Antigen-specific T-T interactions regulate CD4 T-cell expansion.

The regulation of CD4 T cell numbers during an immune response should take account of the amount of antigen (Ag), the initial frequency of Ag-specific T cells, the mix of naive versus experienced cells, and (ideally) the diversity of the repertoire. Here we describe a novel mechanism of T cell regulation that potentially deals with all of these parameters. We found that CD4 T cells establish a negative feedback loop by capturing their cognate MHC/peptide complexes from Ag-presenting cells and presenting them to Ag-experienced CD4 T cells, thereby inhibiting their recruitment into the response while allowing recruitment of naive T cells. The inhibition is Ag specific, begins at day 2 (long before Ag disappearance), and cannot be overcome by providing new Ag-loaded dendritic cells. In this way CD4 T cell proliferation is regulated in a functional relationship to the amount of Ag, while allowing naive T cells to generate repertoire variety.

A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6

Lung T lymphocytes are important in pulmonary immunity and inflammation. it has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5% +/- 1.9% (n = 11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8 +/- 2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells. (C) 2008 Elsevier B.V. All rights reserved.

Suggested Binding Mechanism of the HIV-gp120 to its CD4 Receptor

The molecular recognition and attachment of the CD4 molecule and the HIV envelope glycoprotein (gp120) might be described as a consecutive three-step molecular recognition process. 1. (a) Long range interaction: electrostatic pre-orientation, 2. (b) short range interaction: electronic attachment followed by a ‘Locking-in’ (via aromatic ring orientation) and 3. (c) internal interaction (induced fit): conformational readjustment of the protein molecules. On the basis of the preliminary investigations (X-ray structures of CD4 and biological studies of CD4 and gp120 point mutants) we described a computational model. This approach consists of empirical calculations as well as ab initio level of quantum chemistry. The conformational analysis of the wild type and mutant CD4 molecules was supported by molecular mechanics and dynamics (Amber force field). The latter analysis involves the application of a novel method, the Amino Acid Conformation Assignment of Proteins (ACAP) software, developed for the notation of secondary protein structures. According to the cardinal role of the electrostatic factors during this interaction, several ab initio investigations were performed for better understanding of the recognition process on submolecular level. Using the above mentioned computational model, we could interpret the basic behaviours and predict some additional features of CD4-gp120 interaction, in spite of the missing gp120 X-ray structure.

IL-12R2 promotes the development of CD4+CD25+ regulatory T cells

We have previously shown that mice lacking the IL-12-specific receptor subunit ß2 (IL-12Rß2) develop more severe experimental autoimmune encephalomyelitis than wild-type (WT) mice. The mechanism underlying this phenomenon is not known; nor is it known whether deficiency of IL-12Rß2 impacts other autoimmune disorders similarly. In the present study we demonstrate that IL-12Rß2-/- mice develop earlier onset and more severe disease in the streptozotocin-induced model of diabetes, indicating predisposition of IL-12Rß2-deficient mice to autoimmune diseases. T cells from IL-12Rß2-/- mice exhibited significantly higher proliferative responses upon TCR stimulation. The numbers of naturally occurring CD25+CD4+ regulatory T cells (Tregs) in the thymus and spleen of IL-12Rß2-/- mice were comparable to those of WT mice. However, IL-12Rß2-/- mice exhibited a significantly reduced capacity to develop Tregs upon stimulation with TGF-ß, as shown by significantly lower numbers of CD25+CD4+ T cells that expressed Foxp3. Functionally, CD25+CD4+ Tregs derived from IL-12Rß2-/- mice were less efficient than those from WT mice in suppressing effector T cells. The role of IL-12Rß2 in the induction of Tregs was confirmed using small interfering RNA. These findings suggest that signaling via IL-12Rß2 regulates both the number and functional maturity of Treg cells, which indicates a novel mechanism underlying the regulation of autoimmune diseases by the IL-12 pathway. Copyright © 2008 by The American Association of Immunologists, Inc.

Priming of CD8+ and CD4+ T Cells in Experimental Leishmaniasis Is Initiated by Different Dendritic Cell Subtypes

The biological role of Langerin(+) dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin(+) DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of The biological role of Langerin+ dendritic cells (DCs) such as Langerhans cells and a subset of dermal DCs (dDCs) in adaptive immunity against cutaneous pathogens remains enigmatic. Thus, we analyzed the impact of Langerin+ DCs in adaptive T cell-mediated immunity toward Leishmania major parasites in a Lang-DTR mouse model that allows conditional diphtheria toxin (DT)-induced ablation of Langerin+ DCs in vivo. For the first time, infection experiments with DT-treated Lang-DTR mice revealed that proliferation of L. major-specific CD8+ T cells is significantly reduced during the early phase of the immune response following depletion of Langerin+ DCs. Consequently, the total number of activated CD8+ T cells within the draining lymph node and at the site of infection is diminished. Furthermore, we show that the impaired CD8+ T cell response is due to the absence of Langerin+ dDCs and not Langerhans cells. Nevertheless, the CD4+ T cell response is not altered and the infection is cleared as effectively in DT-treated Lang-DTR mice as in control mice. This clearly demonstrates that Langerin+ DCs are, in general, dispensable for an efficient adaptive immune response against L. major parasites. Thus, we propose a novel concept that, in the experimental model of leishmaniasis, priming of CD4+ T cells is mediated by Langerin− dDCs, whereas Langerin+ dDCs are involved in early priming of CD8+ T cells.

Langerhans cells are critical in the development of atopic dermatitis-like inflammation and symptoms in mice

Genetic or vitamin D3-induced overexpression of thymic stromal lymphopoietin (TSLP) by keratinocytes results in an atopic dermatitis (AD)-like inflammatory phenotype in mice echoing the discovery of high TSLP expression in epidermis from AD patients. Although skin dendritic cells (DC) are suspected to be involved in AD, direct evidence of a pathogenetic role for skin DC in TSLP-induced skin inflammation has not yet been demonstrated. In a mouse model of AD, i.e. mice treated with the low-calcemic vitamin D3 analogue, MC903, we show that epidermal Langerhans cells (LC)-depleted mice treated with MC903 do neither develop AD-like inflammation nor increased serum IgE as compared to vitamin D3 analogue-treated control mice. Accordingly, we show that, in mice treated with MC903 or in K14-TSLP transgenic mice, expression of maturation markers by LC is increased whereas maturation of dermal DC is not altered. Moreover, only LC are responsible for the polarization of naive CD4+ T cells to a Th2 phenotype, i.e. decrease in interferon-gamma and increase in interleukin (IL)-13 production by CD4+ T cells. This effect of LC on T-lymphocytes does not require OX40-L/CD134 and is mediated by a concomitant down-regulation of IL-12 and CD70. Although it was previously stated that TSLP up-regulates the production of thymus and activation-regulated chemokine (TARC)/chemokine (C-C motif) ligand 17 (CCL17) and macrophage-derived chemokine (MDC)/CCL22 by human LC in vitro, our work shows that production of these Th2- cell attracting chemokines is increased only in keratinocytes in response to TSLP overexpression. These results demonstrate that LC are required for the development of AD in mouse models of AD involving epidermal TSLP overexpression.

Induction of Distinct Neurologic Disease Manifestations during Relapsing Fever Requires T Lymphocytes.

Relapsing fever borreliosis is a multisystemic infection characterized primarily by bacteremia but can extend to the CNS. The incidence of CNS disease manifestations in humans depends on the infecting relapsing fever Borrelia species. In the murine model of Borrelia hermsii infection we found high incidence of distinct signs of CNS disease that ranged from a flaccid tail to complete paralysis of hind limbs. Infiltration of large number of T cells into the spinal cord of B. hermsii-infected mice and the upregulation of MHC class II and CD80 on infiltrating macrophages and on microglial cells suggested a role for T cell and Ag-presenting cell interactions in this pathogenesis. Indeed, B. hermsii infection did not induce CNS disease manifestations in T cell-deficient mice (TCR-ß × d-/-), although it resulted in bacteremia comparable to wild-type (Wt) level. Moreover, the infiltration of immune cells into the spinal cord of TCR-ß × d-/- mice was reduced and the resident microglial cells were not activated. Histopathological analysis of lumbar sections of the spinal cord confirmed severe inflammation in Wt but not in TCR-ß × d-/- mice. Induction of CNS disease was dependent on the B. hermsii strain as well as on the ability of the host to control bacteremia. Mice that are impaired in controlling B. hermsii, such as CD14-/- mice, exhibited more severe CNS disease than Wt mice. This study demonstrates that distinct neurologic disease manifestations develop during relapsing fever and that T cells play a critical role in the induction of neuropathogenesis.

Repertoire of HLA-DR1-restricted CD4 T cell responses to capsular Caf1 antigen of Y. pestis HLA-transgenic mice

Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T cell responses for Caf1. We characterized CD4 T cell epitopes of Caf1 in 'humanized'-HLA-DR1 transgenic mice lacking endogenous MHC class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T cell immunity was measured with respect to proliferative and IFNgamma T cell responses and recognition by a panel of T cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased towards a single immunodominant epitope near the C-terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.