1126/science.aav7062

The surprising role of Indoleamine 2,3-dioxygenase (IDO) in supporting the survival of bone marrow resident long lived plasma cells (LLPC)

Abstract Long lived plasma cells (LLPC) are a key component of protective humoral immunity. The ability of these cells to maintain longevity and a durable antibody response is largely dependent on the complex nature of the bone marrow (BM) microenvironment in which these cells reside. Our lab has demonstrated that the co-stimulatory receptor, CD28, is essential for the survival and function of LLPC. Furthermore, when CD28 on LLPC ligates with CD80/CD86 expressed on DC, it induces upregulation of the enzyme indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan (Trp) into kynurenine (kyn), a well-known ligand of the aryl hydrocarbon receptor (AhR). AhR is a nuclear transcription factor that can modulate immune responses and is highly expressed in plasma cells (PC). Our preliminary data reveals that IDO knock-out (KO) mice in comparison to wild type (Wt) mice have a decrease in bone marrow resident PCs and a decrease in overall Ig levels. Additionally, 15 weeks post vaccination IDO KO mice have significantly less antigen-specific long lived antibody titers compared to Wt. It was also observed that interaction with DC both in vitro and in vivo is needed for LLPC survival and function. Moreover, AhR activation by kyn causes an increase in CD28 mRNA in LLPC. This leads us to propose a model where CD28 interaction with CD80/CD86 induces IDO production, which through its metabolite kyn activates AhR which can then regulate CD28 expression- thus creating a mechanism that promotes LLPC survival and function. These findings challenge the traditional idea of IDO as an inhibitory molecule and provide the rationale for investigating its essential role in maintaining LLPC survival and sustained antibody responses.

Staphylococcal and Streptococcal Superantigens Trigger B7/CD28 Costimulatory Receptor Engagement to Hyperinduce Inflammatory Cytokines.

Staphylococcal and streptococcal superantigens are virulence factors that cause toxic shock by hyperinducing inflammatory cytokines. Effective T-cell activation requires interaction between the principal costimulatory receptor CD28 and its two coligands, B7-1 (CD80) and B7-2 (CD86). To elicit an inflammatory cytokine storm, bacterial superantigens must bind directly into the homodimer interfaces of CD28 and B7-2. Recent evidence revealed that by engaging CD28 and B7-2 directly at their dimer interface, staphylococcal enterotoxin B (SEB) potently enhances intercellular synapse formation mediated by B7-2 and CD28, resulting in T-cell hyperactivation. Here, we addressed the question, whether diverse bacterial superantigens share the property of triggering B7-2/CD28 receptor engagement and if so, whether they are capable of enhancing also the interaction between B7-1 and CD28, which occurs with an order-of-magnitude higher affinity. To this end, we compared the ability of distinct staphylococcal and streptococcal superantigens to enhance intercellular B7-2/CD28 engagement. Each of these diverse superantigens promoted B7-2/CD28 engagement to a comparable extent. Moreover, they were capable of triggering the intercellular B7-1/CD28 interaction, analyzed by flow cytometry of co-cultured cell populations transfected separately to express human CD28 or B7-1. Streptococcal mitogenic exotoxin Z (SMEZ), the most potent superantigen known, was as sensitive as SEB, SEA and toxic shock syndrome toxin-1 (TSST-1) to inhibition of inflammatory cytokine induction by CD28 and B7-2 dimer interface mimetic peptides. Thus, superantigens act not only by mediating unconventional interaction between MHC-II molecule and T-cell receptor but especially, by strongly promoting engagement of CD28 by its B7-2 and B7-1 coligands, a critical immune checkpoint, forcing the principal costimulatory axis to signal excessively. Our results show that the diverse superantigens use a common mechanism to subvert the inflammatory response, strongly enhancing B7-1/CD28 and B7-2/CD28 costimulatory receptor engagement.

Restriction of PD-1 function by cis-PD-L1/CD80 interactions is required for optimal T cell responses.

Targeted blockade of PD-1 with immune checkpoint inhibitors can activate T cells to destroy tumors. PD-1 is believed to function mainly at the effector, but not in the activation, phase of T cell responses, yet how PD-1 function is restricted at the activation stage is currently unknown. Here we demonstrate that CD80 interacts with PD-L1 in cis on antigen-presenting cells (APCs) to disrupt PD-L1/PD-1 binding. Subsequently, PD-L1 cannot engage PD-1 to inhibit T cell activation when APCs express substantial amounts of CD80. In knock-in mice in which cis-PD-L1/CD80 interactions do not occur, tumor immunity and autoimmune responses were greatly attenuated by PD-1. These findings indicate that CD80 on APCs limits the PD-1 coinhibitory signal, while promoting CD28-mediated costimulation, and highlight critical components for induction of optimal immune responses.

A novel humanized anti-CTLA-4 antibody compared to ipilimumab in preclinical studies.

32 Background: The efficacy of ipilimumab as a single agent and in combination for the treatment of melanoma and other malignancies has validated the immune checkpoints T-lymphocyte-associated protein 4 (CTLA-4) blockade in immunotherapy. Clinical trials that include anti-CTLA-4 in a combination design have gained traction. However, ipilimumab is the only anti-CTLA-4 antibody therapeutic approved worldwide. Therefore, novel anti-CTLA-4 therapeutics that may overcome the shortcomings of ipilimumab to fulfill the unmet medical needs is desperately desired. Methods: Mouse hybridoma cells were screened, cloned, and lead antibodies were humanized to obtain candidates with high activity in blocking CTLA-4/CD80 interaction. The resulting lead antibody was further characterized with in vitro binding and blocking assays, as well as mixed lymphocyte reaction assay. A humanized CTLA-4-B6 mouse model was used to evaluate the in vivo tumor growth inhibition activity of the antibody. Results: CBT-509, a novel IgG1 humanized monoclonal antibody against CTLA-4 was obtained. EC50 of CBT-509 binding with CTLA-4 and IC50 of CBT-509 blocking CTLA4/CD80 interaction were comparable to ipilimumab. CBT-509 binds to a distinct epitope, and has a high affinity with CTLA-4 (KD = 2.73E-10 M), more than 4-fold stronger than ipilimumab (KD = 1.21E-9 M). Compared with ipilimumab, CBT-509 was more active in mixed lymphocyte reaction, suggesting that it is more potent in human T-cell activation. Importantly, the efficacy of CBT-509 in killing tumor cells in vivo is much stronger than that of ipilimumab. In a MC38 murine colorectal cancer model under huCTLA-4 B6 mice background, CBT-509 eradicated tumors in 7 of 8 mice at a dose of 3 mg/kg, while ipilimumab eradicated tumors in 4 of 8 mice at the same dosage. Conclusions: Taken together, CBT-509 demonstrated an improvement over ipilimumab. Future efforts will focus on developing molecules that can further improve the efficacy and safety of anti-CTLA-4 based therapeutics.

OMIP-051 - 28-color flow cytometry panel to characterize B cells and myeloid cells.

OMIP-051 - 28-color flow cytometry panel to characterize B cells and myeloid cells.

CD40 signaling—germinal center b cells go at their own pace

CD40 signaling—germinal center b cells go at their own pace

CD28 co-stimulation drives memory phenotype (MP) Treg cell and MP CD4+Foxp3− effector T cell homeostatic proliferation.

Abstract Foxp3+Tregs, CD4+Foxp3− effector T cells and CD8+ T cells are composed of naïve (NP, CD44loCD62Lhi) and memory (MP, CD44hiCD62Llo) subsets. About 30% of MP T cells are cycling (Ki-67+) in-vivo. To determine the factors that drive T cell homeostatic proliferation in-vivo, we investigated the roles of cytokines (TNFa, IL-2, IL-7, and IL-33), TCR-MHC-II interactions, co-stimulatory (CD28, ICOS, CD40, GITR, OX40), and co-inhibitory (CTLA-4, PD1, BTLA, TIGIT) receptors. Blockade of CD28-CD80/86 signaling completely inhibited MP Treg and MP CD4+Foxp3− effector T cell proliferation but did not affect MP CD8+ T cell proliferation. Inhibition of cytokine activity did not affect the proliferation of any of the T cell populations. Marked enhancement of homeostatic proliferation of MP Treg and MP CD4+Foxp3−T cells was observed by blocking TCR-MHCII interactions, CTLA4-CD80/CD86 interactions, or by agonistic mAbs to CD40, GITR, and OX40. Blocking PD1-PDL1 interactions modestly enhanced proliferation, while blocking BTLA, TIM3, ICOS and TIGIT did not affect MP T cell proliferation. Importantly, blocking of CD28-CD80/86 signaling completely abrogated the augmentation of proliferation observed after blocking TCR and CTLA4, PD1, or by agonistic mAbs to GITR, OX40, and CD40. Our results support a complex pathway in which CD28-CD80/CD86 interactions play a critical role in Treg and MP CD4+Foxp3−T cells proliferation in-vivo. CTLA4-CD80/CD86 interactions act as a brake, while MHCII-TCR interactions negatively regulate the inhibitory functions of CTLA-4. These findings have important implications for the use of biologic agents to enhance Treg numbers/functions, as well as for the use of checkpoint inhibitors. Supported by DIR, NIAID.

Defective innate immune responses lead to hepatic damage in CD154-deficient mice following infection with attenuated Salmonella typhimurium

Abstract In addition to its established role in immunoglobulin isotype switching, CD40–CD154 interaction plays a role in the regulation of multiple innate immune system cell types. Of note, we have shown that CD154-deficient (CD154−/−) mice are hyper-susceptible to intraperitoneal infection by attenuated strains of Salmonella enterica serovar Typhimurium. Compared to their normal counterparts, CD154−/− mice exhibited progressive morbidity during the early phases of disease and succumbed to infection 3–4 weeks after inoculation. Since the liver is a key target organ in Salmonella infections, we carried out a comparative analysis of infection-associated hepatic changes at different time-points. Bacterial loads in the liver were significantly higher in CD154−/− compared to WT mice, and this correlated with progressively worsening hepatic damage and rising levels of liver enzymes. Liver histomorphology revealed focal necrosis early in the infection that was self-limiting in WT mice. In contrast, infected CD154−/− mice developed large and generalized necrosis coupled with the appearance of large venous thrombi. Immunohistochemical analysis showed a retarded inflammatory response affecting recruitment of myeloid cells in the liver of CD154−/− animals. Moreover, neutrophil recruitment into the peritoneal cavity, as well as the activation of the respiratory burst, was defective in CD154−/− mice. Taken together, our findings demonstrate that CD154-deficiency results in a failure to mount an appropriate innate immune response to contain bacterial proliferation during the acute phase of disease. This renders the animals incapable of limiting the extent of histopathological damage in the liver resulting, ultimately, in their demise.

Expression and Regulation of Thymic Stromal Lymphopoietin and Thymic Stromal Lymphopoietin Receptor Heterocomplex in the Innate-Adaptive Immunity of Pediatric Asthma.

Asthma is a chronic inflammatory disease affecting the airway, and it is characterized by a wheezing breathing sound, variable airflow obstruction and the presence of inflammatory cells in the submucosa of the bronchi. Viral infection, pollutants and sensitivity to aeroallergens damage the epithelium from childhood, which causes asthma. The pathogenesis of asthma includes pathways of innate stimulation by environmental microbes and irritant pathogens. Damaged epithelial cells produce thymic stromal lymphopoietin (TSLP) and stimulate myeloid dendritic cell maturation through the thymic stromal lymphopoietin receptor (TSLPR) heterocomplex. TSLP-activated myeloid dendritic cells promote naive CD4+ T cells to differentiate into T helper type 2 (Th2) phenotype CD4+ T cells. Re-exposure to allergens or environmental stimuli causes an adaptive immune response. TSLP-activated dendritic cells expressing the OX40 ligand (OX40L; CD252) trigger naive CD4+ T cells to differentiate into inflammatory Th2 effector cells secreting the cytokines interleukin-4, 5, 9, and 13 (IL-4, IL-5, IL-9 and IL-13), and the dendritic cells (DCs) promote the proliferation of allergen-specific Th2 memory cells. Allergen presentation by Th2 cells through its interaction with their receptors in the presence of major histocompatibility complex (MHC) class II on B cells and through costimulation involving CD40 and CD40L interactions results in immunoglobulin class switching from IgM to IgE. DCs and other blood cell subsets express the TSLPR heterocomplex. The regulatory mechanism of the TSLPR heterocomplex on these different cell subsets remains unclear. The TSLPR heterocomplex is composed of the IL-7Rα chain and TSLPR chain. Moreover, two isoforms of TSLP, short isoform TSLP (sfTSLP) and long isoform TSLP (lfTSLP), have roles in atopic and allergic development. Identifying and clarifying the regulation of TSLPR and IL-7Rα in pediatric asthma are still difficult, because the type of blood cell and the expression for each blood cell in different stages of atopic diseases are poorly understood. We believe that further integrated assessments of the regulation mechanism of the TSLP–TSLPR heterocomplex axis in vitro and in vivo can provide a faster and earlier diagnosis of pediatric asthma and promote the development of more effective preventive strategies at the onset of allergies.

CD86 Expression by Monocytes Influences an Immunomodulatory Profile in Asymptomatic Patients with Chronic Chagas Disease.

In the chronic phase of Chagas disease, 60% of the patients develop the asymptomatic form known as indeterminate (IND). The remaining 30% of the patients develop a life-threatening form in which digestive and/or cardiac (CARD) alterations take place. The mechanisms underlying the development of severe forms of Chagas disease remain poorly understood. It is well known that interactions between immune cells such as monocytes and lymphocytes drive immune responses. Further, the co-stimulatory molecules CD80 and CD86 expressed by monocytes and subsets induce lymphocyte activation, thereby triggering cellular immune response. Here, we revealed, for the first time, the functional-phenotypic profile of monocytes subsets in Chagas disease. Using flow cytometry, we evaluated the effect of in vitro stimulation with Trypanosoma cruzi antigens on the expression of the co-stimulatory molecules CD80 and CD86 in different monocyte subsets of patients with IND and CARD clinical forms of Chagas disease. We also assessed the expression of toll-like receptor (TLR)-2, TLR-4, TLR-9, HLA-DR, IL-10, and IL-12 in the monocyte subsets and of CTLA-4 and CD28, ligands of CD80 and CD86, in T lymphocytes. CD86 expression in all monocyte subsets was higher in IND patients when compared with non-infected (NI) individuals. After stimulation with T. cruzi, these patients also showed a higher frequency of CD4+CTLA-4+ T lymphocytes than NI individuals. We found an association between CD80 and CD28, and between CD86 and CTLA-4 expression, with a high frequency of regulatory T (Treg) cells in IND patients. We proposed that CD86 may be involved in immunoregulation by its association with CTLA-4 in asymptomatic patients. CD86 and CTLA-4 interaction may influence Treg activation, and this could represent a new strategy to control inflammation and tissue damage.

The CD40-CD40L Dyad in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis.

The CD40–CD40L dyad is an immune checkpoint regulator that promotes both innate and adaptive immune responses and has therefore an essential role in the development of inflammatory diseases, including multiple sclerosis (MS). In MS, CD40 and CD40L are expressed on immune cells present in blood and lymphoid organs, affected resident central nervous system (CNS) cells, and inflammatory cells that have infiltrated the CNS. CD40–CD40L interactions fuel the inflammatory response underlying MS, and both genetic deficiency and antibody-mediated inhibition of the CD40–CD40L dyad reduce disease severity in experimental autoimmune encephalomyelitis (EAE). Both proteins are therefore attractive therapeutic candidates to modulate aberrant inflammatory responses in MS. Here, we discuss the genetic, experimental and clinical studies on the role of CD40 and CD40L interactions in EAE and MS and we explore novel approaches to therapeutically target this dyad to combat neuroinflammatory diseases.

Prospects for modulating the CD40/CD40L pathway in the therapy of the hyper-IgM syndrome.

The critical role of the CD40/CD40L pathway in B-cell proliferation, immunoglobulin (Ig) isotype switching and germinal center formation has been studied and described extensively in previous literature. Interruption of the CD40/CD40L signal causes hyper-IgM (HIGM) syndrome, which has been classified and recognized as a group of rare inherited immune deficiency disorders. Defects in CD40 and CD40L interactions or in downstream signaling molecules, including activation-induced cytidine deaminase, uracyl-DNA-glycosylase, NF-κB and DNA repair enzymes, result in an increased level of serum IgM and a significantly decreased or absent level of IgA, IgG and IgE that is accompanied by severe recurrent infections and autoimmune diseases. Many genetic defects in HIGM have been identified and, as a result, it is possible for patients to be definitively diagnosed by gene sequencing and to delineate the immunological features of the patients. Modifying the CD40/CD40L signaling pathway may offer the possibility of restoring the normal serum Ab production and curing the immunodeficiency. Hematopoietic stem cell transplantation has achieved a high rate of success using a sibling donor. In addition, successful examples of treating other immunodeficiencies using gene therapy indicated that there was a possibility of eradicating HIGM with this approach. In this review, we summarize the current drugs and a variety of therapeutic approaches for the treatment of the HIGM syndrome by interfering with the defective CD40/CD40L pathway.

Conservation of structural and interactional features of CD28 and CD80/86 molecules from Nile tilapia (Oreochromis niloticus)

Interaction of CD28 with CD80 or CD86 molecules provides a costimulatory signals required in T cell activation. In this study, we cloned and analyzed a CD28 gene (On-CD28) and a CD80/86 gene (On-CD80/86) from Nile tilapia (Oreochromis niloticus). Sequence analysis revealed the typical characteristics of On-CD28 protein; for instance, the proline-based motif (117TYPPPL122) is essential in binding of CD28 to CD80/86 ligands. Moreover, an extracellular Ig domain was found in On-CD80/86; this domain is responsible in binding of CD28 to CD80/86 receptors. Subcellular localization analysis showed that both On-CD28 and On-CD80/86 were distributed predominantly in the cytomembrane. Yeast two-hybrid assay showed that On-CD28 directly interacted with On-CD80/86. On-CD28 and On-CD80/86 transcripts were detected in all the examined tissues of healthy Nile tilapia, and the highest expression levels of On-CD28 and On-CD80/86 were detected in the brain and heart, respectively. Following a bacterial challenge using Streptococcus agalactiae in vivo, On-CD28 and On-CD80/86 were upregulated in head kidney, spleen, intestines, and brain. However, they showed different expression profiles in response to stimulation with inactivated S. agalactiae in vitro. These findings indicated that the interaction of On-CD28 with On-CD80/86 provides a costimulatory signals that possibly play an important role in T cell activation during S. agalactiae infection.

Interaction of CD80 with Neph1: a potential mechanism of podocyte injury.

The induction of CD80 on podocytes has been shown in animal models of podocyte injury and in certain cases of nephrotic syndrome. In a lipopolysaccharide (LPS)-induced mouse model of albuminuria, we have recently shown a signalling axis of LPS-myeloid cell activation-TNFα production-podocyte CD80 induction-albuminuria. Therefore, in this report, we investigated the cellular and molecular consequences of TNFα addition and CD80 expression on cultured podocytes. A murine podocyte cell line was used for TNFα treatment and for over-expressing CD80. Expression and localization of various podocyte proteins was analysed by reverse transcriptase-polymerase chain reaction, western blotting and immunofluorescence. HEK293 cells were used to biochemically characterize interactions. Podocytes treated with LPS in vitro did not cause CD80 upregulation but TNFα treatment was associated with an increase in CD80 levels, actin derangement and poor wound healing. Podocytes stably expressing CD80 showed actin derangement and co-localization with Neph1. CD80 and Neph1 interaction was confirmed by pull down assays of CD80 and Neph1 transfected in HEK293 cells. Addition of TNFα to podocytes causes CD80 upregulation, actin reorganization and podocyte injury. Overexpressed CD80 and Neph1 interact via their extracellular domain. This interaction implies a mechanism of slit diaphragm disruption and possible use of small molecules that disrupt CD80-Neph1 interaction as a potential for treatment of nephrotic syndrome associated with CD80 upregulation.

CTLA-4/CD80 pathway regulates T cell infiltration into pancreatic cancer.

The ability of some tumors to exclude effector T cells represents a major challenge to immunotherapy. T cell exclusion is particularly evident in pancreatic ductal adenocarcinoma (PDAC), a disease where blockade of the immune checkpoint molecule CTLA-4 has not produced significant clinical activity. In PDAC, effector T cells are often scarce within tumor tissue and confined to peritumoral lymph nodes and lymphoid aggregates. We hypothesized that CTLA-4 blockade, despite a lack of clinical efficacy seen thus far in PDAC, might still alter T cell immunobiology, which would have therapeutic implications. Using clinically relevant genetic models of PDAC, we found that regulatory T cells (Tregs), which constitutively express CTLA-4, accumulate early during tumor development but are largely confined to peritumoral lymph nodes during disease progression. Tregs were observed to regulate CD4+, but not CD8+, T cell infiltration into tumors through a CTLA-4/CD80 dependent mechanism. Disrupting CTLA-4 interaction with CD80 was sufficient to induce CD4 T cell infiltration into tumors. These data have important implications for T cell immunotherapy in PDAC and demonstrate a novel role for CTLA-4/CD80 interactions in regulating T cell exclusion. In addition, our findings suggest distinct mechanisms govern CD4+ and CD8+ T cell infiltration in PDAC.

PD-L1/CD80 interaction differentially regulates CD8+ T cell metabolism, survival and GVHD capacity in the presence or absence of PD-1

Abstract Purified alloreactive CD8+ T cells induce no acute GVHD, but the mechanisms remain unknown. PD-L1 interacts with PD-1 and CD80. Here we show that, sorted C57BL/6 donor CD8+ T cells (1.25 ×106) expanded in wild-type MHC-mismatched BALB/c recipients but induced no acute GVHD. In PD-L1−/− recipients, donor CD8+ T cells expanded and induced lethal acute GVHD. PD-1−/−CD8+ T cells induced lethal acute GVHD in wild-type recipients. Blockade of PD-L1/CD80 interactions on days 0 and 2 prevented expansion of wild-type and PD-1−/− CD8+ T cells in wild type recipients and prevented acute GVHD caused by PD-1−/− CD8+ T cells. On the other hand, blockade of PD-L1/CD80 interaction after CD8+ T activation on day 5 caused lethal acute GVHD in wild-type recipients that usually have no acute GVHD. Naïve CD8+ T cells express much higher levels of CD80 than PD-1, whereas activated CD8+ T cells express higher levels of PD-1 than CD80. PD-L1/CD80 interaction augmented glycolysis in naïve WT and PD-1−/− CD8+T cells. This interaction was associated with increased phosphorylation of PLC-γ and activation of the AKT/mTOR pathway in a CD28-dependent manner. This interaction increased mitochondrial numbers and volume and augmented ROS production and apoptosis at the peak of CD8+ T cell activation, such that CD8+T cells did not over-expand to cause GVHD. Conversely, blockade of PD-L1/CD80 after CD8+ T activation in the presence of high PD-1 expression led to increased mitophagy, reduced apoptosis and overexpansion of CD8+ T cells, resulting in lethal GVHD. These results indicate that PD-L1/ CD80 interaction is dependent on PD-1 expression to differentially regulate glycolysis and mitophagy as well as expansion, survival and acute GVHD capacity of alloreactive CD8+ T cells.

TCR Signaling and CD28/CTLA-4 Signaling Cooperatively Modulate T Regulatory Cell Homeostasis.

Foxp3+ T regulatory cells (Tregs), conventional CD4+Foxp3- T cells, and CD8+ T cells represent heterogeneous populations composed of naive phenotype (NP, CD44low) and memory phenotype (MP, CD44high) subpopulations. NP and MP subsets differ in their activation state, contribution to immune function, and capacity to proliferate in vivo. To further understand the factors that contribute to the differential homeostasis of NP/MP subsets, we examined the differential effects of CD28 and CTLA-4 interaction with CD80/CD86, as well as MHC class II-TCR interaction within mouse Treg pools and CD4+ and CD8+ T cell pools. Blockade of CD80/CD86 with CTLA-4-Ig markedly reduced the cycling and absolute numbers of MP Tregs and MP CD4+ T cells, with minimal effect on the NP T cell subpopulations. Blockade of MHC class II-TCR interaction led to selective expansion of MP Tregs and MP CD4+ and CD8+ T cells that was reversed upon cotreatment with CTLA-4-Ig. Treatment with anti-CTLA-4 mAb altered MP Treg and MP CD4+ and CD8+ T cell homeostasis in a manner similar to that observed with anti-MHC class II. We postulate a complex pathway in which CD28 is the primary driver of Treg proliferation and CTLA-4 functions as the main brake but is likely dependent on TCR signals and CD80/CD86. These findings have important implications for the use of biologic agents targeting such pathways to modulate autoimmune and neoplastic disease.

Hetero-oligomerization between the TNF receptor superfamily members CD40, Fas and TRAILR2 modulate CD40 signalling

TNF receptor superfamily members (TNFRSF) such as CD40, Fas and TRAIL receptor 2 (TRAILR2) participate to the adaptive immune response by eliciting survival, proliferation, differentiation and/or cell death signals. The balance between these signals determines the fate of the immune response. It was previously reported that these receptors are able to self-assemble in the absence of ligand through their extracellular regions. However, the role of this oligomerization is not well understood, and none of the proposed hypotheses take into account potential hetero-association of receptors. Using CD40 as bait in a flow cytometry Förster resonance energy transfer assay, TNFRSF members with known functions in B cells were probed for interactions. Both Fas and TRAILR2 associated with CD40. Immunoprecipitation experiments confirmed the interaction of CD40 with Fas at the endogenous levels in a BJAB B-cell lymphoma cell line deficient for TRAILR2. TRAILR2-expressing BJAB cells displayed a robust CD40–TRAILR2 interaction at the expense of the CD40–Fas interaction. The same results were obtained by proximity ligation assay, using TRAILR2-positive and -negative BJAB cells and primary human B cells. Expression of the extracellular domains of Fas or TRAILR2 with a glycolipid membrane anchor specifically reduced the intrinsic signalling pathway of CD40 in 293T cells. Conversely, BJAB cells lacking endogenous Fas or TRAILR2 showed an increased NF-κB response to CD40L. Finally, upregulation of TRAILR2 in primary human B cells correlated with reduced NF-κB activation and reduced proliferation in response to CD40L. Altogether, these data reveal that selective interactions between different TNFRSF members may modulate ligand-induced responses upstream signalling events.

CD80 Expressed by CD8+ T Cells Contributes to PD-L1-Induced Apoptosis of Activated CD8+ T Cells.

Tumor cells are capable of limiting antitumor CD8+ T cell responses through their cell surface expression of PD-L1. In addition to PD-1 expressed by CD8+ T cells, PD-L1 also binds to CD80 expressed by CD8+ T cells. The influence of the PD-L1/CD80 interaction on CD8+ T cell function has not been fully characterized, so we sought to investigate the impact of the PD-L1/CD80 interaction on PD-L1-induced apoptosis of activated CD8+ T cells. We found that CD8+ T cells that lacked CD80 expression got activated to the same extent as wild-type CD8+ T cells, but when cultured with anti-CD3 and PD-L1/Fc protein, activated CD8+ T cells that lacked CD80 expression survived better than activated wild-type CD8+ T cells. These findings indicate that PD-L1 induces apoptosis in activated CD8+ T cells in part by signaling through CD80. Thus, in the design and implementation of checkpoint blockade therapies that target PD-L1, it is essential that both binding partners for PD-L1, PD-1, and CD80 are considered.