Negative co-receptors on lymphocytes.

The past year has seen significant advances in our understanding of critical roles of negative immunoregulatory signals delivered through the B7-CD28 superfamily in regulating T cell activation and tolerance. Structural data on CTLA-4 have provided novel insights into the inhibitory functions of CTLA-4. Initial characterization of the PD-1-PD-1-ligand pathway has revealed that this pathway can downregulate TCR- and CD28-mediated signals. Recent studies indicate that ICOS exerts distinct effects at different phases of an immune response: ICOS can inhibit as well as stimulate T cell responses.

Development of autoimmunity in mice lacking DNA topoisomerase 3beta.

Mice lacking DNA topoisomerase 3beta are predisposed to a shortened lifespan, infertility, and lesions in multiple organs resulting from inflammatory responses. Examination of the immune system of 6- and 52-week-old top3beta(-/-) mice revealed no significant aberrations in their central and peripheral tolerance or in T lymphocyte activation. However, the older but not the younger cohort shows a high incidence of serum autoantibodies relative to their TOP3beta(+/+) age-mates. The mutant mice also show an increase in numerical aberrations of chromosomes in splenocytes and bone marrow cells, as well as an increase in apoptotic cells in the thymus. Thus, it appears plausible that the inflammatory lesions in top3beta(-/-) mice are caused by the development of autoimmunity as they age: Chromosomal abnormalities in top3beta(-/-) mice might lead to a persistent increase in apoptotic cells, which might in turn lead to the progression of autoimmunity.

PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis.

To compare the roles of programmed death 1 ligand 1 (PD-L1) and PD-L2 in regulating immunity to infection, we investigated responses of mice lacking PD-L1 or PD-L2 to infection with Leishmania mexicana. PD-L1(-/-) and PD-L2(-/-) mice exhibited distinct disease outcomes following infection with L. mexicana. In comparison to susceptible WT mice, PD-L1(-/-) mice showed resistance to L. mexicana, as demonstrated by reduced growth of cutaneous lesions and parasite burden. In contrast, PD-L2(-/-) mice developed exacerbated disease with increased parasite burden. Host resistance to L. mexicana is partly associated with the development of a Th1 response and down-regulation of the Th2 response. Both PD-L1(-/-) and PD-L2(-/-) mice produced levels of IFN-gamma similar to WT mice. However, the development of IL-4-producing cells was reduced in PD-L1(-/-) mice, demonstrating a role for PD-L1 in regulating Th cell differentiation. This inadequate Th2 response may explain the increased resistance of PD-L1(-/-) mice. Although no alterations in Th1/Th2 skewing were observed in PD-L2(-/-) mice, PD-L2(-/-) mice exhibited a marked increase in L. mexicana-specific antibody production. Increased Leishmania-specific IgG production may suppress the healing response through FcgammaR ligation on macrophages. Taken together, our results demonstrate that PD-L1 and PD-L2 have distinct roles in regulating the immune response to L. mexicana.

The B7 family revisited.

The discovery of new functions for the original B7 family members, together with the identification of additional B7 and CD28 family members, have revealed new ways in which the B7:CD28 family regulates T cell activation and tolerance. B7-1/B7-2:CD28 interactions not only promote initial T cell activation but also regulate self-tolerance by supporting CD4+CD25+ T regulatory cell homeostasis. CTLA-4 can exert its inhibitory effects in both B7-1/B7-2 dependent and independent fashions. B7-1 and B7-2 can signal bidirectionally by engaging CD28 and CTLA-4 on T cells and by delivering signals into B7-expressing cells. The five new B7 family members, ICOS ligand, PD-L1 (B7-H1), PD-L2 (B7-DC), B7-H3, and B7-H4 (B7x/B7-S1) are expressed on professional antigen-presenting cells as well as on cells within nonlymphoid organs, providing new means for regulating T cell activation and tolerance in peripheral tissues. The new CD28 families members, ICOS, PD-1, and BTLA, are inducibly expressed on T cells, and they have important roles in regulating previously activated T cells. PD-1 and BTLA also are expressed on B cells and may have broader immunoregulatory functions. The ICOS:ICOSL pathway appears to be particularly important for stimulating effector T cell responses and T cell-dependent B cell responses, but it also has an important role in regulating T cell tolerance. In addition, the PD-1:PD-L1/PD-L2 pathway plays a critical role in regulating T cell activation and tolerance. In this review, we revisit the roles of the B7:CD28 family members in regulating immune responses, and we discuss their therapeutic potential.

Rap1-GTP is a negative regulator of Th cell function and promotes the generation of CD4+CD103+ regulatory T cells in vivo.

The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.

The threshold pattern of calcineurin-dependent gene expression is altered by loss of the endogenous inhibitor calcipressin.

Calcineurin links calcium signaling to transcriptional responses in the immune, nervous and cardiovascular systems. To determine the function of the calcipressins, a family of putative calcineurin inhibitors, we assessed the calcineurin-dependent process of T cell activation in mice engineered to lack the gene encoding calcipressin 1 (Csp1). Csp1 regulated calcineurin in vivo, and genes triggered in an immune response had unique transactivation thresholds for T cell receptor stimulation. In the absence of Csp1, the apparent transactivation thresholds for all these genes were shifted because of enhanced calcineurin activity. This unbridled calcineurin activity drove Fas ligand expression, which normally requires high T cell receptor stimulation and results in the premature death of T helper type 1 cells. Thus, calcipressins modulate the pattern of calcineurin-dependent transcription, and may influence calcineurin activity beyond calcium to integrate a broad array of signals into the cellular response.

Cutting edge: inducible costimulator protein regulates both Th1 and Th2 responses to cutaneous leishmaniasis.

The CD28 family member inducible costimulator protein (ICOS) has an important role in T cell differentiation and Ig class switching. To investigate the role of ICOS in vivo, ICOS-/- mice were infected s.c. with Leishmania mexicana. While wild-type mice developed large, cutaneous lesions, the growth of lesions and tissue histopathology was significantly delayed in ICOS-/- mice. ICOS-/- mice exhibited marked decreases in both Th1 and Th2 cytokine production and profound defects in L. mexicana-specific Ig isotype class switching to IgG1 and IgG2a and reduced total IgE levels. Our findings indicate that ICOS is a key regulator of both Th1 and Th2 responses and has a role in controlling cutaneous L. mexicana infection.

Cutting edge: CTLA-4 (CD152) differentially regulates mitogen-activated protein kinases (extracellular signal-regulated kinase and c-Jun N-terminal kinase) in CD4+ T cells from receptor/ligand-deficient mice.

Although CTLA-4 (CD152) has potent inhibitory effects on T cell function, the signaling events affected by this coreceptor remain to be fully defined. Mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) act as crucial regulators of multiple aspects of cell function. Ab ligation studies have reported an inhibitory effect of CTLA-4 on TCR-induced ERK and JNK activation. In this study, we have re-examined the specificity of CTLA-4 inhibition of MAPKs by using natural ligand with ex vivo-purified CD4(+) T cells deficient in CD80 and CD86 (double knockout), or CTLA-4, CD80, and CD86 (triple knockout). Under these conditions, CTLA-4 ligation was found to up-regulate and sustain JNK activation, while inhibiting ERK activity. At the same time, JNK activation could not account for CTLA-4 induction of TGF-beta production. Our findings demonstrate that CTLA-4 cosignaling is more complex than previously appreciated, with an ability to differentially regulate members of the MAPK family in T cells.

E mu-BRD2 transgenic mice develop B-cell lymphoma and leukemia.

Transgenic mice with lymphoid-restricted overexpression of the double bromodomain protein bromodomain-containing 2 (Brd2) develop splenic B-cell lymphoma and, upon transplantation, B-cell leukemia with leukemic infiltrates in liver and lung. Brd2 is a nuclear-localized transcription factor kinase that is most closely related to TATA box binding protein-associated factor, 250 kDa (TAF(II)250) and the Drosophila developmental protein female sterile homeotic. Constitutive expression of BRD2 in the lymphoid compartment increases cyclin A transcription, "priming" transgenic B cells for proliferation. Mice stochastically develop an aggressive B-cell lymphoma with the features of B-1 cells, including CD5 and surface IgM expression. The B-cell lymphoma is monoclonal for immunoglobulin gene rearrangement and is phenotypically stable. The lymphoblasts are very large and express a transcriptome that is similar to human non-Hodgkin lymphomas. Both a wild-type BRD2 transgene and a kinase-null point mutant drive lymphomagenesis; therefore we propose that, rather than kinase activity, Brd2-mediated recruitment of E2 promoter binding factors (E2Fs) and a specific histone acetyltransferase to the cyclin A promoter by both types of transgene is a mechanistic basis for neoplasia. This report is the first to describe a transgenic mouse model for constitutive expression of a protein with more than one bromodomain.

CTLA-4 regulates cell cycle progression during a primary immune response.

Engagement of CTLA-4 is critical for inhibiting T cell immune responses. Recent studies have shown that CTLA-4 plays a key role in regulating peripheral T cell tolerance. It has been suggested that one mechanism by which CTLA-4 performs this function is by regulating cell cycle progression. Here, we investigate in depth the role of CTLA-4 in regulating cell cycle progression in naive T cells by comparing the immune responses in the absence or presence of CTLA-4. In the absence of CLTA-4, T cells exhibit marked increases in T cell proliferation, IL-2 mRNA and protein secretion, and cells cycling in the S and G2-M phase. Analyses of cyclins, cyclin-dependent kinases, and cell cycle inhibitors involved in the transition from the G1 to S phase reveal that cell cycle progression is prolonged in the absence of CTLA-4. This is due to the early exit from the G1 phase, entry into the S phase, and prolonged S phase period. Re-expression of the cell cycle inhibitor p27(kip1) is delayed in the absence of CTLA-4. These studies demonstrate that the B7 : CTLA-4 pathway exerts its major effects on T cell immune responses via regulation of the cell cycle.

Deletion of a conserved Il4 silencer impairs T helper type 1-mediated immunity.

Helper T cell differentiation involves silencing as well as activation of gene expression. We have identified a conserved silencer of the gene encoding interleukin 4 (Il4) marked by DNase I hypersensitivity (HS IV) and permissive chromatin structure in all helper T cells. Deletion of HS IV increased Il4 and Il13 transcription by naive T cells and led to T helper type 2 skewing in vitro. HS IV controlled Il4 silencing during T helper type 1 differentiation, as HS IV-deficient T helper type 1 cells that expressed interferon-gamma also produced abundant interleukin 4 in vitro and in vivo. Despite mounting a vigorous interferon-gamma response, HS IV-deficient mice were more susceptible to Leishmania major infection than were wild-type littermate control mice, showing a critical function for Il4 silencing in T helper type 1-mediated immunity.

An autoimmune disease-associated CTLA-4 splice variant lacking the B7 binding domain signals negatively in T cells.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulating T cell responses. A number of autoimmune diseases have shown genetic linkage to the CTLA-4 locus. We have cloned and expressed an alternatively spliced form of CTLA-4 that has genetic linkage with type I diabetes in the NOD mice. This splice variant of CTLA-4, named ligand-independent CTLA-4 (liCTLA-4), lacks exon2 including the MYPPPY motif essential for binding to the costimulatory ligands B7-1 and B7-2. Here we show that liCTLA-4 is expressed as a protein in primary T cells and strongly inhibits T cell responses by binding and dephosphorylating the TcRzeta chain. Expression of liCTLA-4, but not full-length CTLA-4 (flCTLA-4), was higher in memory/regulatory T cells from diabetes-resistant NOD congenic mice compared to susceptible NOD mice. These data suggest that increased expression and negative signaling delivered by the liCTLA-4 may regulate development of T cell-mediated autoimmune diseases.

T helper differentiation in resistant and susceptible B7-deficient mice infected with Leishmania major.

The contribution of the costimulatory molecules B7-1 and B7-2 to the in vivo differentiation of Th cells remains controversial. The infection of resistant and susceptible strains of mice with the parasite Leishmania major provides a well-established model for studying in vivo differentiation of CD4+ T cells. We have infected B7-1/B7-2-deficient mice on the BALB/c and 129 background with L. major and subsequently examined different parameters of infection and cytokine responses upon restimulation of lymph node cells in vitro. BALB/c B7-2-deficient and B7-1/B7-2-double deficient mice are resistant to L. major, whereas BALB/c B7-1-deficient mice remain as susceptible as wild-type BALB/c mice. Differential expression of B7-1 and B7-2 can explain the distinct roles observed for these B7 costimulators in L. major infection.