Distinct T cell clones are deleted during the two waves of thymic negative selection

T cells are the central mediators of adaptive immunity and provide diverse receptors to recognize pathogens. While this diversity protects the host from a wide variety of pathogens, it comes with the potential for harmful autoreactivity. To circumvent autoimmune disease, thymocytes bearing T cell receptors (TCRs) that bind with high affinity to self-peptide/MHC are removed in a process called negative selection, while cells with a low degree of affinity mature into conventional T cells. Recently, it has been shown that the process of negative selection occurs in two stages during thymocyte development, corresponding to medullary or cortical localization in the thymus. Little is known about the self-antigens that are presented and selected against in these locations, and thereby the corresponding T cell repertoires. We examined the T cell repertoires of these two deletion stages by sequencing thymocyte TCRs from mice with impaired negative selection (Bim−/−). These mice were crossed with transgenic Nur77GFP mice, in which GFP expression correlates with TCR signaling strength. Therefore, the rescued autoreactive clones can be identified in Bim deficient mice based on high GFP expression. The two pools of autoreactive clones – CD4+CD8+ double positive thymocytes (largely cortical and CCR7−) and CD4+ single positive thymocytes (predominantly medullary and CCR7+) – had only partially overlapping TCR sequences. As deletion selectively pruned clones, these observations indicate that thymocytes encounter different self-peptide/MHC molecules in the thymic cortex and thymic medulla. To further test if negative selection is TCR intrinsic, we are creating 'retrogenic' mice with auto-reactive TCRs identified from each wave

Strong agonist ligands for the T cell receptor do not mediate positive selection of functional CD8+ T cells

AbstractPositive selection of functional CD8+ T cells expressing an MHC class I-restricted T cell receptor can be induced in fetal thymus organ culture by class I-binding peptides related to the antigenic peptide ligand. Peptides that act as antagonist or weak agonist/antagonist ligands for mature T cells work efficiently in this regard. In the present study, we have investigated whether low concentrations of the original agonist peptide, or variants that still have a strong agonist activity can also mediate positive selection. The antigenic peptide did not Induce positive selection at any concentration tested. A strong agonist variant was capable of stimulating the differentiation of TCRhl CD8+ cells, giving the appearance of phenotypic positive selection. However, these cells lacked biological function, since they could not proliferate In response to antigen. The most efficient positive selection resulted with Ilgands that did not activate mature T cells or stimulate negative selection

Thymic emigration revisited

Conventional αβ T cell precursors undergo positive selection in the thymic cortex. When this is successful, they migrate to the medulla and are exposed to tissue-specific antigens (TSA) for purposes of central tolerance, and they undergo maturation to become functionally responsive T cells. It is commonly understood that thymocytes spend up to 2 wk in the medulla undergoing these final maturation steps before emigrating to peripheral lymphoid tissues. In addition, emigration is thought to occur via a stochastic mechanism whereby some progenitors leave early and others leave late—a so-called “lucky dip” process. However, recent research has revealed that medullary thymocytes are a heterogeneous mix of naive αβ T cell precursors, memory T cells, natural killer T cells, and regulatory T cells. Given this, we revisited the question of how long it takes naive αβ T cell precursors to emigrate. We combined the following three approaches to study this question: BrdU labeling, intrathymic injection of a cellular tag, and RAG2p-GFP reporter mice. We established that, on average, naive αβ T cell precursors emigrate only 4–5 d after becoming single-positive (SP) thymocytes. Furthermore, emigration occurs via a strict “conveyor belt” mechanism, where the oldest thymocytes leave first

How Lipid-Specific T Cells Become Effectors: The Differentiation of iNKT Subsets

In contrast to peptide-recognizing T cells, invariant natural killer T (iNKT) cells express a semi-invariant T cell receptor that specifically recognizes self- or foreign-lipids presented by CD1d molecules. There are three major functionally distinct effector states for iNKT cells. Owning to these innate-like effector states, iNKT cells have been implicated in early protective immunity against pathogens. Yet, growing evidence suggests that iNKT cells play a role in tissue homeostasis as well. In this review, we discuss current knowledge about the underlying mechanisms that regulate the effector states of iNKT subsets, with a highlight on the roles of a variety of transcription factors and describe how each subset influences different facets of thymus homeostasis

Peptide-MHC heterodimers show that thymic positive selection requires a more restricted set of self-peptides than negative selection

T cell selection and maturation in the thymus depends on the interactions between T cell receptors (TCRs) and different self-peptide–major histocompatibility complex (pMHC) molecules. We show that the affinity of the OT-I TCR for its endogenous positively selecting ligands, Catnb-H-2Kb and Cappa1-H-2Kb, is significantly lower than for previously reported positively selecting altered peptide ligands. To understand how these extremely weak endogenous ligands produce signals in maturing thymocytes, we generated soluble monomeric and dimeric peptide–H-2Kb ligands. Soluble monomeric ovalbumin (OVA)-Kb molecules elicited no detectable signaling in OT-I thymocytes, whereas heterodimers of OVA-Kb paired with positively selecting or nonselecting endogenous peptides, but not an engineered null peptide, induced deletion. In contrast, dimer-induced positive selection was much more sensitive to the identity of the partner peptide. Catnb-Kb–Catnb-Kb homodimers, but not heterodimers of Catnb-Kb paired with a nonselecting peptide-Kb, induced positive selection, even though both ligands bind the OT-I TCR with detectable affinity. Thus, both positive and negative selection can be driven by dimeric but not monomeric ligands. In addition, positive selection has much more stringent requirements for the partner self-pMHC

The timing of TCRα expression critically influences T cell development and selection

Sequential rearrangement of the T cell receptor for antigen (TCR) β and α chains is a hallmark of thymocyte development. This temporal control is lost in TCR transgenics because the α chain is expressed prematurely at the CD4−CD8− double negative (DN) stage. To test the importance of this, we expressed the HYα chain at the physiological CD4+CD8+ double positive (DP) stage. The reduced DP and increased DN cellularity typically seen in TCR transgenics was not observed when the α chain was expressed at the appropriate stage. Surprisingly, antigen-driven selection events were also altered. In male mice, thymocyte deletion now occurred at the single positive or medullary stage. In addition, no expansion of CD8αα intestinal intraepithelial lymphocytes (IELs) was observed, despite the fact that HY transgenics have been used to model IEL development. Collectively, these data establish the importance of proper timing of TCR expression in thymic development and selection and emphasize the need to use models that most accurately reflect the physiologic process

CD8αα intestinal intraepithelial lymphocytes derived from two thymic precursors seed the intestine in early life

TCRαβ CD4 CD8αα intestinal intraepithelial lymphocytes (CD8αα IEL) descend from thymic precursors. To better define this IEL precursor (IELp) population, we analyzed their maturation, localization, and emigration. Using rigorous lineage exclusion criteria, we defined two precursors among DN TCRβ thymocytes: a nascent PD-1 population and a T-bet population that accumulates with age. Both gave rise to intestinal CD8αα IEL upon adoptive transfer. In adult mice, PD-1 cells contained more self-reactive clones, localized to the cortex, and were dominant in S1PR1-dependent thymic egress. Gut homing α4β7 was already expressed by these IELp at a thymic stage. To understand the kinetics of CD8αα IEL seeding the intestine, we performed "timestamp" experiments: We crossed Cd4 with Rosa26 (stop-floxed tdTomato) mice. In these mice, tamoxifen or its metabolite 4-OHT permanently labels every CD4 expressing cell. As TCRαβ T cells (including CD8αα IEL) go through a CD4 CD8 stage during thymic development, a single dose of tamoxifen or 4-OHT will label thymic IEL precursors permanently, so that they can be tracked when seeding the gut. Our results indicate that these cells enter the intestine during a narrow time window in early life and that this influx is almost completely shut down by the age of 3 weeks. These data provide an important foundation for understanding the biology of this abundant population of barrier surface T cells

Identification of a novel population of Langerin+ dendritic cells

Langerhans cells (LCs) are antigen-presenting cells that reside in the epidermis of the skin and traffic to lymph nodes (LNs). The general role of these cells in skin immune responses is not clear because distinct models of LC depletion resulted in opposite conclusions about their role in contact hypersensitivity (CHS) responses. While comparing these models, we discovered a novel population of LCs that resides in the dermis and does not represent migrating epidermal LCs, as previously thought. Unlike epidermal LCs, dermal Langerin+ dendritic cells (DCs) were radiosensitive and displayed a distinct cell surface phenotype. Dermal Langerin+ DCs migrate from the skin to the LNs after inflammation and in the steady state, and represent the majority of Langerin+ DCs in skin draining LNs. Both epidermal and dermal Langerin+ DCs were depleted by treatment with diphtheria toxin in Lang-DTREGFP knock-in mice. In contrast, transgenic hLang-DTA mice lack epidermal LCs, but have normal numbers of dermal Langerin+ DCs. CHS responses were abrogated upon depletion of both epidermal and dermal LCs, but were unaffected in the absence of only epidermal LCs. This suggests that dermal LCs can mediate CHS and provides an explanation for previous differences observed in the two-model systems

IL-4 sensitivity shapes the peripheral CD8\u3csup\u3e+\u3c/sup\u3e T cell pool and response to infection

Previous studies have revealed that a population of innate memory CD8+ T cells is generated in response to IL-4, first appearing in the thymus and bearing high expression levels of Eomesodermin (Eomes) but not T-bet. However, the antigen specificity and functional properties of these cells is poorly defined. In this study, we show that IL-4 regulates not only the frequency and function of innate memory CD8+ T cells, but also regulates Eomes expression levels and functional reactivity of naive CD8+ T cells. Lack of IL-4 responsiveness attenuates the capacity of CD8+ T cells to mount a robust response to lymphocytic choriomeningitis virus infection, with both quantitative and qualitative effects on effector and memory antigen-specific CD8+ T cells. Unexpectedly, we found that, although numerically rare, memory phenotype CD8+ T cells in IL-4Rα–deficient mice exhibited enhanced reactivity after in vitro and in vivo stimulation. Importantly, our data revealed that these effects of IL-4 exposure occur before, not during, infection. Together, these data show that IL-4 influences the entire peripheral CD8+ T cell pool, influencing expression of T-box transcription factors, functional reactivity, and the capacity to respond to infection. These findings indicate that IL-4, a canonical Th2 cell cytokine, can sometimes promote rather than impair Th1 cell–type immune responses