Lasse Boding

Protein Kinase C (PKC)α and PKCθ Are the Major PKC Isotypes Involved in TCR Down-Regulation

It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKCα and PKCθ were the only PKC isotypes able to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser126 and the di-leucine-based receptor-sorting motif of the CD3γ chain. Finally, we found that PKCθ was mainly implicated in down-regulation of directly engaged TCR, whereas PKCα was involved in down-regulation of nonengaged TCR.

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.

Vitamin D Up-Regulates the Vitamin D Receptor by Protecting It from Proteasomal Degradation in Human CD4+ T Cells

The active form of vitamin D3, 1,25(OH)2D3, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4+ effector T cells. The biological actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR) and are believed to correlate with the VDR protein expression level in a given cell. The aim of this study was to determine if and how 1,25(OH)2D3 by itself regulates VDR expression in human CD4+ T cells. We found that activated CD4+ T cells have the capacity to convert the inactive 25(OH)D3 to the active 1,25(OH)2D3 that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)2D3 does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4+ T cells. Furthermore, 1,25(OH)2D3 induces a significant intracellular redistribution of the VDR. We show that 1,25(OH)2D3 stabilizes the VDR by protecting it from proteasomal degradation. Finally, we demonstrate that proteasome inhibition leads to up-regulation of VDR protein expression and increases 1,25(OH)2D3-induced gene activation. In conclusion, our study shows that activated CD4+ T cells can produce 1,25(OH)2D3, and that 1,25(OH)2D3 induces a 2-fold up-regulation of the VDR protein expression in activated CD4+ T cells by protecting the VDR against proteasomal degradation.

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.

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.