Expression Of Early Activation Markers Research Articles

Antioxidation protein Nrf2 modulates glutamine metabolism to regulate activation-driven expansion of CD4 +T-cells

Abstract Nutrient metabolism governs the effector functions like activation, expansion, and subset differentiation of CD4 +T-cells, the orchestrators of adaptive immunity. The levels of reactive oxygen species (ROS), the metabolic byproducts of cellular metabolism are regulated by the antioxidation pathway, which is composed of Keap1 and Nrf2 proteins. We observed that, the expression of Nrf2 is temporally regulated post-activation in CD4 +T-cells (high on day 1 and low on days 2 and 3) indicating its role in CD4 +T-cell effector functions. It is yet unknown if and how the antioxidation pathway crosstalks with cellular metabolism to shape CD4 +T-cell effector responses. Here, using mice with T-cell specific deletion of Keap1 (Keap1-KO) or Nrf2 (Nrf2-KO) we deciphered the effects of high and low levels of Nrf2 (antioxidation) levels, respectively in CD4 +T-cells. Upon activation in vitro, Keap1-KO CD4 +T-cells with constitutively high levels of Nrf2 showed higher proliferation, increased expression of early activation markers and elevated TCR-mediated signaling compared to wildtype (WT) cells. In contrast, decreased proliferation and lower TCR-mediated signaling was noted in activated Nrf2-KO CD4 +T-cells. To dissect the underlying mechanisms, we performed metabolite analyses and activation in nutrient-limiting conditions. Interestingly, activated Keap1-KO CD4 +T-cells show upregulated glutamine metabolism as measured by metabolic intermediates and rely on glutamine for optimum activation and increased expansion. Together, our findings identify a mechanism by which antioxidation protein Nrf2 metabolically reprograms CD4 +T-cells and modulates their metabolic dependencies to support their activation and expansion.

Planktonic and Biofilm-Associated Pseudomonas aeruginosa and Staphylococcus epidermidis Elicit Differential Human Peripheral Blood Cell Responses.

Despite the considerable progress made in recent years, our understanding of the human immune response to microbial biofilms is still poor. The aim of the present study was to compare the in vitro response of human peripheral blood mononuclear cells (PBMC) to biofilms and planktonic cells of Pseudomonas aeruginosa and Staphylococcus epidermidis, two bacterial species particularly relevant in patients with cystic fibrosis or undergoing endovascular catheterization, respectively. PBMC isolated from healthy donors were co-cultured with 24 h-old biofilms or with exponentially growing cells of both species. Following 24 h of co-culture, the expression of early activation markers and the levels of cytokines in the culture supernatants were assessed by flow cytometry, while biofilm biomass and architecture were evaluated by crystal violet staining, CFU count, and confocal microscopy. Around 20% of PBMC was activated in response to both biofilms and planktonic cells of P. aeruginosa. In contrast, planktonic cells of S. epidermidis induced a statistically higher degree of activation than their biofilm counterpart (25% versus 15%; p < 0.01). P. aeruginosa biofilms stimulated pro-inflammatory (TNF-α, IL-1β, IFN-γ, and IL-6) and anti-inflammatory (IL-10) cytokine production at statistically significant levels higher than its planktonic counterpart, while an opposite trend was observed with S. epidermidis. Differences in the architecture of the biofilms and in the number of PBMC infiltrating the biofilms between the two bacterial species may at least partially explain these findings. Collectively, the results obtained highlighted marked differences in the host–cell response depending on the species and the mode of growth (biofilms versus planktonic cultures), allowing speculations on the different strategies adopted by P. aeruginosa and S. epidermidis to persist in the host during the course of chronic infections.

Acly promotes metabolic reprogramming and induction of IRF4 during early CD8+ T cell activation.

CD8+ T cells, a fundamental part of the adaptive immune system, employ cytotoxic responses important for targeting pathogenic bacteria, viruses, and tumor cells. During early activation, CD8+ T cells undergo many changes in metabolism and gene expression. The bridge between epigenetic and metabolic influences on gene expression and cell fate has yet to be fully understood. Here, we investigated the importance of ATP citrate lyase (Acly), an enzyme involved in both metabolism and histone acetylation, for early stages of CD8+ T cell activation. We performed polyclonal activation of murine CD8+ T cells in vitro in the presence or absence of the Acly inhibitor BMS303141. We found that inhibiting Acly during early activation results in decreased expression of early activation markers. Consistent with impaired early activation, we found that inhibition also resulted in increased uptake of fatty acids and decreased glucose uptake without changing mitochondrial ATP levels. On an epigenetic and transcriptional level, early stage Acly inhibition specifically downregulated promoter histone H3 acetylation (H3ac) and expression of the key transcription factor IRF4; however, global levels of H3ac remained similar. Most importantly, the study was able to highlight the importance of Acly in early stages of CD8+ T cell activation and histone regulation.

Novel role for gelatinases in modulating metabolic reprogramming associated with T cell receptor stimulation in CD4+ T cell

Abstract The gelatinases, MMP-2 and MMP-9, are proteolytic enzymes with diverse physiological roles. They are linked with various aspects of the immune response, such as leukocyte trafficking and cytokine activation. However, their dysregulation is associated with various pathologies such as metastasis in cancer progression and blood-brain barrier disruption in multiple sclerosis. Much of what is known about the gelatinases is in context of their extracellular activities. However, recent studies including this study suggest an intracellular role in immune cell activation. Our findings suggest, that gelatinase activity can modulate T cell bioenergetics, affecting both oxidative phosphorylation and the glycolytic pathways with a greater influence on the latter. This results in a mitigated CD4+ T cell response to TCR stimulation. Upon stimulation, CD4+ T cells lacking gelatinase activity, demonstrate a reduced capacity to be activated as determined by the expression of early activation markers, proliferation, and the production of effector cytokines. In addition, RNA-seq analysis of control and gelatinase inhibited CD4+ T cells highlight key proteins associated with both the immune system and metabolic processes as predicted by STRING. We confirm our pharmacological studies with the genetic ablation of the gelatinases and further analysis of these shRNA treated T cells using the Nanostring metabolic panel. In addition, we demonstrate in-vivo relevancy using a murine model for multiple sclerosis known to be initiated by CD4+ T cells; gelatinase inhibition resulted in reduced clinical severity. These results suggest an intracellular role for gelatinases in CD4+ T cell mediated diseases and their potential as therapeutic targets.

Initial Viral Inoculum Determines Kinapse-and Synapse-Like T Cell Motility in Reactive Lymph Nodes

T cell activation in lymphoid tissue occurs through interactions with cognate peptide-major histocompatibility complex (pMHC)-presenting dendritic cells (DCs). Intravital imaging studies using ex vivo peptide-pulsed DCs have uncovered that cognate pMHC levels imprint a wide range of dynamic contacts between these two cell types. T cell—DC interactions vary between transient, “kinapse-like” contacts at low to moderate pMHC levels to immediate “synapse-like” arrest at DCs displaying high pMHC levels. To date, it remains unclear whether this pattern is recapitulated when the immune system faces a replicative agent, such as a virus, at low and high inoculum. Here, we locally administered low and high inoculum of lymphocytic choriomeningitis virus (LCMV) in mice to follow activation parameters of Ag-specific CD4+ and CD8+ T cells in draining lymph nodes (LNs) during the first 72 h post infection. We correlated these data with kinapse- and synapse-like motility patterns of Ag-specific T cells obtained by intravital imaging of draining LNs. Our data show that initial viral inoculum controls immediate synapse-like T cell arrest vs. continuous kinapse-like motility. This remains the case when the viral inoculum and thus the inflammatory microenvironment in draining LNs remains identical but cognate pMHC levels vary. Our data imply that the Ag-processing capacity of draining LNs is equipped to rapidly present high levels of cognate pMHC when antigenic material is abundant. Our findings further suggest that widespread T cell arrest during the first 72 h of an antimicrobial immune responses is not required to trigger proliferation. In sum, T cells adapt their scanning behavior according to available antigen levels during viral infections, with dynamic changes in motility occurring before detectable expression of early activation markers.

Selective inhibition of gelatinases modulates cellular energetics in CD4+ T-cells

Abstract The gelatinases, MMP-2 and MMP-9, are proteolytic enzymes with diverse physiological roles. They are linked with various aspects of the immune response, such as leukocyte trafficking and cytokine activation. However, their dysregulation is associated with various pathologies such as metastasis in cancer progression and blood-brain barrier disruption in multiple sclerosis. Much of what is known about the gelatinases is affiliated with their activities in the extracellular milieu. However, recent studies have suggested that they contribute to normal intracellular function of various immune cell types, including T-cells. CD4+ T-cells play a key role in linking the innate and adaptive arms of the immune response, as well as determining the character of the immune response. Our findings show, for the first time, that intracellular gelatinase activity can modulate T-cell bioenergetics, affecting both oxidative phosphorylation and glycolytic pathways and resulting in a mitigated CD4+ T-cell response to stimulation. Upon stimulation, CD4+ T-cells lacking gelatinase activity demonstrate a reduced capacity to function as determined by the expression of early activation markers, proliferation, and the production of effector cytokines. In addition, RNA-seq analysis of control and gelatinase inhibited CD4+ T-cells highlight key proteins associated with T-cell receptor signaling, cytokine production, and metabolism. These results suggest that gelatinases can be immunomodulatory agents in CD4+ T-cell mediated diseases, such as multiple sclerosis, and thus have therapeutic potential.

LPS stimulates IgM production invivo without help from non-B cells.

Gram-negative bacterial LPS induce murine B-cell activation and innate (polyclonal) Ab production. Mouse B cells express the LPS signaling receptor (TLR4), yet how LPS activates B-cell responses invivo is not known. Can LPS directly stimulate B cells to induce innate Ab production? Is activation of non-B cells also required? To address these questions, we transfused LPS-responsive (Tlr4(+/+)) or non-responsive (Tlr4(-/-)) B cells into LPS-responsive or non-responsive mice. Increased expression of the early activation markers CD69 and CD86 could be induced on transfused Tlr4(-/-) B cells by injecting LPS subcutaneously into Tlr4(+/+) mice, demonstrating indirect activation of B cells by TLR4-responsive non-B cells invivo, but the Tlr4(-) (/) (-) B cells did not increase serum IgM levels. In contrast, when Tlr4(-/-) recipients were transfused with Tlr4(+/+) B cells, LPS induced large amounts of serum IgM and LPS could also enhance specific Ab production to a protein that was co-injected with it (adjuvant response). Thus, LPS-exposed non-B cells mediated increased surface expression of early B-cell activation markers, but this response did not predict innate Ab responses or LPS adjuvanticity invivo Direct stimulation of B cells by LPS via TLR4 was necessary and sufficient to induce B cells to produce Ab invivo.

A critical role for alpha-synuclein in development and function of T lymphocytes

Alpha-synuclein is highly expressed in the central nervous system and plays an important role in pathogenesis of neurodegenerative disorders such as Parkinson’s disease and Lewy body dementia. Previous studies have demonstrated the expression of α-synuclein in hematopoietic elements and peripheral blood mononuclear cells, although its roles in hematopoiesis and adaptive immunity are not studied. Using an α-synuclein knock out (KO) mouse model, we have recently shown that α-synuclein deficiency is associated with a mild defect in late stages of hematopoiesis. More importantly, we demonstrated a marked defect in B lymphocyte development and IgG, but not IgM production in these mice. Here we show a marked defect in development of T lymphocytes in α-synuclein KO mice demonstrated by a significant increase in the number of CD4 and CD8 double negative thymocytes and significant decreases in the number of CD4 single positive and CD8 single positive T cells. This resulted in markedly reduced peripheral T lymphocytes. Interestingly, splenic CD4+ and CD8+ T cells that developed in α-synuclein KO mice had a hyperactivated state with higher expression of early activation markers and increased IL-2 production. Moreover, splenic CD4+ T cells from α-synuclein KO mice produced lower levels of IL-4 upon antigenic stimulation suggesting a defective Th2 differentiation. Our data demonstrate an important role for α-synuclein in development of T lymphocytes and regulation of their phenotype and function.

482. Editing of the CD40L Gene Restores Regulated CD40L Expression in X-HIGM Patient T Cells

X-linked hyper-IgM syndrome (X-HIGM) is a primary immunodeficiency syndrome characterized by insufficient CD40 Ligand (CD40L) expression, resulting in defective T-cell help, impaired immunoglobulin class-switching and recurrent opportunistic infections. Previously, constitutive CD40L expression in murine bone marrow cells and thymocytes was achieved by retroviral gene transfer and successfully corrected immune function. However, treated animals developed severe lymphoproliferative disease, likely due to constitutive CD40L surface expression. This demonstrated the importance of preserving endogenous gene regulation in this and other gene therapy endeavors. Here, we report efficient, on-target Homology Directed Repair (HDR) editing of the CD40L locus in primary human T cells using a combination of TALEN gene targeting and a donor template delivered transiently by recombinant Adeno-Associated Virus (rAAV). Our TALEN and donor template reagents were designed to insert a coding sequence (GFP or CD40L) upstream of the endogenous translation start site within Exon 1, thus allowing transgene expression to be regulated by the endogenous CD40L promoter, and included the CD40L 3’UTR to preserve known post-transcriptional regulation features. The kinetics of GFP and edited CD40L surface expression after PMA-Ionomycin stimulation paralleled that of endogenous CD40L in unedited T-cells. Additionally, activated T-cells expressing edited CD40L were able to bind CD40-Ig similar to unedited cells. When T-cells from X-HIGM patients were edited in this fashion, CD40L expression and CD40-Ig binding was restored, and repaired activated X-HIGM T-cells induced the expression of early activation markers and class switching of naive B-cells in vitro. Finally, we found no differences in TCR repertoire or engraftment and stability in NSG mice between T cells that underwent gene editing vs. unedited cells. These results demonstrate the feasibility of site-directed gene repair to restore normally regulated CD40L protein expression and functional T-cell help.

In the absence of its cytosolic domain, the CD28 molecule still contributes to T cell activation.

The CD28 costimulatory receptor has a pivotal role in T cell biology as this molecule amplifies T cell receptor (TCR) signals to provide an efficient immune T cell response. There is a large debate about how CD28 mediates these signals. Here, we designed a CD28 gene-targeted knock-in mouse strain lacking the cytoplasmic tail of CD28. As is the case in CD28-deficient (CD28 knock-out) mice, regulatory T cell homeostasis and T cell activation are altered in these CD28 knock-in mice. Unexpectedly, the presence of a CD28 molecule deprived of its cytoplasmic tail could partially induce some early activation events in T cells such as signaling events or expression of early activation markers. These results unravel a new mechanism of T cell costimulation by CD28, independent of its cytoplasmic tail.

Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1

BackgroundMalaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans.MethodsFluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated.ResultsAnalyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity.ConclusionsThe results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.

Anti-TNF treatment blocks the induction of T cell-dependent humoral responses

ObjectiveExperimental and human data suggest that tumour necrosis factor (TNF) blockade may affect B cell responses, in particular the induction of T cell-dependent (TD) humoral immunity. This study aimed to...

Mycobacterium tuberculosis ESX-1 system-secreted protein ESAT-6 but not CFP10 inhibits human T-cell immune responses

The secreted proteins of M. tuberculosis, early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein 10 kDa (CFP10), have been identified as antigenic proteins with potent T-cell stimulatory effects, and therefore have been the focus of tuberculosis vaccine studies. However, recent work showed that secretion of these proteins by the specialized ESAT-6 secretion system (ESX)-1 of M. tuberculosis is associated with virulence and pathogenesis. The studies demonstrated that ESAT-6 inhibits antigen-presenting cell function by reducing IL-12 production by macrophages through interrupting TLR2 signaling pathways and inducing macrophage apoptosis. However, the effect of ESAT-6 on T cells remains unexplored. To address this question, we studied the effect of recombinant ESAT-6 and CFP10 on human primary T-cell IFN-gamma secretion and proliferation. ESAT-6, but not CFP10, inhibited IFN-gamma production by T cells stimulated with M. tuberculosis or with anti-CD3 plus anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T-cell production of IL-17 and TNF-a, but not IL-2. Presence of CFP10 as part of the ESAT-6/CFP10 heterodimer did not affect ESAT-6 inhibition of T-cell IFN-gamma production. ESAT-6 inhibited the proliferation of CD3+ cells in response to TCR stimulation. ESAT-6 decreased T-cell IFN-gamma secretion by mechanisms independent of cytotoxicity or apoptosis. ESAT-6 reduced IFN-gamma mRNA levels by inhibiting the expression of the transcription factors, ATF-2, c-Jun and CREB, which upregulate IFN-gamma gene expression in T cells through binding to the IFN-gamma proximal promoter. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing phosphorylation of ZAP70, a proximal TCR signaling molecule. We conclude that ESAT-6 directly inhibits human T-cell responses by affecting TCR signaling pathways downstream of ZAP70.

CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

AbstractCD4+ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)–transgenic (tg) CD4+ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3KδD910A/D910A or PI3Kγ-deficient TCR-tg CD4+ T cells showed similar responsiveness to CCL21 costimulation as control CD4+ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4+ T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca2+ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

ESAT-6 Inhibits Production of IFN-γ by Mycobacterium tuberculosis-Responsive Human T Cells

The Mycobacterium tuberculosis early secreted Ag of 6 kDa (ESAT-6) is a potent Ag for human T cells and is a putative vaccine candidate. However, ESAT-6 also contributes to virulence in animal models, mediates cellular cytolysis, and inhibits IL-12 production by mononuclear phagocytes. We evaluated the effects of ESAT-6 and its molecular chaperone, culture filtrate protein of 10 kDa (CFP10), on the capacity of human T cells to produce IFN-gamma and proliferate in response to TCR activation. Recombinant ESAT-6, but not CFP10, markedly inhibited IFN-gamma production by T cells stimulated with M. tuberculosis or with the combination of anti-CD3 and anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T cell production of IL-17 and TNF-alpha but not IL-2. Preincubation of ESAT-6 with CFP10 under conditions that favor dimer formation did not affect inhibition of IFN-gamma. ESAT-6 decreased IFN-gamma transcription and reduced expression of the transcription factors, ATF-2 and c-Jun, which normally bind to the IFN-gamma proximal promoter and stimulate mRNA expression. ESAT-6 inhibited T cell IFN-gamma secretion through mechanisms that did not involve cellular cytotoxicity or apoptosis. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing activation of ZAP70. We conclude that ESAT-6 directly inhibits human T cell responses to mycobacterial Ags by affecting TCR signaling pathways downstream of ZAP70.

The immunosuppressive effects of human bone marrow-derived mesenchymal stem cells target T cell proliferation but not its effector function

Mesenchymal stem cells (MSC) are non-haematopoietic stem cells that are capable of differentiating into tissues of mesodermal origin. MSC play an important role in supporting the development of fetal and adult haematopoiesis. More recently, MSC have also been found to exhibit inhibitory effect on T cell responses. However, there is little information on the mechanism of this immunosuppression and our study addresses this issue by targeting T cell functions at various level of immune responses. We have generated MSC from human adult bone marrow (BM) and investigated their immunoregulatory function at different phases of T cell responses. MSC showed the ability to inhibit mitogen (CD3/CD28 microbeads)-activated T cell proliferation in a dose-dependent manner. In order to evaluate the specificity of this immunosuppression, the proliferation of CD4 + and CD8 + cells were measured. MSC equally inhibit CD4 + and CD8 + subpopulations of T cells in response to PHA stimulation. However, the antiproliferative effect of MSC is not due to the inhibition of T cell activation. The expression of early activation markers of T cells, namely CD25 and CD69 were not significantly altered by MSC at 24, 48 and 72 h. Furthermore, the immunosuppressive effect of MSC mainly targets T cell proliferation rather than their effector function since cytotoxicity of T cells is not affected. This work demonstrates that the immunosuppressive effect of MSC is exclusively a consequence of an anti-proliferative activity, which targets T cells of different subpopulations. For this reason, they have the potential to be exploited in the control of unwanted immune responses such as graft versus host disease (GVHD) and autoimmunity.

Endogenous Transforming Growth Factor β1 Suppresses Inflammation and Promotes Survival in Adult CNS

Transforming growth factor beta1 (TGFbeta1) is a pleiotropic cytokine with potent neurotrophic and immunosuppressive properties that is upregulated after injury, but also expressed in the normal nervous system. In the current study, we examined the regulation of TGFbeta1 and the effects of TGFbeta1 deletion on cellular response in the uninjured adult brain and in the injured and regenerating facial motor nucleus. To avoid lethal autoimmune inflammation within 3 weeks after birth in TGFbeta1-deficient mice, this study was performed on a T- and B-cell-deficient RAG2-/- background. Compared with wild-type siblings, homozygous deletion of TGFbeta1 resulted in an extensive inflammatory response in otherwise uninjured brain parenchyma. Astrocytes increased in GFAP and CD44 immunoreactivity; microglia showed proliferative activity, expression of phagocytosis-associated markers [alphaXbeta2, B7.2, and MHC1 (major histocompatibility complex type 1)], and reduced branching. Ultrastructural analysis revealed focal blockade of axonal transport, perinodal damming of axonal organelles, focal demyelination, and myelin debris in granule-rich, phagocytic microglia. After facial axotomy, absence of TGFbeta1 led to a fourfold increase in neuronal cell death (52 vs 13%), decreased central axonal sprouting, and significant delay in functional recovery. It also interfered with the microglial response, resulting in a diminished expression of early activation markers [ICAM1 (intercellular adhesion molecule 1), alpha6beta1, and alphaMbeta2] and reduced proliferation. In line with axonal and glial findings in the otherwise uninjured CNS, absence of endogenous TGFbeta1 also caused an approximately 10% reduction in the number of normal motoneurons, pointing to an ongoing and potent trophic role of this anti-inflammatory cytokine in the normal as well as in the injured brain.

The Role of B Cells in the Development of CD4 Effector T Cells during a Polarized Th2 Immune Response

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.

Regulation of the tonsil cytokine milieu favors HIV susceptibility

Mucosal associated lymphoid tissues are major targets of HIV during early infection and disease progression but can also provide a viral safe haven during highly active antiretroviral therapy. Among these tissues, the tonsils remain enigmatic regarding their status as primary and/or secondary sites of retroviral infection. To dissect the mechanisms underlying susceptibility to HIV in this compartment, isolated tonsil cells were studied for phenotypic and functional characteristics, which may account for their permissiveness to infection. For this, tonsil cells and PBMC were infected in parallel with HIV, and viral replication was monitored by p24 ELISA. Our results demonstrate that unstimulated tonsil cells were more readily infected than PBMC with HIV. Phenotypic characterization of the tonsil cells revealed heterogeneous lymphoid populations but with increased expression of early activation markers and the viral co-receptor CXCR4, relative to PBMC, all of which may contribute to viral susceptibility. Furthermore, the cytokine microenvironment appeared to be key in facilitating HIV infection and tonsil-secreted products enhanced HIV infection in PBMC. Of the cytokines detected in the tonsil supernatants, TH2 cytokines, particularly IL-4, promoted HIV infection and replication. Interestingly, this TH2 profile appeared to dominate, even in the presence of the TH1 cytokine IFNgamma and the anti-viral factor IFNalpha, likely due to the enhanced expression of suppressor of cytokine signaling (SOCS) proteins, which may disengage IFN signaling. These and other local environmental factors may render tonsil cells increasingly susceptible to HIV infection.

P59 fyn is upregulated in anergic CD8 + T cells

The ultimate goal in clinical transplantation is achievement of graft tolerance. Despite long-term immunosuppression, alloantigens on transplants elicit alloresponses that can initiate organ rejection. Acute rejection is mediated by CD8 + cytotoxic T cells, whereas chronic rejection is a result of many factors including non-immunological events. The aim of this study was to examine the molecular requirements of T cell anergy, a cellular state that is an integral component of tolerance in vivo. In vitro, the tolerant state is usually best represented by T cell anergy, which is defined by loss of the ability of T cells to produce and secrete interleukin-2 upon restimulation. In the literature, molecular changes in anergic CD4 + T cells have been studied in great detail, but only little is known about functional and biochemical characteristics of anergic CD8 + T lymphocytes. In this study, we demonstrate, that CD8 + T cells are rendered anergic by TCR stimulation without costimulation. They exhibit impaired interleukin-2 production and tyrosine-phosphorylation, but markedly upregulated p59 fyn expression, which could be shown to be an early event during anergization. Anergic CD8 + T lymphocytes show elevated surface expression of early activation markers as well as costimulatory molecules, especially that of CTLA4. These results, are an important component for the discovery of potential molecular targets, which contribute to the development and maintenance of tolerance.