Selected Toll-like receptor ligands and viruses promote helper-independent cytotoxic T cell priming by upregulating CD40L on dendritic cells.

CD40L (CD154) on CD4(+) T cells has been shown to license dendritic cells (DCs) via CD40 to prime cytotoxic T lymphocyte (CTL) responses. We found that the converse (CD40L on DCs) was also important. Anti-CD40L treatment decreased endogenous CTL responses to both ovalbumin and influenza infection even in the absence of CD4(+) T cells. DCs expressed CD40L upon stimulation with agonists to Toll-like receptor 3 (TLR3) and TLR9. Moreover, influenza infection, which stimulates CTLs without help, upregulated CD40L on DCs, but herpes simplex infection, which elicits CTLs through help, did not. CD40L-deficient (Cd40lg(-/-)) DCs are suboptimal both in vivo in bone marrow chimera experiments and in vitro in mixed lymphocyte reactions. In contrast, Cd40lg(-/-) CD8(+) T cells killed as effectively as wild-type cells. Thus, CD40L upregulation on DCs promoted optimal priming of CD8(+) T cells without CD4(+) T cells, providing a mechanism by which pathogens may elicit helper-independent CTL immunity.

Monocyte-Derived Dendritic Cells Promote Th Polarization, whereas Conventional Dendritic Cells Promote Th Proliferation.

Monocyte-derived dendritic cells (moDCs) dramatically increase in numbers upon infection and inflammation; accordingly, we found that this also occurs during allogeneic responses. Despite their prominence, how emergent moDCs and resident conventional DCs (cDCs) divide their labor as APCs remain undefined. Hence, we compared both direct and indirect presentation by murine moDCs versus cDCs. We found that, despite having equivalent MHC class II expression and in vitro survival, moDCs were 20-fold less efficient than cDCs at inducing CD4(+) T cell proliferation through both direct and indirect Ag presentation. Despite this, moDCs were more potent at inducing Th1 and Th17 differentiation (e.g., 8-fold higher IFN-γ and 2-fold higher IL-17A in T cell cocultures), whereas cDCs induced 10-fold higher IL-2 production. Intriguingly, moDCs potently reduced the ability of cDCs to stimulate T cell proliferation in vitro and in vivo, partially through NO production. We surmise that such division of labor between moDCs and cDCs has implications for their respective roles in the immune response.

Innate Allorecognition Results in Rapid Accumulation of Monocyte-Derived Dendritic Cells.

Although the mechanisms governing the innate recognition of pathogen-associated molecular patterns have been well defined, how allogeneic cellular stimuli evoke innate responses remains less so. In this article, we report that upon i.v. transfer (to avoid major iatrogenic interference), allogeneic but not syngeneic leukocytes could induce a rapid (after 1 d) accumulation of host monocyte-derived dendritic cells (moDCs) without any increase in conventional DCs. This occurred in various donor-host strain combinations, did not require MHC mismatch, and could be induced by various donor cell types including B cells, T cells, or NK cells. Using RAG(-/-)γc(-/-) and scid γc(-/-)mice with different MHC, we found that the presence of either donor or host lymphoid cells was required. Alloinduced moDC accumulation was significantly reduced when splenocytes from mice deficient in NK cells by genetic ablation were used as donors. A major component of this moDC accumulation appears to be recruitment. Our findings provide new insights into how the innate and adaptive immune system may interact during allogeneic encounters and thus transplant rejection.

Prosurvival Bcl-2 family members reveal a distinct apoptotic identity between conventional and plasmacytoid dendritic cells.

Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.

Heterogeneity, functional specialization and differentiation of monocyte-derived dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells that consist of functionally and phenotypically heterogeneous populations. Monocyte-derived DCs (moDCs) are a DC subset that have been attracting increasing interest owing to their potent influence on adaptive immune function and their rapid accumulation upon an inflammatory stimulus. Although early studies on moDCs mainly addressed infection, their emergence and function in other settings such as autoimmunity and allogeneic organ transplantation are now being increasingly appreciated. In this review, the relationship between murine monocyte subsets and the moDCs that arise from them is discussed. Their role in initiating and modulating innate and adaptive immune responses in various pathophysiological scenarios is also explored, including how they may separate their labour from conventional DCs. How these findings might relate to their human counterparts is also discussed. Overall, monocytes and moDCs exhibit complex and heterogeneous behaviours that are critical in responses against microbial invasion, autoimmunity and allograft rejection.

Effector-memory T cells develop in islets and report islet pathology in type 1 diabetes.

CD8(+) T cells are critical in human type 1 diabetes and in the NOD mouse. In this study, we elucidated the natural history of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific CD8(+) T cells in NOD diabetes using MHC-tetramer technology. IGRP206-214-specific T cells in the peripheral lymphoid tissue increased with age, and their numbers correlated with insulitis progression. IGRP206-214-specific T cells in the peripheral lymphoid tissue expressed markers of chronic Ag stimulation, and their numbers were stable after diagnosis of diabetes, consistent with their memory phenotype. IGRP206-214-specific T cells in NOD mice expand, acquire the phenotype of effector-memory T cells in the islets, and emigrate to the peripheral lymphoid tissue. Our observations suggest that enumeration of effector-memory T cells of multiple autoantigen specificities in the periphery of type 1 diabetic subjects could be a reliable reporter for progression of islet pathology.

Functional cytotoxic T lymphocytes against IGRP206-214 predict diabetes in the non-obese diabetic mouse.

CD8(+) T cells are prominent in autoimmune diabetes of both humans and non-obese diabetic (NOD) mice. For example, CD8(+) T cells against islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP) can be detected readily in older NOD mice. It has been suggested that the enumeration of islet-specific CD8(+) T cells in the peripheral blood may be a predictive biomarker for autoimmune type 1 diabetes (T1D). Here, we determined the natural history of the functional endogenous IGRP(206-214)-specific cytotoxic T lymphocytes (CTLs) in NOD mice with regard to age (3- to 15-week-old pre-diabetic mice and diabetic mice) and sex. We demonstrated that in vivo IGRP(206-214)-specific CTLs significantly increased after 12 weeks of age and in vivo cytotoxicity in female NOD mice was significantly higher than in male NOD mice. To determine the in vivo IGRP(206-214)-specific CTL frequency without killing the mice, we performed splenectomies on a cohort of mice after injecting IGRP(206-214)-coated targets and then followed their diabetes progression. We found that CTL frequency correlated with future of disease onset. Thus, our data support that IGRP(206-214)-specific CTLs may be a potent biomarker for T1D.

Unlike CD4+ T-cell help, CD28 costimulation is necessary for effective primary CD8+ T-cell influenza-specific immunity.

The importance of costimulation on CD4(+) T cells has been well documented. However, primary CTLs against many infections including influenza can be generated in the absence of CD4(+) T-cell help. The role of costimulation under such "helpless" circumstances is not fully elucidated. Here, we investigated such a role for CD28 using CTLA4Ig transgenic (Tg) mice. To ensure valid comparison across the genotypes, we showed that all mice had similar naïve precursor frequencies and similar peak viral loads. In the absence of help, viral clearance was significantly reduced in CTLA4Ig Tg mice compared with WT mice. CD44(+) BrdU(+) influenza-specific CD8(+) T cells were diminished in CTLA4Ig Tg mice at days 5 and 8 postinfection. Adoptive transfer of ovalbumin-specific transgenic CD8(+) T cells (OT-I)-I cells into WT or CTLA4Ig Tg mice revealed that loss of CD28 costimulation resulted in impairment in OT-I cell division. As shown previously, neither viral clearance nor the generation of influenza-specific CD8(+) T cells was affected by the absence of CD4(+) T cells alone. In contrast, both were markedly impaired by CD28 blockade of "helpless" CD8(+) T cells. We suggest that direct CD28 costimulation of CD8(+) T cells is more critical in their priming during primary influenza infection than previously appreciated.

Anti-CD2 producing pig xenografts effect localized depletion of human T cells in a huSCID model.

BACKGROUND: We investigated whether graft produced anti-human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters (pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local. METHODS: A mouse anti-human CD2 Ab (CD2hb11) previously generated by us was genetically engineered to produce chimeric and humanized versions. The three forms of CD2hb11 were named dilimomab (mouse), diliximab (chimeric) and dilizumab (humanized). All 3 forms of CD2hb11 Ab were tested for their ability to bind CD3(+) human T cells and to inhibit a human anti-pig xenogeneic mixed lymphocyte reaction (MLR). They were administered systemically in a humanized mouse model in order to test their ability to deplete human CD3(+) T cells and whether they induced a cytokine storm. An adenoviral vector expressing diliximab was generated for transduction of pNICC. Humanized mice were transplanted with either control-transduced pNICC or diliximab-transduced pNICC and human T cells within grafts and spleens were enumerated by flow cytometry. RESULTS: Dilimomab and diliximab inhibited a human anti-pig xenogeneic response but dilizumab did not. All 3 forms of CD2hb11 Ab bound human T cells in vitro though dilimomab and diliximab exhibited 300-fold higher avidity than dilizumab. All 3 anti-CD2 Abs could deplete human CD3(+) T cells in vivo in a humanized mouse model without inducing upregulation of activation markers or significant release of cytokines. Humanized mice transplanted with diliximab-transduced pNICC afforded depletion of CD3(+) T cells at the graft site leaving the peripheral immune system intact. CONCLUSIONS: Local production of a single Ab against T cells can reduce graft infiltration at the xenograft site and may reduce the need for conventional, systemic immunosuppression.

BH3 mimetics antagonizing restricted prosurvival Bcl-2 proteins represent another class of selective immune modulatory drugs.

Death by apoptosis shapes tissue homeostasis. Apoptotic mechanisms are so universal that harnessing them for tailored immune intervention would seem challenging; however, the range and different expression levels of pro- and anti-apoptotic molecules among tissues offer hope that targeting only a subset of such molecules may be therapeutically useful. We examined the effects of the drug ABT-737, a mimetic of the killer BH3 domain of the Bcl-2 family of proteins that induces apoptosis by antagonizing Bcl-2, Bcl-X(L), and Bcl-W (but not Mcl-1 and A1), on the mouse immune system. Treatment with ABT-737 reduced the numbers of selected lymphocyte and dendritic cell subpopulations, most markedly in lymph nodes. It inhibited the persistence of memory B cells, the establishment of newly arising bone marrow plasma cells, and the induction of a cytotoxic T cell response. Preexisting plasma cells and germinal centers were unaffected. Notably, ABT-737 was sufficiently immunomodulatory to allow long-term survival of pancreatic allografts, reversing established diabetes in this model. These results provide an insight into the selective mechanisms of immune cell survival and how this selectivity avails a different strategy for immune modulation.

Tissue destruction caused by cytotoxic T lymphocytes induces deletional tolerance.

Autoimmune diseases tend to be chronic and progressive, but how these responses are sustained is not clear. One cell type that might contribute to autoimmunity is the cytotoxic T lymphocyte (CTL), which, as a consequence of causing tissue destruction and production of cytokines, could provide a sustained supply of antigen and inflammatory signals for dendritic cells to maintain immune stimulation. Here we examined whether such CTL-mediated tissue damage alone could provide antigen in the right context to recruit immune effectors and sustain autoimmunity. We show that while CTL-mediated tissue damage caused the release of self-antigens that stimulated the proliferation of naive autoreactive CD8(+) T cells, such responses failed to precipitate disease and, instead, led to deletional tolerance. These findings indicate that despite the capacity of CTLs to produce inflammatory cytokines and to cause tissue damage, their responses are not sustaining, but instead favor induction of self-tolerance.

BCL-XL antagonism selectively reduces neutrophil life span within inflamed tissues without causing neutropenia.

Neutrophils help to clear pathogens and cellular debris, but can also cause collateral damage within inflamed tissues. Prolonged neutrophil residency within an inflammatory niche can exacerbate tissue pathology. Using both genetic and pharmacological approaches, we show that BCL-XL is required for the persistence of neutrophils within inflammatory sites in mice. We demonstrate that a selective BCL-XL inhibitor (A-1331852) has therapeutic potential by causing apoptosis in inflammatory human neutrophils ex vivo. Moreover, in murine models of acute and chronic inflammatory disease, it reduced inflammatory neutrophil numbers and ameliorated tissue pathology. In contrast, there was minimal effect on circulating neutrophils. Thus, we show a differential survival requirement in activated neutrophils for BCL-XL and reveal a new therapeutic approach to neutrophil-mediated diseases.

Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways.

Cytotoxic T-cells are the major mediators of beta-cell destruction in type 1 diabetes, but the molecular mechanisms are not definitively established. We have examined the contribution of perforin and Fas ligand to beta-cell destruction using islet-specific CD8(+) T-cells from T-cell receptor transgenic NOD8.3 mice. NOD8.3 T-cells killed Fas-deficient islets in vitro and in vivo. Perforin-deficient NOD8.3 T-cells were able to destroy wild-type but not Fas-deficient islets in vitro. These results imply that NOD8.3 T-cells use both pathways and that Fas is required for beta-cell killing only when perforin is missing. Consistent with this theory, transgenic NOD8.3 mice with beta-cells that do not respond to Fas ligation were not protected from diabetes. We next investigated the mechanism of protection provided by overexpression of suppressor of cytokine signaling-1 (SOCS-1) in beta-cells of NOD8.3 mice. SOCS-1 islets remained intact when grafted into NOD8.3 mice and were less efficiently killed in vitro. However, addition of exogenous peptide rendered SOCS-1 islets susceptible to 8.3 T-cell-mediated lysis. Therefore, NOD8.3 T-cells use both perforin and Fas pathways to kill beta-cells and the surprising blockade of NOD8.3 T-cell-mediated beta-cell death by SOCS-1 overexpression may be due in part to reduced target cell recognition.

Monocyte-Derived Dendritic Cells Impair Early Graft Function Following Allogeneic Islet Transplantation.

Islet transplantation can cure type 1 diabetes but is limited by lack of donor organs and early graft dysfunction, such that many patients require multiple transplants to achieve insulin independence. Monocyte-derived dendritic cells (moDCs) arise during inflammation and allograft encounters where they can promote various innate and adaptive immune responses. To determine whether moDCs impair early graft function following allogeneic islet transplantation, we transplanted MHC-mismatched BALB/c (H-2d) islets into diabetic C57BL/6-CCR2.DTR recipients (H-2b) treated with either saline (control) or diphtheria toxin (DT) to deplete moDCs. Graft function was assessed by blood glucose (BG) measurement. DT treatment resulted in specific depletion of graft site moDCs posttransplant. Despite equivalent pretransplant BG levels [27.0 ± 1.3 vs. 29.6 ± 1.1 mM, not significant (ns)], DT recipients achieved lower posttransplant BG levels and better rates of normoglycemia than control recipients (11.0 ± 1.9 vs. 19.1 ± 1.4 mM, p = 0.004) at 1 day posttransplant in diabetic recipients. When a suboptimal donor dose of 200 islets was transplanted, DT-induced moDC depletion resulted in normoglycemia in 78% compared to 25% of control recipients (p = 0.03). As well as amelioration of graft dysfunction in the immediate peritransplant period, prolonged DT administration (15 days posttransplant) resulted in improved graft survival (21 vs. 11 days, p = 0.005). moDCs impair early graft function post-allogeneic islet transplantation. moDC depletion may allow for improved early graft function, permit transplantation with lower islet masses, and enhance graft survival.

Cognate antigen engagement on parenchymal cells stimulates CD8+ T cell proliferation in situ.

T-cell responses are initiated upon cognate presentation by professional antigen presenting cells in lymphoid tissue. T cells then migrate to inflamed tissues, but further T-cell stimulation in these parenchymal target sites is not well understood. Here we show that T-cell expansion within inflamed tissues is a distinct phase that is neither a classical primary nor classical secondary response. This response, which we term 'the mezzanine response', commences within days after initial antigen encounter, unlike the secondary response that usually occurs weeks after priming. A further distinction of this response is that T-cell proliferation is driven by parenchymal cell antigen presentation, without requiring professional antigen presenting cells, but with increased dependence on IL-2. The mezzanine response might, therefore, be a new target for inhibiting T-cell responses in allograft rejection and autoimmunity or for enhancing T-cell responses in the context of microbial or tumour immunity.

Repurposed JAK1/JAK2 Inhibitor Reverses Established Autoimmune Insulitis in NOD Mice.

Recent advances in immunotherapeutics have not yet changed the routine management of autoimmune type 1 diabetes. There is an opportunity to repurpose therapeutics used to treat other diseases to treat type 1 diabetes, especially when there is evidence for overlapping mechanisms. Janus kinase (JAK) 1/JAK2 inhibitors are in development or clinical use for indications including rheumatoid arthritis. There is good evidence for activation of the JAK1/JAK2 and signal transducer and activator of transcription (STAT) 1 pathway in human type 1 diabetes and in mouse models, especially in ß-cells. We tested the hypothesis that using these drugs to block the JAK-STAT pathway would prevent autoimmune diabetes. The JAK1/JAK2 inhibitor AZD1480 blocked the effect of cytokines on mouse and human ß-cells by inhibiting MHC class I upregulation. This prevented the direct interaction between CD8+ T cells and ß-cells, and reduced immune cell infiltration into islets. NOD mice treated with AZD1480 were protected from autoimmune diabetes, and diabetes was reversed in newly diagnosed NOD mice. This provides mechanistic groundwork for repurposing clinically approved JAK1/JAK2 inhibitors for type 1 diabetes.

Anti-apoptotic proteins BCL-2, MCL-1 and A1 summate collectively to maintain survival of immune cell populations both in vitro and in vivo.

Survival of various immune cell populations has been proposed to preferentially rely on a particular anti-apoptotic BCL-2 family member, for example, naive T cells require BCL-2, while regulatory T cells require MCL-1. Here we examined the survival requirements of multiple immune cell subsets in vitro and in vivo, using both genetic and pharmacological approaches. Our findings support a model in which survival is determined by quantitative participation of multiple anti-apoptotic proteins rather than by a single anti-apoptotic protein. This model provides both an insight into how the sum of relative levels of anti-apoptotic proteins BCL-2, MCL-1 and A1 influence survival of T cells, B cells and dendritic cells, and a framework for ascertaining how these different immune cells can be optimally targeted in treatment of immunopathology, transplantation rejection or hematological cancers.

Bcl-2 protection of islet allografts is unmasked by costimulation blockade.

One major limitation in pancreatic islet transplantation is availability of donor tissue. Donor shortage is exacerbated by islet apoptosis from the stresses of islet isolation and transplantation. Furthermore, the side effects of immunosuppressive drugs preclude transplants into patients whose diabetes is controlled by parenteral insulin. We hypothesised that over-expressing anti-apoptotic Bcl-2 or secretion of immunomodulatory CTLA4Ig molecules in islet beta cells would enhance survival of transplanted islets while minimizing systemic side effects. Over-expression of Bcl-2 neither significantly increased preservation of islet cell mass after transplantation into immunocompromised recipients nor decreased cytokine-mediated apoptosis in vitro. Although Bcl-2 over-expression alone was insufficient in protecting islet allografts from rejection, its beneficence was shown by the enhancement of protection when the adaptive immune response was inhibited by locally produced CTLA4Ig. Thus, the combination of anti-apoptotic and immunosuppressive intervention has additive or synergistic efficacy and may reduce the level of systemic immunosuppression or quantity of donor tissue required.