Ann Kathrine Hansen

Cellular dynamics in the draining lymph nodes during sensitization and elicitation phases of contact hypersensitivity

Background:  The different role of various immunological effector cells in contact hypersensitivity (CHS) is receiving increased attention. During the past decade, the involvement of different cell types in CHS has been investigated by the use of antibody‐induced depletion of specific subtypes of immunological cells and by studying knockout mice lacking one or more of these immunological cell populations.

Activated human CD4 + T cells express transporters for both cysteine and cystine

Because naïve T cells are unable to import cystine due to the absence of cystine transporters, it has been suggested that T cell activation is dependent on cysteine generated by antigen presenting cells. The aim of this study was to determine at which phases during T cell activation exogenous cystine/cysteine is required and how T cells meet this requirement. We found that early activation of T cells is independent of exogenous cystine/cysteine, whereas T cell proliferation is strictly dependent of uptake of exogenous cystine/cysteine. Naïve T cells express no or very low levels of both cystine and cysteine transporters. However, we found that these transporters become strongly up-regulated during T cell activation and provide activated T cells with the required amount of cystine/cysteine needed for T cell proliferation. Thus, T cells are equipped with mechanisms that allow T cell activation and proliferation independently of cysteine generated by antigen presenting cells.

TCR Down-Regulation Controls Virus-Specific CD8+ T Cell Responses

The CD3γ di-leucine-based motif plays a central role in TCR down-regulation. However, little is understood about the role of the CD3γ di-leucine-based motif in physiological T cell responses. In this study, we show that the expansion in numbers of virus-specific CD8+ T cells is impaired in mice with a mutated CD3γ di-leucine-based motif. The CD3γ mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic molecule Bcl-2. This resulted in a 2-fold reduction in the clonal expansion of virus-specific CD8+ T cells during the acute phase of vesicular stomatitis virus and lymphocytic choriomeningitis virus infections. These results identify an important role of CD3γ-mediated TCR down-regulation in virus-specific CD8+ T cell responses.

PKC-θ exists in an oxidized inactive form in naive human T cells.

PKC‐θ plays a central role in TCR‐induced IL‐2 production and T‐cell proliferation. The aim of the present study was to analyse how PKC‐θ is regulated in human T cells during T‐cell activation and differentiation. We show that PKC‐θ is found in a high‐molecular disulfide‐linked complex in naïve T cells, and that PKC‐θ most likely is inactive in this form. In parallel with the accumulation of the major redox regulators, glutathione and thioredoxin, PKC‐θ is gradually reduced to the 82 kDa active form during T‐cell activation. We demonstrate that PKC‐θ is recruited to the plasma membrane in the disulfide‐linked form in naïve T cells, and that activation of PKC‐θ is redox dependent and requires de novo synthesis of glutathione. This is the first study that shows that the activity of PKC‐θ is regulated by the intracellular redox state, and that PKC‐θ is recruited to the plasma membrane in an inactive form in naïve T cells. Our observations underscore the existence of major differences in TCR signaling in naïve versus primed T cells.

TCR down‐regulation boosts T‐cell‐mediated cytotoxicity and protection against poxvirus infections

Cytotoxic T (Tc) cells play a key role in the defense against virus infections. Tc cells recognize infected cells via the T‐cell receptor (TCR) and subsequently kill the target cells by one or more cytotoxic mechanisms. Induction of the cytotoxic mechanisms is finely tuned by the activation signals from the TCR. To determine whether TCR down‐regulation affects the cytotoxicity of Tc cells, we studied TCR down‐regulation‐deficient CD3γLLAA mice. We found that Tc cells from CD3γLLAA mice have reduced cytotoxicity due to a specific deficiency in exocytosis of lytic granules. To determine whether this defect was reflected in an increased susceptibility to virus infections, we studied the course of ectromelia virus (ECTV) infection. We found that the susceptibility to ECTV infection was significantly increased in CD3γLLAA mice with a mortality rate almost as high as in granzyme B knock‐out mice. Finally, we found that TCR signaling in CD3γLLAA Tc cells caused highly increased tyrosine phosphorylation and activation of the c‐Cbl ubiquitin ligase, and that the impaired exocytosis of lytic granules could be rescued by the knockdown of c‐Cbl. Thus, our work demonstrates that TCR down‐regulation critically increases Tc cell cytotoxicity and protection against poxvirus infection.

TCR down-regulation controls T cell homeostasis

TCR and cytokine receptor signaling play key roles in the complex homeostatic mechanisms that maintain a relative stable number of T cells throughout life. Despite the homeostatic mechanisms, a slow decline in naive T cells is typically observed with age. The CD3γ di-leucine-based motif controls TCR down-regulation and plays a central role in fine-tuning TCR expression and signaling in T cells. In this study, we show that the age-associated decline of naive T cells is strongly accelerated in CD3γLLAA knock-in mice homozygous for a double leucine to alanine mutation in the CD3γ di-leucine-based motif, whereas the number of memory T cells is unaffected by the mutation. This results in premature T cell population senescence with a severe dominance of memory T cells and very few naive T cells in middle-aged to old CD3γ mutant mice. The reduced number of naive T cells in CD3γ mutant mice was caused by the combination of reduced thymic output, decreased T cell apoptosis, and increased transition of naive T cells to memory T cells. Experiments with bone marrow chimeric mice confirmed that the CD3γLLAA mutation exerted a T cell intrinsic effect on T cell homeostasis that resulted in an increased transition of CD3γLLAA naive T cells to memory T cells and a survival advantage of CD3γLLAA T cells compared with wild-type T cells. The experimental observations were further supported by mathematical modeling of T cell homeostasis. Our study thus identifies an important role of CD3γ-mediated TCR down-regulation in T cell homeostasis.

TL1A regulates TCRγδ+ intraepithelial lymphocytes and gut microbial composition.

TL1A is a proinflammatory cytokine, which is prevalent in the gut. High TL1A concentrations are present in patients with inflammatory bowel disease (IBD) and in IBD mouse models. However, the role of TL1A during steady‐state conditions is relatively unknown. Here, we used TL1A knockout (KO) mice to analyse the impact of TL1A on the intestinal immune system and gut microbiota. The TL1A KO mice showed reduced amounts of small intestinal intraepithelial TCRγδ+ and CD8+ T cells, and reduced expression of the activating receptor NKG2D. Moreover, the TL1A KO mice had significantly reduced body weight and visceral adipose tissue deposits, as well as lower levels of leptin and CXCL1, compared with wild‐type mice. Analysis of the gut microbial composition of TL1A KO mice revealed a reduction of caecal Clostridial cluster IV, a change in the Firmicutes/Bacteroidetes ratio in caecum and less Lactobacillus spp. in the mucosal ileum. Our results show that TL1A deficiency impacts on the gut microbial composition and the mucosal immune system, especially the intraepithelial TCRγδ+ T‐cell subset, and that TL1A is involved in the establishment of adipose tissue. This research contributes to a broader understanding of TL1A inhibition, which is increasingly considered for treatment of IBD.

Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells

Midline 1 (MID1) is a microtubule‐associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss‐of‐function mutations in MID1 lead to the X‐linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here, we show that MID1 is strongly upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking, and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1−/− CTLs is impaired. Transfection of MID1 into MID1−/− CTLs completely rescued lytic granule exocytosis, and vice versa, knockdown of MID1 inhibited exocytosis of lytic granules in WT CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity.

Midline 1 controls polarization and migration of murine cytotoxic T cells

Midline 1 (MID1) is a microtubule‐associated ubiquitin ligase that regulates protein phosphatase 2 A levels. Loss‐of‐function mutations in MID1 lead to the human X‐linked Opitz G/BBB (OS) syndrome characterized by defective midline development during embryogenesis. We have recently shown that MID1 is strongly up‐regulated in murine cytotoxic T lymphocytes (CTLs), and that it has a significant impact on exocytosis of lytic granules and the killing capacity of CTLs. The aims of the present study were to determine the localization of MID1 in migrating CTLs, and to investigate whether MID1 affects CTL polarization and migration. We found that MID1 mainly localizes to the uropod of migrating CTLs and that it has a substantial impact on CTL polarization and migration in vitro. Furthermore, analysis of contact hypersensitivity responses supported that MID1 controls effector functions of CTLs in hapten‐challenged skin in vivo. These results provide significant new knowledge on the role of MID1 in CTL biology.

MID2 can substitute for MID1 and control exocytosis of lytic granules in cytotoxic T cells

We have recently shown that the E3 ubiquitin ligase midline 1 (MID1) is upregulated in murine cytotoxic lymphocytes (CTL), where it controls exocytosis of lytic granules and the killing capacity. Accordingly, CTL from MID1 knock‐out (MID1−/−) mice have a 25–30% reduction in exocytosis of lytic granules and cytotoxicity compared to CTL from wild‐type (WT) mice. We wondered why the MID1 gene knock‐out did not affect exocytosis and cytotoxicity more severely and speculated whether MID2, a close homologue of MID1, might partially compensate for the loss of MID1 in MID1−/− CTL. Here, we showed that MID2, like MID1, is upregulated in activated murine T cells. Furthermore, MID1−/− CTL upregulated MID2 two–twenty‐fold stronger than CTL from WT mice, suggesting that MID2 might compensate for MID1. In agreement, transfection of MID2 into MID1−/− CTL completely rescued exocytosis of lytic granules in MID1−/− CTL, and vice versa, knock‐down of MID2 inhibited exocytosis of lytic granules in both WT and MID1−/− CTL, demonstrating that both MID1 and MID2 play a central role in the regulation of granule exocytosis and that functional redundancy exists between MID1 and MID2 in CTL.