Hans-Willi Mittrücker

Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA-4.

The absence of CTLA-4 results in uncontrolled T cell proliferation. The T cell receptor-specific kinases FYN, LCK, and ZAP-70 as well as the RAS pathway were found to be activated in T cells of Ctla-4−/− mutant mice. In addition, CTLA-4 specifically associated with the tyrosine phosphatase SYP, an interaction mediated by the SRC homology 2 (SH2) domains of SYP and the phosphotyrosine sequence Tyr-Val-Lys-Met within the CTLA-4 cytoplasmic tail. The CTLA-4-associated SYP had phosphatase activity toward the RAS regulator p52SHC. Thus, the RAS pathway and T cell activation through the T cell receptor are regulated by CTLA-4-associated SYP.

Duration of TCR stimulation determines costimulatory requirement of T cells.

Current models suggest that T cells that receive only signal-1 through antigenic stimulation of the T cell receptor (TCR) become anergic, but will mount an immune response when a costimulatory signal-2 is provided. Using mice deficient for an important costimulatory molecule, CD28, we show that a transient signal-1 alone, either through infection with an abortively replicating virus, or through injection of viral peptide, anergizes CD8+ T cells, demonstrating the biological relevance of T cell anergy in vivo. However, in the absence of CD28, continued presence of signal-1 alone, either through prolonged viral replication or repeated injection of peptide, prevents the induction of anergy and generates a functional T cell response in vivo.

Interferon Regulatory Factor-1 Is Required for a T Helper 1 Immune Response In Vivo

The transcription factor interferon regulatory factor-1 (IRF-1) mediates the effects of IFN. No information exists on its role in lymphokine production. Protection against the intracellular pathogen Leishmania major depends on a Th1 response. Here, we show that CD4+ T cells from Leishmania-infected mice lacking one (+/-) or both (-/-) alleles of the IRF-1 gene developed a profound, gene dose-dependent decrease in IFNgamma production. IRF-1(-/-) mice showed dramatically exacerbated Leishmaniasis. They produced increased Leishmania-specific IgG1 and IgE, and their CD4+ T cells produced increased IL-4, characteristics of the non-protective Th2 response. In cell transfer experiments, IRF-1(-/-) CD4+ T cells mounted normal Th1 responses. However, the ability of IRF-1(-/-) mice to produce IL-12 was severely compromised. Thus, IRF-1 is a determining factor for Th1 responses.

Immune response to infection with Salmonella typhimurium in mice

Infection of mice with Salmonella typhimurium results in systemic infection and a disease similar to that seen in humans after infection with S. typhi. The innate immune system can restrict replication of S. typhimurium to a certain degree, but for effective control and eradication of bacteria, acquired immunity is essential. Salmonella infection induces the generation of specific CD4+ and CD8+ T cells, and both T cell populations are important for protection during primary and secondary responses, although the mechanisms underlying T cell‐mediated protection are not yet completely understood. Infection with S. typhimurium also results in a strong antibody response to Salmonella antigens and, in contrast to most other intracellular bacteria, this antibody response participates in protection. In summary, the response to S. typhimurium involves both T and B cell‐mediated immunity, and mechanisms mediated by both lymphocyte populations are important for control of primary infection and protection against secondary infection. J. Leukoc. Biol. 67: 457–463; 2000.

Cutting edge: Regulatory T cells prevent efficient clearance of Mycobacterium tuberculosis

Mycobacterium tuberculosis remains one of the top microbial killers of humans causing ∼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-Rag1tm1Mom 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-γ- or TNF-α-expressing cells or general expression levels of IFN-γ 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.

Dysregulated T helper cell differentiation in the absence of interferon regulatory factor 4

Certain IFN regulatory factor (IRF) transcription factors indirectly influence T helper (Th) cell differentiation by regulating the production of IL-12. Here, we show that IRF4 directly regulates Th cell differentiation in vitro and in vivo during murine leishmaniasis. In the absence of IRF4, IL-12-induced Th1 cell differentiation was compromised, while IL-4 failed to induce Th2 cell differentiation. Instead, IL-4 tended to induce Th1 cells, defined by production of IFN-γ and TNF. Although early IL-4 signaling was normal in IRF4−/− Th cells, the protein GATA-3, a transcription factor critical for Th2 development, was not up-regulated following IL-4 treatment. Retroviral overexpression of GATA-3 rescued Th2 differentiation. Therefore, IRF4 deficiency manifests itself as severely dysregulated Th cell differentiation.

Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis

Mycobacterium bovis bacille Calmette–Guérin (BCG) is the most widely used live bacterial vaccine. However, limited information is available correlating route and dose of vaccination and induction of specific T cell responses with protection against tuberculosis. We compared efficacy of oral and systemic vaccination and correlated vaccine-induced T cell responses with protection in experimental tuberculosis of mice. After oral and systemic vaccination, we observed profound differences in persistence and dissemination of BCG and frequencies and location of specific IFN-γ-secreting CD4+ and CD8+ T cells. Yet, both vaccination routes caused comparable levels of protection against aerosol challenge with Mycobacterium tuberculosis. Protection correlated best with rapid accumulation of specific CD8+ T cells in infected tissues of challenged mice. In contrast, specific IFN-γ production by CD4+ T cells reflected the load of M. tuberculosis rather than the strength of protection. Our data question the measurement of IFN-γ secretion by CD4+ T cells and emphasize the need for new biomarkers for evaluation of tuberculosis vaccine efficacies.

Systemic NKG2D Down-Regulation Impairs NK and CD8 T Cell Responses In Vivo

The immunoreceptor NKG2D stimulates activation of cytotoxic lymphocytes upon engagement with MHC class I-related NKG2D ligands of which at least some are expressed inducibly upon exposure to carcinogens, cell stress, or viruses. In this study, we investigated consequences of a persistent NKG2D ligand expression in vivo by using transgenic mice expressing MHC class I chain-related protein A (MICA) under control of the H2-Kb promoter. Although MICA functions as a potent activating ligand of mouse NKG2D, H2-Kb-MICA mice appear healthy without aberrations in lymphocyte subsets. However, NKG2D-mediated cytotoxicity of H2-Kb-MICA NK cells is severely impaired in vitro and in vivo. This deficiency concurs with a pronounced down-regulation of surface NKG2D that is also seen on activated CD8 T cells. As a consequence, H2-Kb-MICA mice fail to reject MICA-expressing tumors and to mount normal CD8 T cell responses upon Listeria infection emphasizing the importance of NKG2D in immunity against tumors and intracellular infectious agents.

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.

A Th17‐like developmental process leads to CD8+ Tc17 cells with reduced cytotoxic activity

Activation of naive CD8+ T cells with antigen in the absence of skewing cytokines triggers their differentiation into effector CTL, which induces death of target cells. We show that CD8+ T cells activated in the presence of the cytokines IL‐6 or IL‐21 plus TGF‐β similar to CD4+ T cells, develop into IL‐17‐producing (Tc17) cells. These cells display greatly suppressed cytotoxic function along with low levels of the CTL markers: T‐box transcription factor Eomesodermin, granzyme B and IFN‐γ. Instead, these cells express hallmark molecules of Th17 program including retinoic acid receptor‐related orphan receptor (ROR)γt, RORα, IL‐21 and IL‐23R. The expression of the type 17 master regulator RORγt is causally linked to Tc17 generation, because its overexpression stimulates production of IL‐17 in the presence of IL‐6 or IL‐21. Both, upregulation of the type 17 program as well as suppression of CTL differentiation are STAT3 dependent. Furthermore, Tc17 cells producing IL‐17 but not granzyme B are also detectable in EAE, a mouse model for multiple sclerosis. Our data point to the existence of mutually exclusive CTL and Tc17 developmental pathways in vitro and in vivo.