Regulatory T cells control the transition from acute into chronic inflammation in glucose-6-phosphate isomerase-induced arthritis.

OBJECTIVES: Glucose-6-phosphate isomerase (G6PI)-induced arthritis is a spontaneously remitting experimental arthritis model. It was hypothesised that regulatory T cells (Tregs) are involved in remission and their role in G6PI-induced arthritis was investigated. METHODS: Tregs were depleted by injection of anti-CD25 before immunisation of DBA/1 mice with G6PI. The severity of arthritis was assessed clinically and histologically and the number and function of G6PI-specific T helper (Th) cells were determined by flow cytometry. Th cells and monocytes/macrophages were depleted using anti-CD4 or clodronate-containing liposomes. RESULTS: Injection of anti-CD25 depleted Tregs transiently. Normal numbers of Tregs were restored 5 weeks after G6PI immunisation. Whereas arthritis started to resolve in control mice 3 weeks after immunisation with G6PI, severe arthritis was still present in the anti-CD25-treated mice 12 weeks after immunisation. The most striking ex vivo correlate of non-remitting arthritis was a strong increase in G6PI-specific Th cells 3 days after G6PI immunisation. This difference between treated and control mice declined at later time points. Depletion of CD4 cells ameliorated arthritis in controls but not in anti-CD25-treated mice. In contrast, clodronate-containing liposomes were an effective treatment in both groups. CONCLUSIONS: Tregs control the transition from acute self-limiting to non-remitting destructive G6PI-induced arthritis already in the preclinical disease stage. Once established, non-remitting destructive arthritis is not controlled by restoration of normal Treg numbers. These findings question the rationale of therapeutic approaches augmenting Treg number or function in established arthritis.

Inducible costimulator (ICOS) blockade inhibits accumulation of polyfunctional T helper 1/T helper 17 cells and mitigates autoimmune arthritis.

OBJECTIVES: Inducible costimulator (ICOS) and its ligand (ICOSL) regulate T and B cell responses. Glucose-6-phosphate isomerase (G6PI)-induced arthritis requires T and B lymphocytes. It was hypothesised that blocking ICOS/ICOSL interactions ameliorates G6PI-induced arthritis and reduces G6PI-specific B and T lymphocyte responses. METHODS: DBA/1 mice were injected with a blocking, non-depleting anti-ICOSL monoclonal antibodies (mAbs) during the induction or effector phase of G6PI-induced arthritis. G6PI-specific antibody responses were measured by ELISA. G6PI-specific T helper (Th) cell responses were assayed by polychromatic flow cytometry. RESULTS: Transient blockade of ICOS/ICOSL interactions profoundly reduced the severity of G6PI-induced arthritis. ELISA and proliferation assays showed no clear ex vivo correlates of protection. Polychromatic flow cytometry revealed two major findings: the absolute number of G6PI-specific Th cells was markedly diminished in secondary lymphatic organs from mice with blocked ICOS/ICOSL interactions. Within the pool of G6PI-specific Th cells the frequency of interleukin 17 (IL17), interferon gamma or tumour necrosis factor alpha producers or polyfunctional Th cells (expressing two or more of these cytokines) was higher in treated than in control mice. CONCLUSIONS: ICOS costimulation is not mandatory for the differentiation of Th1 or Th17 cells. Instead, the lack of ICOS costimulation results in reduced survival of G6PI-specific Th cells irrespective of their functional differentiation. This study demonstrates that a thorough examination of the quantity and the quality of antigen-specific immune responses is useful to determine ex vivo correlates of efficacy for immunomodulating treatments.

Autoregulation of Th1-mediated inflammation by twist1.

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor kappaB (NF-kappaB)-dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-kappaB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-gamma, IL-2, and tumor necrosis factor-alpha, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

Cutting Edge: Regulatory T cells prevent efficient clearance of Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains one of the top microbial killers of humans causing approximately 2 million deaths annually. More than 90% of the 2 billion individuals infected never develop active disease, indicating that the immune system is able to generate mechanisms that control infection. However, the immune response generally fails to achieve sterile clearance of bacilli. Using adoptive cell transfer into C57BL/6J-Rag1(tm1Mom) mice (Rag1(-/-)), we show that regulatory T cells prevent eradication of tubercle bacilli by suppressing an otherwise efficient CD4+ T cell response. This protective CD4+ T cell response was not correlated with increased numbers of IFN-gamma- or TNF-alpha-expressing cells or general expression levels of IFN-gamma or inducible NO synthase in infected organs compared with wild-type C57BL/6 animals. Furthermore, suppression of protection by cotransferred regulatory T cells was neither accompanied by a general increase of IL-10 expression nor by higher numbers of IL-10-producing CD4+ T cells.

Chemotactic responses of IL-4-, IL-10-, and IFN-gamma-producing CD4+ T cells depend on tissue origin and microbial stimulus.

Th1- and Th2-polarized immune responses are crucial in the defense against pathogens but can also promote autoimmunity and allergy. The chemokine receptors CXCR3 and CCR4 have been implicated in differential trafficking of IFN-gamma- and IL-4-producing T cells, respectively, but also in tissue and inflammation-specific homing independent of cytokine responses. Here, we tested whether CD4+ T cells isolated from murine tissues under homeostatic or inflammatory conditions exhibit restricted patterns of chemotactic responses that correlate with their production of IFN-gamma, IL-4, or IL-10. In uninfected mice, IL-4-producing T cells preferentially migrated to the CCR4 ligand, CCL17, whereas IFN-gamma-expressing T cells as well as populations of IL-4+ or IL-10+ T cells migrated to the CXCR3 ligand, CXCL9. All cytokine-producing T cell subsets strongly migrated to the CXCR4 ligand, CXCL12. We assessed chemotaxis of T cells isolated from mice infected with influenza A virus or the nematode Nippostrongylus brasiliensis, which induce a strong Th1 or Th2 response in the lung, respectively. Unexpectedly, the chemotactic responses of IL-4+ T cells and T cells expressing the immunosuppressive cytokine IL-10 were influenced not only by the strongly Th1- or Th2-polarized environments but also by their anatomical localization, i.e., lung or spleen. In contrast, IFN-gamma+ T cells exhibited robust chemotaxis toward CXCL9 and had the most consistent migration pattern in both infection models. The results support a model in which the trafficking responses of many effector and regulatory T cells are regulated as a function of the infectious and tissue environments.

Lipopolysaccharide injection induces relapses of experimental autoimmune encephalomyelitis in nontransgenic mice via bystander activation of autoreactive CD4+ cells.

Infections sometimes associate with exacerbations of autoimmune diseases through pathways that are poorly understood. Ag-specific mechanisms such as cross-reactivity between a microbial Ag and a self-Ag have received no direct support. In this study, we show that injection of LPS induces experimental autoimmune encephalomyelitis in TCR-transgenic mice and relapse of encephalomyelitis in normal mice. This form of treatment induces proliferation and cytokine production in a fraction of effector/memory Th lymphocytes in vitro via physical contact of Th cells with CD4(-) LPS-responsive cells. TCR-mediated signals are not necessary; rather what is required is ligation of costimulatory receptors on Th cells by costimulatory molecules on the CD4(-) cells. This form of bystander activation provides an Ag-independent link between infection and autoimmunity that might fit the clinical and epidemiological data on the connection between infection and autoimmunity better than the Ag-specific models.

Expression of selectin ligands on murine effector and IL-10-producing CD4+ T cells from non-infected and infected tissues.

Endothelial selectins are crucial for the recruitment of leukocytes into sites of inflammation. On T cells, ligands for selectins become induced upon differentiation into the effector/memory stage. Initial in vitro studies suggested a correlation between the Th1 phenotype and ligand expression, but whether this also holds true in vivo remained uncertain. We here analyzed selectin ligands on CD4+ T cells producing IFN-gamma, IL-4 or IL-10, prototypic cytokines of the Th1, Th2 and Tr1 subset, respectively. We analyzed mice infected with influenza virus, the bacterium Listeria, and the parasites Toxoplasma (all Th1 models) or Nippostrongylus (Th2 model). A link between the Th1 phenotype and ligand expression was not found in vivo. Surprisingly, the potentially regulatory IL-10-producing T cells displayed the highest frequency of ligand-positive cells in general. Within the inflamed tissues, the frequencies of P-selectin-binding cells increased in the dominant subset, either Th1 or Th2. Up-regulation was also found for E-selectin ligands during influenza, but not Nippostrongylus infection. In conclusion, conditions driving T cell polarization into either Th1 or Th2 in vivo do not affect the expression of selectin ligands, but acquisition of P-selectin binding and hence migration into inflamed tissues is boosted by an inflammatory milieu.

CC chemokine receptor 7 expression by effector/memory CD4+ T cells depends on antigen specificity and tissue localization during influenza A virus infection.

The lung is an important entry site for respiratory pathogens such as influenza A virus. In order to combat such invading infectious agents, effector/memory T cells home to the lung and other peripheral tissues as well as lymphoid organs. In this process, chemokines and their receptors fulfill important roles in the guidance of T cells into such organs and specialized microenvironments within tissues. In this study, we determined if CD4(+) T cells residing in different lung compartments and draining lymph nodes of influenza A virus-infected and naïve mice express receptors allowing their recirculation into secondary lymphoid tissues. We found high levels of l-selectin and CC chemokine receptor 7 (CCR7) expression in lung-derived CD4(+) T cells, similar to that detected on T cells in secondary lymphoid organs. Upon influenza A virus infection, the bulk of gamma interferon-positive (IFN-gamma(+)) and IFN-gamma(-) CD4(+) T cells recovered from lung parenchyma retained functional CCR7, whereas virus-specific IFN-gamma-producing T cells were CCR7(-). In contrast, a majority of virus-specific IFN-gamma(+) T cells in the lung draining lymph node were CCR7(+). Independent of infection, CD4(+) T cells obtained from the lung airways exhibited the lowest expression level of l-selectin and CCR7, indicating that T cells at this anatomical site represent the most differentiated effector cell type, lacking the ability to recirculate. Our results suggest that effector/memory T cells that enter inflammatory sites retain functional CCR7 expression, which is lost only upon response to viral antigen and after localization to the final effector site.

Two waves of antigen-containing dendritic cells in vivo in experimental Leishmania major infection.

Dendritic cells (DC) can induce Th1 cell differentiation by producing IL-12. In experimental infection with Leishmania major, DC could differently respond to infection and induce Th1 cells in C57BL/6 but not BALB/c mice, and thus determine the resistance or susceptibility of these mice. We characterized L. major antigen-containing DC in vivo in draining lymph nodes of both strains. Conventional experimental infection is shown to result in two waves of these DC and our data argue against a relevant genetic difference in the DC initiating the anti-parasite Th cell response in these mice. In both strains the first wave of DC presented L. major antigens but was not infected, produced IL-12 but induced disease-mediating Th2 cells upon adoptive transfer. In contrast to current belief, this response was therefore not initiated by infected DC, which were only detected in the second wave. The kinetics of the two waves suggests that DC turnover has an important impact on antigen presentation during infections with complex pathogens.

Antigen-specific CD8+ T cell responses in intestinal tissues during murine listeriosis.

Infection of mice with Listeria monocytogenes induces a strong CD8+ T cell response, which is critical for the control of bacteria and for protection against re-infection. We analyzed the CD8+ T cell response in different intestinal tissues following oral and intravenous (i.v.) L. monocytogenes infection. After oral infection, bacterial titers in small intestine and large intestine, and the listeria-specific CD8+ T cell response in the mucosa of both parts of the intestine, were highly correlated. Oral infection of CD28-deficient mice revealed that this response was strictly dependent on CD28 costimulation. Significant listeria-specific CD8+ T cell responses also occurred in all intestinal tissues analyzed after i.v. infection or after DNA vaccination, indicating that the accumulation of listeria-specific CD8+ T cells in these tissues only partially depends on local antigen presentation and inflammation.

ICOS+ Th cells produce distinct cytokines in different mucosal immune responses.

T cell activation, differentiation and effector functions depend on signals delivered through the antigen-specific TCR and non-clonal costimulatory receptors on the T cell. Activated T cells express the inducible costimulator (ICOS). We examined the co-expression of ICOS with Th cytokines in mucosal immune responses. ICOS+CD4+ Th cells expressed strikingly different cytokines depending on the type of infection encountered and the cells' anatomical localization. In the Th2-dominated response to Schistosoma mansoni, ICOS expression of CD4+ cells isolated from the liver was strongly associated with the expression of IL-5, IL-10, IL-13, and T1/ST2, but not with the chemokine receptor CXCR5, a pattern consistent with Th2 effector cells. In the secondary lymphatic organs of schistosome-infected mice, ICOS expression was randomly correlated with Th2 effector-cytokines, but positively correlated with CXCR5 expression; a pattern consistent with follicular Th cells. In Th cells isolated from gut or liver of mice infected with Toxoplasma gondii, ICOS expression was positively correlated with IFN-gamma production. Finally, in the severe combined immunodeficiency transfer colitis model, ICOS expression was strongly positively associated with IFN-gamma and IL-2. Thus, ICOS appears to costimulate distinct effector functions in different immune responses, depending on factors such as the nature of the antigen encountered and localization and chronicity of the immune response.

Expression of ICOS in vivo defines CD4+ effector T cells with high inflammatory potential and a strong bias for secretion of interleukin 10.

The studies performed to date analyzed the overall participation of the inducible costimulator (ICOS) in model diseases, but did not yield information on the nature and function of ICOS-expressing T cells in vivo. We examined ICOS(+) T cells in the secondary lymphoid organs of nonmanipulated mice, in the context of an "unbiased" immune system shaped by environmental antigens. Using single cell analysis, ICOS(low) cells were found to be loosely associated with the early cytokines interleukin (IL)-2, IL-3, IL-6, and interferon (IFN)-gamma. ICOS(medium) cells, the large majority of ICOS(+) T cells in vivo, were very tightly associated with the synthesis of the T helper type 2 (Th2) cytokines IL-4, IL-5, and IL-13, and these cells exhibited potent inflammatory effects in vivo. In contrast, ICOS(high) T cells were highly and selectively linked to the anti-inflammatory cytokine IL-10. Overall, these data seem to indicate that ICOS cell surface density serves as a regulatory mechanism for the release of cytokines with different immunological properties. Further in vivo functional experiments with in vitro-activated T cells strongly suggested that the ICOS(+) population, although representing in vivo only around 10% of T cells bearing early or late activation markers, nevertheless encompasses virtually all effector T cells, a finding with major diagnostic and therapeutic implications.

Regulatory CD4+CD25+ T cells restrict memory CD8+ T cell responses.

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.

Organ-specific CD4+ T cell response during Listeria monocytogenes infection.

The immune response against the intracellular bacterium Listeria monocytogenes involves both CD4(+) and CD8(+) T cells. We used the MHC class II-presented peptide listeriolysin(189-201) to characterize the organ-specific CD4(+) T cell response during infection. Systemic listeriosis resulted in a strong peptide-specific CD4(+) T cell response with frequencies of 1/100 and 1/30 CD4(+) splenocytes at the peak of primary and secondary response, respectively. This response was not restricted to lymphoid organs, because we detected specific CD4(+) T cells in all tissues analyzed. However, the tissue distribution of the T cell response was dependent on the route of infection. After i.v. infection, the strongest CD4(+) T cell response and the highest levels of memory cells were observed in spleen and liver, the major sites of L. monocytogenes replication. After oral infection, we detected a strong response in the liver, the lamina propria, and the intestinal epithelium. These tissues also harbored the highest frequencies of listeriolysin(189-201)-specific CD4(+) memory T cells 5-8 wk post oral infection. Our results show that kinetics and magnitude of the CD4(+) T cell response and the accumulation of CD4(+) memory T cells depend on the route of infection and are regulated in a tissue-specific way.