Experimental myositis inducible with transfer of dendritic cells presenting a skeletal muscle C protein-derived CD8 epitope peptide.

It is suggested that polymyositis, an autoimmune inflammatory myopathy, is mediated by autoaggressive CD8 T cells. Skeletal muscle C protein is a self-antigen that induces C protein-induced myositis, a murine model of polymyositis. To establish a new murine model of myositis inducible with a single CD8 T-cell epitope peptide that derives from the C protein, three internet-based prediction systems were employed to identify 24 candidate peptides of the immunogenic fragment of the C protein and bind theoretically to major histocompatibility complex class I molecules of C57BL/6 (B6) mice. RMA-S cell assay revealed that a HILIYSDV peptide, amino acid position 399-406 of the C protein, had the highest affinity to the H2-K(b) molecules. Transfer of mature bone marrow-derived dendritic cells pulsed with HILIYSDV induced myositis in naive B6 mice. This myositis was suppressed by anti-CD8-depleting antibodies but not by anti-CD4-depleting antibodies. Because this myositis model is mediated by CD8 T cells independently of CD4 T cells, it should be a useful tool to investigate pathology of polymyositis and develop therapies targeting CD8 T cells.

T lymphocytes and muscle condition act like seeds and soil in a murine polymyositis model.

OBJECTIVE: It has been reported that polymyositis (PM) is driven by CD8+ cytotoxic T lymphocytes. The C protein-induced myositis (CIM) model we have established is similar to PM in pathology except that it undergoes spontaneous remission. We undertook the present study to delineate the roles of innate and acquired immunity in myositis. METHODS: C57BL/6 mice were immunized with recombinant C protein fragments together with Freund's complete adjuvant (CFA) and Toll-like receptor (TLR) ligands at hind leg footpads and tail bases. CIM mediated by adoptive transfer of T cells to naive mice was treated with cytokine antagonists. RESULTS: Second immunization with C protein fragments revealed no induction of tolerance. Injection of CFA and TLR ligands at the hind leg footpads reinduced myositis in the same legs. Interestingly, initial myositis was observed only in the CFA-treated forelegs. Transfer of C protein fragment-specific T cells from mice with CIM induced myositis in CFA- and TLR ligand-treated legs of recipient mice. CFA treatment resulted in the recruitment of macrophages producing inflammatory cytokines. Induction of myositis was inhibited by blocking interleukin-1 receptor or tumor necrosis factor α. CONCLUSION: Myositis development requires activation of autoaggressive T cells and conditioning of muscle tissue. CIM regression is due to attenuation of local CFA-induced immune activation. These results are in accordance with a "seed and soil" model of disease development and might offer clues to decipher clinical aspects of PM.

Murine CD4 T cells produce a new form of TGF-ß as measured by a newly developed TGF-ß bioassay.

BACKGROUND: It is generally assumed that T cells do not produce active TGF-ß since active TGF-ß as measured in supernatants by ELISA without acidification is usually not detectable. However, it is possible that active TGF-ß from T cells may take a special form which is not detectable by ELISA. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a TGF-ß bioassay which can detect both soluble and membrane-bound forms of TGF-ß from T cells. For this bioassay, 293T cells were transduced with (caga)(12) Smad binding element-luciferase along with CD32 (Fc receptor) and CD86. The resulting cells act as artificial antigen presenting cells in the presence of anti-CD3 and produce luciferase in response to biologically active TGF-ß. We co-cultured pre-activated murine CD4(+)CD25(-) T cells or CD4(+)CD25(+) T cells with the 293T-caga-Luc-CD32-CD86 reporter cells in the presence of anti-CD3 and IL-2. CD4(+)CD25(-) T cells induced higher luciferase in the reporter cells than CD4(+)CD25(+) T cells. This T cell-produced TGF-ß is in a soluble form since T cell culture supernatants contained the TGF-ß activity. The TGF-ß activity was neutralized with an anti-mouse LAP mAb or an anti-latent TGF-ß/pro-TGF-ß mAb, but not with anti-active TGF-ß Abs. An anti-mouse LAP mAb removed virtually all acid activatable latent TGF-ß from the T cell culture supernatant, but not the ability to induce TGF-ß signaling in the reporter cells. The induction of TGF-ß signaling by T cell culture supernatants was cell type-specific. CONCLUSIONS/SIGNIFICANCE: A newly developed 293T-caga-Luc-CD32-CD86 reporter cell bioassay demonstrated that murine CD4 T cells produce an unconventional form of TGF-ß which can induce TGF-ß signaling. This new form of TGF-ß contains LAP as a component. Our finding of a new form of T cell-produced TGF-ß and the newly developed TGF-ß bioassay system will provide a new avenue to investigate T cell function of the immune system.

TGF-ß induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction.

BACKGROUND: It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3(+) Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs. METHODOLOGY/PRINCIPAL FINDINGS: We generated anti-mouse LAP mAbs by immunizing TGF-ß(-/-) animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3(+) CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4(+)CD25(-) T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-ß and found that TGF-ß induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-ß treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells. CONCLUSIONS/SIGNIFICANCE: Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-ß. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

Depletion of TGF-ß from fetal bovine serum.

TGF-ß is one of the key cytokines controlling immune responses. Measuring TGF-ß from culture supernatants in vitro is an important index of immune function. However, fetal bovine serum (FBS) contains a high level of latent TGF-ß that often hampers measuring T cell-derived TGF-ß in culture using FBS-supplemented medium. In this report, we generated anti-latency associated peptide (LAP) monoclonal antibodies which cross-react with bovine LAP, and developed a protocol to deplete TGF-ß from FBS. This provides the ability to reliably quantify TGF-ß in vitro without relying on serum-free media which do not support growth of murine T cells.

Overexpression of TGF-ß 1 gene induces cell surface localized glucose-regulated protein 78-associated latency-associated peptide/TGF-ß.

TGF-beta plays a crucial role in immune regulation. It has been reported that pro-TGF-beta, latency-associated peptide (LAP), latent TGF-beta and/or active TGF-beta (LAP/TGF-beta) is localized on the cell surface of Foxp3(+) regulatory T cells. However, the molecular mechanism(s) of how LAP/TGF-beta is anchored on the cell membrane is unknown. In this study, we show that forced expression of human TGF-beta(1) gene by retrovirus transduction into P3U1 mouse myeloma cells, and other cell types including murine CD4(+)CD25(-) T cells, makes these cells surface LAP/TGF-beta-positive. The surface LAP/TGF-beta contains high-glycosylated, furin-processed latent TGF-beta, which is different from the low-glycosylated, furin-unprocessed intracellular form or the high-glycosylated, furin-unprocessed secreted form. Furthermore, surface LAP/TGF-beta forms a complex with the molecular chaperone glucose-regulated protein 78 (GRP78, also known as BiP), and knockdown of GRP78 reduced the expression levels of surface LAP/TGF-beta. GRP78, however, is not involved in GARP-mediated surface LAP/TGF-beta. Our results suggest that GRP78 provides an additional surface localization mechanism for LAP/TGF-beta, which may play an important role in controlling TGF-beta activity.

Differential control of allo-antigen-specific regulatory T cells and effector T cells by anti-CD4 and other agents in establishing transplantation tolerance.

Donor-specific graft tolerance can be established by a combination of allo-antigen exposure and manipulation of T cell function, for example by donor-specific transfusion (DST) under the cover of a non-depleting anti-CD4 mAb. Yet, the cellular basis of this graft tolerance is still obscure. This report shows that T cell-deficient BALB/c nude mice reconstituted with naive unfractionated T cells are specifically tolerized to DBA/2 skin grafts by DST and anti-CD4 mAb treatment, whereas those transferred with T cell suspensions depleted of all Foxp3(+)CD25(+)CD4(+) natural regulatory T cells (Tregs) are not. The treatment inhibits Mls-1(a) allo-antigen-specific expansion of CD4(+) non-Tregs expressing Vbeta6 TCR subfamily but leaves the expansion of Vbeta6-expressing Tregs unaffected, allowing the latter to selectively expand and establish donor-specific tolerance. Furthermore, anti-CD4 mAb inhibits in vitro the selective expansion of allo-antigen-specific CD4(+) non-Tregs but not natural Tregs, as observed with in vitro anti-CD154 [CD40 ligand (CD40L)] mAb or rapamycin treatment. The results collectively indicate that the differential effect of biologicals and pharmacological substances on the expansion of allo-antigen-specific Tregs and effector T cells and resulting dominance of the former can be a key general mechanism underlying dominant transplantation tolerance.

TGF-beta-mediated suppression by CD4+CD25+ T cells is facilitated by CTLA-4 signaling.

CD4+CD25+ T cells play a pivotal role in immunological homeostasis by their capacity to exert immunosuppressive activity. However, the mechanism by which these cells function is still a subject for debate. We previously reported that surface (membrane) TGF-beta produced by CD4+CD25+ T cells was an effector molecule mediating suppressor function. We now support this finding by imaging surface TGF-beta on Foxp3+CD4+CD25+ T cells in confocal fluorescence microscopy. Then, using a TGF-beta-sensitive mink lung epithelial cell (luciferase) reporter system, we show that surface TGF-beta can be activated to signal upon cell-cell contact. Moreover, if such TGF-beta signaling is blocked in an in vitro assay of CD4+CD25+ T cell suppression by a specific inhibitor of TGF-betaRI, suppressor function is also blocked. Finally, we address the role of CTLA-4 in CD4+CD25+ T cell suppression, showing first that whereas anti-CTLA-4 does not block in vitro suppressor function, it does complement the blocking activity of anti-TGF-beta. We then show with confocal fluorescence microscopy that incubation of CD4+CD25+ T cells with anti-CTLA-4- and rB7-1/Fc-coated beads results in accumulation of TGF-beta at the cell-bead contact site. This suggests that CTLA-4 signaling facilitates TGF-beta-mediated suppression by intensifying the TGF-beta signal at the point of suppressor cell-target cell interaction.

Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis.

Naturally occurring CD25(+)CD4(+) regulatory T cells (Tregs) actively engage in the maintenance of immunologic self-tolerance and immunoregulation. They specifically express the transcription factor Forkhead box P3 (Foxp3) as a master control molecule for their development and function. Although several cell-surface molecules have been reported as Treg-specific markers, such as CD25, glucocorticoid-induced TNFR family-related gene/protein and CTL-associated molecule-4, they are also expressed on activated T cells derived from CD25(-)CD4(+) naive T cells. To identify Treg-specific molecules controlled by Foxp3, we performed DNA microarray analysis by comparing the following pairs of cell populations: fresh CD25(+)CD4(+) T cells versus fresh CD25(-)CD4(+) T cells, activated CD25(+)CD4(+) T cells versus activated CD25(-)CD4(+) T cells and retrovirally Foxp3-transduced CD25(-)CD4(+) T cells versus mock-transduced CD25(-)CD4(+) T cells. We found that the Gpr83, Ecm1, Cmtm7, Nkg7, Socs2 and glutaredoxin genes are predominantly transcribed in fresh and activated natural Treg as well as in Foxp3-transduced cells, while insulin-like 7, galectin-1, granzyme B and helios genes are natural Treg specific but Foxp3 independent. G protein-coupled receptor 83 (Gpr83) expression on the cell surface of natural Treg was confirmed by staining with Gpr83-specific antibody. Retroviral transduction of either group of genes in CD25(-)CD4(+) T cells failed to confer in vitro suppressive activity. Thus, there are several genes that are expressed in a highly Treg-specific fashion. Some of these genes are controlled by Foxp3, and others are not. These genes, in particular, Gpr83, Ecm1 and Helios, could potentially be used as specific markers for natural Treg.

Microglia-mediated nitric oxide cytotoxicity of T cells following amyloid beta-peptide presentation to Th1 cells.

Alzheimer's disease is marked by progressive accumulation of amyloid beta-peptide (Abeta) which appears to trigger neurotoxic and inflammatory cascades. Substantial activation of microglia as part of a local innate immune response is prominent at sites of Abeta plaques in the CNS. However, the role of activated microglia as Abeta APCs and the induction of adaptive immune responses has not been investigated. We have used primary microglial cultures to characterize Abeta-Ag presentation and interaction with Abeta-specific T cells. We found that IFN-gamma-treated microglia serve as efficient Abeta APCs of both Abeta1-40 and Abeta1-42, mediating CD86-dependent proliferation of Abeta-reactive T cells. When cultured with Th1 and Th2 subsets of Abeta-reactive T cells, Th1, but not Th2, cells, underwent apoptosis after stimulation, which was accompanied by increased levels of IFN-gamma, NO, and caspase-3. T cell apoptosis was prevented in the presence of an inducible NO synthase type 2 inhibitor. Microglia-mediated proliferation of Abeta-reactive Th2 cells was associated with expression of the Th2 cytokines IL-4 and IL-10, which counterbalanced the toxic levels of NO induced by Abeta. Our results demonstrate NO-dependent apoptosis of T cells by Abeta-stimulated microglia which may enhance CNS innate immune responses and neurotoxicity in Alzheimer's disease. Secretion of NO by stimulated microglia may underlie a more general pathway of T cell death in the CNS seen in neurodegenerative diseases. Furthermore, Th2 type T cell responses may have a beneficial effect on this process by down-regulation of NO and the proinflammatory environment.

CD4+CD25- T cells that express latency-associated peptide on the surface suppress CD4+CD45RBhigh-induced colitis by a TGF-beta-dependent mechanism.

Murine CD4(+)CD25(+) regulatory cells have been reported to express latency-associated peptide (LAP) and TGF-beta on the surface after activation, and exert regulatory function by the membrane-bound TGF-beta in vitro. We have now found that a small population of CD4(+) T cells, both CD25(+) and CD25(-), can be stained with a goat anti-LAP polyclonal Ab without being stimulated. Virtually all these LAP(+) cells are also positive for thrombospondin, which has the ability to convert latent TGF-beta to the active form. In the CD4(+)CD45RB(high)-induced colitis model of SCID mice, regulatory activity was exhibited not only by CD25(+)LAP(+) and CD25(+)LAP(-) cells, but also by CD25(-)LAP(+) cells. CD4(+)CD25(-)LAP(+) T cells were part of the CD45RB(low) cell fraction. CD4(+)CD25(-)LAP(-)CD45RB(low) cells had minimal, if any, regulatory activity in the colitis model. The regulatory function of CD25(-)LAP(+) cells was abrogated in vivo by anti-TGF-beta mAb. These results identify a new TGF-beta-dependent regulatory CD4(+) T cell phenotype that is CD25(-) and LAP(+).