Nelfinavir monotherapy increases naïve T-cell numbers in HIV-negative healthy young adults.

Although patients treated with HIV protease inhibitor (PI) containing regimens manifest increases in naïve T cell number, it is unclear whether this is due to reduction in viral replication or a direct drug effect. We questioned whether Nelfinavir monotherapy directly impacted naïve T-cell number in HIV-negative individuals. HIV-negative volunteers received Nelfinavir, 1250 mg orally, BID for 3 weeks, and T-cell receptor recombination excision circles (TREC) content in peripheral blood were assessed. Whereas TREC copies did not change over 3 weeks in untreated controls, TREC copies/copies CCR5 increased following Nelfinavir monotherapy in 8 patients (p < 0.02), and did not change in 7 patients (p = NS). Those patients who responded were younger than those who did not with a median age of 55 years for responders and 71 years for non-responders (p < 0.03). The increase in TREC was most pronounced in those patients less than 40-years old (p < 0.01). Moreover, the patients who did not increase TREC levels were more likely to have suffered a medical illness previously shown to reduce thymic function. In HIV-negative patients, monotherapy with the HIV PI Nelfinavir for 21 days increases TREC-positive naïve T cell number, particularly in individuals who are healthy and young.

CD28 ligation costimulates cell death but not maturation of double-positive thymocytes due to defective ERK MAPK signaling.

The differentiation of double-positive (DP) CD4(+)CD8(+) thymocytes to single-positive CD4(+) or CD8(+) T cells is regulated by signals that are initiated by coengagement of the Ag (TCR) and costimulatory receptors. CD28 costimulatory receptors, which augment differentiation and antiapoptotic responses in mature T lymphocytes, have been reported to stimulate both differentiation and apoptotic responses in TCR-activated DP thymocytes. We have used artificial APCs that express ligands for TCR and CD28 to show that CD28 signals increase expression of CD69, Bim, and cell death in TCR-activated DP thymocytes but do not costimulate DP thymocytes to initiate the differentiation program. The lack of a differentiation response is not due to defects in CD28-initiated TCR proximal signaling events but by a selective defect in the activation of ERK MAPK. To characterize signals needed to initiate the death response, a mutational analysis was performed on the CD28 cytoplasmic domain. Although mutation of all of CD28 cytoplasmic domain signaling motifs blocks cell death, the presence of any single motif is able to signal a death response. Thus, there is functional redundancy in the CD28 cytoplasmic domain signaling motifs that initiate the thymocyte death response. In contrast, immobilized Abs can initiate differentiation responses and cell death in DP thymocytes. However, because Ab-mediated differentiation occurs through CD28 receptors with no cytoplasmic domain, the response may be mediated by increased adhesion to immobilized anti-TCR Abs.

Ly9 (CD229)-deficient mice exhibit T cell defects yet do not share several phenotypic characteristics associated with SLAM- and SAP-deficient mice.

Signaling lymphocyte activation molecule (SLAM) family receptors are critically involved in modulating innate and adaptive immune responses. Several SLAM family receptors have been shown to interact with the adaptor molecule SAP; however, subsequent intracellular signaling is poorly defined. Notably, mutations in SLAM-associated protein (SAP) lead to X-linked lymphoproliferative disease, a rare but fatal immunodeficiency. Although the SLAM family member Ly9 (CD229) is known to interact with SAP, the functions of this receptor have remained elusive. Therefore, we have generated Ly9-/- mice and compared their phenotype with that of SLAM-/- and SAP-/- mice. We report that Ly9-/- T cells exhibit a mild Th2 defect associated with reduced IL-4 production after stimulation with anti-TCR and anti-CD28 in vitro. This defect is similar in magnitude to the previously reported Th2 defect in SLAM-/- mice but is more subtle than that observed in SAP-/- mice. In contrast to SLAM-/- and SAP-/- mice, T cells from Ly9-/- mice proliferate poorly and produce little IL-2 after suboptimal stimulation with anti-CD3 in vitro. We have also found that Ly9-/- macrophages exhibit no defects in cytokine production or bacterial killing as was observed in SLAM-/- macrophages. Additionally, Ly9-/- mice differ from SAP-/- mice in that they foster normal development of NKT cells and mount appropriate T and B cell responses to lymphocytic choriomeningitis virus. We have identified significant phenotypic differences between Ly-9-/- mice as compared with both SLAM-/- and SAP-/- mice. Although Ly9, SLAM, and SAP play a common role in promoting Th2 polarization, Ly-9 is uniquely involved in enhancing T cell activation.

Increased thymic output in HIV-negative patients after antiretroviral therapy.

OBJECTIVE: To determine the effects of antiretroviral therapy on thymic output independent of HIV infection. METHODS: Thymic output was evaluated by quantifying signal joint T-cell receptor (TCR) recombination excision circles in peripheral blood lymphocytes from HIV-negative patients undergoing prophylactic antiretroviral therapy. Additionally, effects of the HIV protease inhibitor nelfinavir were assessed in vivo on TCR-induced death of murine double-positive thymocytes. RESULTS: Five out of seven HIV-negative patients undergoing prophylactic antiretroviral therapy exhibited a dramatic increase (1-3 log10) in recent thymic emigrants containing signal joint TCR recombination excision circles while their peripheral T cell compartments remained relatively unaffected. None of the patients developed subsequent HIV infections. Interestingly, nelfinavir did not have significant effects on TCR-induced apoptosis of murine thymocytes in vivo. CONCLUSION: Antiretroviral therapy augments thymic output independent of HIV. Furthermore, nelfinavir does not dramatically affect TCR-induced thymocyte death in mice, thus central tolerance remains intact.

Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse.

Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.

Selective inhibition of inflammatory gene expression in activated T lymphocytes: a mechanism of immune suppression by thiopurines.

Azathioprine and 6-mercaptopurine are antimetabolite thiopurine drugs that play important roles in the treatment of leukemia and in the management of conditions requiring immunosuppression, such as inflammatory bowel disease. The biochemical pharmacology of these drugs suggests that inhibition of purine nucleotide formation through the 6-thioguanine nucleotide metabolites is their key molecular mechanism. However, it is unclear how these metabolites suppress immunity. We hypothesized that azathioprine produces a selective inhibitory effect on activated but not quiescent T lymphocytes. We first established a model system of T lymphocyte culture with azathioprine that produced pharmacologically relevant concentrations of 6-thioguanine nucleotides. Using genome-wide expression profiling, we identified a group of azathioprine-regulated genes in quiescent and activated T lymphocytes. Several genes involved in immunity and inflammation were selectively down-regulated by azathioprine in stimulated but not quiescent cells. Quantitative reverse transcription-polymerase chain reaction for three of these genes, tumor necrosis factor-related apoptosis-inducing ligand, tumor necrosis factor receptor superfamily member 7, and alpha4-integrin, confirmed down-regulated expression of transcript levels. Tumor necrosis factor-related apoptosis-inducing ligand protein expression was further studied and found to be inhibited by azathioprine, 6-mercaptopurine, and 6-thioguanine, implying that the inhibitory effects of azathioprine on expression are mediated by 6-thioguanine nucleotides. These results therefore provide a previously unrecognized molecular mechanism for the immunosuppressive properties of thiopurine antimetabolite drugs.

Augmentation of T cell levels and responses induced by androgen deprivation.

Androgen has been implicated as a negative regulator of host immune function and a factor contributing to the gender dimorphism of autoimmunity. Conversely, androgen deprivation has been suggested to potentiate male host immunity. Studies have shown that removal of androgen in postpubertal male mice produces an increase in size and cellularity of primary and peripheral lymphoid organs, and enhances a variety of immune responses. Yet, few details are known about the effect of androgen removal on T cell-mediated immunity. In this study, we demonstrate two pronounced and independent alterations in T cell immunity that occur in response to androgen deprivation, provided by castration, in postpubertal male mice. First, we show that levels of T cells in peripheral lymphoid tissues of mice are increased by androgen deprivation. Second, T cells from these mice transiently proliferate more vigorously to TCR- and CD28-mediated costimulation as well as to Ag-specific activation. In addition, androgen deprivation accelerates normalization of host T and B cell levels following chemotherapy-induced lymphocyte depletion. Such alterations induced by androgen deprivation may have implications for enhancing immune responses to immunotherapy and for accelerating the recovery of the immune system following chemotherapy.

Direct transcriptional regulation of RelB by 1alpha,25-dihydroxyvitamin D3 and its analogs: physiologic and therapeutic implications for dendritic cell function.

The nuclear factor-kappaB (NF-kappaB) protein RelB plays a unique role in dendritic cell (DC) function and, as such, is an important regulator of antigen presentation and immune regulation. In this study, inhibition of RelB expression in DCs exposed to an analog of the active form of vitamin D3 (1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3)) was observed and shown to be mediated by the vitamin D receptor (VDR). Potential vitamin D response elements were identified within promoter regions of human and mouse relB genes. In gel shift experiments, these motifs specifically bound VDR.retinoid X receptor-alpha complexes. Reporter assays confirmed that transcriptional activity of human and mouse relB promoters was inhibited by 1alpha,25-(OH)2D3 agonists in a DC-derived cell line. The inhibition was abolished by mutagenesis of the putative vitamin D response elements and was enhanced by overexpression of VDR. Mutagenesis of NF-kappaB response elements within the relB promoter did not affect the magnitude of 1alpha,25-(OH)2D3 analog-mediated inhibition, ruling out an indirect effect on NF-kappaB signaling. Glucocorticoid caused additional inhibition of relB promoter activity when combined with the 1alpha,25-(OH)2D3 analog. This effect was dependent on the integrity of the NF-kappaB response elements, suggesting separate regulatory mechanisms for the two steroid pathways on this promoter. We conclude that relB is a direct target for 1alpha,25-(OH)2D3-mediated negative transcriptional regulation via binding of VDR.retinoid X receptor-alpha to discrete DNA motifs. This mechanism has important implications for the inhibitory effect of 1alpha,25-(OH)2D3 on DC maturation and for the potential immunotherapeutic use of 1alpha,25-(OH)2D3 analogs alone or combined with other agents.

Distinctive dendritic cell modulation by vitamin D(3) and glucocorticoid pathways.

Dendritic cell (DC) maturation plays a central role in regulating immunity. We show that glucocorticoid and 1alpha,25(OH)(2)D(3) agonists modulate DCs via distinct and additive signaling pathways. Phenotypic and functional indices were examined in DCs treated with dexamethasone (DEX) and/or a 1alpha,25(OH)(2)D(3) analog (D(3) analog). DEX potently attenuated pro-inflammatory cytokines and chemokines but had modest, reversible effects on T-cell stimulatory capacity. D(3) analog produced significantly greater inhibition of T-cell stimulation in vitro and in vivo and, unlike DEX, increased expression of the chemokines MCP-1 and MIP-1alpha. Both DEX and D(3) analog were associated with reduced expression of the NF-kappaB proteins c-Rel and Rel B but not Rel A. Combined DEX and D(3) analog treatment of DCs resulted in significant additive inhibition of pro-inflammatory cytokines, T-cell stimulation, chemokines, chemokine receptors, and NF-kappaB components. Additive inhibition was most striking for RANTES, CCR5, CCR7, and Rel B. The combined effects of the two hormonal pathways on DCs have unique immunomodulatory potential.