Troels R. Petersen

Myelin-specific regulatory T cells accumulate in the CNS but fail to control autoimmune inflammation.

Treatment with ex vivo–generated regulatory T cells (T-reg) has been regarded as a potentially attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp knock-in (Foxp3gfp.KI) mice and myelin oligodendrocyte glycoprotein (MOG)35–55/IAb (MHC class II) tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. MOG tetramer–reactive, Foxp3+ T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. Foxp3+ T cells isolated from the CNS were effective in suppressing naive MOG-specific T cells, but failed to control CNS-derived encephalitogenic T-eff that secreted interleukin (IL)-6 and tumor necrosis factor (TNF). Our data suggest that in order for CD4+Foxp3+ T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation.

Langerin+CD8α+ Dendritic Cells Are Critical for Cross-Priming and IL-12 Production in Response to Systemic Antigens

Distinct dendritic cell (DC) subsets differ with respect to pathways of Ag uptake and intracellular routing to MHC class I or MHC class II molecules. Murine studies suggest a specialized role for CD8α+ DC in cross-presentation, where exogenous Ags are presented on MHC class I molecules to CD8+ T cells, while CD8α− DC are more likely to present extracellular Ags on MHC class II molecules to CD4+ T cells. As a proportion of CD8α+ DC have been shown to express langerin (CD207), we investigated the role of langerin+CD8α+ DC in presenting Ag and priming T cell responses to soluble Ags. When splenic DC populations were sorted from animals administered protein i.v., the ability to cross-present Ag was restricted to the langerin+ compartment of the CD8α+ DC population. The langerin+CD8α+ DC population was also susceptible to depletion following administration of cytochrome c, which is known to trigger apoptosis if diverted to the cytosol. Cross-priming of CTL in the presence of the adjuvant activity of the TLR2 ligand N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Serl-[S]-Lys4-trihydrochloride or the invariant NKT cell ligand α-galactosylceramide was severely impaired in animals selectively depleted of langerin+ cells in vivo. The production of IL-12p40 in response to these systemic activation stimuli was restricted to langerin+CD8α+ DC, and the release of IL-12p70 into the serum following invariant NKT cell activation was ablated in the absence of langerin+ cells. These data suggest a critical role for the langerin+ compartment of the CD8α+ DC population in cross-priming and IL-12 production.

Vaccination with irradiated tumor cells pulsed with an adjuvant that stimulates NKT cells is an effective treatment for glioma.

Purpose: The prognosis for patients with glioblastoma multiforme (GBM) remains extremely poor despite recent treatment advances. There is an urgent need to develop novel therapies for this disease. Experimental Design: We used the implantable GL261 murine glioma model to investigate the therapeutic potential of a vaccine consisting of intravenous injection of irradiated whole tumor cells pulsed with the immuno-adjuvant α-galactosylceramide (α-GalCer). Results: Vaccine treatment alone was highly effective in a prophylactic setting. In a more stringent therapeutic setting, administration of one dose of vaccine combined with depletion of regulatory T cells (Treg) resulted in 43% long-term survival and the disappearance of mass lesions detected by MRI. Mechanistically, the α-GalCer component was shown to act by stimulating “invariant” natural killer–like T cells (iNKT cells) in a CD1d-restricted manner, which in turn supported the development of a CD4+ T-cell–mediated adaptive immune response. Pulsing α-GalCer onto tumor cells avoided the profound iNKT cell anergy induced by free α-GalCer. To investigate the potential for clinical application of this vaccine, the number and function of iNKT cells was assessed in patients with GBM and shown to be similar to age-matched healthy volunteers. Furthermore, irradiated GBM tumor cells pulsed with α-GalCer were able to stimulate iNKT cells and augment a T-cell response in vitro. Conclusions: Injection of irradiated tumor cells loaded with α-GalCer is a simple procedure that could provide effective immunotherapy for patients with high-grade glioma. Clin Cancer Res; 18(23); 6446–59. ©2012 AACR.

Characterization of MHC- and TCR-binding residues of the myelin oligodendrocyte glycoprotein 38-51 peptide

Myelin oligodendrocyte glycoprotein (MOG) is a major experimental autoimmune encephalomyelitis (EAE) antigen in H‐2b mice and a potential autoantigen in multiple sclerosis. How well MOG peptides bind to MHC and how TCR recognize the peptide/MHC complex have important implications for thymic selection as well as T cell activation in the periphery. In this study, we have characterized amino acids in the MOG38–51 peptide important for peptide binding to I‐Ab, and for TCR recognition of the peptide/MHC complex. We found that the amino acids R41, F44, R46 and V47 constituted the major TCR contact residues, as alanine substitution at these positions abrogated T cell responses without decreasing their binding affinity to I‐Ab. In addition, G38 andW39 were found to be minor TCR contact residues. Finally, substituting tyrosine for alanine at position 40 decreased binding to I‐Ab by approximately 50% and prevented induction of T cell responses in C57BL/6J mice upon immunization. Thus, Y40 is the dominant MHC‐binding residue of the MOG38–51 peptide and most likely occupies the p1 pocket of I‐Ab. Our results could be useful to design peptides with altered agretopes and epitopes of the MOG38–51 peptide to study their therapeutic potential in the EAE model.

Potent anti-tumor responses to immunization with dendritic cells loaded with tumor tissue and an NKT cell ligand

Cancer immunotherapy is well tolerated and specific, but its efficacy remains variable. To enhance anti‐tumor CD8+ T‐cell responses induced by immunization with antigen‐loaded dendritic cells (DCs), we explored the impact of eliciting a potent source of T‐cell help from activated invariant natural killer (NK)‐like T cells (iNKT cells) using the specific glycolipid ligand α‐galactosylceramide (α‐GalCer). As cytokines released by iNKT cells may drive proliferation of CD4+CD25+ regulatory T cells (Tregs), we assessed this immunization strategy in animals treated with anti‐CD25 antibody to inactivate Treg function. Combining DC immunization with iNKT cell activation was found to significantly enhance anti‐tumor activity, which was improved further by the prior inactivation of Tregs. The improved anti‐tumor activity with Treg inactivation was associated with a prolonged proliferative burst of responding CD8+ T cells. We could find no evidence that inclusion of α‐GalCer in the vaccine enhanced Treg numbers, or that the ‘helper’ function of iNKT cells was improved in the absence of Treg activity. Rather, the two activities appeared to act independently to improve the tumor‐specific T‐cell response. Inactivating regulatory T cells and eliciting iNKT cell activation are therefore two useful strategies that can be used in combination to improve anti‐tumor immunization with antigen‐loaded DCs.

Exploiting the Role of Endogenous Lymphoid-Resident Dendritic Cells in the Priming of NKT Cells and CD8+ T Cells to Dendritic Cell-Based Vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d−/− BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.

An autologous leukemia cell vaccine prevents murine acute leukemia relapse after cytarabine treatment

Acute leukemias with adverse prognostic features carry a high relapse rate without allogeneic stem cell transplantation (allo-SCT). Allo-SCT has a high morbidity and is precluded for many patients because of advanced age or comorbidities. Postremission therapies with reduced toxicities are urgently needed. The murine acute leukemia model C1498 was used to study the efficacy of an intravenously administered vaccine consisting of irradiated leukemia cells loaded with the natural killer T (NKT)-cell agonist α-galactosylceramide (α-GalCer). Prophylactically, the vaccine was highly effective at preventing leukemia development through the downstream activities of activated NKT cells, which were dependent on splenic langerin(+)CD8α(+) dendritic cells and which led to stimulation of antileukemia CD4(+) and CD8(+) T cells. However, hosts with established leukemia received no protective benefit from the vaccine, despite inducing NKT-cell activation. Established leukemia was associated with increases in regulatory T cells and myeloid-derived suppressor cells, and the leukemic cells themselves were highly suppressive in vitro. Although this suppressive environment impaired both effector arms of the immune response, CD4(+) T-cell responses were more severely affected. When cytarabine chemotherapy was administered prior to vaccination, all animals in remission posttherapy were protected against rechallenge with viable leukemia cells.

Activated NKT Cells Can Condition Different Splenic Dendritic Cell Subsets To Respond More Effectively to TLR Engagement and Enhance Cross-Priming

The function of dendritic cells (DCs) can be modulated through multiple signals, including recognition of pathogen-associated molecular patterns, as well as signals provided by rapidly activated leukocytes in the local environment, such as innate-like T cells. In this article, we addressed the possibility that the roles of different murine DC subsets in cross-priming CD8+ T cells can change with the nature and timing of activatory stimuli. We show that CD8α+ DCs play a critical role in cross-priming CD8+ T cell responses to circulating proteins that enter the spleen in close temporal association with ligands for TLRs and/or compounds that activate NKT cells. However, if NKT cells are activated first, then CD8α− DCs become conditioned to respond more vigorously to TLR ligation, and if triggered directly, these cells can also contribute to priming of CD8+ T cell responses. In fact, the initial activation of NKT cells can condition multiple DC subsets to respond more effectively to TLR ligation, with plasmacytoid DCs making more IFN-α and both CD8α+ and CD8α− DCs manufacturing more IL-12. These results suggest that different DC subsets can contribute to T cell priming if provided appropriately phased activatory stimuli, an observation that could be factored into the design of more effective vaccines.

Batf3‐independent langerin− CX3CR1− CD8α+ splenic DCs represent a precursor for classical cross‐presenting CD8α+ DCs

This study tests the hypothesis that CD8α+ DCs in the spleen of mice contain an immature precursor for functionally mature, “classical” cross‐presenting CD8α+ DCs. The lymphoid tissues contain a network of phenotypically distinct DCs with unique roles in surveillance and immunity. Splenic CD8α+ DCs have been shown to exhibit a heightened capacity for phagocytosis of cellular material, secretion of IL‐12, and cross‐priming of CD8+ T cells. However, this population can be subdivided further on the basis of expression of both langerin/CD207 and CX3CR1. We therefore evaluated the functional capacities of these different subsets. The CX3CR1+ CD8α+ DC subset does not express langerin and does not exhibit the classical features above. The CX3CR1− CD8α+ DC can be divided into langerin‐positive and negative populations, both of which express DEC205, Clec9A, and high basal levels of CD86. However, the langerin+ CX3CR1− CD8α+ subset has a superior capacity for acquiring cellular material and producing IL‐12 and is more susceptible to activation‐induced cell death. Significantly, following purification and adoptive transfer into new hosts, the langerin− CX3CR1− CD8α+ subset survives longer, up‐regulates expression of langerin, and becomes more susceptible to activation‐induced cell death. Last, in contrast to langerin+ CX3CR1− CD8α+, the langerin− CX3CR1− CD8α+ are still present in Batf3−/− mice. We conclude that the classical attributes of CD8α+ DC are confined primarily to the langerin+ CX3CR1− CD8α+ DC population and that the langerin− CX3CR1− subset represents a Batf3‐independent precursor to this mature population.

The control of CD8+ T cell responses is preserved in perforin-deficient mice and released by depletion of CD4+CD25+ regulatory T cells

Immune suppression by Treg has been demonstrated in a number of models, but the mechanisms of this suppression are only partly understood. Recent work has suggested that Tregs may suppress by directly killing immune cell populations in vivo in a perforin‐ and granzyme B‐dependent manner. To establish whether perforin is necessary for the regulation of immune responses in vivo, we examined OVA‐specific CD8+ T cell responses in WT and PKO mice immunized with OVA and α‐GalCer and the expansion of WT OT‐I CD8+ T cells adoptively transferred into WT or PKO mice immunized with DC‐OVA. We observed similar expansion, phenotype, and effector function of CD8+ T cells in WT and PKO mice, suggesting that CD8+ T cells were subjected to a similar amount of regulation in the two mouse strains. In addition, when WT and PKO mice were depleted of Tregs by anti‐CD25 mAb treatment before DC‐OVA immunization, CD8+ T cell proliferation, cytotoxicity, and cytokine production were increased similarly, suggesting a comparable involvement of CD25+ Tregs in controlling T cell proliferation and effector function in these two mouse strains. These data suggest that perforin expression is not required for normal immune regulation in these models of in vivo CD8+ T cell responses induced by immunization with OVA and α‐GalCer or DC‐OVA.