Themis controls T cell activation, effector functions, and metabolism of peripheral CD8+ T cells.

Themis is important in regulating positive selection of thymocytes during T cell development, but its role in peripheral T cells is less understood. Here, we investigated T cell activation and its sequelae using a tamoxifen-mediated, acute Themis deletion mouse model. We find that proliferation, effector functions including anti-tumor killing, and up-regulation of energy metabolism are severely compromised. This study reveals the phenomenon of peripheral adaptation to loss of Themis, by demonstrating direct TCR-induced defects after acute deletion of Themis that were not evident in peripheral T cells chronically deprived of Themis in dLck-Cre deletion model. Peripheral adaptation to long-term loss was compared using chronic versus acute tamoxifen-mediated deletion and with the (chronic) dLck-Cre deletion model. We found that upon chronic tamoxifen-mediated Themis deletion, there was modulation in the gene expression profile for both TCR and cytokine signaling pathways. This profile overlapped with (chronic) dLck-Cre deletion model. Hence, we found that peripheral adaptation induced changes to both TCR and cytokine signaling modules. Our data highlight the importance of Themis in the activation of CD8+ T cells.

IL12/18/21 Preactivation Enhances the Antitumor Efficacy of Expanded γδT Cells and Overcomes Resistance to Anti-PD-L1 Treatment.

γδT cells are promising candidates for cellular immunotherapy due to their immune regulation through cytokine production and MHC-independent direct cytotoxicity against a broad spectrum of tumors. However, current γδT cell-based cancer immunotherapy has limited efficacy, and novel strategies are needed to improve clinical outcomes. Here, we report that cytokine pretreatment with IL12/18, IL12/15/18, IL12/18/21, and IL12/15/18/21 effectively enhanced the activation and cytotoxicity of in vitro-expanded murine and human γδT cells. However, only adoptive transfer of IL12/18/21 preactivated γδT cells significantly inhibited tumor growth in a murine melanoma model and a hepatocellular carcinoma model. Both IL12/18/21 preactivated antibody-expanded and zoledronate-expanded human γδT cells effectively controlled tumor growth in a humanized mouse model. IL12/18/21 preactivation promoted γδT cell proliferation and cytokine production in vivo and enhanced IFNγ production and activation of endogenous CD8+ T cells in a cell-cell contact- and ICAM-1-dependent manner. Furthermore, the adoptive transfer of IL12/18/21 preactivated γδT cells could overcome the resistance to anti-PD-L1 therapy, and the combination therapy had a synergistic effect on the therapeutic outcomes. Moreover, the enhanced antitumor function of adoptively transferred IL12/18/21 preactivated γδT cells was largely diminished in the absence of endogenous CD8+ T cells when administered alone or in combination with anti-PD-L1, suggesting a CD8+ T cell-dependent mechanism. Taken together, IL12/18/21 preactivation can promote γδT cell antitumor function and overcome the resistance to checkpoint blockade therapy, indicating an effective combinational cancer immunotherapeutic strategy.

CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance.

Signal transduction induced by chimeric antigen receptors (CARs) is generally believed to rely on the activity of the SRC family kinase (SFK) LCK, as is the case with T cell receptor (TCR) signaling. Here, we show that CAR signaling occurs in the absence of LCK. This LCK-independent signaling requires the related SFK FYN and a CD28 intracellular domain within the CAR. LCK-deficient CAR-T cells are strongly signaled through CAR and have better in vivo efficacy with reduced exhaustion phenotype and enhanced induction of memory and proliferation. These distinctions can be attributed to the fact that FYN signaling tends to promote proliferation and survival, whereas LCK signaling promotes strong signaling that tends to lead to exhaustion. This non-canonical signaling of CAR-T cells provides insight into the initiation of both TCR and CAR signaling and has important clinical implications for improvement of CAR function.

Regulation of adipogenic differentiation and adipose tissue inflammation by interferon regulatory factor 3.

Dysfunction of adipocytes and adipose tissue is a primary defect in obesity and obesity-associated metabolic diseases. Interferon regulatory factor 3 (IRF3) has been implicated in adipogenesis. However, the role of IRF3 in obesity and obesity-associated disorders remains unclear. Here, we show that IRF3 expression in human adipose tissues is positively associated with insulin sensitivity and negatively associated with type 2 diabetes. In mouse pre-adipocytes, deficiency of IRF3 results in increased expression of PPARγ and PPARγ-mediated adipogenic genes, leading to increased adipogenesis and altered adipocyte functionality. The IRF3 knockout (KO) mice develop obesity, insulin resistance, glucose intolerance, and eventually type 2 diabetes with aging, which is associated with the development of white adipose tissue (WAT) inflammation. Increased macrophage accumulation with M1 phenotype which is due to the loss of IFNß-mediated IL-10 expression is observed in WAT of the KO mice compared to that in wild-type mice. Bone-marrow reconstitution experiments demonstrate that the nonhematopoietic cells are the primary contributors to the development of obesity and both hematopoietic and nonhematopoietic cells contribute to the development of obesity-related complications in IRF3 KO mice. This study demonstrates that IRF3 regulates the biology of multiple cell types including adipocytes and macrophages to prevent the development of obesity and obesity-related complications and hence, could be a potential target for therapeutic interventions for the prevention and treatment of obesity-associated metabolic disorders.

Blomia tropicalis allergen 5 (Blo t 5) T-cell epitopes and their ability to suppress the allergic immune response.

Blomia tropicalis is the major asthma allergen in the tropics comparable to Dermatophagoides pteronyssinus. However, little is known about the B. tropicalis epitopes recognized by T cells. Our aim was to identify the T-cell epitopes in the major B. tropicalis allergen, Blo t 5, and investigate the potential of the corresponding peptides to inhibit the allergic inflammatory lung response. C57BL/6 mice were immunized with plasmid DNA encoding Blo t 5 and T-cell epitopes identified using the interferon-γ ELISPOT assay with 15-mer overlapping peptides. C57BL/6 mice were sensitized with bone-marrow-derived dendritic cells (BMDC) pulsed with Blo t 5 allergen followed by intranasal Blo t 5 challenge. Two H-2b restricted epitopes (Bt576-90 and Bt5106-115 ) were recognized by CD4 T cells specific for Blo t 5, but no CD8 epitopes were identified. In mice sensitized with Blo t 5-pulsed BMDC and challenged with intranasal Blo t 5 Bt576-90 and Bt5106-115 , peptide-specific CD4 T cells were found to secrete the T helper type 2 cytokines interleukin-5 and interleukin-13. Intradermal administration of synthetic peptides encoding the identified T-cell epitopes suppressed allergic airway inflammation to further allergen challenges. Hence, we have identified novel CD4 T-cell epitopes specific for Blo t 5 and demonstrated that these peptides could be employed therapeutically to suppress the T-cell response in a murine model of allergic airway inflammation.

GM-CSF-licensed CD11b+ lung dendritic cells orchestrate Th2 immunity to Blomia tropicalis.

The Blomia tropicalis dust mite is prevalent in tropical and subtropical regions of the world. Although it is a leading cause of asthma, little is known how it induces allergy. Using a novel murine asthma model induced by intranasal exposure to B. tropicalis, we observed that a single intranasal sensitization to B. tropicalis extract induces strong Th2 priming in the lung draining lymph node. Resident CD11b(+) dendritic cells (DCs) preferentially transport Ag from the lung to the draining lymph node and are crucial for the initiation of Th2 CD4(+) T cell responses. As a consequence, mice selectively deficient in CD11b(+) DCs exhibited attenuated Th2 responses and more importantly did not develop any allergic inflammation. Conversely, mice deficient in CD103(+) DCs and CCR2-dependent monocyte-derived DCs exhibited similar allergic inflammation compared with their wild-type counterparts. We also show that CD11b(+) DCs constitutively express higher levels of GM-CSF receptor compared with CD103(+) DCs and are thus selectively licensed by lung epithelial-derived GM-CSF to induce Th2 immunity. Taken together, our study identifies GM-CSF-licensed CD11b(+) lung DCs as a key component for induction of Th2 responses and represents a potential target for therapeutic intervention in allergy.

CD8 T cells regulate allergic contact dermatitis by modulating CCR2-dependent TNF/iNOS-expressing Ly6C+ CD11b+ monocytic cells.

Monocytes and their derived cells have critical roles in inflammation and immune defense. However, their function in skin diseases such as allergic contact dermatitis remains poorly defined. Using a model of contact hypersensitivity (CHS) toward 2,4-dinitrochlorobenzene, we show that Ly6C+ CD11b+ monocytic cells participate in the pathophysiology of CHS and their accumulation is regulated by effector CD8 T cells. These Ly6C+ CD11b+ monocytic cells are the primary contributors of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) and derive from Ly6C(hi)CCR2+ monocytes, as they were absent in non-inflamed skin and accumulate as a consequence of inflammation in a C-C chemokine receptor type 2 (CCR2)-dependent manner. Importantly, CCR2(-/-) mice, or wild-type mice depleted of monocytes via clodronate liposomes, display a marked decrease in TNF-α and iNOS expression accompanied by attenuated skin inflammation. Using transgenic mice and antibody depletion, we show that effector CD8 T cells regulate the accumulation of Ly6C+ CD11b+ monocytic cells through IL-17 and activate them for TNF-α and iNOS through IFN-γ. CD8 T cell-derived IFN-γ was also critical for the accumulation of the major histocompatibility complex II-expressing Ly6C+ CD11b+ subset, which expressed intermediate levels of CD11c and costimulatory molecules. Taken together, our findings provide further insight into the pathophysiology of allergic contact dermatitis by showing that CD8 T cells regulate the inflammatory cascade through TNF/iNOS-expressing Ly6C+ CD11b+ monocytic cells.