Microbiology

Most developing thymocytes undergo apoptosis because they cannot interact productively with molecules encoded by the major histocompatibility complex. Here, we show that mice lacking the orphan nuclear hormone receptor ROR␥ lose thymic expression of the anti-apoptotic factor Bcl-xL. ROR␥ thus regulates the survival of CD4 ϩ 8 ϩ thymocytes and may control the temporal window during which thymocytes can undergo positive selection. ROR␥ was also required for development of lymph nodes and Peyer's patches, but not splenic follicles. In its absence, there was loss of a population of CD3 -CD4 ϩ CD45 ϩ cells that normally express ROR␥ and that are likely early progenitors of lymphoid organs. Hence, ROR␥ has critical functions in T cell repertoire selection and lymphoid organogenesis.

We investigated whether human resting T cells could be activated to proliferate and display effector function in the absence of T cell receptor occupancy. We report that combination of interleukin 2 (IL-2), tumor necrosis factor or, and IL-6 activated highly purified naive (CD45RA +) and memory (CD45RO +) resting CD4 + T cells to proliferate. Under this condition, memory resting T cells could also display effector function as measured by lymphokine synthesis and help for immunoglobulin production by B cells. This novel Ag-independent pathway of T cell activation may play an important role in vivo in recruiting effector T cells at the site of immune response and in maintaining the clonal size of memory T cells in the absence of antigenic stimulation. Moreover, cytokines can induce proliferation of naive T cells without switch to memory phenotype and this may help the maintenance of the peripheral pool of naive T cells.

The adult liver is an organ without constitutive lymphoid components. Therefore, any intrahepatic T cell found in chronic hepatitis should have migrated to the liver after infection and inflammation. Because of the little information available on the differences between intrahepatic and peripheral T cells, we used recombinant proteins of the hepatitis C virus (HCV) to establish specific T cell lines and clones from liver biopsies of patients with chronic hepatitis C and compared them with those present in peripheral blood mononuclear cells (PBMC). We found that the protein nonstructural 4 (NS4) was able to stimulate CD4 + T cells isolated from liver biopsies, whereas with all the other HCV proteins we consistently failed to establish liver-derived T cell lines from 16 biopsies. We then compared NS4-specific T cell clones obtained on the same day from PBMC and liver of the same patient. We found that the 22 PBMC-derived T cell clones represent, at least, six distinct clonal populations that differ in major histocompatibility complex restriction and response to superantigens, whereas the 27 liver-derived T cell clones appear all identical, as further confirmed by cloning and sequencing of the T cell receptor (TCK) variable and hypervariable regions. Remarkably, none of the PBMC-derived clones has a TCK identical to the liver-derived clone, and even with polymerase chain reaction oligotyping we did not find the liver-derived clonotypic TCK transcript in the PBMC, indicating a preferential intrahepatic localization of these T cells. Functionally, the liver-derived T cells provided help for polyclonal immunoglobulin (Ig)A production by B cells in vitro that is 10-fold more effective than that provided by the PBMC-derived clones, whereas there is no difference in the help provided for IgM and IgG production. Altogether these results demonstrate that the protein Ng4 is highly immunogenic for intrahepatic CD4 + T cells primed by HCV in vivo, and that there can he compartmentalization of some NS4-specific CD4 + T cells to the liver of patients with chronic hepatitis C.

Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope

Studies of mammalian gene function are hampered by temporal limitations in which phenotypes occurring at one stage of development interfere with analysis at later stages. Moreover, phenotypes resulting from altered gene activity include both direct and indirect effects that may be difficult to distinguish. In the present study, recombinant fusion proteins bearing the 12 amino acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF-4) were used to transduce enzymatically active Cre proteins directly into mammalian cells. High levels of recombination were observed in a variety of cultured cell types and in all tissues examined in mice following intraperitoneal administration. This represents the first use of protein transduction to induce the enzymatic conversion of a substrate in living cells and animals and provides a rapid and efficient means to manipulate mammalian gene structure and function.

Regulatory T (T reg ) cells control immune activation and maintain tolerance. How T regs mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in T regs activated through the T cell receptor (TCR). Ectopic expression of GARP in human naïve T (T N ) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in T N cells induced expression of T reg master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human T reg cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.

Dendritic cells (DC) serve a key function in host defense, linking innate detection of microbes to the activation of pathogen-specific adaptive immune responses(1,2). Whether there is cell-intrinsic recognition of HIV-1 by host innate pattern-recognition receptors and subsequent coupling to antiviral T cell responses is not yet known(3). DC are largely resistant to infection with HIV-1(4), but facilitate infection of co-cultured T-helper cells through a process of trans-enhancement(5,6). We show here that, when DC resistance to infection is circumvented(7,8), HIV-1 induces DC maturation, an antiviral type I interferon response and activation of T cells. This innate response is dependent on the interaction of newly-synthesized HIV-1 capsid (CA) with cellular cyclophilin A (CypA) and the subsequent activation of the transcription factor IRF3. Because the peptidyl-prolyl isomerase CypA also interacts with CA to promote HIV-1 infectivity, our results suggest that CA conformation has evolved under opposing selective pressures for infectivity versus furtiveness. Thus, a cell intrinsic sensor for HIV-1 exists in DC and mediates an antiviral immune response, but it is not typically engaged due to absence of DC infection. The virulence of HIV-1 may be related to evasion of this response, whose manipulation may be necessary to generate an effective HIV-1 vaccine.

The purpose of this study is to identify invariant natural killer T cells (NKT cells) in cellular infiltrate of human allergic contact dermatitis (ACD) skin challenge sites. Skin biopsy specimens were taken from positive patch test reactions from 10 different patients (9 different allergens) and studied by immunochemistry, real-time PCR, nested PCR, and in situ hybridization to identify NKT cells and the cytokines associated with this cell type. Invariant NKT cells were identified in all the 10 skin biopsy specimens studied, ranging from 1.72 to 33% of the cellular infiltrate. These NKT cells were activated in all cases, as they expressed cytokine transcripts for IFN-γ and IL-4. Invariant NKT cells are present in ACD, regardless of the allergen that triggers the reaction, and are in an activated state. We conclude that innate immunity plays a role in late phases of type IV hypersensitivity reactions and may be responding to self-lipids released during allergic inflammation. These data complement the previous work by other investigators that suggest that NKT cells are important in the early cellular response during primary immune responses to allergens. Herein, it is demonstrated that NKT cells are constantly present during the late elicitation phase of human type IV hypersensitivity reactions.

Background. Natural killer T (NKT) cells are a subset of T cells that help potentiate and regulate immune responses. Although human NKT cell subsets with distinct effector functions have been identified, it is unclear whether the effector functions of these subsets are imprinted during development or can be selectively reprogrammed in the periphery. Results. We found that neonatal NKT cells are predominantly CD4+ and express higher levels of CCR7 and CD62L and lower levels of CD94 and CD161 than adult CD4+ or CD42 NKT cell subsets. Accordingly, neonatal NKT cells were more flexible than adult CD4+ NKT cells in their capacity to acquire Th1-or Th2-like functions upon either cytokine-mediated polarization or ectopic expression of the Th1 or Th2 transcription factors T-bet and GATA-3, respectively. Consistent with their more differentiated phenotype, CD4-NKT cells were predominantly resistant to functional reprogramming and displayed higher cytotoxic function. In contrast to conventional T cells, neither the expression of CXCR3 nor the cytotoxic capacity of neonatal NKT cells could be reprogrammed. Conclusions and Significance. Together, these results suggest that neonatal CD4+, adult CD4+, and adult CD42 NKT may represent unique states of maturation and that some functions of human NKT cells may be developmentally imprinted, while others are acquired similar to conventional T cell subsets during peripheral maturation and differentiation. Given the potent immuno-regulatory functions of NKT cells, these findings have important implications for the development of novel NKT cellbased therapeutics and vaccines.

Protein transduction has been widely used to analyze biochemical processes in living cells quantitatively and under non-steady-state conditions. The present study analyzed the effects of cell cycle on the uptake and activity of cell-permeant Cre recombinase proteins. Previous studies had suggested that the efficiency of recombination and/or protein transduction varied among individual cells, even within a clonal population. We report here that cells in the G1 phase of the cell cycle undergo recombination at a lower rate than cells at other phases of the cell cycle, and that this variation results largely from differences in protein uptake, associated with differences in cell size. These results have implications regarding the mechanism of protein transduction and identify a source of heterogeneity that can influence the response of individual cells to cell-permeant proteins.

Regulatory T cells (Tregs) suppress immune responses against both self and non-self antigens. Tregs require activation through the T cell receptor (TCR) and IL-2 to exert their suppressive functions. However, how strength of TCR signals modulate the potency of Treg-mediated suppression of antigen-specific T cell activation remain unclear. We found that both strength of TCR signals and ratios of Tregs to target cells, either through superantigen, allogeneic antigens or HIVspecific peptides, modified the suppressive ability of Tregs. While human Tregs were able to mediate suppression in the presence of only autologous antigen-presenting cells, this was much less efficient as compared to when Tregs were activated by allogeneic dendritic cells. In another physiologically relevant system, we show that the strength of peptide stimulation, high frequency of responder CD8+ T cells or presence of high IL-2 can override the suppression of HIV-specific CD8+ T cells by Tregs. These findings suggest that ratios and TCR activation of human Tregs, are important parameters to overcome robust immune responses to pathogens or allogeneic antigens. Modulating the strength of T cell signals and selective enhancement or depletion of antigen-specific Tregs thus may have implications for designing potent vaccines and regulating immune responses during allogeneic transplantation and chronic infections.

Pore-forming toxins are critical virulence factors for many bacterial pathogens and are central to Staphylococcus aureus-mediated killing of host cells. S. aureus encodes pore-forming bi-component leukotoxins that are toxic towards neutrophils, but also specifically target other immune cells. Despite decades since the first description of staphylococcal leukocidal activity, the host factors responsible for the selectivity of leukotoxins towards different immune cells remain unknown.

Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections.

Abstract Background: A poor vitamin D status has been associated with a high disease activity of multiple sclerosis (MS). Recently, we described associations between vitamin D status and peripheral T cell characteristics in relapsing remitting MS (RRMS) patients. In the present study, we studied the effects of high dose vitamin D3 supplementation on safety and T cell related outcome measures.

Background: According to the international consensus statement on antiphospholipid syndrome (APS), antiphospholipid antbodies (aPL) of the IgG or the IgM isotype have to be positive for the classification of APS. The IgA isotype (either anticardiolipin antibodies [aCL] or anti-β2-glycoprotein I antibodies [antiβ2GPI]) do not fulfil laboratory criteria for APS classification. However, they are discussed in the consensus paper.