Deficiency of CBL and CBLB ubiquitin ligases leads to hyper T follicular helper cell responses and lupus by reducing BCL6 degradation.

Recent evidence reveals hyper T follicular helper (Tfh) cell responses in systemic lupus erythematosus (SLE); however, molecular mechanisms responsible for hyper Tfh cell responses and whether they cause SLE are unclear. We found that SLE patients downregulated both ubiquitin ligases, casitas B-lineage lymphoma (CBL) and CBLB (CBLs), in CD4+ T cells. T cell-specific CBLs-deficient mice developed hyper Tfh cell responses and SLE, whereas blockade of Tfh cell development in the mutant mice was sufficient to prevent SLE. ICOS was upregulated in SLE Tfh cells, whose signaling increased BCL6 by attenuating BCL6 degradation via chaperone-mediated autophagy (CMA). Conversely, CBLs restrained BCL6 expression by ubiquitinating ICOS. Blockade of BCL6 degradation was sufficient to enhance Tfh cell responses. Thus, the compromised expression of CBLs is a prevalent risk trait shared by SLE patients and causative to hyper Tfh cell responses and SLE. The ICOS-CBLs axis may be a target to treat SLE.

ICOS-expressing Regulatory T Cells Influence the Composition of Antitumor CTL Populations.

The role of ICOS in antitumor T cell responses and overall tumor progression has been controversial. In this study, we compared tumor progression in mice lacking ICOS selectively in regulatory T (Treg) cells or in all T cells. Using an experimental melanoma lung metastasis model, we found that Treg cell-specific ICOS knockout reduces the overall tumor burden compared with Cre control mice, with increased CD4+-to-Treg cell and CD8+-to-Treg cell ratios in the tumor. In contrast, there was no difference in the tumor burden in mice lacking ICOS in all of the T cell compartments. This suggests a dual role of ICOS costimulation in promoting protumor and antitumor T cell responses. Consistent with reduced tumor burden, we found that Treg cell-specific deletion of ICOS leads to an increase of CD8+ CTLs that express high levels of granzyme B and perforin. Moreover, single-cell transcriptome analysis revealed an increase of Ly108+Eomeshi CD8+ T cells at the cost of the Ly108+T-bethi subset in Treg cell-specific knockout mice. These results suggest that ICOS-expressing Treg cells suppress the CTL maturation process at the level of Eomes upregulation, a critical step known to drive perforin expression and cytotoxicity. Collectively, our data imply that cancer immunotherapies using ICOS agonist Abs may work better in Treg cell-low tumors or when they are combined with regimens that deplete tumor-infiltrating Treg cells.

ICOS-Deficient Regulatory T Cells Can Prevent Spontaneous Autoimmunity but Are Impaired in Controlling Acute Inflammation.

ICOS is induced in activated T cells and its main role is to boost differentiation and function of effector T cells. ICOS is also constitutively expressed in a subpopulation of Foxp3+ regulatory T cells under steady-state condition. Studies using ICOS germline knockout mice or ICOS-blocking reagents suggested that ICOS has supportive roles in regulatory T (Treg) cell homeostasis, migration, and function. To avoid any compounding effects that may arise from ICOS-deficient non-Treg cells, we generated a conditional knockout system in which ICOS expression is selectively abrogated in Foxp3-expressing cells (ICOS FC mice). Compared to Foxp3-Cre control mice, ICOS FC mice showed a minor numerical deficit of steady-state Treg cells but did not show any signs of spontaneous autoimmunity, indicating that tissue-protective Treg populations do not heavily rely on ICOS costimulation. However, ICOS FC mice showed more severe inflammation in oxazolone-induced contact hypersensitivity, a model of atopic dermatitis. This correlated with elevated numbers of inflammatory T cells expressing IFN-γ and/or TNF-α in ICOS FC mice compared with the control group. In contrast, elimination of ICOS in all T cell compartments negated the differences, confirming that ICOS has a dual positive role in effector and Treg cells. Single-cell transcriptome analysis suggested that ICOS-deficient Treg cells fail to mature into T-bet+CXCR3+ "Th1-Treg" cells in the draining lymph node. Our results suggest that regimens that preferentially stimulate ICOS pathways in Treg cells might be beneficial for the treatment of Th1-driven inflammation.

Inducible T-cell co-stimulator: Signaling mechanisms in T follicular helper cells and beyond.

Human patients with homozygous null mutations in the ICOS gene suffer from recurrent infections due to humoral immune defects. Studies on human patients and mouse models have shown that inducible T-cell co-stimulator (ICOS)-deficient individuals cannot form T follicular helper (Tfh) cells, a group of CD4 T cells that migrate into B cell follicles and facilitate germinal center (GC) reactions. ICOS-induced phosphoinositide 3-kinase signaling pathways have been shown to play critical roles in Tfh programming, migration of Tfh cells into the GC, and delivery of T cell help during Tfh-GC B cell conjugation. These processes are also assisted by ICOS-mediated intracellular calcium mobilization and TANK-binding kinase 1 signaling. However, ICOS signaling also has stimulatory roles in T regulatory cells and innate lymphoid cells (ILCs), providing another layer of complexity. In this review, we discuss cell-type-specific signaling mechanisms utilized by ICOS in Tfh cells, T regulatory cells, and ILCs. Whenever relevant, we compare the roles and signaling pathways of ICOS and CD28. Understanding ICOS signal transduction mechanisms used by distinct immune subsets at different stages of immune responses or disease progression may help improve vaccination protocols, treat autoimmune diseases, and enhance cancer immunotherapy.

The ICOS-ICOSL pathway tunes thymic selection.

Negative selection of developing T cells plays a significant role in T-cell tolerance to self-antigen. This process relies on thymic antigen-presenting cells which express both self-antigens and cosignaling molecules. Inducible T-cell costimulator (ICOS) belongs to the CD28 family of cosignaling molecules and binds to ICOS ligand (ICOSL). The ICOS signaling pathway plays important roles in shaping the immune response to infections, but its role in central tolerance is less well understood. Here we show that ICOSL is expressed by subsets of thymic dendritic cells and medullary thymic epithelial cells as well as thymic B cells. ICOS expression is upregulated as T cells mature in the thymus and correlates with T-cell receptor signal strength during thymic selection. We also provide evidence of a role for ICOS signaling in mediating negative selection. Our findings suggest that ICOS may fine-tune T-cell receptor signals during thymic selection contributing to the generation of a tolerant T-cell population.

Hippo Pathway Kinase Mst1 Is Required for Long-Lived Humoral Immunity.

The protein kinase Mst1 is a key component of the evolutionarily conserved Hippo pathway that regulates cell survival, proliferation, differentiation, and migration. In humans, Mst1 deficiency causes primary immunodeficiency. Patients with MST1-null mutations show progressive loss of naive T cells but, paradoxically, mildly elevated serum Ab titers. Nonetheless, the role of Mst1 in humoral immunity remains poorly understood. In this study, we found that early T cell-dependent IgG1 responses in young adult Mst1-deficient mice were largely intact with signs of impaired affinity maturation. However, the established Ag-specific IgG1 titers in Mst1-deficient mice decayed more readily because of a loss of Ag-specific but not the overall bone marrow plasma cells. Despite the impaired affinity and longevity of Ag-specific Abs, Mst1-deficient mice produced plasma cells displaying apparently normal maturation markers with intact migratory and secretory capacities. Intriguingly, in immunized Mst1-deficient mice, T follicular helper cells were hyperactive, expressing higher levels of IL-21, IL-4, and surface CD40L. Accordingly, germinal center B cells progressed more rapidly into the plasma cell lineage, presumably forgoing rigorous affinity maturation processes. Importantly, Mst1-deficient mice had elevated levels of CD138+Blimp1+ splenic plasma cell populations, yet the size of the bone marrow plasma cell population remained normal. Thus, overproduced low-affinity plasma cells from dysregulated germinal centers seem to underlie humoral immune defects in Mst1-deficiency. Our findings imply that vaccination of Mst1-deficient human patients, even at the early stage of life, may fail to establish long-lived high-affinity humoral immunity and that prophylactic Ab replacement therapy can be beneficial to the patients.

ICOS Signaling Controls Induction and Maintenance of Collagen-Induced Arthritis.

ICOS is a key costimulatory receptor facilitating differentiation and function of follicular helper T cells and inflammatory T cells. Rheumatoid arthritis patients were shown to have elevated levels of ICOS+ T cells in the synovial fluid, suggesting a potential role of ICOS-mediated T cell costimulation in autoimmune joint inflammation. In this study, using ICOS knockout and knockin mouse models, we found that ICOS signaling is required for the induction and maintenance of collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis. For the initiation of CIA, the Tyr181-based SH2-binding motif of ICOS that is known to activate PI3K was critical for Ab production and expansion of inflammatory T cells. Furthermore, we found that Tyr181-dependent ICOS signaling is important for maintenance of CIA in an Ab-independent manner. Importantly, we found that a small molecule inhibitor of glycolysis, 3-bromopyruvate, ameliorates established CIA, suggesting an overlap between ICOS signaling, PI3K signaling, and glucose metabolism. Thus, we identified ICOS as a key costimulatory pathway that controls induction and maintenance of CIA and provide evidence that T cell glycolytic pathways can be potential therapeutic targets for rheumatoid arthritis.

The TLR4-TRIF pathway can protect against the development of experimental allergic asthma.

The Toll-like receptor (TLR) adaptor proteins myeloid differentiating factor 88 (MyD88) and Toll, interleukin-1 receptor and resistance protein (TIR) domain-containing adaptor inducing interferon-ß (TRIF) comprise the two principal limbs of the TLR signalling network. We studied the role of these adaptors in the TLR4-dependent inhibition of allergic airway disease and induction of CD4+ ICOS+ T cells by nasal application of Protollin™, a mucosal adjuvant composed of TLR2 and TLR4 agonists. Wild-type (WT), Trif-/- or Myd88-/- mice were sensitized to birch pollen extract (BPEx), then received intranasal Protollin followed by consecutive BPEx challenges. Protollin's protection against allergic airway disease was TRIF-dependent and MyD88-independent. TRIF deficiency diminished the CD4+ ICOS+ T-cell subsets in the lymph nodes draining the nasal mucosa, as well as their recruitment to the lungs. Overall, TRIF deficiency reduced the proportion of cervical lymph node and lung CD4+ ICOS+ Foxp3- cells, in particular. Adoptive transfer of cervical lymph node cells supported a role for Protollin-induced CD4+ ICOS+ cells in the TRIF-dependent inhibition of airway hyper-responsiveness. Hence, our data demonstrate that stimulation of the TLR4-TRIF pathway can protect against the development of allergic airway disease and that a TRIF-dependent adjuvant effect on CD4+ ICOS+ T-cell responses may be a contributing mechanism.

Synergistic effects of host B7-H4 deficiency and gemcitabine treatment on tumor regression and anti-tumor T cell immunity in a mouse model.

B7-H4 (B7x/B7S1), a B7 family inhibitor of T cell activity, is expressed in multiple human cancers and correlates with decreased infiltrating lymphocytes and poor prognosis. In murine models, tumor-expressed B7-H4 enhances tumor growth and reduces T cell immunity, and blockade of tumor-B7-H4 rescues T cell activity and lowers tumor burden. This implicates B7-H4 as a target for cancer immunotherapy, yet limits the efficacy of B7-H4 blockade exclusively to patients with B7-H4+ tumors. Given the expression of B7-H4 on host immune cells, we have previously shown that BALB/c mice lacking host B7-H4 have enhanced anti-tumor profiles, yet similar 4T1 tumor growth relative to control. Given that T cell-mediated immunotherapies work best for tumors presenting tumor-associated neoantigens, we further investigated the function of host B7-H4 in the growth of a more immunogenic derivative, 4T1-12B, which is known to elicit strong anti-tumor CD8 T cell responses due to expression of a surrogate tumor-specific antigen, firefly luciferase. Notably, B7-H4 knockout hosts not only mounted greater tumor-associated anti-tumor T cell responses, but also displayed reduced tumors. Additionally, B7-H4-deficiency synergized with gemcitabine to further inhibit tumor growth, often leading to tumor eradication and the generation of protective T cell immunity. These findings imply that inhibition of host B7-H4 can enhance anti-tumor T cell immunity in immunogenic cancers, and can be combined with other anti-cancer therapies to further reduce tumor burden regardless of tumor-B7-H4 positivity.

Miz-1 regulates translation of Trp53 via ribosomal protein L22 in cells undergoing V(D)J recombination.

To be effective, the adaptive immune response requires a large repertoire of antigen receptors, which are generated through V(D)J recombination in lymphoid precursors. These precursors must be protected from DNA damage-induced cell death, however, because V(D)J recombination generates double-strand breaks and may activate p53. Here we show that the BTB/POZ domain protein Miz-1 restricts p53-dependent induction of apoptosis in both pro-B and DN3a pre-T cells that actively rearrange antigen receptor genes. Miz-1 exerts this function by directly activating the gene for ribosomal protein L22 (Rpl22), which binds to p53 mRNA and negatively regulates its translation. This mechanism limits p53 expression levels and thus contains its apoptosis-inducing functions in lymphocytes, precisely at differentiation stages in which V(D)J recombination occurs.

Host B7-H4 regulates antitumor T cell responses through inhibition of myeloid-derived suppressor cells in a 4T1 tumor transplantation model.

B7-H4, a member of the B7 family of T cell immunomodulatory proteins, has been shown to inhibit T cell responses and neutrophil expansion during bacterial infections. However, the role of B7-H4 in the immune response during tumor growth has been unclear. In this study, we examined the host immune responses in B7-H4-deficient (knockout [KO]) or sufficient (wild-type [WT]) BALB/cJ mice upon transplantation of murine 4T1 carcinoma cells that had little B7-H4 expression. We reveal that host B7-H4 not only dampens the antitumor Th1 responses, but also inhibits the protumor function of myeloid-derived suppressor cells (MDSC). We observed increased expression of both antitumor immune effectors and protumor MDSC-associated transcripts in 4T1 tumors grown in B7-H4 KO mice compared with those grown in WT hosts. Consistently, MDSCs derived from B7-H4 KO mice suppressed T cell proliferation more potently than their WT counterparts. Although the primary growth of 4T1 tumors in B7-H4 KO hosts was similar to that in WT mice, tumors that had grown in B7-H4 KO hosts grew much slower than those from WT mice when subsequently transplanted into WT hosts. Importantly, this differential tumor growth during the secondary transplantation was abrogated when recipient mice lacked T cells, indicating that the immune environment in B7-H4 KO hosts allowed outgrowth of 4T1 tumors with reduced immune-evasive capacities against T cells. Thus, B7-H4 can inhibit both antitumor T cells and protumor MDSCs, influencing the immune-evasive character of the outgrowing tumors. These factors should be considered if B7-H4 blockade is to be used for cancer immunotherapy.

Phosphatidylinositol 3-kinase-independent signaling pathways contribute to ICOS-mediated T cell costimulation in acute graft-versus-host disease in mice.

We and others have previously shown that ICOS plays an important role in inducing acute graft-versus-host disease (GVHD) in murine models of allogeneic bone marrow transplantation. ICOS potentiates TCR-mediated PI3K activation and intracellular calcium mobilization. However, ICOS signal transduction pathways involved in GVHD remain unknown. In this study, we examined the contribution of ICOS-PI3K signaling in the pathogenic potential of T cells using a knock-in mouse strain, ICOS-YF, which selectively lost the ability to activate PI3K. We found that when total T cells were used as alloreactive T cells, ICOS-YF T cells caused less severe GVHD compared with ICOS wild-type T cells, but they induced much more aggressive disease than ICOS knockout T cells. This intermediate level of pathogenic capacity of ICOS-YF T cells was correlated with similar levels of IFN-γ-producing CD8 T cells that developed in the recipients of ICOS-WT or ICOS-YF T cells. We further evaluated the role of ICOS-PI3K signaling in CD4 versus CD8 T cell compartment using GVHD models that are exclusively driven by CD4 or CD8 T cells. Remarkably, ICOS-YF CD8 T cells caused disease similar to ICOS wild-type CD8 T cells, whereas ICOS-YF CD4 T cells behaved very similarly to their ICOS knockout counterparts. Consistent with their in vivo pathogenic potential, CD8 T cells responded to ICOS ligation in vitro by PI3K-independent calcium flux, T cell activation, and proliferation. Thus, in acute GVHD in mice, CD4 T cells heavily rely on ICOS-PI3K signaling pathways; in contrast, CD8 T cells can use PI3K-independent ICOS signaling pathways, possibly through calcium.

ICOS costimulation is indispensable for the differentiation of T follicular regulatory cells.

ICOS is a T-cell costimulatory receptor critical for Tfh cell generation and function. However, the role of ICOS in Tfr cell differentiation remains unclear. Using Foxp3-Cre-mediated ICOS knockout (ICOS FC) mice, we show that ICOS deficiency in Treg-lineage cells drastically reduces the number of Tfr cells during GC reactions but has a minimal impact on conventional Treg cells. Single-cell transcriptome analysis of Foxp3+ cells at an early stage of the GC reaction suggests that ICOS normally inhibits Klf2 expression to promote follicular features including Bcl6 up-regulation. Furthermore, ICOS costimulation promotes nuclear localization of NFAT2, a known driver of CXCR5 expression. Notably, ICOS FC mice had an unaltered overall GC B-cell output but showed signs of expanded autoreactive B cells along with elevated autoantibody titers. Thus, our study demonstrates that ICOS costimulation is critical for Tfr cell differentiation and highlights the importance of Tfr cells in maintaining humoral immune tolerance during GC reactions.

Inducible costimulator promotes helper T-cell differentiation through phosphoinositide 3-kinase.

The T-cell costimulatory receptors, CD28 and the inducible costimulator (ICOS), are required for the generation of follicular B helper T cells (T(FH)) and germinal center (GC) reaction. A common signal transducer used by CD28 and ICOS is the phosphoinositide 3-kinase (PI3K). Although it is known that CD28-mediated PI3K activation is dispensable for GC reaction, the role of ICOS-driven PI3K signaling has not been defined. We show here that knock-in mice that selectively lost the ability to activate PI3K through ICOS had severe defects in T(FH) generation, GC reaction, antibody class switch, and antibody affinity maturation. In preactivated CD4(+) T cells, ICOS delivered a potent PI3K signal that was critical for the induction of the key T(FH) cytokines, IL-21 and IL-4. Under the same settings, CD28 was unable to activate PI3K but supported a robust secondary expansion of T cells. Thus, our results demonstrate a nonredundant function of ICOS-PI3K pathway in the generation of T(FH) and suggest that CD28 and ICOS play differential roles during a multistep process of T(FH) differentiation.

Roles of CD28, CTLA4, and inducible costimulator in acute graft-versus-host disease in mice.

T cells deficient for CD28 have reduced ability to expand and survive, but still cause graft-versus-host disease (GVHD). Inducible costimulator (ICOS), a member of the CD28 family, is expressed on antigen-activated T cells and plays unique roles in T cell activation and effector function. We hypothesized that ICOS contributes to the development of GVHD in the absence of B7:CD28/CTLA4 costimulation. In this study, we evaluated the roles of CD28, CTLA4, and ICOS in the pathogenesis of acute GVHD after myeloablative allogeneic bone marrow transplantation. Unexpectedly, we found that blocking CD28 and CTLA4 signals using the clinically relevant reagent CTLA4-Ig increases the severity of GVHD mediated by CD4(+) T cells, and that such treatment does not add any benefit to the blockade of ICOS. In contrast, selectively blocking CD28 and ICOS, but not CTLA4, prevents GVHD more effectively than blocking either CD28 or ICOS alone. Taken together, these results indicate that CD28 and ICOS are synergistic in promoting GVHD, whereas the CTLA4 signal is required for T cell tolerance regardless of ICOS signaling. Thus, blocking CD28 and ICOS while sparing CTLA4 represents a promising approach for abrogating pathogenic T cell responses after allogeneic bone marrow transplantation.

Survivin loss in thymocytes triggers p53-mediated growth arrest and p53-independent cell death.

Because survivin-null embryos die at an early embryonic stage, the role of survivin in thymocyte development is unknown. We have investigated the role by deleting the survivin gene only in the T lineage and show here that loss of survivin blocks the transition from CD4- CD8- double negative (DN) thymocytes to CD4+ CD8+ double positive cells. Although the pre-T cell receptor signaling pathway is intact in survivin-deficient thymocytes, the cells cannot respond to its signals. In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death. Strikingly, loss of survivin activates the tumor suppressor p53. However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2. These lines of evidence indicate that developing thymocytes depend on the cytoprotective function of survivin and that this function is tightly coupled to cell proliferation but independent of p53 and Bcl-2. Thus, survivin plays a critical role in early thymocyte development.

CD28 controls differentiation of regulatory T cells from naive CD4 T cells.

CD28 is required for the development of regulatory T cells (Tregs; CD4(+)CD25(+)Foxp3(+)) in the thymus and also contributes to their survival and homeostasis in the periphery. We studied whether and how CD28 and ICOS control the differentiation of Tregs from naive T cells. By using wild-type, CD28-, ICOS-, or CD28/ICOS-double knockout mice on C57BL/6 background as T cell sources, we found that CD28 is essential, whereas ICOS is dispensable, for the development and homeostasis of Tregs. Furthermore, the differentiation of Tregs from naive CD4(+)CD25(-) T cells in vivo also depends on CD28. The requirement of CD28 for Treg differentiation was mediated by IL-2, because neutralization of IL-2 with its specific mAb-blocked Treg differentiation from wild-type CD4(+)CD25(-) T cells and addition of IL-2 restored Treg differentiation from CD28(-/-) T cells. Other common gamma-chain cytokines, IL-4, IL-7, or IL-15, do not share such a role with IL-2. Although CD28 is required for the differentiation of Tregs from naive T cells, already generated Tregs do not depend on CD28 to exert their suppressive function. Our study reveals a new aspect of CD28 function in regulating T cell response.

Hippo Signal Transduction Mechanisms in T Cell Immunity.

Hippo signaling pathways are evolutionarily conserved signal transduction mechanisms mainly involved in organ size control, tissue regeneration, and tumor suppression. However, in mammals, the primary role of Hippo signaling seems to be regulation of immunity. As such, humans with null mutations in STK4 (mammalian homologue of Drosophila Hippo; also known as MST1) suffer from recurrent infections and autoimmune symptoms. Although dysregulated T cell homeostasis and functions have been identified in MST1-deficient human patients and mouse models, detailed cellular and molecular bases of the immune dysfunction remain to be elucidated. Although the canonical Hippo signaling pathway involves transcriptional co-activator Yes-associated protein (YAP) or transcriptional coactivator with PDZ motif (TAZ), the major Hippo downstream signaling pathways in T cells are YAP/TAZ-independent and they widely differ between T cell subsets. Here we will review Hippo signaling mechanisms in T cell immunity and describe their implications for immune defects found in MST1-deficient patients and animals. Further, we propose that mutual inhibition of Mst and Akt kinases and their opposing roles on the stability and function of forkhead box O and ß-catenin may explain various immune defects discovered in mutant mice lacking Hippo signaling components. Understanding these diverse Hippo signaling pathways and their interplay with other evolutionarily-conserved signaling components in T cells may uncover molecular targets relevant to vaccination, autoimmune diseases, and cancer immunotherapies.

Progression of AITL-like tumors in mice is driven by Tfh signature proteins and T-B cross talk.

Angioimmunoblastic T-cell lymphoma (AITL) is an aggressive peripheral T-cell lymphoma driven by a pool of neoplastic cells originating from T follicular helper (Tfh) cells and concomitant expansion of B cells. Conventional chemotherapies for AITL have shown limited efficacy, and as such, there is a need for improved therapeutic options. Because AITL originates from Tfh cells, we hypothesized that AITL tumors continue to rely on essential Tfh components and intimate T-cell-B-cell (T-B) interactions. Using a spontaneous AITL mouse model (Roquinsan/+ mice), we found that acute loss of Bcl6 activity in growing tumors drastically reduced tumor size, demonstrating that AITL-like tumors critically depend on the Tfh lineage-defining transcription factor Bcl6. Because Bcl6 can upregulate expression of signaling lymphocytic activation molecule-associated protein (SAP), which is known to promote T-B conjugation, we next targeted the SAP-encoding Sh2d1a gene. We observed that Sh2d1a deletion from CD4+ T cells in fully developed tumors also led to tumor regression. Further, we provide evidence that tumor progression depends on T-B cross talk facilitated by SAP and high-affinity LFA-1. In our study, AITL-like tumors relied heavily on molecular pathways that support Tfh cell identity and T-B collaboration, revealing potential therapeutic targets for AITL.

Generation and characterization of B7-H4/B7S1/B7x-deficient mice.

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.