Rebecca J. Greenwald

ICOS is critical for CD40-mediated antibody class switching

The inducible co-stimulatory molecule (ICOS) is a CD28 homologue implicated in regulating T-cell differentiation. Because co-stimulatory signals are critical for regulating T-cell activation, an understanding of co-stimulatory signals may enable the design of rational therapies for immune-mediated diseases. According to the two-signal model for T-cell activation, T cells require an antigen-specific signal and a second, co-stimulatory, signal for optimal T-cell activation. The co-stimulatory signal promotes T-cell proliferation, lymphokine secretion and effector function. The B7–CD28 pathway provides essential signals for T-cell activation, but does not account for all co-stimulation. We have generated mice lacking ICOS (ICOS-/- ) to determine the essential functions of ICOS. Here we report that ICOS-/- mice exhibit profound deficits in immunoglobulin isotype class switching, accompanied by impaired germinal centre formation. Class switching was restored in ICOS-/- mice by CD40 stimulation, showing that ICOS promotes T-cell/B-cell collaboration through the CD40/CD40L pathway.

CTLA-4 Regulates Induction of Anergy In Vivo

The requirement for CTLA-4 during the induction of peripheral T cell tolerance in vivo was investigated using naive TCR transgenic T cells lacking CTLA-4. CTLA-4(-/-) T cells are resistant to tolerance induction, as demonstrated by their proliferative responses, IL-2 production, and progression into the cell cycle. Following exposure to a tolerogenic stimulus in vivo and restimulation in vitro, wild-type T cells are blocked at the late G1 to S restriction point of the cell cycle. In contrast, CTLA-4(-/-) T cells enter into the S phase of the cell cycle, as shown by downregulation of p27(kip1), elevated cdk2 kinase activity, and Rb hyperphosphorylation. Thus, CTLA-4 has an essential role in determining the outcome of T cell encounter with a tolerogenic stimulus.

Blockade of CTLA-4 on CD4+CD25+ regulatory T cells abrogates their function in vivo.

Naturally occurring CD4+ regulatory T cells (TR) that express CD25 and the transcription factor FoxP3 play a key role in immune homeostasis, preventing immune pathological responses to self and foreign Ags. CTLA-4 is expressed by a high percentage of these cells, and is often considered as a marker for TR in experimental and clinical analysis. However, it has not yet been proven that CTLA-4 has a direct role in TR function. In this study, using a T cell-mediated colitis model, we demonstrate that anti-CTLA-4 mAb treatment inhibits TR function in vivo via direct effects on CTLA-4-expressing TR, and not via hyperactivation of colitogenic effector T cells. Although anti-CTLA-4 mAb treatment completely inhibits TR function, it does not reduce TR numbers or their homing to the GALT, suggesting the Ab mediates its function by blockade of a signal required for TR activity. In contrast to the striking effect of the Ab, CTLA-4-deficient mice can produce functional TR, suggesting that under some circumstances other immune regulatory mechanisms, including the production of IL-10, are able to compensate for the loss of the CTLA-4-mediated pathway. This study provides direct evidence that CTLA-4 has a specific, nonredundant role in the function of normal TR. This role has to be taken into account when targeting CTLA-4 for therapeutic purposes, as such a strategy will not only boost effector T cell responses, but might also break TR-mediated self-tolerance.

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.

CTLA-4 and T cell activation

The past year has seen significant advances in our understanding of the role of cytotoxic T lymphocyte antigen 4 (CTLA-4) in regulating T cell activation and tolerance. Recent studies indicate that CTLA-4 not only counterbalances CD28 signals but also can inhibit T cell responses independently of CD28. Recent work has also revealed a role for CTLA-4 in regulating Th1/Th2 differentiation. Manipulation of CTLA-4 in animal models of autoimmunity has shown that CTLA-4 regulates both the initiation and the progression of autoimmune diseases.

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.

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.

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-γ also produced abundant interleukin 4 in vitro and in vivo. Despite mounting a vigorous interferon-γ 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.

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 suggestedthat 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 p27kip1 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.

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.