Regulation of T-cell migration and effector functions: insights from in vivo imaging studies

Summary: Studies of the immune system are providing us with ever more detailed information on the cellular and molecular mechanisms that underlie our evolutionarily conserved ability to fend off infectious pathogens. Progress has probably been fastest at two levels: the various basic biological functions of isolated cells on one side and the significance of individual molecules or cells to the organism as a whole on the other. In both cases, direct phenomenological observation has been an invaluable methodological approach. Where we know least is the middle ground, i.e. how immune functions are integrated through the dynamic interplay of immune cell subsets within the organism. Most of our knowledge in this area has been obtained through inference from static snapshots of dynamic processes, such as histological sections, or from surrogate cell co-culture models. The latter are employed under the assumption that an in vivo equivalent exists for each type of cellular contact artificially enforced in absence of anatomical compartmentalization. In this review, we summarize recent insights on migration and effector functions of T cells, focusing on observations gained from their dynamic microscopic visualization in physiological tissue environments.     

Murine models of transplantation tolerance through mixed chimerism: advances and roadblocks

Organ transplantation is the treatment of choice for patients with end‐stage organ failure, but chronic immunosuppression is taking its toll in terms of morbidity and poor efficacy in preventing late graft loss. Therefore, a drug‐free state would be desirable where the recipient permanently accepts a donor organ while remaining otherwise fully immunologically competent. Mouse studies unveiled mixed chimerism as an effective approach to induce such donor‐specific tolerance deliberately and laid the foundation for a series of clinical pilot trials. Nevertheless, its widespread clinical implementation is currently prevented by cytotoxic conditioning and limited efficacy. Therefore, the use of mouse studies remains an indispensable tool for the development of novel concepts with potential for translation and for the delineation of underlying tolerance mechanisms. Recent innovations developed in mice include the use of pro‐apoptotic drugs or regulatory T cell (T_reg) transfer for promoting bone marrow engraftment in the absence of myelosuppression and new insight gained in the role of innate immunity and the interplay between deletion and regulation in maintaining tolerance in chimeras. Here, we review these and other recent advances in murine studies inducing transplantation tolerance through mixed chimerism and discuss both the advances and roadblocks of this approach.     

Immature dendritic cells convert anergic nonregulatory T cells into Foxp3−IL‐10+ regulatory T cells by engaging CD28 and CTLA‐4

Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide‐specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL‐10^?Foxp3^?CTLA‐4⁺CD25^lowEgr2⁺ T cells could be converted into suppressive IL‐10⁺Foxp3^?CTLA‐4⁺CD25^highEgr2⁺ cells resembling type‐1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF‐β during anergy induction favored Foxp3⁺ Treg‐cell induction, while TGF‐β had little effect when added to the second stimulation. Expression of both CD28 and CTLA‐4 molecules on anergic T cells was required to allow their conversion into Tr1‐like cells. Suppressor activity was enabled via CD28‐mediated CD25 upregulation, acting as an IL‐2 sink, together with a CTLA‐4‐mediated inhibition of NFATc1/α activation to shut down IL‐2‐mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1‐like cells.     

E3 ubiquitin ligase GRAIL controls primary T cell activation and oral tolerance

T cell unresponsiveness or anergy is one of the mechanisms that maintain inactivity of self-reactive lymphocytes. E3 ubiquitin ligases are important mediators of the anergic state. The RING finger E3 ligase GRAIL is thought to selectively function in anergic T cells but its mechanism of action and its role in vivo are largely unknown. We show here that genetic deletion of Grail in mice leads not only to loss of an anergic phenotype in various models but also to hyperactivation of primary CD4⁺ T cells. Grail^−/− CD4⁺ T cells hyperproliferate in vitro to TCR stimulation alone or with concomitant anti-CD28 costimulation, with transient increased survival. In vitro differentiated T helper 1 cells show slight but significant hypersecretion of IFN-γ in Grail^−/− mice whereas Th2 and Th17 cytokine secretions are unchanged. Consistent with defective in vitro anergy, oral tolerance is abolished in vivo in OT-II TCR transgenic Grail^−/− mice fed with ovalbumin. In experimental allergic encephalitis, a model of organ-specific autoimmunity, oral tolerization with myelin basic protein was abrogated as well in Grail^−/− mice. On the protein level, Grail^−/− naïve T cells show no significant differences of total and phosphorylated levels of ZAP70, phospholipase Cγ1, and MAP kinases p38 and JNK but elevated baseline levels of MAP kinase ERK1/2. In summary, we define a role for GRAIL in primary T cell activation, survival, and differentiation. In addition, we formally prove an indispensable role for GRAIL in T cell anergy and oral tolerance—a promising, antigen-specific strategy to treat autoimmune diseases.     

CD4+CD25?LAG3+ regulatory T cells controlled by the transcription factor Egr-2

Regulatory T cells (Tregs) are engaged in the maintenance of immunological self-tolerance and immune homeostasis. IL-10 has an important role in maintaining the normal immune state. Here, we show that IL-10-secreting Tregs can be delineated in normal mice as CD4⁺CD25⁻Foxp3⁻ T cells that express lymphocyte activation gene 3 (LAG-3), an MHC-class-II-binding CD4 homolog. Although ≈2% of the CD4⁺CD25⁻ T cell population consisted of CD4⁺CD25⁻LAG3⁺ T cells in the spleen, CD4⁺CD25⁻LAG3⁺ T cells are enriched to ≈8% in the Peyer''s patch. They are hypoproliferative upon in vitro antigenic stimulation and suppress in vivo development of colitis. Gene expression analysis reveals that CD4⁺CD25⁻LAG3⁺ Tregs characteristically express early growth response gene 2 (Egr-2), a key molecule for anergy induction. Retroviral gene transfer of Egr-2 converts naïve CD4⁺ T cells into the IL-10-secreting and LAG-3-expressing phenotype, and Egr-2-transduced CD4⁺ T cells exhibit antigen-specific immunosuppressive capacity in vivo. Unlike Foxp3⁺ natural Tregs, high-affinity interactions with selecting peptide/MHC ligands expressed in the thymus do not induce the development of CD4⁺CD25⁻LAG3⁺ Tregs. In contrast, the number of CD4⁺CD25⁻LAG3⁺ Tregs is influenced by the presence of environmental microbiota. Thus, IL-10-secreting Egr-2⁺LAG3⁺CD4⁺ Tregs can be exploited for the control of peripheral immunity.     

Impaired function of CD4+/CD25+ T regulatory lymphocytes characterizes the self-limited hepatitis A virus infection

Background and Aim:  Hepatitis A virus (HAV) causes a transient illness leaving permanent protection against reinfection. Few data are available on the regulatory mechanisms involved in the CD4+ T helper activation. We aimed to investigate the frequency and function of CD3+/CD4+/CD25+ T cells with regulatory function (Tregs) during acute HAV infection.
Methods:  We enrolled 35 consecutive patients and 15 healthy donors, enumerated Tregs by flow cytometry assay and evaluated, after immunomagnetical sorting with magnetic beads, their ability to inhibit the proliferation of CD4+/CD25– T lymphocytes at different ratios (1:1, 1:10, 1:20).
Results:  All patients had the usual course of infection. Our immunological analysis showed Tregs frequency in these patients (6.5% [range, 5–8.8%]; 36 [range, 10–87] cells) did not have any statistical difference compared with healthy donors (6% [range, 5–8%]; 48 (range, 23–71) cells), while their ability to suppress CD4+/CD25– was drastically reduced at different ratios (Mann–Whitney U -test; ratio 1:1, 93% vs 72%, z = −3.34, P  < 0.0001; ratio 1:10, 86% vs 51%, z = −4.04, P  < 0.001; ratio 1:20, 56% vs 30%, z = −3.43, P  < 0.0001). After the seroconversion, CD4+/CD25+ frequency and function in HAV-infected patients did not differ from healthy individuals.
Conclusion:  CD4+/CD25+ T cells seem to be impaired in their function during the HAV acute infection. This evidence might help to determine an optimal T helper cell immune network that is a predisposing factor for a self-limiting disease.     

Anti-4-1BB monoclonal antibodies abrogate T cell-dependent humoral immune responses in vivo through the induction of helper T cell anergy

The 4-1BB receptor (CDw137), a member of the tumor necrosis factor receptor superfamily, has been shown to costimulate the activation of T cells. Here we show that anti-mouse 4-1BB monoclonal antibodies (mAbs) inhibit thymus-dependent antibody production by B cells. Injection of anti-4-1BB mAbs into mice being immunized with cellular or soluble protein antigens induced long-term anergy of antigen-specific T cells. The immune response to the type II T cell-independent antigen trinintrophenol-conjugated Ficoll, however, was not suppressed. Inhibition of humoral immunity occurred only when anti-4-1BB mAb was given within 1 wk after immunization. Anti-4-1BB inhibition was observed in mice lacking functional CD8(+) T cells, indicating that CD8(+) T cells were not required for the induction of anergy. Analysis of the requirements for the anti-4-1BB-mediated inhibition of humoral immunity revealed that suppression could not be adoptively transferred with T cells from anti-4-1BB-treated mice. Transfer of BALB/c splenic T cells from sheep red blood cell (SRBC)-immunized and anti-4-1BB-treated mice together with normal BALB/c B cells into C.B-17 severe combined immunodeficient mice failed to generate an anti-SRBC response. However, B cells from the SRBC-immunized, anti-4-1BB-treated BALB/c mice, together with normal naive T cells, exhibited a normal humoral immune response against SRBC after transfer, demonstrating that SRBC-specific B cells were left unaffected by anti-4-1BB mAbs.     

Blockade of T cell costimulatory signals using adenovirus vectors prevents both the induction and the progression of experimental autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) has been used as a model for human myocarditis in relation to the autoimmune mechanism and proved to be a T cell-mediated autoimmune disease. Interactions of T cell surface receptors CD28 and CD40L with their ligands B7 and CD40, respectively, on APCs are critical for antigen-specific T cell activation under physiological and pathological conditions. To achieve effective inhibition of these interactions, we have constructed adenovirus vectors containing CTLA4Ig (AdexCTLA4Ig) and CD40Ig (AdexCD40Ig) and examined the effects of these adenovirus vectors in preventing EAM. AdexLacZ as a control, or AdexCTLA4Ig and/or AdexCD40Ig were injected intravenously into rats on day 0 or 14 after immunization to study the preventive effects on EAM in the T cell activation phase or inflammatory phase. Disease severity was estimated by the macroscopic and microscopic findings of the heart, heart weight to body weight ratios, and cellular and humoral immune responses on day 21. The onset of EAM after AdexCTLA4Ig or AdexCD40Ig treatment on day 0 was completely inhibited and antigen-specific lymphocyte proliferation was significantly reduced in those adenovirus-treatment groups, suggesting that those therapies induce antigen-specific T cell anergy. Moreover, significant reduction in disease severity was achieved after the adenovirus vector treatment even on day 14 compared with EAM rats. This study indicates the therapeutic potential of costimulatory pathway blockade by gene-transfer in myocarditis.