Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.

Flare-up reactions in severe drug hypersensitivity: infection or ongoing T-cell hyperresponsiveness

"Flare-up" reactions are late manifestations of severe T-cell-mediated drug hypersensitivity reactions. Management is anti-inflammatory treatment and avoiding unnecessary medicines. Symptoms like fever, lymph node swelling, and blood count abnormalities may lead to confusion with bacterial infections. For prompt recognition it is important to keep the differential diagnosis in mind.

Methodologies for the Analysis of HCV-Specific CD4(+) T Cells

Virus-specific CD4(+) T cells play a major role in viral infections, such as hepatitis C virus (HCV). Viral clearance is associated with vigorous and multi-specific CD4(+) T-cell responses, while chronic infection has been shown to be associated with weak or absent T-cell responses. Most of these studies have used functional assays to analyze virus-specific CD4(+) T-cell responses; however, these and other detection methods have various limitations. Therefore, the important question of whether virus-specific CD4(+) T cells are completely absent or primarily impaired in specific effector functions during chronic infection, has yet to be analyzed in detail. A novel assay, in which virus-specific CD4(+) T-cell frequencies can be determined by de novo CD154 (CD40 ligand) expression in response to viral antigens, can help to overcome some of the limitations of functional assays and restrictions of multimer-based methods. This and other current established methods for the detection of HCV-specific CD4(+) T cells will be discussed in this review.

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

The role of MEKK3 in T cell homeostasis and IFNgamma production

MEKK3, a member of the MAP3K family, is involved in regulating multiple MAPK and NF-κB pathways. The MAPK and NF-κB signaling pathways are important in regulating T cell functions. MEKK3 is expressed through the development of T cell and also in subsets of T cell in the peripheral. However, the specific role of MEKK3 in T cell function is unknown. To reveal the in vivo function of MEKK3 in T cells, I have generated MEKK3 T cell conditional knock-out mice. Despite a normal thymus development in the conditional knock-out mice, I observed a decrease in the number of peripheral T-cells and impaired T-cell function in response to antigen stimulation. T cells undergo homeostatic proliferation under lymphopenia condition, a process called lymphopenia-induced proliferation (LIP). Using a LIP model, I demonstrated that the reduction of peripheral T cell number is largely due to a severe impairment of the self-antigen/MHC mediated T cell homeostasis. Upon anti-CD3 stimulation, the proliferation of MEKK3-deficient T cell is not significantly affected, but the production of IFNγ by naïve and effector CD4 T cells are markedly decreased. Interestingly, the IL-12/IL-18 driven IFNγ production and MAPK activation in MEKK3-deficient T cells is not affected, suggesting that MEKK3 selectively mediates the TCR induced MAPK signaling. Furthermore, I found that MEKK3 is activated by TCR stimulation in a RAC1/2 dependent manner, but not by IL-12/IL-18 stimulation. Finally, I showed that basal level of ERK and JNK activation is defective under LIP condition. I showed that the TCR induced ERK, JNK and p38 MAPK activation is also defective in MEKK3 deficient CD4 T cells. Taken together, my data demonstrate a crucial role of MEKK3 in T cell homeostasis and IFNγ production through regulating the TCR mediated MAPK pathway. ^

Effects of thymus size and involution on the contribution of recent thymic emigrants to the peripheral T cell pool

The contribution of recent thymic emigrants (RTEs) to the peripheral naïve T cell population is necessary to maintain diversity of the T cell receptor (TCR) repertoire and produce immune responses against newly encountered antigens. The thymus involutes with age, after irradiation or chemotherapy, and due to severe viral infections. Thymus involution results in decreased thymopoiesis and RTE output leading to a reduced diversity of peripheral T cells. This increases susceptibility to disease and impairs immune responsiveness to vaccines. Therefore, studies aimed at maintaining or regenerating thymic function are integral for maintaining and restoring peripheral TCR diversity. Mice that express a K5.CyclinD1 transgene expression have a severely hyperplastic thymus that fails to undergo involution. Both thymocyte and TEC development appear normal in these mice. We have used the K5.CyclinD1 transgenic model to test the hypothesis that preventing thymus involution will sustain RTE output and incorporation into the peripheral T cell pool to prevent naïve T cell depletion with age. The K5.CyclinD1 transgene was crossed to the RAG2p-GFP transgenic model so that RTEs could be tracked by the intensity of the GFP signal. The frequency and number of RTEs in naïve CD4 splenic T cells was analyzed at monthly intervals to 5 months of age. Using this double transgenic approach, we determined that preventing thymus involution does maintain or enhance the number of RTEs in the peripheral T cell pool before and after thymus involution.

IMMUNOLOGICAL MECHANISMS OF EXTRACORPOREAL PHOTOPHERESIS IN CUTANEOUS T CELL LYMPHOMA AND GRAFT VERSUS HOST DISEASE

IMMUNOLOGICAL MECHANISMS OF EXTRACORPOREAL PHOTOPHERESIS IN CUTANEOUS T CELL LYMPHOMA AND GRAFT VERSUS HOST DISEASE Publication No.___________ Lisa Harn-Ging Shiue, B.S. Supervisory Professor: Madeleine Duvic, M.D. Extracorporeal photopheresis (ECP) is an effective, low-risk immunomodulating therapy for leukemic cutaneous T cell lymphoma (L-CTCL) and graft versus host disease (GVHD), but whether the mechanism(s) of action in these two diseases is (are) identical or different is unclear. To determine the effects of ECP in vivo, we studied regulatory T cells (T-regs), cytotoxic T lymphocytes (CTLs), and dendritic cells (DCs) by immunofluorescence flow cytometry in 18 L-CTCL and 11 GVHD patients before and after ECP at Day 2, 1 month, 3 months, and 6 months. In this study, ECP was effective in 12/18 L-CTCL patients with a 66.7% overall response rate (ORR) and 6/11 GVHD patients with a 54.5% ORR. Prior to ECP, the percentages of CD4+Foxp3+ T cells in 9 L-CTCL patients were either lower (L-CTCL-Low, n=2) or higher (L-CTCL-High, n=7) than normal. Five of the 7 GVHD patients had high percentages of CD4+Foxp3+ T cells (GVHD-High). Six of 7 L-CTCL-High patients had >80% CD4+Foxp3+ T cells which were correlated with tumor cells, and were responders. Both L-CTCL-High and GVHD-High patients had decreased percentages of CD4+Foxp3+ and CD4+Foxp3+CD25- T cells after 3 months of treatment. CD4+Foxp3+CD25+ T cells increased in GVHD-High patients but decreased in L-CTCL-High patients after 3 months of ECP. In addition, numbers of CTLs were abnormal. We confirmed that numbers of CTLs were low in L-CTCL patients, but high in GVHD patients prior to ECP. After ECP, CTLs increased after 1 month in 4/6 L-CTCL patients whereas CTLs decreased after 6 months in 3/3 GVHD patients. Myeloid (mDCs) and plasmacytoid DCs (pDCs) were also low at baseline in L-CTCL and GVHD patients confirming the DC defect. After 6 months of ECP, numbers and percentages of mDCs and pDCs increased in L-CTCL and GVHD. MDCs were favorably increased in 8/12 L-CTCL responders whereas pDCs were favorably increased in GVHD patients. These data suggest that ECP is favorably modulating the DC subsets. In L-CTCL patients, the mDCs may orchestrate Th1 cell responses to overcome immune suppression and facilitate disease regression. However, in GVHD patients, ECP is favorably down-regulating the immune system and may be facilitating immune tolerance to auto-or allo-antigens. In both L-CTCL and GVHD patients, DCs are modulated, but the T cell responses orchestrated by the DCs are different, suggesting that ECP modulates depending on the immune milieu. _______________

Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 − T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

Porewater concentrations, pH, single cell number, anaerobic oxidation of methane (AOM) rate, sulfate reduction (SR) rate of samples during METEOR cruise M76/3b at the REGAB cold seep site from the West African margin in 2008

The giant pockmark REGAB (West African margin, 3160 m water depth) is an active methane-emitting cold seep ecosystem, where the energy derived from microbially mediated oxidation of methane supports high biomass and diversity of chemosynthetic communities. Bare sediments interspersed with heterogeneous chemosynthetic assemblages of mytilid mussels, vesicomyid clams and siboglinid tubeworms form a complex seep ecosystem. To better understand if benthic bacterial communities reflect the patchy distribution of chemosynthetic fauna, all major chemosynthetic habitats at REGAB were investigated using an interdisciplinary approach combining porewater geochemistry, in situ quantification of fluxes and consumption of methane, as well bacterial community fingerprinting. This study revealed that sediments populated by different fauna assemblages show distinct biogeochemical activities and are associated with distinct sediment bacterial communities. The methane consumption and methane effluxes ranged over one to two orders of magnitude across habitats, and reached highest values at the mussel habitat, which hosted a different bacterial community compared to the other habitats. Clam assemblages had a profound impact on the sediment geochemistry, but less so on the bacterial community structure. Moreover, all clam assemblages at REGAB were restricted to sediments characterized by complete methane consumption in the seafloor, and intermediate biogeochemical activity. Overall, variations in the sediment geochemistry were reflected in the distribution of both fauna and microbial communities; and were mostly determined by methane flux.

Disruption of the CD4–major histocompatibility complex class II interaction blocks the development of CD4+ T cells in vivo

The experiments presented in this report were designed to specifically examine the role of CD4–major histocompatibility complex (MHC) class II interactions during T cell development in vivo. We have generated transgenic mice expressing class II molecules that cannot interact with CD4 but that are otherwise competent to present peptides to the T cell receptor. MHC class II expression was reconstituted in Aβ gene knock-out mice by injection of a transgenic construct encoding either the wild-type I-Aβb protein or a construct encoding a mutation designed to specifically disrupt binding to the CD4 molecule. We demonstrate that the mutation, EA137 and VA142 in the β2 domain of I-Ab, is sufficient to disrupt CD4–MHC class II interactions in vivo. Furthermore, we show that this interaction is critical for the efficient selection of a complete repertoire of mature CD4+ T helper cells as evidenced by drastically reduced numbers of conventional CD4+ T cells in animals expressing the EA137/VA142 mutant I-Ab and by the failure to positively select the transgenic AND T cell receptor on the mutated I-Ab. These results underscore the importance of the CD4–class II interaction in the development of mature peripheral CD4+ T cells.

T cell vaccination induces T cell receptor Vβ-specific Qa-1-restricted regulatory CD8+ T cells

Vaccination of mice with activated autoantigen-reactive CD4+ T cells (T cell vaccination, TCV) has been shown to induce protection from the subsequent induction of a variety of experimental autoimmune diseases, including experimental allergic encephalomyelitis (EAE). Although the mechanisms involved in TCV-mediated protection are not completely known, there is some evidence that TCV induces CD8+ regulatory T cells that are specific for pathogenic CD4+ T cells. Previously, we demonstrated that, after superantigen administration in vivo, CD8+ T cells emerge that preferentially lyse and regulate activated autologous CD4+ T cells in a T cell receptor (TCR) Vβ-specific manner. This TCR Vβ-specific regulation is not observed in β2-microglobulin-deficient mice and is inhibited, in vitro, by antibody to Qa-1. We now show that similar Vβ8-specific Qa-1-restricted CD8+ T cells are also induced by TCV with activated CD4+ Vβ8+ T cells. These CD8+ T cells specifically lyse murine or human transfectants coexpressing Qa-1 and murine TCR Vβ8. Further, CD8+ T cell hybridoma clones generated from B10.PL mice vaccinated with a myelin basic protein-specific CD4+Vβ8+ T cell clone specifically recognize other CD4+ T cells and T cell tumors that express Vβ8 and the syngeneic Qa-1a but not the allogeneic Qa-1b molecule. Thus, Vβ-specific Qa-1-restricted CD8+ T cells are induced by activated CD4+ T cells. We suggest that these CD8+ T cells may function to specifically regulate activated CD4+ T cells during immune responses.