Central tolerance shapes the neutralizing B cell repertoire against a persisting virus in its natural host.

Viral mimicry of host cell structures has been postulated to curtail the B cell receptor (BCR) repertoire against persisting viruses through tolerance mechanisms. This concept awaits, however, experimental testing in a setting of natural virus-host relationship. We engineered mouse models expressing a monoclonal BCR specific for the envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV), a naturally persisting mouse pathogen. When the heavy chain of the LCMV-neutralizing antibody KL25 was paired with its unmutated ancestor light chain, most B cells underwent receptor editing, a behavior reminiscent of autoreactive clones. In contrast, monoclonal B cells expressing the same heavy chain in conjunction with the hypermutated KL25 light chain did not undergo receptor editing but exhibited low levels of surface IgM, suggesting that light chain hypermutation had lessened KL25 autoreactivity. Upon viral challenge, these IgMlow cells were not anergic but up-regulated IgM, participated in germinal center reactions, produced antiviral antibodies, and underwent immunoglobulin class switch as well as further affinity maturation. These studies on a persisting virus in its natural host species suggest that central tolerance mechanisms prune the protective antiviral B cell repertoire.

T-cell-B-cell collaboration in the lung.

Collaboration between T and B cells in secondary lymphoid organs is a crucial component of adaptive immunity, but lymphocytes also persist in other tissues. Recent studies have examined T-cell-B-cell interactions in nonlymphoid tissues such as the lung. CD4+ T- resident helper cells (TRH) remain in the lung after influenza infection and support both resident CD8 T cells and B cells. Multiple lung-resident B-cell subsets (B-resident memory (BRM)) that exhibit spatial and phenotypic diversity have also been described. Though not generated by all types of infection, inducible bronchus-associated lymphoid tissue offers a logical place for T and B cells to interact. Perturbations to BRM and TRH cells elicit effects specific to Immunoglobulin A (IgA) production, an antibody isotype with privileged access to mucosa. Understanding the interplay of lymphocytes in mucosal tissues, which can be insulated from systemic immune responses, may improve the design of future vaccines and therapies.

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.

Opposing effects of T cell receptor signal strength on CD4 T cells responding to acute versus chronic viral infection.

A hallmark of adaptive immunity is CD4 T cells' ability to differentiate into specialized effectors. A long-standing question is whether T cell receptor (TCR) signal strength can dominantly instruct the development of Th1 and T follicular helper (Tfh) cells across distinct infectious contexts. We characterized the differentiation of murine CD4 TCR transgenic T cells responding to altered peptide ligand lymphocytic choriomeningitis viruses (LCMV) derived from acute and chronic parental strains. We found that TCR signal strength exerts opposite and hierarchical effects on the balance of Th1 and Tfh cells responding to acute versus persistent infection. TCR signal strength correlates positively with Th1 generation during acute but negatively during chronic infection. Weakly activated T cells express lower levels of markers associated with chronic T cell stimulation and may resist functional inactivation. We anticipate that the panel of recombinant viruses described herein will be valuable for investigating a wide range of CD4 T cell responses.

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.

TB or not to be: what specificities and impact do antibodies have during tuberculosis?

Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis (Mtb), is a major cause of global morbidity and mortality. The primary barrier to the development of an effective tuberculosis vaccine is our failure to fully understand the fundamental characteristics of a protective immune response. There is an increasing evidence that mobilization of antibody and B cell responses during natural Mtb infection and vaccination play a role in host protection. Several studies have assessed the levels of Mtb-specific antibodies induced during active disease as well as the potential of monoclonal antibodies to modulate bacterial growth in vitro and in vivo. A major limitation of these studies, however, is that the specific antigens capable of eliciting humoral responses are largely unknown. As a result, information about antibody dynamics and function, which might fundamentally transform our understanding of host Mtb immunity, is missing. Importantly, Mtb infection also induces the recruitment, accumulation and colocalization of B and T cells in the lung, which are positively correlated with protection in humans and animal models of disease. These ectopic lymphoid tissues generally support local germinal center reactions for the proliferation and ongoing selection of effector and memory B cells in the mucosa. Efforts to leverage such responses for human health, however, require a more complete understanding of how antibodies and B cells contribute to the local and systemic host Mtb immunity.

Resident Memory T Cells Escape 'Home Quarantine'.

T resident memory (Trm) cells provide a first line of defense in non-lymphoid tissues. A new report in Nature Immunology by Fonseca et al. reveals that CD8+ Trm cells can give rise to circulating effector and memory T cells, but remain predisposed to migrate back into their tissue of origin.

Long-lived T follicular helper cells retain plasticity and help sustain humoral immunity.

CD4+ memory T cells play an important role in protective immunity and are a key target in vaccine development. Many studies have focused on T central memory (Tcm) cells, whereas the existence and functional significance of long-lived T follicular helper (Tfh) cells are controversial. Here, we show that Tfh cells are highly susceptible to NAD-induced cell death (NICD) during isolation from tissues, leading to their underrepresentation in prior studies. NICD blockade reveals the persistence of abundant Tfh cells with high expression of hallmark Tfh markers to at least 400 days after infection, by which time Tcm cells are no longer found. Using single-cell RNA-seq, we demonstrate that long-lived Tfh cells are transcriptionally distinct from Tcm cells, maintain stemness and self-renewal gene expression, and, in contrast to Tcm cells, are multipotent after recall. At the protein level, we show that folate receptor 4 (FR4) robustly discriminates long-lived Tfh cells from Tcm cells. Unexpectedly, long-lived Tfh cells concurrently express a distinct glycolytic signature similar to trained immune cells, including elevated expression of mTOR-, HIF-1-, and cAMP-regulated genes. Late disruption of glycolysis/ICOS signaling leads to Tfh cell depletion concomitant with decreased splenic plasma cells and circulating antibody titers, demonstrating both unique homeostatic regulation of Tfh and their sustained function during the memory phase of the immune response. These results highlight the metabolic heterogeneity underlying distinct long-lived T cell subsets and establish Tfh cells as an attractive target for the induction of durable adaptive immunity.

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.

CD4+ Memory T Cells at Home in the Tissue: Mechanisms for Health and Disease.

During the last 10 years, a population of clonally expanded T cells that take up permanent residence in non-lymphoid tissues has been identified. The localization of these tissue resident memory (TRM) cells allows them to rapidly respond at the site of antigen exposure, making them an attractive therapeutic target for various immune interventions. Although most studies have focused on understanding the biology underlying CD8 TRMs, CD4 T cells actually far outnumber CD8 T cells in barrier tissues such as lung and skin. Depending on the immune context, CD4 TRM can contribute to immune protection, pathology, or tissue remodeling. Although the ability of CD4 T cells to differentiate into heterogeneous effector and memory subsets has been well-established, how this heterogeneity manifests within the TRM compartment and within different tissues is just beginning to be elucidated. In this review we will discuss our current understanding of how CD4 TRMs are generated and maintained as well as a potential role for CD4 TRM plasticity in mediating the balance between beneficial and pathogenic immune responses.

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.

Memory CD8(+) T Cells Require Increased Concentrations of Acetate Induced by Stress for Optimal Function.

How systemic metabolic alterations during acute infections impact immune cell function remains poorly understood. We found that acetate accumulates in the serum within hours of systemic bacterial infections and that these increased acetate concentrations are required for optimal memory CD8(+) T cell function in vitro and in vivo. Mechanistically, upon uptake by memory CD8(+) T cells, stress levels of acetate expanded the cellular acetyl-coenzyme A pool via ATP citrate lyase and promoted acetylation of the enzyme GAPDH. This context-dependent post-translational modification enhanced GAPDH activity, catalyzing glycolysis and thus boosting rapid memory CD8(+) T cell responses. Accordingly, in a murine Listeria monocytogenes model, transfer of acetate-augmented memory CD8(+) T cells exerted superior immune control compared to control cells. Our results demonstrate that increased systemic acetate concentrations are functionally integrated by CD8(+) T cells and translate into increased glycolytic and functional capacity. The immune system thus directly relates systemic metabolism with immune alertness.

IL-7R signaling in regulatory T cells maintains peripheral and allograft tolerance in mice.

Foxp3(+)CD4(+) regulatory T cells (Treg) have a crucial role in controlling CD4(+) T-cell activation, proliferation, and effector function. However, the molecular mechanisms regulating Treg function remain poorly understood. Here we assessed the role of IL-7, a key cytokine regulating T-cell homeostasis, in suppressor capacity of Treg. Using a skin allograft model in which transplant acceptance is controlled by the number of transferred Treg, we find that Treg impair the proliferation of allogeneic CD4(+) T cells, decrease production of IFNγ by effector T cells, and prevent early and increase late IL-7 induction by lymph node stromal cells. Increased IL-7 availability enhanced Treg survival, stabilized Treg molecular signature, enhanced surface IL-2Rα expression, and improved IL-2 binding of Treg, which diminished proliferation of alloreactive CD4(+) T cells. Sequestration of IL-7 or impairment of IL-7R signaling after allograft transplantation abolished Treg-mediated tolerance by limiting their suppressive capacity. Aged Il7rα-ΔTreg mice displayed mild symptoms of autoimmunity correlating with impaired expansion of effector Treg in response to IL-2. Thus, IL-7R signaling on Treg supports the functional activity of effector Treg by increasing their IL-2 sensitivity in the lymph node during peripheral and allograft tolerance.

Antigen affinity and antigen dose exert distinct influences on CD4 T-cell differentiation.

Cumulative T-cell receptor signal strength and ensuing T-cell responses are affected by both antigen affinity and antigen dose. Here we examined the distinct contributions of these parameters to CD4 T-cell differentiation during infection. We found that high antigen affinity positively correlates with T helper (Th)1 differentiation at both high and low doses of antigen. In contrast, follicular helper T cell (TFH) effectors are generated after priming with high, intermediate, and low affinity ligand. Unexpectedly, memory T cells generated after priming with very low affinity antigen remain impaired in their ability to generate secondary Th1 effectors, despite being recalled with high affinity antigen. These data challenge the view that only strongly stimulated CD4 T cells are capable of differentiating into the TFH and memory T-cell compartments and reveal that differential strength of stimulation during primary T-cell activation imprints unique and long lasting T-cell differentiation programs.

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.

T cell affinity regulates asymmetric division, effector cell differentiation, and tissue pathology.

The strength of interactions between T cell receptors and the peptide-major histocompatibility complex (pMHC) directly modulates T cell fitness, clonal expansion, and acquisition of effector properties. Here we show that asymmetric T cell division is an important mechanistic link between increased signal strength, effector differentiation, and the ability to induce tissue pathology. Recognition of pMHC above a threshold affinity drove responding T cells into asymmetric cell division. The ensuing proximal daughters underwent extensive division and differentiated into short-lived effector cells expressing the integrin VLA-4, allowing the activated T cell to infiltrate and mediate destruction of peripheral target tissues. In contrast, T cells activated by below-threshold antigens underwent symmetric division, leading to abortive clonal expansion and failure to fully differentiate into tissue-infiltrating effector cells. Antigen affinity and asymmetric division are important factors that regulate fate specification in CD8(+) T cells and predict the potential of a self-reactive T cell to mediate tissue pathology.

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.