CD28 controls the development of innate‐like CD8+ T cells by promoting the functional maturation of NKT cells

NK T cells(NKT cells) share functional characteristics and homing properties that are distinct from conventional T cells. In this study, we investigated the contribution of CD28 in the functional development of γδ NKT and αβ NKT cells in mice. We show that CD28 promotes the thymic maturation of promyelocytic leukemia zinc finger⁺ IL‐4⁺ NKT cells and upregulation of LFA‐1 expression on NKT cells. We demonstrate that the developmental defect of γδ NKT cells in CD28‐deficient mice is cell autonomous. Moreover, we show in both wild‐type C57BL/6 mice and in downstream of tyrosine kinase‐1 transgenic mice, a mouse model with increased numbers of γδ NKT cells, that CD28‐mediated regulation of thymic IL‐4⁺ NKT cells promotes the differentiation of eomesodermin⁺ CD44^high innate‐like CD8⁺ T cells. These findings reveal a previously unappreciated mechanism by which CD28 controls NKT‐cell homeostasis and the size of the innate‐like CD8⁺ T‐cell pool.     

Differences in Bcl‐2 expression by T‐cell subsets alter their balance after in vivo irradiation to favor CD4+Bcl‐2hi NKT cells

Although it is well known that in vivo radiation depletes immune cells via the Bcl‐2 apoptotic pathway, a more nuanced analysis of the changes in the balance of immune‐cell subsets is needed to understand the impact of radiation on immune function. We show the balance of T‐cell subsets changes after increasing single doses of total body irradiation (TBI) or after fractionated irradiation of the lymphoid tissues (TLI) of mice due to differences in radioresistance and Bcl‐2 expression of the NKT‐cell and non‐NKT subsets to favor CD4⁺Bcl‐2^hi NKT cells. Reduction of the Bcl‐2^lo mature T‐cell subsets was at least 100‐fold greater than that of the Bcl‐2^hi subsets. CD4⁺ NKT cells upregulated Bcl‐2 after TBI and TLI and developed a Th2 bias after TLI, whereas non‐NKT cells failed to do so. Our previous studies showed TLI protects against graft versus host disease in wild‐type, but not in NKT‐cell‐deficient mice. The present study shows that NKT cells have a protective function even after TBI, and these cells are tenfold more abundant after an equal dose of TLI. In conclusion, differential expression of Bcl‐2 contributes to the changes in T‐cell subsets and immune function after irradiation.     

TRAF2 regulates peripheral CD8+ T‐cell and NKT‐cell homeostasis by modulating sensitivity to IL‐15

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8⁺ T‐cell and NKT‐cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8⁺ T‐cell subsets. IL‐15‐dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8⁺CD44^hiCD122⁺ T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8⁺CD44^hi T cells exhibited impaired dose‐dependent proliferation to exogenous IL‐15. In contrast, TRAF2TKO CD8⁺ T cells proliferated normally to anti‐CD3 and TRAF2TKO CD8⁺CD44^hi T cells exhibited normal proliferation to exogenous IL‐2. TRAF2TKO CD8⁺ T cells expressed normal levels of IL‐15‐associated receptors and possessed functional IL‐15‐mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8⁺CD44^hiCD122⁺ and NKT cells was mechanistically linked to an inability to respond to IL‐15. The reduced CD8⁺CD44^hiCD122⁺ T‐cell and NKT‐cell populations in TRAF2TKO mice were rescued in the presence of high dose IL‐15 by IL‐15/IL‐15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8⁺ CD44^hiCD122⁺ T‐cell and NKT‐cell homeostasis by modulating sensitivity to T‐cell intrinsic growth factors such as IL‐15.     

DC therapy induces long‐term NK reactivity to tumors via host DC

We vaccinated mice with DC loaded with or without invariant NKT‐cell ligand α‐galactosylceramide and evaluated long‐term resistance against tumor challenge. When mice had been given either DC or DC/galactosylceramide and were challenged with tumor cells even 6–12 months later, both NK and NKT cells were quickly activated to express CD69 and produce IFN‐γ. The NK cells could resist a challenge with several different tumors in vivo. The activated NK and NKT cells could be depleted with anti‐NK1.1 treatment. In spite of this, the activated cells recovered, indicating that tumor‐responsive NK and NKT cells were being generated continuously as a result of vaccination with DC and were not true memory cells. The NK and NKT antitumor response in DC‐vaccinated mice depended on CD4⁺ T cells, but neither CD8⁺T cells nor CD4⁺CD25⁺ regulatory T cells. However, both vaccine DC and host DC were required for the development of long‐term, tumor reactive innate immunity. These results indicate that DC therapy in mice induces long‐lasting innate NK‐ and NKT‐cell activation through a pathway that requires host DC and CD4⁺ T cells and that the continued generation of active NK cells resists the establishment of metastases in vivo.     

Differential requirement for CARMA1 in agonist‐selected T‐cell development

Caspase recruitment domain‐containing membrane‐associated guanylate kinase protein‐1 (CARMA1) is a critical component of the NF‐κB signaling cascade mediated by TCR engagement. In addition to activation of naïve T cells, TCR signaling is important for the development of agonist‐selected T‐cell subsets such as Treg, NKT cells, and CD8‐αα T cells. However, little is known about the role of CARMA1 in the development of these lineages. Here we show that CARMA1‐deficient mice (CARMA1^?/?) have altered populations of specific subsets of agonist‐selected T cells. Specifically, CARMA1^?/? mice have impaired natural and adaptive Treg development, whereas NKT cell numbers are normal compared with wild‐type mice. Interestingly, CD8‐αα T cells, which may also be able to develop through an extrathymic selection pathway, are enriched in the gut of CARMA1^?/? mice, whereas memory‐phenotype CD4⁺ T cells (CD62L^low/CD44^high) are present at reduced numbers in the periphery. These results indicate that CARMA1 is essential for Treg development, but is not necessary for the development of other agonist‐selected T‐cell subsets. Overall, these data reveal an important but differential role for CARMA1‐mediated TCR signaling in T‐cell development.     

IL‐4 and IL‐15 promotion of virtual memory CD8+ T cells is determined by genetic background

Virtual memory (VM) CD8⁺ T cells are present in unimmunized mice, yet possess T‐cell receptors specific for foreign antigens. To date, VM cells have only been characterized in C57BL/6 mice. Here, we assessed the cytokine requirements for VM cells in C57BL/6 and BALB/c mice. As reported previously, VM cells in C57BL/6 mice rely mostly on IL‐15 and marginally on IL‐4. In stark contrast, VM cells in BALB/c mice rely substantially on IL‐4 and marginally on IL‐15. Further, NKT cells are the likely source of IL‐4, because CD1d‐deficient mice on a BALB/c background have significantly fewer VM cells. Notably, this NKT/IL‐4 axis contributes to appropriate effector and memory T‐cell responses to infection in BALB/c mice, but not in C57BL/6 mice. However, the effects of IL‐4 are manifest prior to, rather than during, infection. Thus, cytokine‐mediated control of the precursor population affects the development of virus‐specific CD8⁺ T‐cell memory. Depending upon the genetic background, different cytokines encountered before infection may influence the subsequent ability to mount primary and memory anti‐viral CD8⁺ T‐cell responses.     

mTORC2 regulates multiple aspects of NKT‐cell development and function

Invariant NKT (iNKT) cells bridge innate and adaptive immunity by rapidly secreting cytokines and lysing targets following TCR recognition of lipid antigens. Based on their ability to secrete IFN‐γ, IL‐4 and IL‐17A, iNKT‐cells are classified as NKT‐1, NKT‐2, and NKT‐17 subsets, respectively. The molecular pathways regulating iNKT‐cell fate are not fully defined. Recent studies implicate Rictor, a required component of mTORC2, in the development of select iNKT‐cell subsets, however these reports are conflicting. To resolve these questions, we used Rictor^fl/fl CD4cre⁺ mice and found that Rictor is required for NKT‐17 cell development and normal iNKT‐cell cytolytic function. Conversely, Rictor is not absolutely required for IL‐4 and IFN‐γ production as peripheral iNKT‐cells make copious amounts of these cytokines. Overall iNKT‐cell numbers are dramatically reduced in the absence of Rictor. We provide data indicating Rictor regulates cell survival as well as proliferation of developing and mature iNKT‐cells. Thus, mTORC2 regulates multiple aspects of iNKT‐cell development and function.     

Ex‐vivo analysis of human Natural Killer T cells demonstrates heterogeneity between tissues and within established CD4+ and CD4− subsets

Our understanding of human type 1 natural killer T (NKT) cells has been heavily dependent on studies of cells from peripheral blood. These have identified two functionally distinct subsets defined by expression of CD4, although it is widely believed that this underestimates the true number of subsets. Two recent studies supporting this view have provided more detail about diversity of the human NKT cells, but relied on analysis of NKT cells from human blood that had been expanded in vitro prior to analysis. In this study we extend those findings by assessing the heterogeneity of CD4⁺ and CD4⁻ human NKT cell subsets from peripheral blood, cord blood, thymus and spleen without prior expansion ex vivo, and identifying for the first time cytokines expressed by human NKT cells from spleen and thymus. Our comparative analysis reveals highly heterogeneous expression of surface antigens by CD4⁺ and CD4⁻ NKT cell subsets and identifies several antigens whose differential expression correlates with the cytokine response. Collectively, our findings reveal that the common classification of NKT cells into CD4⁺ and CD4⁻ subsets fails to reflect the diversity of this lineage, and that more studies are needed to establish the functional significance of the antigen expression patterns and tissue residency of human NKT cells.     

Human CD4+ invariant NKT cells are involved in antibacterial immunity against Brucella suis through CD1d‐dependent but CD4‐independent mechanisms

Human invariant NKT (iNKT) cells are a unique subset of T cells, which recognize glycolipids presented by the CD1d. Among the iNKT cells, several functionally distinct subsets have been characterized according to CD4 and/or CD8 co‐receptor expression. The current study is focussed on the CD4⁺ iNKT cell subset and its role in an anti‐infectious response. We have examined the role of CD4⁺ iNKT cells on the intracellular Brucella suis growth. Our results indicate that CD4⁺ iNKT cells impair the intramacrophagic growth of Brucella. This inhibition is due to a combination of soluble and contact‐dependent mechanisms: IFN‐γ is weakly involved while cytotoxic activities such as the induction of the Fas pathway and the release of lytic granules are major mechanisms. The impairment of Brucella growth by CD4⁺ iNKT cells requires an interaction with CD1d on macrophage surface. Also, we have shown that although CD4 regulates several biological responses of CD4⁺ iNKT cells, it is not involved in their antibacterial activity. Here, we have shown for the first time that the CD4⁺ iNKT cell population has antibacterial activity and thus, participates directly in the elimination of bacteria and/or in the control of bacterial growth by killing infected cells.     

IFN-γ production downstream of NKT cell activation in mice infected with influenza virus enhances the cytolytic activities of both NK cells and viral antigen-specific CD8 T cells

Natural killer T (NKT) cell activation is responsible for eliminating pathogens. However, the biological functions of NKT cells against influenza virus are not fully understood. We therefore investigated the effects of NKT cells in viral infection using CD1d knockout (KO) mice. When CD1d KO or wild-type (WT) mice were infected with a sub-lethal dosage of the influenza virus, the survival rate of CD1d KO mice was significantly lower than for WT mice in association with delayed viral clearance in the lungs. Consistently, IFN-γ production in bronchoalveolar lavage fluid of CD1d KO mice was largely reduced compared to WT mice during infection. Moreover, the cytotoxic activities of NK cells and viral antigen-specific CD8⁺ T cells were impaired in CD1d KO mice. It was concluded that activated NKT cell-induced IFN-γ release enhances both NK-cell activity and antigen-specific CD8⁺ T cells to eliminate the influenza virus, thus leading to an enhanced survival.     

Inhibition of R5‐tropic HIV type‐1 replication in CD4+ natural killer T cells by γδ T lymphocytes

After the development of highly active anti‐retroviral therapy, it became clear that the majority of emergent HIV‐1 is macrophage‐tropic and infects CD4⁺, CCR5‐expressing cells (R5‐tropic). There are three distinct cell populations, R5‐tropic, HIV‐1‐susceptible CD4⁺ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue‐associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4⁺ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4⁺ T cells derived from circulating PBMCs, and that R5‐tropic HIV‐1 expands efficiently in the CD4⁺ NKT cells. Moreover, when PBMCs depleted of CD8α⁺ cells were stimulated in the presence of α‐galactosylceramide (α‐GalCer) and R5‐tropic HIV‐1 [NL(AD8)], the production of HIV‐1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8β⁺ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8αα⁺ but not CD8αβ⁺ cells may have the ability to inhibit R5‐tropic HIV‐1 replication in CD4⁺ NKT cells. Here, we show that co‐culturing R5‐tropic HIV‐1‐infected CD4⁺ NKT cells with CD8αα⁺ γδ T cells, in particular Vγ1Vδ1 cells, but not with CD8αα⁺ NKT cells or CD8αα⁺ dendritic cells, inhibits HIV‐1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1α and 1β and RANTES. Collectively, these results indicate the importance of CD8αα⁺ γδ T cells in the control of R5‐tropic HIV‐1 replication and persistence in CD4⁺ NKT cells.