Inhibitory receptors and ligands beyond PD-1, PD-L1 and CTLA-4: breakthroughs or backups.

Although immunotherapeutics targeting the inhibitory receptors (IRs) CTLA-4, PD-1 or PD-L1 have made substantial clinical progress in cancer, a considerable proportion of patients remain unresponsive to treatment. Targeting novel IR-ligand pathways in combination with current immunotherapies may improve clinical outcomes. New clinical immunotherapeutics target T cell-expressed IRs (LAG-3, TIM-3 and TIGIT) as well as inhibitory ligands in the B7 family (B7-H3, B7-H4 and B7-H5), although many of these targets have complex biologies and unclear mechanisms of action. With only modest clinical success in targeting these IRs, current immunotherapeutic design may not be optimal. This Review covers the biology of targeting novel IR-ligand pathways and the current clinical status of their immunotherapeutics, either as monotherapy or in combination with antibody to PD-1 or to its ligand PD-L1. Further understanding of the basic biology of these targets is imperative to the development of effective cancer immunotherapies.

VISTA is an acidic pH-selective ligand for PSGL-1.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.

Large remodeling of the Myc-induced cell surface proteome in B cells and prostate cells creates new opportunities for immunotherapy.

MYC is a powerful transcription factor overexpressed in many human cancers including B cell and prostate cancers. Antibody therapeutics are exciting opportunities to attack cancers but require knowledge of surface proteins that change due to oncogene expression. To identify how MYC overexpression remodels the cell surface proteome in a cell autologous fashion and in different cell types, we investigated the impact of MYC overexpression on 800 surface proteins in three isogenic model cell lines either of B cell or prostate cell origin engineered to have high or low MYC levels. We found that MYC overexpression resulted in dramatic remodeling (both up- and down-regulation) of the cell surfaceome in a cell type-dependent fashion. We found systematic and large increases in distinct sets of >80 transporters including nucleoside transporters and nutrient transporters making cells more sensitive to toxic nucleoside analogs like cytarabine, commonly used for treating hematological cancers. Paradoxically, MYC overexpression also increased expression of surface proteins driving cell turnover such as TNFRSF10B, also known as death receptor 5, and immune cell attacking signals such as the natural killer cell activating ligand NCR3LG1, also known as B7-H6. We generated recombinant antibodies to these two targets and verified their up-regulation in MYC overexpression cell lines and showed they were sensitive to bispecific T cell engagers (BiTEs). Our studies demonstrate how MYC overexpression leads to dramatic bidirectional remodeling of the surfaceome in a cell type-dependent but functionally convergent fashion and identify surface targets or combinations thereof as possible candidates for cytotoxic metabolite or immunotherapy.

High-resolution structural genomics reveals new therapeutic vulnerabilities in glioblastoma.

We investigated the role of 3D genome architecture in instructing functional properties of glioblastoma stem cells (GSCs) by generating sub-5-kb resolution 3D genome maps by in situ Hi-C. Contact maps at sub-5-kb resolution allow identification of individual DNA loops, domain organization, and large-scale genome compartmentalization. We observed differences in looping architectures among GSCs from different patients, suggesting that 3D genome architecture is a further layer of inter-patient heterogeneity for glioblastoma. Integration of DNA contact maps with chromatin and transcriptional profiles identified specific mechanisms of gene regulation, including the convergence of multiple super enhancers to individual stemness genes within individual cells. We show that the number of loops contacting a gene correlates with elevated transcription. These results indicate that stemness genes are hubs of interaction between multiple regulatory regions, likely to ensure their sustained expression. Regions of open chromatin common among the GSCs tested were poised for expression of immune-related genes, including CD276 We demonstrate that this gene is co-expressed with stemness genes in GSCs and that CD276 can be targeted with an antibody-drug conjugate to eliminate self-renewing cells. Our results demonstrate that integrated structural genomics data sets can be employed to rationally identify therapeutic vulnerabilities in self-renewing cells.

Interleukin-7-loaded oncolytic adenovirus improves CAR-T cell therapy for glioblastoma.

BACKGROUND: T cell with chimeric antigen receptors (CAR-T) has presented remarkable efficacy for blood cancer as an emerging immunotherapy. However, for solid tumors, the therapeutic efficacy is much impaired due to the lack of infiltration and persistence of CAR-T in tumor tissue. Thus, we constructed an interleukin-7-loaded oncolytic adenovirus and combined the use of oncolytic virus and CAR-T to improve the therapeutic outcome. METHODS: We constructed an interleukin-7-loaded oncolytic adenovirus (oAD-IL7) and a B7H3-targeted CAR-T and explored the efficacy of the single use of oAD-IL7, B7H3-CAR-T, or the combined therapy for glioblastoma in vitro and in vivo. The improved CAR-T anti-tumor efficacy was evaluated according to the proliferation, survival, persistence, exhaustion of T cells, and tumor regression. RESULTS: Constructed oAD-IL7 and B7H3-CAR-T presented moderate cytotoxicity during in vitro study, but failed to induce a thorough and persistent anti-tumor therapeutic efficacy in vivo. The combination of oAD-IL7 and B7H3-CAR-T in vitro resulted in enhanced T cell proliferation and reduced T cell apoptosis. The joint efficacy was further confirmed using tumor-bearing xenograft mice. During in vivo study, the mice treated with both oAD-IL7 and B7H3-CAR-T showed prolonged survival and reduced tumor burden. According to the ex vivo study, oAD-IL7 improved the proliferation and persistence of tumor-infiltrating B7H3-CAR-T, but failed to reverse the exhaustion. CONCLUSIONS: Our results indicated that oAD-IL7 is a promising auxiliary therapy to improve the therapeutic efficacy of B7H3-CAR-T in glioblastoma by providing the activating signals for tumor-infiltrating T cells. Our results also lay the basis for the future clinical trials for the combination of IL7-loaded oncolytic adenovirus and CAR-T therapy for glioblastoma.

Concomitant inhibition of B7-H3 and PD-L1 expression by a novel and synthetic microRNA delivers potent antitumor activities in colorectal tumor models.

The families of miR-34 and miR-449 share the same seed region. However, the members showed differential effects on the expression of B7-H3 and PD-L1 in HCT-116 cells. Using miR-34a as a template, the non-seed region was modified by nucleotide alteration, yielding four synthetic microRNA (miRNA) analogs. Among those, NS-MX3, with a base alteration from G to C at the 18th locus of miR-34a, showed the most potent inhibition on both B7-H3 and PD-L1 expression. Subsequent investigations demonstrated that NS-MX3 had a broad anti-proliferation activity against several colorectal tumor cell lines and its antitumor effect was consistently reflected by tumor growth inhibition (TGI) in the HCT-116 xenograft model. In addition, NS-MX3 displayed a synergistic effect on TGI when combined with bevacizumab or regorafenib. Further analysis revealed that the superior antitumor activity of NS-MX3 was correlated to concomitant suppression of both B7-H3 and PD-L1 expression in tumor tissues. Taken together, the present study indicates that the non-seed region of miRNAs plays an important role in the regulation of checkpoint genes, thus showcasing single nucleotide alteration of the non-seed region as a promising approach to discover and develop novel immunotherapies.

Mifepristone inhibited the expression of B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis.

BACKGROUND: The immune mechanism was shown to be involved in the development of adenomyosis. The aim of the current study was to evaluate the expression of the immune checkpoints B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and to explore the effect of mifepristone on the expression of these immune checkpoints. METHODS: The expression of B7-H2, B7-H3, B7-H4 and PD-L2 in normal endometria and adenomyosis patient samples treated with or without mifepristone was determined by immunohistochemistry analysis. RESULTS: In adenomyosis patient samples, the expression of B7-H2, B7-H3 and B7-H4 was increased in the eutopic and ectopic endometria compared with normal endometria, both in the proliferative and secretory phases. Moreover, the expression of B7-H2 and B7-H3 was higher in adenomyotic lesions than in the corresponding eutopic endometria, both in the proliferative and secretory phases. The expression of PD-L2 was higher in adenomyotic lesions than in normal endometria in both the proliferative and secretory phases. In the secretory phase but not the proliferative phase, the expression of B7-H4 and PD-L2 in adenomyotic lesions was significantly higher than that in the corresponding eutopic endometria. In normal endometria and eutopic endometria, the expression of B7-H4 was elevated in the proliferative phase compared with that in the secretory phase, while in the ectopic endometria, B7-H4 expression was decreased in the proliferative phase compared with the secretory phase. In addition, the expression of B7-H2, B7-H3, B7-H4 and PD-L2 was significantly decreased in adenomyosis tissues after treatment with mifepristone. CONCLUSIONS: The expression of the immune checkpoint proteins B7-H2, B7-H3, B7-H4 and PD-L2 is upregulated in adenomyosis tissues and is downregulated with mifepristone treatment. The data suggest that B7 immunomodulatory molecules are involved in the pathophysiology of adenomyosis.

The Positive and Negative Immunoregulatory Role of B7 Family: Promising Novel Targets in Gastric Cancer Treatment.

Gastric cancer (GC), with a heterogeneous nature, is the third leading cause of death worldwide. Over the past few decades, stable reductions in the incidence of GC have been observed. However, due to the poor response to common treatments and late diagnosis, this cancer is still considered one of the lethal cancers. Emerging methods such as immunotherapy with immune checkpoint inhibitors (ICIs) have transformed the landscape of treatment for GC patients. There are presently eleven known members of the B7 family as immune checkpoint molecules: B7-1 (CD80), B7-2 (CD86), B7-H1 (PD-L1, CD274), B7-DC (PDCD1LG2, PD-L2, CD273), B7-H2 (B7RP1, ICOS-L, CD275), B7-H3 (CD276), B7-H4 (B7x, B7S1, Vtcn1), B7-H5 (VISTA, Gi24, DD1α, Dies1 SISP1), B7-H6 (NCR3LG1), B7-H7 (HHLA2), and Ig-like domain-containing receptor 2 (ILDR2). Interaction of the B7 family of immune-regulatory ligands with the corresponding receptors resulted in the induction and inhibition of T cell responses by sending co-stimulatory and co-inhibitory signals, respectively. Manipulation of the signals provided by the B7 family has significant potential in the management of GC.

Discovery and Optimization of Small-Molecule Ligands for V-Domain Ig Suppressor of T-Cell Activation (VISTA).

V-domain Ig suppressor of T-cell activation (VISTA) is an immune checkpoint that affects the ability of T-cells to attack tumors. A FRET-based high throughput screening identified NSC622608 as the first small-molecule ligand for VISTA. Investigation of the interaction of NSC622608 with VISTA using STD NMR and molecular modeling enabled the identification of a potential binding site in VISTA for NSC622608. Screening NSC622608 against a library of single-point VISTA mutants revealed the key residues in VISTA interacting with NSC622608. Further structural optimization resulted in a lead with submicromolar VISTA binding affinity. The lead compound blocked VISTA signaling in vitro, enhanced T-cell proliferation, and restored T-cell activation in the presence of VISTA-expressing cancer cell lines. This work would enable future development of small molecules targeting VISTA as immunomodulators and imaging probes.

VISTA: Coming of age as a multi-lineage immune checkpoint.

The immune response is governed by a highly complex set of interactions among cells and mediators. T cells may be rendered dysfunctional by the presence of high levels of antigen in the absence of co-stimulation while myeloid cells may be programmed towards an immunosuppressive state that promotes cancer growth and metastasis while deterring tumor immunity. In addition, inhibitory programs driven by immune checkpoint regulators dampen anti-tumor immunity. The ideal cancer immunotherapy treatment will improve both cross-priming in the tumor microenvironment and relieve suppression by the inhibitory checkpoints. Recently, blockade of programmed cell death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) has elicited impressive results, but not in all patients, so additional targets are under investigation. V-set immunoglobulin domain suppressor of T cell activation (VISTA) is a novel immunoregulatory receptor that is broadly expressed on cells of the myeloid and lymphoid lineages, and is frequently implicated as a poor prognostic indicator in multiple cancers. Importantly, antibody targeting of VISTA uniquely engages both innate and adaptive immunity. This, combined with the expression of VISTA and its non-redundant activities compared to other immune checkpoint regulators, qualifies VISTA to be a promising target for improving cancer immunotherapy.

Frequent B7-H3 overexpression in craniopharyngioma.

Craniopharyngiomas (CPs) are uncommon intracranial benign neoplasms that located in sellar/parasellar region with clinically challenging. B7-H3 is an immune checkpoint molecule highly expressed in many malignant tumors. In this study, we analyzed whether B7-H3 is expressed in 44 CPs samples (adamantinomatous CPs: n = 30 and papillary CPs: n = 14), and whether it could serve as an immunotherapy target in CPs. Immunohistochemical analysis showed that B7-H3 was highly expressed in adamantinomatous CPs (184.3 ±â€¯13.58) and papillary CPs (223.2 ±â€¯11.89), while almost undetectable in normal brain tissue (24 ±â€¯4.9). Besides, B7-H3 expression level was correlated with poor prognosis of patients with CPs. Immunofluorescence and Western blot analysis further suggested that ß-catenin co-localized with B7-H3 and could promote its expression in adaCPs. B7-H3 expression level was positively correlated with staining intensity of IBA1+ cells, but negatively with T cell infiltration in CPs, suggesting that B7-H3 might play a role in the regulation of tumor microenvironment in CPs. Moreover, B7-H3/CD3 bi-specific T cell engager (BiTE) efficiently inhibited the growth of human primary craniopharyngioma cells in a time- and dose-dependent manner. Our results revealed B7-H3 was highly expressed in CPs and targeting B7-H3 might therefore be an effective therapeutic strategy against craniopharyngioma.

Bispecific anti-CD3 x anti-B7-H3 antibody mediates T cell cytotoxic ability to human melanoma in vitro and in vivo.

Inhibition of the B7-H3 immune checkpoint is reported to limit the tumor growth of B7-H3+ tumors. In this study, we demonstrated B7-H3 expression in human melanoma cells, including a primary culture and several cell lines. Furthermore, we investigated whether B7-H3 could serve as a target for T cell-mediated immunotherapy against melanoma. The cytotoxic capacity of activated T cells (ATCs) armed with an anti-CD3 x anti-B7-H3 bispecific antibody (B7-H3Bi-Ab) to melanoma cells was measured using a bioluminescent signal through a luciferase reporter on tumor cells. In contrast to unarmed ATCs, B7-H3Bi-Ab-armed ATCs exhibited increased cytotoxicity against melanoma cells at effector/target ratios from 1:1 to 20:1. Moreover, B7-H3Bi-Ab-armed ATCs secreted more interferin-gamma (IFN-γ), accompanied by higher levels of activating marker CD69 and CD25 expression. Infusion of B7-H3Bi-Ab-armed ATCs suppressed melanoma growth in a xenograft mouse model. Taken together, our results indicate that B7-H3Bi-Ab-armed ATCs may be a promising approach to immunotherapy for melanoma patients.

Enhancing Anti-PD-1/PD-L1 Immune Checkpoint Inhibitory Cancer Therapy by CD276-Targeted Photodynamic Ablation of Tumor Cells and Tumor Vasculature.

Antiangiogenic therapies have been demonstrated to improve the efficacy of immune checkpoint inhibition by overcoming the immunosuppressive status of the tumor microenvironment. However, most of the current antiangiogenic agents cannot discriminate tumor angiogenesis from physiological angiogenesis. The aim of this study was to investigate whether a photodynamic therapy (PDT) agent that targets CD276, a receptor overexpressed in various tumor cells and tumor vasculature but with limited expression in normal tissue vasculature, could improve the tumor inhibitory efficacy of a PD-1/PD-L1 blockade. A CD276-targeting agent (IRD-αCD276/Fab) was synthesized by conjugating the Fab fragment of an anti-CD276 antibody with a photosensitizer IRDye700. The in vivo tumor-targeting efficacy and therapeutic effects of IRD-αCD276/Fab with or without an anti-PD-1/PD-L1 blockade were tested in subcutaneous and lung metastatic tumor models. PDT using IRD-αCD276/Fab significantly suppressed the growth of subcutaneous 4T1 tumor and inhibited its lung metastasis. Moreover, it triggered in vivo antitumor immunity by increasing the activation and maturation of dendritic cells. Tumor PD-L1 levels were also markedly increased after PDT using IRD-αCD276/Fab, as evidenced by noninvasive PD-L1-targeted small-animal PET imaging. In combination with an anti-PD-1/PD-L1 blockade, IRD-αCD276/Fab PDT markedly suppressed the growth of tumors and prevented their metastasis to the lung by recruiting the tumor infiltration of CD8+ T cells. Our data provide evidence for the role of CD276-targeted PDT for local immune modulation, and its combination with PD-L1/PD-1 axis inhibition is a promising strategy for eliminating primary tumors as well as disseminated metastases, by generating local and systemic antitumor responses.

Alterations of the Immunologic Co-Stimulator B7 and TNFR Families Correlate with Hepatocellular Carcinoma Prognosis and Metastasis by Inactivating STAT3.

Blockade of the immunosuppressive checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA4) or programmed death 1 (PD-1) and its cognate ligand, programmed death 1 ligand (PD-L1), has altered the landscape of anti-tumor immunotherapy. B7 family and tumor necrosis factor receptor (TNFR) superfamily play a crucial role in T cell activation, tolerance, and anergy through co-stimulatory and inhibitory signal transduction. Investigating the immune molecular landscapes of the B7 and TNFR families is critical in defining the promising responsive candidates. Herein, we performed comprehensive alteration analysis of the B7 and TNFR family genes across six hepatocellular carcinoma (HCC) datasets with over 1000 patients using cBioPortal TCGA data. About 16% of patients had both B7 and TNFR gene alterations. TNFR gene amplifications were relatively more common (1.73⁻8.82%) than B7 gene amplifications (1.61⁻2.94%). Analysis of 371 sequenced samples revealed that all genes were upregulated: B7 and TNFR mRNA were upregulated in 23% of cases (86/371) and 28% of cases (105/371), respectively. Promoter methylation analysis indicated an epigenetic basis for B7 and TNFR gene regulation. The mRNA levels of B7 and TNFR genes were inversely correlated with promoter methylation status. B7-H6 expression was significantly associated with worse overall survival, and B7-H6 mRNA was increased gradually in cases with gene copy number alterations. B7-H6 overexpression was associated with aggressive clinicopathologic features and poor prognosis in HCC. Downregulation of B7-H6 in HCC cells significantly inhibited cell adhesion, proliferation, migration, and invasion. Knockdown of B7-H6 in HCC cells inhibited tumor growth and metastasis in vivo. B7-H6 promoted HCC metastasis via induction of MMP-9 expression and STAT3 activation. B7-H6 and STAT3 performed functional overlapping roles on enhancing the MMP-9 promoter activity in HCC cells. These results suggest that alterations of the immunologic co-stimulator B7 and TNFR families correlate with HCC metastasis and prognosis, and especially B7-H6 plays a critical role in promoting metastasis of HCC.

CA-170 - A Potent Small-Molecule PD-L1 Inhibitor or Not?

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins - important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 - a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.

Selective blockade of B7-H3 enhances antitumour immune activity by reducing immature myeloid cells in head and neck squamous cell carcinoma.

Immature myeloid cells including myeloid-derived suppressor cells (MDSCs) and tumour-associated macrophages (TAMs) promote tumour growth and metastasis by facilitating tumour transformation and angiogenesis, as well as by suppressing antitumour effector immune responses. Therefore, strategies designed to reduce MDSCs and TAMs accumulation and their activities are potentially valuable therapeutic goals. In this study, we show that negative immune checkpoint molecule B7-H3 is significantly overexpressed in human head and neck squamous cell carcinoma (HNSCC) specimen as compared with normal oral mucosa. Using immunocompetent transgenic HNSCC models, we observed that targeting inhibition of B7-H3 reduced tumour size. Flow cytometry analysis revealed that targeting inhibition of B7-H3 increases antitumour immune response by decreasing immunosuppressive cells and promoting cytotoxic T cell activation in both tumour microenvironment and macroenvironment. Our study provides direct in vivo evidence for a rationale for B7-H3 blockade as a future therapeutic strategy to treat patients with HNSCC.

Development of DS-5573a: A novel afucosylated mAb directed at B7-H3 with potent antitumor activity.

B7-H3 is highly overexpressed in a variety of human clinical tumors, and its expression is significantly associated with poor outcomes. In our study, we aimed to develop new antitumor mAbs by employing cancer cell immunization, and succeeded in generating a mouse anti-human B7-H3 antibody (M30) that shows antitumor activity. M30 was humanized (Hu-M30), and an afucosylated Hu-M30 (DS-5573a) was also generated. To assess the potency of DS-5573a as a therapeutic mAb, we characterized this mAb and evaluated its antitumor activity in vitro and in vivo. Flow cytometry analysis showed that B7-H3 proteins were expressed on various types of cancer cell lines broadly, and DS-5573a binds to IgC1 and IgC2 domains of human B7-H3. Antibody-dependent cellular cytotoxicity activity of DS-5573a was drastically enhanced against medium to high B7-H3-expressing cancer cell lines MDA-MB-231 and NCI-H322. DS-5573a also induced high antibody-dependent cellular cytotoxicity activity against low B7-H3-expressing cancer cell line COLO205, whereas Hu-M30 induced little activity against it. In addition, DS-5573a was found to be a novel anti-B7-H3 antibody which showed antibody-dependent cellular phagocytosis activity. Furthermore, DS-5573a showed dose-dependent and significant antitumor efficacy (0.03-3 mg/kg) in MDA-MB-231-bearing SCID mice (which have functional natural killer cells and macrophages), but little antitumor efficacy in NOG mice (which lack natural killer cells and have reduced macrophage function). These results suggest that antitumor activity of DS-5573a is mediated by effector cells, and this mAb could be a promising antitumor therapy for patients with a wide range of B7-H3-expressing tumors.

The interaction of MMP-2/B7-H3 in human osteoporosis.

The immune costimulatory molecule B7-H3 has been shown to be involved in the regulation of murine bone formation. However, the role of B7-H3 in bone metabolic diseases remains unknown. In our study, matrix metalloproteinase 2 (MMP-2) and soluble B7-H3 (sB7-H3) were found to be correlatively up-regulated in the sera of osteoporosis patients. Furthermore, our results showed that MG63 cells treated with MMP-2 inhibitors produced lower amounts of sB7-H3 while cells with recombinant MMP-2 had an increased membrane B7-H3 (mB7-H3) shedding. Therefore, elevated MMP-2 levels resulted in an elevation of serum sB7-H3 and reduction of osteoblastic mB7-H3. B7-H3 knockdown in MG63 cells significantly decreased osteoblastic markers and substantially decreased the number of mineralized nodules after 21days. Thus, B7-H3-deficient MG63 cells exhibited impaired bone formation. These results suggest that mB7-H3 is required for the later phases of osteoblast differentiation and that MMP-2/B7-H3 plays a negative regulatory role in osteoporosis.

CD28/CTLA-4/B7 costimulatory pathway blockade affects regulatory T-cell function in autoimmunity.

Naïve T cells require B7/CD28 costimulation in order to be fully activated. Attempts to block this pathway have been effective in preventing unwanted immune reactions. As B7 blockade might also affect Treg cells and interfere with negative signaling through membrane CTLA-4 on effector T (Teff) cells, its immune-modulatory effects are potentially more complex. Here, we used the mouse model of multiple sclerosis (MS), EAE, to study the effect of B7 blockade. An effective therapy for MS patients has to interfere with ongoing inflammation, and therefore we injected CTLA-4Ig at day 7 and 9 after immunization, when myelin-reactive T cells have been primed and start migrating toward the CNS. Surprisingly, B7 blockade exacerbated disease signs and resulted in more severe CNS inflammation and demyelination, and was associated with an enhanced production of the inflammatory cytokines IL-17 and IFN-γ. Importantly, CTLA-4Ig treatment resulted in a transient reduction of Ki67 and CTLA-4 expression and function of peripheral Treg cells. Taken together, B7 blockade at a particular stage of the autoimmune response can result in the suppression of Treg cells, leading to a more severe disease.

Foxp3+ regulatory T cells are activated in spite of B7-CD28 and CD40-CD40L blockade.

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)-cell activity. We previously reported that the blockade of the B7-CD28 and CD40L-CD40 interaction efficiently suppresses allogeneic T-cell activation in vivo. This was characterized by an initial rise in Foxp3(+) cells, followed by depletion of host-reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4(+) cell activation. When CTLA-4Ig and anti-CD40L mAb (MR1) were added to the cultures, T-cell proliferation and IL-2 production were strongly reduced. However, Foxp3(+) cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4(+) cells much more efficiently than did freshly isolated Treg cells. CD4(+) cells activated by allogeneic cells in the presence of MR1 and CTLA-4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4(+) cells when Foxp3(+) Treg cells were removed. We conclude that natural Treg cells are less dependent on B7-CD28 or CD40-CD40L costimulation compared with Foxp3(-) T cells. Reduced costimulation therefore alters the balance between Teff and Treg-cell activation in favor of Treg-cell activity.