T lymphocytes control microbial composition by regulating the abundance of Vibrio in the zebrafish gut.

Dysbiosis of the intestinal microbial community is considered a risk factor for development of chronic intestinal inflammation as well as other diseases such as diabetes, obesity and even cancer. Study of the innate and adaptive immune pathways controlling bacterial colonization has however proven difficult in rodents, considering the extensive cross-talk between bacteria and innate and adaptive immunity. Here, we used the zebrafish to study innate and adaptive immune processes controlling the microbial community. Zebrafish lack a functional adaptive immune system in the first weeks of life, enabling study of the innate immune system in the absence of adaptive immunity. We show that in wild type zebrafish, the initial lack of adaptive immunity associates with overgrowth of Vibrio species (a group encompassing fish and human pathogens), which is overcome upon adaptive immune development. In Rag1-deficient zebrafish (lacking adaptive immunity) Vibrio abundance remains high, suggesting that adaptive immune processes indeed control Vibrio species. Using cell transfer experiments, we confirm that adoptive transfer of T lymphocytes, but not B lymphocytes into Rag1-deficient recipients suppresses outgrowth of Vibrio. In addition, ex vivo exposure of intestinal T lymphocytes to Rag1-deficient microbiota results in increased interferon-gamma expression by these T lymphocytes, compared to exposure to wild type microbiota. In conclusion, we show that T lymphocytes control microbial composition by effectively suppressing the outgrowth of Vibrio species in the zebrafish intestine.

T lymphocyte-dependent and -independent regulation of Cxcl8 expression in zebrafish intestines.

CXCL8 is a potent neutrophil recruiting chemokine. CXCL8 is produced by several innate immune cells, including neutrophils, macrophages, as well as epithelial cells. Although previously considered only to be produced as a result of TLR signaling in these cells, recent reports show that T cell-derived cytokines also induce CXCL8 in epithelial cells. Likewise, we observed that T cell inhibition diminished intestinal production of functional mouse homologs of CXCL8 in the early phase of enterocolitis. In this study, we specifically investigated whether adaptive cells contribute to innate cxcl8 expression in the intestines. To this end, we used the zebrafish as our model system. Unlike murine models that lack CXCL8, zebrafish have two CXCL8 chemokines that are both elevated after an acute inflammatory stimulus and recruit neutrophils. Furthermore, zebrafish develop innate and adaptive immunity sequentially, enabling analysis of intestinal cxcl8 expression in the absence (<3 wk of age) and presence (>3 wk of age) of adaptive immunity. In this study, we show that intestinal cxcl8-l1 but not cxcl8-l2 expression is regulated by T lymphocytes under homeostatic conditions. In contrast, during intestinal inflammation especially, cxcl8-l1 expression is upregulated independent of T lymphocyte presence. Furthermore, we show that human CXCL8 is able to induce intestinal zebrafish neutrophil recruitment and cxcl8-l1 expression, demonstrating that zebrafish can be used as a model to study CXCL8 function and regulation. In conclusion, these data provide evidence that Cxcl8-l1 and Cxcl8-l2 are differentially regulated via T lymphocyte-dependent and -independent mechanisms during homeostasis and inflammation.

Selection of perforin expressing CD4+ adenovirus-specific T-cells with artificial antigen presenting cells.

Adenoviruses (HAdV) can cause life threatening infections, especially in paediatric patients after allogeneic stem cell transplantation (SCT). Yet, no effective antiviral medication is available. One treatment option is adoptive transfer of HAdV-specific T-cells from the graft donor into the patient. Especially CD4+ T-cells are critical to control HAdV infection. To allow for applicability of CD4+ T-cells in adoptive therapy, sufficient numbers of HAdV-specific T-cells with low levels of residual alloreactive T-cells are required. In this study, we explored the possibility to selectively expand and isolate functional HAdV-specific T-cells from PBMCs in response to 15-mer peptides using artificial antigen-presenting cells (aAPCs), composed of liposomes harbouring HAdV-peptide/HLA-Class-II complexes. HAdV-specific T-cells generated using this method produce mainly pro-inflammatory cytokines, express perforin and granzyme B, kill HAdV-infected cells effectively and are not alloreactive. Thus, the generation and isolation of HAdV-specific CD4+ T-cells seem a critical step towards specific adoptive therapy for HAdV infections after allogeneic SCT.

Recognition of self-heat shock protein 60 by T cells from patients with atopic dermatitis.

Heat shock protein 60 (hsp60) is a highly conserved stress protein and target of self-reactive T cells in various inflammatory diseases. Not much is known about a possible role in atopic disease. As atopic diseases are considered to be the result of a disturbance in the balance between T helper cells type 2 and regulatory T cells, it is of interest to know whether hsp60 acts as a bystander antigen in atopic disease. Our aim was to investigate whether hsp60 is involved in the chronicity of inflammation of atopic dermatitis (AD). We studied the expression of hsp60 in skin tissue of adults with AD by immunohistochemistry. Peripheral blood mononuclear cells (PBMC) of children with AD were cultured with hsp60 and proliferative responses, cytokine secretion, surface markers, and functional assays were compared to responses of PBMC of healthy controls (HC). Hsp60 was detected more in lesional skin of AD patients compared to nonlesional skin. Furthermore, PBMC of children with AD proliferated more strongly in response to hsp60 compared to HC. hsp60-reactive T cells of atopic children produced high levels of IFNγ and low levels of IL-10. In vitro activation with hsp60 leads to the induction of CD4(+)CD25(bright) T cells expressing FOXP3 in both HC as well as in atopic children. However, despite their regulatory phenotype, hsp60-induced CD4(+)CD25(bright)CD127(-)FOXP3(+) T cells of AD patients were incapable of suppressing effector T cells in vitro. hsp60 is recognized by proinflammatory (IFNγ high, IL-10 low) T cells in atopic patients and is more present in lesional AD skin. This suggests that hsp60-specific T cell responses contribute to local inflammation in AD.

Cord blood CD4+ T cells respond to self heat shock protein 60 (HSP60).

BACKGROUND: To prevent harmful autoimmunity most immune responses to self proteins are controlled by central and peripheral tolerance. T cells specific for a limited set of self-proteins such as human heat shock protein 60 (HSP60) may contribute to peripheral tolerance. It is not known whether HSP60-specific T cells are present at birth and thus may play a role in neonatal tolerance. We studied whether self-HSP60 reactive T cells are present in cord blood, and if so, what phenotype these cells have. METHODOLOGY/PRINCIPAL FINDINGS: Cord blood mononuclear cells (CBMC) of healthy, full term neonates (n = 21), were cultured with HSP60 and Tetanus Toxoid (TT) to study antigen specific proliferation, cytokine secretion and up-regulation of surface markers. The functional capacity of HSP60-induced T cells was determined with in vitro suppression assays. Stimulation of CBMC with HSP60 led to CD4(+) T cell proliferation and the production of various cytokines, most notably IL-10, Interferon-gamma, and IL-6. HSP60-induced T cells expressed FOXP3 and suppressed effector T cell responses in vitro. CONCLUSION: Self-reactive HSP60 specific T cells are already present at birth. Upon stimulation with self-HSP60 these cells proliferate, produce cytokines and express FOXP3. These cells function as suppressor cells in vitro and thus they may be involved in the regulation of neonatal immune responses.

Vaccination leads to an aberrant FOXP3 T-cell response in non-remitting juvenile idiopathic arthritis.

OBJECTIVE: To investigate how meningococcal C vaccination in patients with remitting (oligoarticular) or progressive (polyarticular) juvenile idiopathic arthritis (JIA) influences the specific T-cell response to both the vaccine and heat shock protein 60, a regulatory auto-antigen in JIA. METHODS: Twenty six oligoarticular, 28 polyarticular JIA patients and 20 healthy adults were studied before and after MenC vaccination in a prospective follow-up study. T-cell proliferation assay, flow cytometry, carboxyfluorescein diacetate succinimidyl ester staining and multiplex immunoassay were performed to quantify and qualify the antigen-specific immune responses. RESULTS: Peripheral blood mononuclear cells (PBMC) from polyarticular JIA exemplified higher antigen-specific CD4 T-cell proliferation, interleukin 2 (IL-2) and tumour necrosis factor alpha (TNFα) production when compared with oligoarticular JIA or healthy individuals after vaccination. Furthermore, in polyarticular JIA antigen-induced CD4+CD25(bright) or CD4+FOXP3+ T cells did not increase upon vaccination. CONCLUSION: Polyarticular JIA CD4+FOXP3+ T cells did not respond to vaccination and demonstrated a higher percentage of cells irrespective of vaccination when compared with oligoarticular JIA. These cells are either activated T cells and/or regulatory cells unable to regulate the antigen-specific immune response after vaccination. When compared with oligoarticular JIA, the increased IL-2 and TNFα production underline the immune hyperresponsiveness of polyarticular JIA PBMC to an antigenic trigger. As this may hold a risk for derailment, these findings could provide a cellular basis for the presumed relationship between environmental triggers and disease in human autoimmune diseases.

Novel self-epitopes derived from aggrecan, fibrillin, and matrix metalloproteinase-3 drive distinct autoreactive T-cell responses in juvenile idiopathic arthritis and in health.

Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. Knowing which antigens drive the autoreactive T-cell response in JIA is crucial for the understanding of disease pathogenesis and additionally may provide targets for antigen-specific immune therapy. In this study, we tested 9 self-peptides derived from joint-related autoantigens for T-cell recognition (T-cell proliferative responses and cytokine production) in 36 JIA patients and 15 healthy controls. Positive T-cell proliferative responses (stimulation index > or =2) to one or more peptides were detected in peripheral blood mononuclear cells (PBMC) of 69% of JIA patients irrespective of major histocompatibility complex (MHC) genotype. The peptides derived from aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3 yielded the highest frequency of T-cell proliferative responses in JIA patients. In both the oligoarticular and polyarticular subtypes of JIA, the aggrecan peptide induced T-cell proliferative responses that were inversely related with disease duration. The fibrillin peptide, to our knowledge, is the first identified autoantigen that is primarily recognized in polyarticular JIA patients. Finally, the epitope derived from MMP-3 elicited immune responses in both subtypes of JIA and in healthy controls. Cytokine production in short-term peptide-specific T-cell lines revealed production of interferon-gamma (aggrecan/MMP-3) and interleukin (IL)-17 (aggrecan) and inhibition of IL-10 production (aggrecan). Here, we have identified a triplet of self-epitopes, each with distinct patterns of T-cell recognition in JIA patients. Additional experiments need to be performed to explore their qualities and role in disease pathogenesis in further detail.

Novel pan-DR-binding T cell epitopes of adenovirus induce pro-inflammatory cytokines and chemokines in healthy donors.

Adenovirus can cause fatal infections in the immunocompromised host. To date, no effective anti-viral therapy is available. Adoptive therapy with adenovirus-specific T cells could be a promising treatment, but requires the identification of such T cells. Aim of this study was to identify conserved adenoviral T cell epitopes recognized in a majority of healthy individuals. By using a computer algorithm designed to predict pan-HLA-DR-binding T cell epitopes, we selected 19 peptides of adenovirus serotype 5. PBMCs from 26 healthy subjects were isolated and incubated with these peptides to test epitope-specific T cell proliferation. Six epitopes derived from E1B protein, hexon protein (two epitopes), DNA polymerase, E3A glycoprotein and fiber protein induced a proliferative T cell response in the majority of healthy controls. In vitro MHC binding assays confirmed the potential capacity of the adenovirus epitopes to bind multiple MHC alleles. The cytokine and chemokine profile induced by these epitopes was determined with a multiplex immunoassay and revealed a predominant pro-inflammatory pattern. Based on the broad recognition and the induced cytokine and chemokine profile, the detected epitopes can be regarded as potential candidates to select adenovirus-specific T cells for immune intervention in the immunocompromised host.

Clustering of T cell ligands on artificial APC membranes influences T cell activation and protein kinase C theta translocation to the T cell plasma membrane.

T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase theta, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.