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.

Expansion of an Unusual Virtual Memory CD8+ Subpopulation Bearing Vα3.2 TCR in Themis-Deficient Mice.

Deletion of the gene for Themis affects T cell selection in the thymus, which would be expected to affect the TCR repertoire. We found an increased proportion of cells expressing Vα3.2 (TRAV9N-3) in the peripheral CD8+ T cell population in mice with germline Themis deficiency. Analysis of the TCRα repertoire indicated it was generally reduced in diversity in the absence of Themis, whereas the diversity of sequences using the TRAV9N-3 V-region element was increased. In wild type mice, Vα3.2+ cells showed higher CD5, CD6 and CD44 expression than non-Vα3-expressing cells, and this was more marked in cells from Themis-deficient mice. This suggested a virtual memory phenotype, as well as a stronger response to self-pMHC. The Vα3.2+ cells responded more strongly to IL-15, as well as showing bystander effector capability in a Listeria infection. Thus, the unusually large population of Vα3.2+ CD8+ T cells found in the periphery of Themis-deficient mice reflects not only altered thymic selection, but also allowed identification of a subset of bystander-competent cells that are also present in wild-type mice.

Relative contribution of nonstructural protein 1 in dengue pathogenesis.

Dengue is a major public health concern in the tropical and subtropical world, with no effective treatment. The controversial live attenuated virus vaccine Dengvaxia has boosted the pursuit of subunit vaccine approaches, and nonstructural protein 1 (NS1) has recently emerged as a promising candidate. However, we found that NS1 immunization or passive transfer of NS1 antibodies failed to confer protection in symptomatic dengue mouse models using two non-mouse-adapted DENV2 strains that are highly virulent. Exogenous administration of purified NS1 also failed to worsen in vivo vascular leakage in sublethally infected mice. Neither method of NS1 immune neutralization changed the disease outcome of a chimeric strain expressing a vascular leak-potent NS1. Instead, virus chimerization involving the prME structural region indicated that these proteins play a critical role in driving in vivo fitness and virulence of the virus, through induction of key proinflammatory cytokines. This work highlights that the pathogenic role of NS1 is DENV strain dependent, which warrants reevaluation of NS1 as a universal dengue vaccine candidate.

T cell receptor and cytokine signal integration in CD8+ T cells is mediated by the protein Themis.

T cell homeostasis and functional responsiveness require signals from self-peptide-major histocompatibility complex (self-pMHC) and cytokines, but the mechanisms controlling this signal integration are unknown. Using a conditional deletion of the T cell lineage-specific protein Themis, we show that Themis is required for the maintenance of peripheral CD8+ T cells and for proliferative CD8+ T cell responses to low-affinity pMHC aided by cytokines. Themis-deficient peripheral T cells show a phenotype indicative of reduced tonic signaling from self-pMHC, strongly suggesting that Themis is a positive regulator of T cell receptor signal strength in response to low-affinity self-pMHC in peripheral T cells. Signals from low-affinity pMHC and cytokines synergistically induce phosphorylation of the kinase Akt, metabolic changes and c-Myc transcription factor induction in CD8+ T cells only in the presence of Themis. This function of Themis is mediated through Shp1 phosphatase, as peripheral Themis and Shp1 double deletion rescues the peripheral CD8+ T cell maintenance.

Ap4A Regulates Directional Mobility and Antigen Presentation in Dendritic Cells.

The significance of intracellular Ap4A levels over immune activity of dendritic cells (DCs) has been studied in Nudt2fl/fl/CD11c-cre mice. The transgenic mice have been generated by crossing floxed NUDT2 gene mice with DC marker CD11c recombinase (cre) mice. The DCs derived from these mice have higher levels of Ap4A (≈30-fold) compared with those derived from Nudt2+/+ mice. Interestingly, the elevated Ap4A in DCs has led them to possess higher motility and lower directional variability. In addition, the DCs are able to enhance immune protection indicated by the higher cross-presentation of antigen and priming of CD8+ OT-I T cells. Overall, the study denotes prominent impact of Ap4A over the functionality of DCs. The Nudt2fl/fl/CD11c-cre mice could serve as a useful tool to study the influence of Ap4A in the critical immune mechanisms of DCs.

Dengue vaccine-induced CD8+ T cell immunity confers protection in the context of enhancing, interfering maternal antibodies.

Declining levels of maternal antibodies were shown to sensitize infants born to dengue-immune mothers to severe disease during primary infection, through the process of antibody-dependent enhancement of infection (ADE). With the recent approval for human use of Sanofi-Pasteur's chimeric dengue vaccine CYD-TDV and several vaccine candidates in clinical development, the scenario of infants born to vaccinated mothers has become a reality. This raises 2 questions: will declining levels of maternal vaccine-induced antibodies cause ADE; and, will maternal antibodies interfere with vaccination efficacy in the infant? To address these questions, the above scenario was modeled in mice. Type I IFN-deficient female mice were immunized with live attenuated DENV2 PDK53, the core component of the tetravalent DENVax candidate currently under clinical development. Pups born to PDK53-immunized dams acquired maternal antibodies that strongly neutralized parental strain 16681, but not the heterologous DENV2 strain D2Y98P-PP1, and instead caused ADE during primary infection with this strain. Furthermore, pups failed to seroconvert after PDK53 vaccination, owing to maternal antibody interference. However, a cross-protective multifunctional CD8+ T cell response did develop. Thus, our work advocates for the development of dengue vaccine candidates that induce protective CD8+ T cells despite the presence of enhancing, interfering maternal antibodies.

CD40L Expression Allows CD8+ T Cells to Promote Their Own Expansion and Differentiation through Dendritic Cells.

CD8+ T cells play an important role in providing protective immunity against a wide range of pathogens, and a number of different factors control their activation. Although CD40L-mediated CD40 licensing of dendritic cells (DCs) by CD4+ T cells is known to be necessary for the generation of a robust CD8+ T cell response, the contribution of CD8+ T cell-expressed CD40L on DC licensing is less clear. We have previously shown that CD8+ T cells are able to induce the production of IL-12 p70 by DCs in a CD40L-dependent manner, providing some evidence that CD8+ T cell-mediated activation of DCs is possible. To better understand the role of CD40L on CD8+ T cell responses, we generated and characterized CD40L-expressing CD8+ T cells both in vitro and in vivo. We found that CD40L was expressed on 30-50% of effector CD8+ T cells when stimulated and that this expression was transient. The expression of CD40L on CD8+ T cells promoted the proliferation and differentiation of both the CD40L-expressing CD8+ T cells and the bystander effector CD8+ T cells. This process occurred via a cell-extrinsic manner and was mediated by DCs. These data demonstrate the existence of a mechanism where CD8+ T cells and DCs cooperate to maximize CD8+ T cell responses.

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.

Blomia tropicalis-Specific TCR Transgenic Th2 Cells Induce Inducible BALT and Severe Asthma in Mice by an IL-4/IL-13-Dependent Mechanism.

Previous studies have highlighted the importance of lung-draining lymph nodes in the respiratory allergic immune response, whereas the lung parenchymal immune system has been largely neglected. We describe a new in vivo model of respiratory sensitization to Blomia tropicalis, the principal asthma allergen in the tropics, in which the immune response is focused on the lung parenchyma by transfer of Th2 cells from a novel TCR transgenic mouse, specific for the major B. tropicalis allergen Blo t 5, that targets the lung rather than the draining lymph nodes. Transfer of highly polarized transgenic CD4 effector Th2 cells, termed BT-II, followed by repeated inhalation of Blo t 5 expands these cells in the lung >100-fold, and subsequent Blo t 5 challenge induced decreased body temperature, reduction in movement, and a fall in specific lung compliance unseen in conventional mouse asthma models following a physiological allergen challenge. These mice exhibit lung eosinophilia; smooth muscle cell, collagen, and goblet cell hyperplasia; hyper IgE syndrome; mucus plugging; and extensive inducible BALT. In addition, there is a fall in total lung volume and forced expiratory volume at 100 ms. These pathophysiological changes were substantially reduced and, in some cases, completely abolished by administration of neutralizing mAbs specific for IL-4 and IL-13 on weeks 1, 2, and 3. This IL-4/IL-13-dependent inducible BALT model will be useful for investigating the pathophysiological mechanisms that underlie asthma and the development of more effective drugs for treating severe asthma.

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.

Gamma interferon regulates contraction of the influenza virus-specific CD8 T cell response and limits the size of the memory population.

The factors that regulate the contraction of the CD8 T cell response and the magnitude of the memory cell population against localized mucosal infections such as influenza are important for generation of efficient vaccines but are currently undefined. In this study, we used a mouse model of influenza to demonstrate that the absence of gamma interferon (IFN-γ) or IFN-γ receptor 1 (IFN-γR1) leads to aberrant contraction of antigen-specific CD8 T cell responses. The increased accumulation of the effector CD8 T cell population was independent of viral load. Reduced contraction was associated with an increased fraction of CD8 T cells expressing the interleukin-7 receptor (IL-7R) at the peak of the response, resulting in enhanced numbers of memory/memory precursor cells in IFN-γ(-/-) and IFN-γR(-/-) compared to wild-type (WT) mice. Blockade of IL-7 within the lungs of IFN-γ(-/-) mice restored the contraction of influenza virus-specific CD8 T cells, indicating that IL-7R is important for survival and is not simply a consequence of the lack of IFN-γ signaling. Finally, enhanced CD8 T cell recall responses and accelerated viral clearance were observed in the IFN-γ(-/-) and IFN-γR(-/-) mice after rechallenge with a heterologous strain of influenza virus, confirming that higher frequencies of memory precursors are formed in the absence of IFN-γ signaling. In summary, we have identified IFN-γ as an important regulator of localized viral immunity that promotes the contraction of antigen-specific CD8 T cells and inhibits memory precursor formation, thereby limiting the size of the memory cell population after an influenza virus infection.

CD44high memory CD8 T cells synergize with CpG DNA to activate dendritic cell IL-12p70 production.

Protective memory CD8 T cell responses are generally associated with the rapid and efficient acquisition of CTL function. However, the ability of memory CD8 T cells to modulate immune responses through interactions with dendritic cells (DCs) during the early states of secondary Ag exposure is poorly understood. In this study, we show that murine Ag-specific CD44(high) CD8 T cells, representing CD8 T cells of the memory phenotype, potently activate DCs to produce high levels of IL-12p70 in conjunction with stimulation of DCs with the TLR 9 ligand, unmethylated CpG DNA. IL-12p70 production was produced predominantly by CD8alpha(+) DCs and plasmacytoid DCs, and mediated by CD8 T cell-derived cytokines IFN-gamma, GM-CSF, TNF-alpha, and surface CD40L. We also find that CD44(high) memory phenotype CD8 T cells were better DC IL-12p70 stimulators than CD44(low) naive phenotype CD8 T cells, and this was attributed to higher levels of IFN-gamma and GM-CSF produced by CD44(high) memory phenotype CD8 T cells during their Ag specific interaction with DCs. Our study identifies CpG DNA as the most effective TLR ligand that cooperates with CD8 T cells for DC IL-12p70 production, and suggests that effectiveness of memory CD8 T cells could be attributed to their ability to rapidly and effectively induce protective Th1 immunity during early stages of pathogen reinfection.