Virus-specific CD4+ T helper lymphocytes are critical to the maintenance of effective immunity in a number of chronic viral infections, but are characteristically undetectable in chronic human immunodeficiency virus-type 1 (HIV-1) infection. In individuals who control viremia in the absence of antiviral therapy, polyclonal, persistent, and vigorous HIV-1-specific CD4+ T cell proliferative responses were present, resulting in the elaboration of interferon-gamma and antiviral beta chemokines. In persons with chronic infection, HIV-1-specific proliferative responses to p24 were inversely related to viral load. Strong HIV-1-specific proliferative responses were also detected following treatment of acutely infected persons with potent antiviral therapy. The HIV-1-specific helper cells are likely to be important in immunotherapeutic interventions and vaccine development.

Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca(2+) binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.

CRACM1 (Orai1) constitutes the pore subunit of CRAC channels that are crucial for many physiological processes 1-6 . A point mutation in CRACM1 has been associated with SCID disease in humans 2 . We have generated CRACM1 deficient mice using gene trap, where β-galactosidase (LacZ) activity identifies CRACM1 expression in tissues. We show here that the homozygous CRACM1 deficient mice are considerably smaller in size and are grossly defective in mast cell degranulation and cytokine secretion. FcεRI-mediated in vivo allergic reactions were also inhibited in CRACM1-/-mice. Other tissues expressing truncated CRACM1-LacZ fusion protein include skeletal muscles, kidney and regions in the brain and heart. Surprisingly, no CRACM1 expression was seen in the lymphoid regions of thymus. Accordingly, we found no defect in T cell development. Thus, our data reveal novel crucial roles for CRAC channels including a putative role in excitable cells.

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Dysfunction of virus-specific CD4 + T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4 + T cells specific for human immunodeficiency virus (HIV) from HIV-infected people prior and after initiation of antiretroviral therapy (ART). A follicular helper T cell (T FH cell)–like profile characterized HIV-specific CD4 + T cells in viraemic infection. HIV-specific CD4 + T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the T H 1, T H 17 and T H 22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with T FH cells but persistently low expression of genes associated with T H 1, T H 17 and T H 22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4 + T cells and involves both gain of function and loss of function.

Cellular immune responses are thought to be an important antiviral host defense, but the relationship between virus-specific T-helper and cytotoxic-T-lymphocyte (CTL) responses has not been defined. To investigate a potential link between these responses, we examined functional human immunodeficiency virus type 1 (HIV-1)-specific memory CTL precursor frequencies and p24-specific proliferative responses in a cohort of infected untreated persons with a wide range of viral loads and CD4 cell counts. Levels of p24-specific proliferative responses positively correlated with levels of Gag-specific CTL precursors and negatively correlated with levels of plasma HIV-1 RNA. These data linking the levels of HIV-specific CTL with virus-specific helper cell function during chronic viral infection provide cellular immunologic parameters to guide therapeutic and prophylactic vaccine development.

High-affinity IgE receptor (FcɛRI) cross-linking on mast cells (MCs) induces secretion of preformed allergy mediators (degranulation) and synthesis of lipid mediators and cytokines. Degranulation produces many symptoms of immediate-type allergic reactions and is modulated by adhesion to surfaces coated with specific extracellular matrix (ECM) proteins. The signals involved in this modulation are mostly unknown and their contribution to allergic reactions in vivo is unclear. Here we report the generation of monoclonal antibodies that potently suppress FcɛRI-induced degranulation, but not leukotriene synthesis. We identified the antibody target as the tetraspanin CD63. Tetraspanins are membrane molecules that form multimolecular complexes with a broad array of molecules including ECM protein-binding β integrins. We found that anti-CD63 inhibits MC adhesion to fibronectin and vitronectin. Furthermore, anti-CD63 inhibits FcɛRI-mediated degranulation in cells adherent to those ECM proteins but not in nonadherent cells. Thus the inhibition of degranulation by anti-CD63 correlates with its effect on adhesion. In support of a mechanistic linkage between the two types of inhibition, anti-CD63 had no effect on FcɛRI-induced global tyrosine phosphorylation and calcium mobilization but impaired the Gab2–PI3K pathway that is known to be essential for both degranulation and adhesion. Finally, we showed that these antibodies inhibited FcɛRI-mediated allergic reactions in vivo. These properties raise the possibility that anti-CD63 could be used as therapeutic agents in MC-dependent diseases.

Human high affinity IgE receptors are expressed as two different isoforms: the tetrameric isoform, alphabetagamma(2), or the trimeric isoform, alphagamma(2). The alpha chain is the IgE binding subunit, whereas the FcRbeta and FcRgamma chains are the signaling modules. Both FcRbeta and FcRgamma contain immunoreceptor tyrosine-based activation motifs (ITAM), but the beta ITAM differs from canonical ITAMs in two ways; the spacing between the two canonical tyrosines harbors a third tyrosine, and it is one amino acid shorter than in canonical ITAMs, making it unfit to bind the tandem SH2 of Syk. We have shown that FcRbeta functions as an amplifier of the FcRgamma signaling function. However, the molecular mechanism of this amplification remains unclear. Here we show that mutation of the three tyrosines (Tyr-219, Tyr-225, and Tyr-229) in the beta ITAM essentially converts alphabetagamma(2)into an alphagamma(2) complex in terms of Lyn recruitment, FcRgamma phosphorylation, Syk activation, and calcium mobilization. Tyr-219 is the most critical residue in this regard. In addition, a detailed analysis of the dynamics of calcium mobilization suggests a possible inhibitory role for Tyr-225, which becomes apparent when Tyr-219 is mutated. Thus, the signaling amplification function of FcRbeta is mainly encoded in Tyr-219 and in its capacity to recruit Lyn. In turn, this Tyr-219-mediated Lyn recruitment enhances gamma chain phosphorylation, Syk activation, and calcium mobilization. The two other tyrosines appear to have a modulating function that remains to be fully assessed.