Cutting Edge: Tissue Antigen Expression Levels Fine-Tune T Cell Differentiation Decisions In Vivo.

Immune homeostasis in peripheral tissues is, to a large degree, maintained by the differentiation and action of regulatory T cells (Treg) specific for tissue Ags. Using a novel mouse model, we have studied the differentiation of naive CD4+ T cells into Foxp3+ Treg in response to a cutaneous Ag (OVA). We found that expression of OVA resulted in fatal autoimmunity and in prevention of peripheral Treg generation. Inhibiting mTOR activity with rapamycin rescued the generation of Foxp3+ T cells. When we varied the level of Ag expression to modulate TCR signaling, we found that low Ag concentrations promoted the generation of Foxp3+ T cells, whereas high levels expanded effector T cells and caused severe autoimmunity. Our findings indicate that the expression level of tissue Ag is a key determinant of the balance between tissue-reactive effector and peripheral Foxp3+ T cells, which determines the choice between tolerance and autoimmunity.

Mutational landscape of the transcriptome offers putative targets for immunotherapy of myeloproliferative neoplasms.

Ph-negative myeloproliferative neoplasms (MPNs) are hematological cancers that can be subdivided into entities with distinct clinical features. Somatic mutations in JAK2, CALR, and MPL have been described as drivers of the disease, together with a variable landscape of nondriver mutations. Despite detailed knowledge of disease mechanisms, targeted therapies effective enough to eliminate MPN cells are still missing. In this study of 113 MPN patients, we aimed to comprehensively characterize the mutational landscape of the granulocyte transcriptome using RNA sequencing data and subsequently examine the applicability of immunotherapeutic strategies for MPN patients. Following implementation of customized workflows and data filtering, we identified a total of 13 (12/13 novel) gene fusions, 231 nonsynonymous single nucleotide variants, and 21 insertions and deletions in 106 of 113 patients. We found a high frequency of SF3B1-mutated primary myelofibrosis patients (14%) with distinct 3' splicing patterns, many of these with a protein-altering potential. Finally, from all mutations detected, we generated a virtual peptide library and used NetMHC to predict 149 unique neoantigens in 62% of MPN patients. Peptides from CALR and MPL mutations provide a rich source of neoantigens as a result of their unique ability to bind many common MHC class I molecules. Finally, we propose that mutations derived from splicing defects present in SF3B1-mutated patients may offer an unexplored neoantigen repertoire in MPNs. We validated 35 predicted peptides to be strong MHC class I binders through direct binding of predicted peptides to MHC proteins in vitro. Our results may serve as a resource for personalized vaccine or adoptive cell-based therapy development.

CDK6 coordinates JAK2 V617F mutant MPN via NF-κB and apoptotic networks.

Over 80% of patients with myeloproliferative neoplasms (MPNs) harbor the acquired somatic JAK2 V617F mutation. JAK inhibition is not curative and fails to induce a persistent response in most patients, illustrating the need for the development of novel therapeutic approaches. We describe a critical role for CDK6 in MPN evolution. The absence of Cdk6 ameliorates clinical symptoms and prolongs survival. The CDK6 protein interferes with 3 hallmarks of disease: besides regulating malignant stem cell quiescence, it promotes nuclear factor κB (NF-κB) signaling and contributes to cytokine production while inhibiting apoptosis. The effects are not mirrored by palbociclib, showing that the functions of CDK6 in MPN pathogenesis are largely kinase independent. Our findings thus provide a rationale for targeting CDK6 in MPN.

A novel humanized mouse model to study the function of human cutaneous memory T cells in vivo in human skin.

Human skin contains a population of memory T cells that supports tissue homeostasis and provides protective immunity. The study of human memory T cells is often restricted to in vitro studies and to human PBMC serving as primary cell source. Because the tissue environment impacts the phenotype and function of memory T cells, it is crucial to study these cells within their tissue. Here we utilized immunodeficient NOD-scid IL2rγnull (NSG) mice that carried in vivo-generated engineered human skin (ES). ES was generated from human keratinocytes and fibroblasts and was initially devoid of skin-resident immune cells. Upon adoptive transfer of human PBMC, this reductionist system allowed us to study human T cell recruitment from a circulating pool of T cells into non-inflamed human skin in vivo. Circulating human memory T cells preferentially infiltrated ES and showed diverse functional profiles of T cells found in fresh human skin. The chemokine and cytokine microenvironment of ES closely resembled that of non-inflamed human skin. Upon entering the ES T cells assumed a resident memory T cell-like phenotype in the absence of infection, and a proportion of these cutaneous T cells can be locally activated upon injection of monocyte derived dendritic cells (moDCs) that presented Candida albicans. Interestingly, we found that CD69+ memory T cells produced higher levels of effector cytokines in response to Candida albicans, compared to CD69- T cells. Overall, this model has broad utility in many areas of human skin immunology research, including the study of immune-mediated skin diseases.

Anti-Interleukin-6 Promotes Allogeneic Bone Marrow Engraftment and Prolonged Graft Survival in an Irradiation-Free Murine Transplant Model.

Transfer of recipient regulatory T cells (Tregs) induces mixed chimerism and tolerance in an irradiation-free bone marrow (BM) transplantation (BMT) model involving short-course co-stimulation blockade and mTOR inhibition. Boosting endogenous Tregs pharmacologically in vivo would be an attractive alternative avoiding the current limitations of performing adoptive cell therapy in the routine clinical setting. Interleukin-6 (IL-6) potently inhibits Treg differentiation and its blockade was shown to increase Treg numbers in vivo. Therefore, we investigated whether IL-6 blockade can replace adoptive Treg transfer in irradiation-free allogeneic BMT. Treatment with anti-IL-6 instead of Treg transfer led to multi-lineage chimerism (persisting for ~12 weeks) in recipients of fully mismatched BM and significantly prolonged donor skin (MST 58 days) and heart (MST > 100 days) graft survival. Endogenous Foxp3+ Tregs expanded in anti-IL-6-treated BMT recipients, while dendritic cell (DC) activation and memory CD8+ T cell development were inhibited. Adding anti-IL-17 to anti-IL-6 treatment increased Treg frequencies, but did not further prolong donor skin graft survival significantly. These results demonstrate that IL-6 blockade promotes BM engraftment and donor graft survival in non-irradiated recipients and might provide an alternative to Treg cell therapy in the clinical setting.