Magnesium sensing via LFA-1 regulates CD8+ T cell effector function.

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.

Vaccine-elicited CD4 T cells prevent the deletion of antiviral B cells in chronic infection.

Chronic viral infections subvert protective B cell immunity. An early type I interferon (IFN-I)-driven bias to short-lived plasmablast differentiation leads to clonal deletion, so-called "decimation," of antiviral memory B cells. Therefore, prophylactic countermeasures against decimation remain an unmet need. We show that vaccination-induced CD4 T cells prevented the decimation of naïve and memory B cells in chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. Although these B cell responses were largely T independent when IFN-I was blocked, preexisting T help assured their sustainability under conditions of IFN-I-driven inflammation by instructing a germinal center B cell transcriptional program. Prevention of decimation depended on T cell-intrinsic Bcl6 and Tfh progeny formation. Antigen presentation by B cells, interactions with antigen-specific T helper cells, and costimulation by CD40 and ICOS were also required. Importantly, B cell-mediated virus control averted Th1-driven immunopathology in LCMV-challenged animals with preexisting CD4 T cell immunity. Our findings show that vaccination-induced Tfh cells represent a cornerstone of effective B cell immunity to chronic virus challenge, pointing the way toward more effective B cell-based vaccination against persistent viral diseases.

T resident helper cells promote humoral responses in the lung.

Influenza is a deadly and costly infectious disease, even during flu seasons when an effective vaccine has been developed. To improve vaccines against respiratory viruses, a better understanding of the immune response at the site of infection is crucial. After influenza infection, clonally expanded T cells take up permanent residence in the lung, poised to rapidly respond to subsequent infection. Here, we characterized the dynamics and transcriptional regulation of lung-resident CD4+ T cells during influenza infection and identified a long-lived, Bcl6-dependent population that we have termed T resident helper (TRH) cells. TRH cells arise in the lung independently of lymph node T follicular helper cells but are dependent on B cells, with which they tightly colocalize in inducible bronchus-associated lymphoid tissue (iBALT). Deletion of Bcl6 in CD4+ T cells before heterotypic challenge infection resulted in redistribution of CD4+ T cells outside of iBALT areas and impaired local antibody production. These results highlight iBALT as a homeostatic niche for TRH cells and advocate for vaccination strategies that induce TRH cells in the lung.

Dynamics of the Coreceptor-LCK Interactions during T Cell Development Shape the Self-Reactivity of Peripheral CD4 and CD8 T Cells.

Overtly self-reactive T cells are removed during thymic selection. However, it has been recently established that T cell self-reactivity promotes protective immune responses. Apparently, the level of self-reactivity of mature T cells must be tightly balanced. Our mathematical model and experimental data show that the dynamic regulation of CD4- and CD8-LCK coupling establish the self-reactivity of the peripheral T cell pool. The stoichiometry of the interaction between CD8 and LCK, but not between CD4 and LCK, substantially increases upon T cell maturation. As a result, peripheral CD8+ T cells are more self-reactive than CD4+ T cells. The different levels of self-reactivity of mature CD8+ and CD4+ T cells likely reflect the unique roles of these subsets in immunity. These results indicate that the evolutionary selection pressure tuned the CD4-LCK and CD8-LCK stoichiometries, as they represent the unique parts of the proximal T cell receptor (TCR) signaling pathway, which differ between CD4+ and CD8+ T cells.

Affinity for self antigen selects Treg cells with distinct functional properties.

The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

Coreceptor scanning by the T cell receptor provides a mechanism for T cell tolerance.

In the thymus, high-affinity, self-reactive thymocytes are eliminated from the pool of developing T cells, generating central tolerance. Here, we investigate how developing T cells measure self-antigen affinity. We show that very few CD4 or CD8 coreceptor molecules are coupled with the signal-initiating kinase, Lck. To initiate signaling, an antigen-engaged T cell receptor (TCR) scans multiple coreceptor molecules to find one that is coupled to Lck; this is the first and rate-limiting step in a kinetic proofreading chain of events that eventually leads to TCR triggering and negative selection. MHCII-restricted TCRs require a shorter antigen dwell time (0.2 s) to initiate negative selection compared to MHCI-restricted TCRs (0.9 s) because more CD4 coreceptors are Lck-loaded compared to CD8. We generated a model (Lck come&stay/signal duration) that accurately predicts the observed differences in antigen dwell-time thresholds used by MHCI- and MHCII-restricted thymocytes to initiate negative selection and generate self-tolerance.

Cutting edge: requirement for TRAF6 in the induction of T cell anergy.

TRAF6, TNFR-associated factor 6, is a key adaptor downstream from the TNF receptor and TLR superfamily members. T cell-specific deletion of TRAF6 (TRAF6-DeltaT) was recently shown to result in the development of multiorgan inflammatory disease and the resistance of responder T cells to suppression by CD4+CD25+ regulatory T cells. In this study we examined the role of TRAF6 in an additional mechanism of peripheral tolerance, anergy. We have determined that the loss of TRAF6 restores the ability of CD28-/- T cells to proliferate and produce IL-2. Consistent with this, TRAF6-DeltaT T cells were resistant to anergizing signals both in vitro and in vivo. Resistance to anergy was correlated with decreased expression of Cbl-b. These findings reveal that in addition to its role in rendering T cells susceptible to control by CD4+CD25+ regulatory T cells, TRAF6 is essential for the induction of T cell anergy, implicating TRAF6 as a critical mediator of peripheral tolerance.

TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immune homeostasis.

TRAF6 has a key role in the regulation of innate immune responses by mediating signals from both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Here we show that T cell-specific deletion of TRAF6 unexpectedly results in multiorgan inflammatory disease. TRAF6-deficient T cells exhibit hyperactivation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway compared with wild-type T cells and, as a result, become resistant to suppression by CD4+ CD25+ regulatory T cells. These data identify a previously unrecognized role for TRAF6 in the maintenance of peripheral tolerance, and suggest the presence of a T cell-intrinsic control mechanism to render responder T cells susceptible to tolerizing signals.

TNF receptor-associated factor 6 deficiency during hemopoiesis induces Th2-polarized inflammatory disease.

Toll-like receptors (TLR) initiate rapid innate immune responses by recognizing microbial products. These events in turn lead to the development of an efficient adaptive immune response through the up-regulation of a number of costimulatory molecules, including members of the TNF/TNFR superfamily, on the surface of an APC. TNFR-associated factor 6 (TRAF6) is a common signaling adapter used by members of both the TNFR and the TLR/IL-1R superfamilies, and as such plays a critical role in the development of immune responses. As TRAF6-deficient mice die prematurely, we generated chimeras reconstituted with TRAF6-deficient fetal liver cells to analyze functions of TRAF6 in vivo in the hemopoietic compartment. We found that TRAF6-deficient chimeras develop a progressive lethal inflammatory disease associated with massive organ infiltration and activation of CD4(+) T cells in a Th2-polarized phenotype, and a defect in IL-18 responsiveness. When recombination-activating gene 2(-/-) blastocysts were complemented with TRAF6-deficient embryonic stem cells, a marked elevation of activated CD4(+) T cells and progressive inflammatory disease were also observed. Moreover, T cell activation and lethal inflammation were not reversed in mixed chimeric mice generated from normal and TRAF6-deficient fetal liver cells. These results suggest that deletion of TRAF6 induces a dominant Th2-type polarized autoimmune response. Therefore, in addition to playing a critical role in innate and adaptive immunity, TRAF6 is likely to play a previously unrecognized role in the maintenance of self-tolerance.

TRAF6 is a critical factor for dendritic cell maturation and development.

IL-1 receptor (IL-1R)/Toll-like receptor (TLR) family and TNF receptor (TNFR) superfamily members are critical for regulating multiple aspects of dendritic cell (DC) biology. Several signaling pathways associated with each family utilize the adapter molecule, TRAF6, but its role in DCs is unclear. By examining TRAF6-deficient mice and bone marrow (BM) chimeras reconstituted with TRAF6-deficient fetal liver cells, we show that proper DC maturation requires TRAF6. In response to either microbial components or CD40L, TRAF6-deficient DCs fail to upregulate surface expression of MHCII and B7.2, or produce inflammatory cytokines. Moreover, LPS-treated TRAF6-deficient DCs do not exhibit an enhanced capacity to stimulate naive T cells. Interestingly, a major population of splenic DCs, the CD4(+)CD8alpha(-) subset, is nearly absent in both TRAF6-deficient mice and BM chimeras. Together these results indicate that TRAF6 regulates the critical processes required for maturation, activation, and development of DCs, the primary cellular bridge between innate and adaptive immunity.