Colleen J. Winstead

IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells.

Naive T cells undergo slow homeostatic proliferation in response to T cell lymphopenia, which is also called lymphopenia-induced proliferation (LIP). IL-7 is critically required for this process, but previous studies suggested IL-15 was expendable for LIP of naive CD8 T cells. In contrast, we show that IL-15 is important for sustained CD8 T cell proliferation and accumulation in a lymphopenic setting, as revealed by truncated LIP in IL-15−/− hosts. At the same time, we find that IL-12 enhances LIP by acting directly on the CD8 T cells and independently of IL-15, suggesting distinct pathways by which cytokines can regulate homeostatic proliferation. Interestingly, the memory-phenotype CD8 T cell generated by LIP in IL-15−/− hosts are phenotypically distinct from the rare endogenous memory-phenotype cells found in IL-15−/− animals, suggesting these cells are generated by different means. These findings demonstrate that cytokine requirements for LIP change during the process itself, illustrating the need to identify factors that regulate successive stages of lymphopenia-driven proliferation.

CD4+CD25+Foxp3+ Regulatory T Cells Optimize Diversity of the Conventional T Cell Repertoire during Reconstitution from Lymphopenia

The functional capacity of the adaptive immune system is dependent on the size and the diversity of the T cell population. In states of lymphopenia, T cells are driven to proliferate to restore the T cell population size. However, different T cell clones proliferate at different rates, and some T cells experience burst-like expansion called spontaneous lymphopenia-induced proliferation (LIP). These T cells are likely receiving stimulation from cognate Ags and are most responsible for inflammatory pathology that can emerge in lymphopenic states. Foxp3+ regulatory T cells (Tregs) selectively inhibit spontaneous LIP, which may contribute to their ability to prevent lymphopenia-associated autoimmunity. We hypothesized that another potential negative consequence of unrestrained spontaneous LIP is constriction of the total T cell repertoire. We demonstrate that the absence of Foxp3+ Tregs during the period of immune reconstitution results in the development of TCR repertoire “holes” and the loss of Ag-specific responsiveness to infectious microorganisms. In contrast, the presence of Tregs during the period of immune reconstitution preserves optimal TCR diversity and foreign Ag responsiveness. This finding contrasts with the generally accepted immunosuppressive role of Tregs and provides another example of Treg activity that actually enhances immune function.

The matrikine N-α-PGP couples extracellular matrix fragmentation to endothelial permeability.

Organ tissue breakdown can induce vascular leak in lung injury. The compartmentalization and transport of proteins and solutes across the endothelium is a critical biologic function altered during inflammation and disease, leading to pathology in multiple disorders. The impact of tissue damage and subsequent extracellular matrix (ECM) fragmentation in regulating this process is unknown. We demonstrate that the collagen-derived matrikine acetylated proline-glycine-proline (N-α-PGP) serves as a critical regulator of endothelial permeability. N-α-PGP activates human endothelial cells via CXC-chemokine receptor 2 (CXCR2), triggering monolayer permeability through a discrete intracellular signaling pathway. In vivo, N-α-PGP induces local vascular leak after subcutaneous administration and pulmonary vascular permeability after systemic administration. Furthermore, neutralization of N-α-PGP attenuates lipopolysaccharide-induced lung leak. Finally, we demonstrate that plasma from patients with acute respiratory distress syndrome (ARDS) induces VE-cadherin phosphorylation in human endothelial cells, and this activation is attenuated by N-α-PGP blockade with a concomitant improvement in endothelial monolayer impedance. These results identify N-α-PGP as a novel ECM-derived matrikine regulating paracellular permeability during inflammatory disease and demonstrate the potential to target this ligand in various disorders characterized by excessive matrix turnover and vascular leak such as ARDS.

Deletion of a Conserved cis-Element in the Ifng Locus Highlights the Role of Acute Histone Acetylation in Modulating Inducible Gene Transcription

Differentiation-dependent regulation of the Ifng cytokine gene locus in T helper (Th) cells has emerged as an excellent model for functional study of distal elements that control lineage-specific gene expression. We previously identified a cis-regulatory element located 22 kb upstream of the Ifng gene (Conserved Non-coding Sequence -22, or CNS-22) that is a site for recruitment of the transcription factors T-bet, Runx3, NF-κB and STAT4, which act to regulate transcription of the Ifng gene in Th1 cells. Here, we report the generation of mice with a conditional deletion of CNS-22 that has enabled us to define the epigenetic and functional consequences of its absence. Deletion of CNS-22 led to a defect in induction of Ifng by the cytokines IL-12 and IL-18, with a more modest effect on induction via T-cell receptor activation. To better understand how CNS-22 and other Ifng CNSs regulated Ifng transcription in response to these distinct stimuli, we examined activation-dependent changes in epigenetic modifications across the extended Ifng locus in CNS-22-deficient T cells. We demonstrate that in response to both cytokine and TCR driven activation signals, CNS-22 and other Ifng CNSs recruit increased activity of histone acetyl transferases (HATs) that transiently enhance levels of histones H3 and H4 acetylation across the extended Ifng locus. We also demonstrate that activation-responsive increases in histone acetylation levels are directly linked to the ability of Ifng CNSs to acutely enhance Pol II recruitment to the Ifng promoter. Finally, we show that impairment in IL-12+IL-18 dependent induction of Ifng stems from the importance of CNS-22 in coordinating locus-wide levels of histone acetylation in response to these cytokines. These findings identify a role for acute histone acetylation in the enhancer function of distal conserved cis-elements that regulate of Ifng gene expression.

Follicular helper T cell-mediated mucosal barrier maintenance

The basic functions of the immune system are protection from pathogens and maintenance of tolerance to self. The maintenance of commensal microbiota at mucosal surfaces adds a layer of complexity to these basic functions. Recent reports suggest follicular helper T cells (Tfh), a CD4(+) T cell subset specialized to provide help to B cells undergoing isotype switching and affinity maturation in germinal centers (GC), interact with the microbiota and are essential to maintenance of mucosal barriers. Complicating the issue is ongoing controversy in the field regarding origin of the Tfh subset and its distinction from other effector CD4 T cell phenotypes (Th1/Th17/Treg). This review focuses on the differentiation, phenotypic plasticity, and function of CD4 T cells, with an emphasis on commensal-specific GC responses in the gut.

Dwelling on T Cell Fate Decisions

Defining determinants of T cell fate is central to understanding adaptive immunity and the design of effective vaccines. Tubo et al. demonstrate that intrinsic properties of T cell receptor signaling dictate whether CD4 T cells adopt predominantly type 1 helper or follicular helper T cell phenotypes in response to bacterial or viral infection.

Fully human anti-BAFF inhibitory monoclonal antibody tabalumab does not adversely affect T-dependent antibody responses in cynomolgus monkey (Macaca fasicularis): A summary of three pre-clinical immunotoxicology evaluations.

Abstract The potential immunotoxicity of tabalumab was assessed as a component of standard pre-clinical toxicology studies in cynomolgus monkeys. To evaluate potential tabalumab-associated immunosuppression after antigen challenge, cynomolgus monkeys were administered placebo control or tabalumab in three immunotoxicological safety studies. Study 1, a 4-week pilot study, evaluated biweekly intravenous (IV) control, and 0.3, 1.0, 5.0, and 15.0 mg/kg tabalumab doses. Study 2 evaluated IV control, and 0.1, 1.0, and 30.0 mg/kg tabalumab doses biweekly for 6 weeks. Study 3 evaluated IV control and 0.1, 1.0, 30.0 mg/kg, and subcutaneous (SC) 30.0 mg/kg tabalumab biweekly for 6 months, with recovery (16 weeks) to monitor standard immunotoxicity endpoints. T-cell dependent primary and secondary antibody responses to tetanus toxoid antigen challenge (4-week and 6-week studies) or keyhole limpet hemocyanin (KLH; 6-week and 6-month studies) were evaluated as a measure of immunocompetence, together with quantitation of T- and B-cell subsets. In addition, anti-tabalumab antibody formation (6-week and 6-month studies) was assessed. The results indicated that, despite expected decreases in circulating B-cell populations, no changes in follicle histopathology or organ weights, except decreases in spleen weight (after 6-months of 30 mg/kg IV/SC treatment only), were attributed to tabalumab. Non-adverse microscopic decreases in size or number of germinal centers in spleen, mesenteric, and mandibular lymph nodes occurred, but without an effect on antibody responses to KLH or tetanus. At 16-weeks recovery, microscopic compound-related changes observed after 6 months of treatment were completely reversed (0.1 mg/kg group) and partially reversed (1.0 and 30.0 mg/kg groups), while peripheral blood B cells remained 66–72% reduced from baseline. Despite reduced germinal centres in lymphoid organs, and reductions in circulating B cells, T-cell-dependent humoral immunity was maintained following tabalumab administration in three safety studies in cynomolgus monkeys.

Differential IL-2 expression defines developmental fates of follicular versus nonfollicular helper T cells

(IL-)2 be or not to be? Immunological T follicular helper (TFH) cells are a subpopulation of CD4+ T cells that support B cell antibody production and the establishment of B cell memory. By contrast, non-TFH cells orchestrate enhanced innate immune cell functions at sites of pathogen encounter. The factors underlying differentiation into a TFH or non-TFH cell remain poorly understood, though there is evidence to suggest that the T cell growth factor interleukin-2 (IL-2) may play a role. Using IL-2 reporter mice, DiToro et al. show that naïve CD4+ T cells that produce IL-2 are fated to become TFH cells, whereas nonproducers, which receive IL-2, become non-TFH cells. The CD4+ T cell–fate decision was linked to T cell receptor strength—only those naïve CD4+ T cells that received the highest T cell receptor signals were able to produce IL-2. Science, this issue p. eaao2933 Expression of the cytokine IL-2 is linked with cell fate choice in immunological T cells. INTRODUCTION The adaptive immune system has evolved to mount different types of responses that are matched to the type of invading pathogen. For CD4+ T cells, this is predicated on the multipotentiality of clonally restricted naïve T cells, which differentiate into distinct subsets of effector T cells contingent on recognition of cognate antigen and cytokine cues from cells of the innate immune system. There are two broad divisions of effector CD4+ T cells: T follicular helper (TFH) cells, which are programmed to interact with B cells within lymphoid tissues to support production of high-affinity, class-switched antibodies, and non-TFH effector cells, including T helper 1 (TH1), TH2, and TH17 cells, which are programmed to egress from lymphoid tissues to orchestrate heightened innate immune cell function at sites of pathogen entry. The mechanisms controlling bifurcation into TFH versus non-TFH effector cell pathways are incompletely understood. RATIONALE An impediment to understanding mechanisms controlling TFH–non-TFH cell divergence is an absence of early markers to define cells destined for these alternative fates. Unlike effector CD4+ T cells, which produce a diversity of cytokines that define their phenotype and function, naïve CD4+ T cells are largely limited to the rapid production of interleukin-2 (IL-2) when activated by antigen. IL-2 is only produced by a subset of activated naïve T cells, suggesting a possible relationship between IL-2 production and effector cell fate determination. To explore this, we developed two IL-2 reporter mice strains with complementary features that enabled the tracking and deletion of T cells on the basis of differential IL-2 expression. This allowed us to determine whether naïve T cells that do, or do not, produce IL-2 are biased in their developmental programming and, if so, how. RESULTS RNA sequencing of naïve T cells sorted on the basis of IL-2 reporter expression identified cosegregation of transcripts encoding IL-2 and Bcl6—the signature transcription factor of TFH cells. Conversely, IL-2–negative (IL-2–) cells preferentially expressed the gene Prdm1, which encodes the transcriptional repressor Blimp1. Blimp1, in turn, antagonizes Bcl6 and the TFH developmental program. This suggested that IL-2 producers give rise to TFH cells, whereas IL-2 nonproducers give rise to non-TFH effector cells. Moreover, the fact that IL-2 receptor signaling induces expression of Prdm1 via Stat5 suggested that IL-2 producers resisted IL-2 signaling and activated IL-2 signaling in nonproducers in trans. Indeed, in vivo studies established that IL-2 signaling was mostly paracrine and that depletion of IL-2–producing cells selectively impaired TFH cell development. Finally, IL-2 expression was limited to a subset of naïve T cells that received the strongest T cell receptor (TCR) signals, establishing a link between TCR signal strength, IL-2 production, and TFH versus non-TFH differentiation. CONCLUSION This study provides new insights into the mechanisms that control early bifurcation of CD4+ T cells into TFH and non-TFH effectors. Naïve T cells that receive differing strengths of TCR signals stratify into those that exceed a threshold predisposing them to IL-2 production and early TFH commitment and those that do not express IL-2 yet receive IL-2 signaling, which reinforces non-TFH effector commitment. IL-2–producing CD4+ T cells become TFH cells, whereas IL-2 nonproducers become non-TFH cells. (Left) Strong TCR signaling via an antigen presenting cell induces Il2 and Bcl6 gene expression (red pathway); weaker signaling induces expression of non-TFH genes (blue pathway), including Prdm1 and S1pr1, which encodes the S1P receptor S1PR1. Bcl6+ cells (red) secrete IL-2 in trans to T regulatory (Treg) cells (yellow) and recently activated IL-2– cells (blue), up-regulating IL-2 receptor IL2rα and reinforcing Prdm1. (Top right) Bcl6+ cells engage cognate B cells (green) and migrate to germinal centers (GCs); Bcl6+ TFH cells mature into GC-TFH cells. (Bottom right) Prdm1+ cells migrate to efferent lymphatics and mature into non-TFH effectors in nonlymphoid tissues. MHCII, major histocompatibility complex class II; ICOS, inducible costimulator; CXCR5, a receptor for chemokine CXCL13. In response to infection, naïve CD4+ T cells differentiate into two subpopulations: T follicular helper (TFH) cells, which support B cell antibody production, and non-TFH cells, which enhance innate immune cell functions. Interleukin-2 (IL-2), the major cytokine produced by naïve T cells, plays an important role in the developmental divergence of these populations. However, the relationship between IL-2 production and fate determination remains unclear. Using reporter mice, we found that differential production of IL-2 by naïve CD4+ T cells defined precursors fated for different immune functions. IL-2 producers, which were fated to become TFH cells, delivered IL-2 to nonproducers destined to become non-TFH cells. Because IL-2 production was limited to cells receiving the strongest T cell receptor (TCR) signals, a direct link between TCR-signal strength, IL-2 production, and T cell fate determination has been established.