Calcium channel blocker prevents T helper type 2 cell-mediated airway inflammation.

RATIONALE: Ca(2+) signaling controls the production of T helper (Th) type 2 cytokines known to be deleterious in asthma. Recently, we showed that Ca(2+) signaling was dihydropyridine (DHP)-sensitive in Th2 lymphocytes and that the DHP derivate, nicardipine, used in the treatment of cardiovascular pathologies, prevents Th2-dependent B cell polyclonal activation. OBJECTIVES: We tested the effect of nicardipine in experimental allergic asthma. METHODS: BALB/c mice immunized with ovalbumin (OVA) in alum and challenged with intranasal OVA were treated with nicardipine once the Th2 response, or even airway inflammation, was induced. We also tested the effect of nicardipine in asthma induced by transferring OVA-specific Th2 cells in BALB/c mice exposed to intranasal OVA. We checked the impact of nicardipine on T-cell responses and airway inflammation. MEASUREMENTS AND MAIN RESULTS: Nicardipine inhibited in vitro Ca(2+) response in Th2 cells. In vivo, it impeded the development of Th2-mediated airway inflammation and reduced the capacity of lymphocytes from lung-draining lymph nodes to secrete Th2, but not Th1, cytokines. Nicardipine did not affect antigen presentation to CD4(+) T lymphocytes, nor the initial localization of Th2 cells into the lungs of mice exposed to intranasal OVA; however, it reduced the production of type 2 cytokines and the amplification of the Th2 response in mice with asthma. Conversely, nicardipine had no effect on Th1-mediated airway inflammation. CONCLUSIONS: Nicardipine improves experimental asthma by impairing Th2-dependent inflammation. This study could provide a rationale for developing drugs selectively targeting DHP receptors of Th2 lymphocytes, potentially beneficial in the treatment of asthma.

Dihydropyridine receptors are selective markers of Th2 cells and can be targeted to prevent Th2-dependent immunopathological disorders.

Th1 cells that produce IFN-gamma are essential in the elimination of intracellular pathogens, and Th2 cells that synthetize IL-4 control the eradication of helminths. However, highly polarized Th1 or Th2 responses may be harmful and even lethal. Thus, the development of strategies to selectively down-modulate Th1 or Th2 responses is of therapeutic importance. Herein, we demonstrate that dihydropyridine receptors (DHPR) are expressed on Th2 and not on Th1 murine cells. By using selective agonists and antagonists of DHPR, we show that DHPR are involved in TCR-dependent calcium response in Th2 cells as well as in IL-4, IL-5, and IL-10 synthesis. Nicardipine, an inhibitor of DHPR, is beneficial in experimental models of Th2-dependent pathologies in rats. It strongly inhibits the Th2-mediated autoimmune glomerulonephritis induced by injecting Brown Norway (BN) rats with heavy metals. This drug also prevents the chronic graft vs host reaction induced by injecting CD4(+) T cells from BN rats into (LEW x BN)F(1) hybrids. By contrast, treatment with nicardipine has no effect on the Th1-dependent experimental autoimmune encephalomyelitis triggered in LEW rats immunized with myelin. These data indicate that 1) DHPR are a selective marker of Th2 cells, 2) these calcium channels contribute to calcium signaling in Th2 cells, and 3) blockers of these channels are beneficial in the treatment of Th2-mediated pathologies.

Selection of similar naive T cell repertoires but induction of distinct T cell responses by native and modified antigen.

To study the T cell responses induced by native and modified Ag, we have followed in vivo TCR selection and cytokine profile of T cells, as well as the isotype of induced Abs, in response to the model Ag hen egg-white lysozyme (HEL) and its reduced and carboxymethylated form (RCM-HEL). RCM-HEL induces in vivo a T cell response focused on the same immunodominant determinant characterizing the response to native HEL, but further skewed to the Th1 pathway. No difference between HEL and RCM-HEL could be observed in the efficiency of processing, nor in the type of APCs involved. In vivo experiments show that coimmunization with HEL and RCM-HEL generates distinct Th2 or Th1 responses in naive mice, but the two forms of Ag expand the same HEL-specific public clonotype, characterized by the Vbeta8.2-Jbeta1.5 rearrangement, indicating that the populations of naive T cells activated by the two Ag forms overlap. T cells primed by RCM-HEL are restimulated by soluble HEL in vivo, but divert the phenotype of the HEL-specific response to Th1, implying that priming of naive T cells by a structurally modified Ag can induce Th1-type memory/effector T cells more efficiently than native Ag.

Tracking T cell clonotypes in complex T lymphocyte populations by real-time quantitative PCR using fluorogenic complementarity-determining region-3-specific probes.

The T cell receptor (TCR) alpha and beta chains are encoded by a series of stochastic rearrangements between variable (V), diversity (D) for TCR beta chain only, and joining (J) gene segments, creating hypervariable complementarity-determining region 3 (CDR3) regions that contact the peptide/MHC complex and confer specificity. In the present paper, we applied the recently developed real-time quantitative RT-PCR technique to the detection of rearranged TCR beta chain mRNA transcripts. We designed BV- and BJ-specific primers together with TaqMan probes specific for the CDR3 regions of the clones of interest. As an external reference, we used plasmids containing the entire TCR beta chains, making it possible to normalize the number of specific rearranged BV-J mRNA copies among the total number of TCR beta chains. Here, we present data validating this fluorogenic PCR-based method for the quantification of several TCR clonotypes characteristic of the CD4 T cell response to hen egg white lysozyme (HEL) in mice of the H-2d haplotype. This accurate and sensitive procedure permits the precise determination of T cell clone frequencies ranging from 10(-2) to less than 10(-5) in normal biological samples; it may provide an alternative approach when frequencies are too low to be assessed by flow cytometry.

Chronic soluble antigen sensitization primes a unique memory/effector T cell repertoire associated with th2 phenotype acquisition in vivo.

Although much progress has been made in characterization of the signaling pathways that control Th cell commitment, little is known about the early events that govern differentiation of IL-4-producing T lymphocytes in vivo. We have previously shown that chronic administration of low dose, soluble hen egg white lysozyme (HEL) induced the selective development of Ag-specific Th2 in genetically predisposed BALB/c mice. Here, we show that these memory/effector Th2 cells express a unique TCR Vbeta repertoire, different from the TCR Vbeta profile of primary effector cells from HEL-adjuvant-primed mice. This Th2-associated repertoire contains a highly frequent public clonotype characterized by preferred TCR AV and BV gene segment usage along with conserved sequences in the third hypervariable regions of both TCR chains. This Th2 clonotype, which is not recruited in primary effector T cells from HEL-adjuvant-immunized mice, recognized an IA(d)-restricted HEL determinant, preferentially processed by dendritic cells, but not by B cells. Thus, IL-4-producing CD4 T cells that expand following chronic Ag sensitization emerge from a distinct pool of precursors, supporting the hypothesis that ligand-TCR interactions play a crucial role in the regulation of Ag-specific Th2 cell development in vivo.

Blockade of CD86 in BALB/c mice infected with Leishmania major does not prevent the expansion of low avidity T cells.

The interactions between CD28 and its ligand CD86 are critical for the regulation of T cell responses. However, it is not clear whether CD4+ T cells expressing low and high avidity TCR are equally dependent on CD28 costimulation for their activation and expansion. To address this issue, we have used multimers of I-Ad molecules linked to a peptide derived from the Leishmania major homolog for the receptor of activated C kinase (LACK) antigen to compare the fate of LACK-specific CD4+ T cells in Leishmania-infected BALB/c mice which have been treated or not with anti-CD86 mAb. Although the administration of anti-CD86 mAb did not completely prevent the expansion of LACK-specific T cells, their frequency and number were markedly reduced. In mice treated with anti-CD86 mAb as well as in control animals, L. major induced the clonal expansion of LACK-specific T cells which expressed a canonical low avidity Valpha8/Vbeta4 TCR. Taken together, our results suggest that the molecular interactions between CD28 on T cells and CD86 on APC serve to amplify and modulate T cell responses without promoting breadth in the TCR repertoire.