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1.  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-g–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.
PMCID: PMC4318500  PMID: 23729441
2.  The CD28-B7 Family in Anti-Tumor Immunity: Emerging Concepts in Cancer Immunotherapy 
Immune Network  2014;14(6):265-276.
The interactions between B7 molecules and CD28-family receptors are crucial in the regulation of adaptive cellular immunity. In cancer, the aberrant expression of co-inhibitory B7 molecules has been attributed to reduced anti-tumor immunity and cancer immune evasion, prompting the development of cancer therapeutics that can restore T cell function. Murine tumor models have provided significant support for the targeting of multiple immune checkpoints involving CTLA-4, PD-1, ICOS, B7-H3 and B7-H4 during tumor growth, and clinical studies investigating the therapeutic effects of CTLA-4 and PD-1 blockade have shown exceptionally promising results in patients with advanced melanoma and other cancers. The expression pattern of co-inhibitory B7 ligands in the tumor microenvironment has also been largely correlated with poor patient prognosis, and recent evidence suggests that the presence of several B7 molecules may predict the responsiveness of immunotherapies that rely on pre-existing tumor-associated immune responses. While monotherapies blocking T cell co-inhibition have beneficial effects in reducing tumor burden, combinatorial immunotherapy targeting multiple immune checkpoints involved in various stages of the anti-tumor response has led to the most substantial impact on tumor reduction. In this review, we will examine the contributions of B7- and CD28-family members in the context of cancer development, and discuss the implications of current human findings in cancer immunotherapy.
PMCID: PMC4275384  PMID: 25550693
B7 family; CD28 family; Co-stimulation; Co-inhibition; Cancer immunotherapy; Immune evasion
3.  Generation and Characterization of B7-H4/B7S1/B7x-Deficient Mice 
Molecular and Cellular Biology  2006;26(17):6403-6411.
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.
PMCID: PMC1592821  PMID: 16914726
4.  Life of T Follicular Helper Cells 
Molecules and Cells  2014;38(3):195-201.
Antibodies are powerful defense tools against pathogens but may cause autoimmune diseases when erroneously directed toward self-antigens. Thus, antibody producing cells are carefully selected, refined, and expanded in a highly regulated microenvironment (germinal center) in the peripheral lymphoid organs. A subset of T cells termed T follicular helper cells (Tfh) play a central role in instructing B cells to form a repertoire of antibody producing cells that provide life-long supply of high affinity, pathogen-specific antibodies. Therefore, understanding how Tfh cells arise and how they facilitate B cell selection and differentiation during germinal center reaction is critical to improve vaccines and better treat autoimmune diseases. In this review, I will summarise recent findings on molecular and cellular mechanisms underlying Tfh generation and function with an emphasis on T cell costimulation.
PMCID: PMC4363718  PMID: 25537859
antibody; costimulation; germinal center; T follicular helper
5.  Anti-Chlamydial Th17 Responses Are Controlled by the Inducible Costimulator Partially through Phosphoinositide 3-Kinase Signaling 
PLoS ONE  2012;7(12):e52657.
We previously showed that mice deficient in the Inducible Costimulator ligand (ICOSL-KO) develop more severe disease and lung pathology with delayed bacterial clearance upon respiratory infection of Chlamydia muridarum. Importantly, the exacerbation of disease in ICOSL-KO mice was seen despite heightened IFN-γ/Th1 responses, the major defense mechanisms against Chlamydia. To gain insight into the mechanism of ICOS function in this model, we presently analyzed anti-Chlamydia immune responses in mice lacking the entire ICOS (ICOS-KO) versus knock-in mice expressing a mutant ICOS (ICOS-Y181F) that has selectively lost the ability to activate phosphoinositide 3-kinase (PI3K). Like ICOSL-KO mice, ICOS-KO mice showed worse disease with elevated IFN-γ/Th1 responses compared to wild-type (WT) mice. ICOS-Y181F mice developed much milder disease compared to ICOS-KO mice, yet they were still not fully protected to the WT level. This partial protection in ICOS-Y181F mice could not be explained by the magnitude of IFN-γ/Th1 responses since these mice developed a similar level of IFN-γ response compared to WT mice. It was rather IL-17/Th17 responses that reflected disease severity: IL-17/Th17 response was partially impaired in ICOS-Y181F mice compared to WT, but was substantially stronger than that of ICOS-KO mice. Consistently, we found that both polarization and expansion of Th17 cells were partially impaired in ICOS-Y181F CD4 T cells, and was further reduced in ICOS-KO CD4 T cells in vitro. Our results indicate that once the IFN-γ/Th1 response is above a threshold level, the IL-17/Th17 response becomes a limiting factor in controlling Chlamydia lung infection, and that ICOS plays an important role in promoting Th17 responses in part through the activation of PI3K.
PMCID: PMC3527575  PMID: 23285133
6.  Coordinate activation of inflammatory gene networks, alveolar destruction and neonatal death in AKNA deficient mice 
Cell Research  2011;21(11):1564-1577.
Gene expression can be regulated by chromatin modifiers, transcription factors and proteins that modulate DNA architecture. Among the latter, AT-hook transcription factors have emerged as multifaceted regulators that can activate or repress broad A/T-rich gene networks. Thus, alterations of AT-hook genes could affect the transcription of multiple genes causing global cell dysfunction. Here we report that targeted deletions of mouse AKNA, a hypothetical AT-hook-like transcription factor, sensitize mice to pathogen-induced inflammation and cause sudden neonatal death. Compared with wild-type littermates, AKNA KO mice appeared weak, failed to thrive and most died by postnatal day 10. Systemic inflammation, predominantly in the lungs, was accompanied by enhanced leukocyte infiltration and alveolar destruction. Cytologic, immunohistochemical and molecular analyses revealed CD11b+Gr1+ neutrophils as major tissue infiltrators, neutrophilic granule protein, cathelin-related antimicrobial peptide and S100A8/9 as neutrophil-specific chemoattracting factors, interleukin-1β and interferon-γ as proinflammatory mediators, and matrix metalloprotease 9 as a plausible proteolytic trigger of alveolar damage. AKNA KO bone marrow transplants in wild-type recipients reproduced the severe pathogen-induced reactions and confirmed the involvement of neutrophils in acute inflammation. Moreover, promoter/reporter experiments showed that AKNA could act as a gene repressor. Our results support the concept of coordinated pathway-specific gene regulation functions modulating the intensity of inflammatory responses, reveal neutrophils as prominent mediators of acute inflammation and suggest mechanisms underlying the triggering of acute and potentially fatal immune reactions.
PMCID: PMC3365639  PMID: 21606955
in vivo; mouse genetics; gene network regulation; inflammation; pathogens; innate reactions
7.  Role of CD28, CTLA4 and ICOS costimulation 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), the third member of the CD28 family, is expressed on antigen-activated T cells and has 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 under myeloablative allogeneic bone marrow transplantation (BMT). Unexpectedly, we found that blocking CD28 and CTLA4 signals using the clinically relevant reagent, CTLA4-Ig, results in the enhancement of GVHD severity mediated by CD4+ T cells, and such treatment does not add any benefit to 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 BMT.
PMCID: PMC3131782  PMID: 21447398
8.  CD28 Controls Differentiation of Regulatory T Cells from Naive CD4 T Cells1 
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 γ-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.
PMCID: PMC2688779  PMID: 18684917
9.  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.
PMCID: PMC2211792  PMID: 14757745
pre–T cell; cell death; development; thymus; mitosis
10.  Generation and Characterization of Smac/DIABLO-Deficient Mice 
Molecular and Cellular Biology  2002;22(10):3509-3517.
The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac−/−) mice by using homologous recombination in embryonic stem (ES) cells. Smac−/− mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac−/− cells, all types of cultured Smac−/− cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.
PMCID: PMC133802  PMID: 11971981
11.  Tumor Necrosis Factor Receptor–Associated Factor (Traf)2 Represses the T Helper Cell Type 2 Response through Interaction with Nfat-Interacting Protein (Nip45) 
Recently we have identified a novel protein NIP45 (nuclear factor of activated T cells [NFAT]-interacting protein) which substantially augments interleukin (IL)-4 gene transcription. The provision of NIP45 together with NFAT and the T helper cell type 2 (Th2)-specific transcription factor c-Maf to cells normally refractory to IL-4 production, such as B cells or Th1 clones, results in substantial IL-4 secretion to levels that approximate those produced by primary Th2 cells. In studies designed to further our understanding of NIP45 activity, we have uncovered a novel facet of IL-4 gene regulation. We present evidence that members of the tumor necrosis factor receptor–associated factor (TRAF) family of proteins, generally known to function as adapter proteins that transduce signals from the tumor necrosis factor receptor superfamily, contribute to the repression of IL-4 gene transcription and that this effect is mediated through their interaction with NIP45.
PMCID: PMC2193447  PMID: 11435475
NIP45; interleukin-4; cytokines; TRAF2; transcription

Results 1-11 (11)