An Opposite Pattern of Selection of a Single T Cell Antigen Receptor in the Thymus and among Intraepithelial Lymphocytes

The differentiation of intestinal intraepithelial lymphocytes (IEL) remains controversial, which may be due in part to the phenotypic complexity of these T cells. We have investigated here the development of IEL in mice on the recombination activating gene (RAG)-2^−/− background which express a T cell antigen receptor (TCR) transgene specific for an H-Y peptide presented by D^b (H-Y/D^b × RAG-2⁻ mice). In contrast to the thymus, the small intestine in female H-Y/D^b × RAG-2⁻ mice is severely deficient in the number of IEL; TCR transgene⁺ CD8αα and CD8αβ are virtually absent. This is similar to the number and phenotype of IEL in transgenic mice that do not express the D^b class I molecule, and which therefore fail positive selection. Paradoxically, in male mice, the small intestine contains large numbers of TCR⁺ IEL that express high levels of CD8αα homodimers. The IEL isolated from male mice are functional, as they respond upon TCR cross-linking, although they are not autoreactive to stimulator cells from male mice. We hypothesize that the H-Y/D^b TCR fails to undergo selection in IEL of female mice due to the reduced avidity of the TCR for major histocompatibility complex peptide in conjunction with the CD8αα homodimers expressed by many cells in this lineage. By contrast, this reduced TCR/CD8αα avidity may permit positive rather than negative selection of this TCR in male mice. Therefore, the data presented provide conclusive evidence that a TCR which is positively selected in the thymus will not necessarily be selected in IEL, and furthermore, that the expression of a distinct CD8 isoform by IEL may be a critical determinant of the differential pattern of selection of these T cells.     

Regulatory CD4+ T Cells Expressing Endogenous T Cell Receptor Chains Protect Myelin Basic Protein–specific Transgenic Mice from Spontaneous Autoimmune Encephalomyelitis

The development of T cell–mediated autoimmune diseases hinges on the balance between effector and regulatory mechanisms. Using two transgenic mouse lines expressing identical myelin basic protein (MBP)–specific T cell receptor (TCR) genes, we have previously shown that mice bearing exclusively MBP-specific T cells (designated T/R⁻) spontaneously develop experimental autoimmune encephalomyelitis (EAE), whereas mice bearing MBP-specific T cells as well as other lymphocytes (designated T/R⁺) did not. Here we demonstrate that T/R⁻ mice can be protected from EAE by the early transfer of total splenocytes or purified CD4⁺ T cells from normal donors. Moreover, whereas T/R⁺ mice crossed with B cell–deficient, γ/δ T cell–deficient, or major histocompatibility complex class I–deficient mice did not develop EAE spontaneously, T/R⁺ mice crossed with TCR-α and -β knockout mice developed EAE with the same incidence and severity as T/R⁻ mice. In addition, MBP-specific transgenic mice that lack only endogenous TCR-α chains developed EAE with high incidence but reduced severity. Surprisingly, two-thirds of MBP-specific transgenic mice lacking only endogenous TCR-β chains also developed EAE, suggesting that in T/R⁺ mice, cells with high protective activity escape TCR-β chain allelic exclusion. Our study identifies CD4⁺ T cells bearing endogenous α and β TCR chains as the lymphocytes that prevent spontaneous EAE in T/R⁺ mice.     

CD4+ T Cells Prevent Spontaneous Experimental Autoimmune Encephalomyelitis in Anti–Myelin Basic Protein T Cell Receptor Transgenic Mice

Autoimmune diseases result from a failure of tolerance. Although many self-reactive T cells are present in animals and humans, their activation appears to be prevented normally by regulatory T cells. In this study, we show that regulatory CD4⁺ T cells do protect mice against the spontaneous occurrence of experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. Anti–myelin basic protein (MBP) TCR transgenic mice (T/R⁺) do not spontaneously develop EAE although many self-reactive T cells are present in their thymi and peripheral lymphoid organs. However, the disease develops in all crosses of T/R⁺ mice with recombination-activating gene (RAG)-1 knockout mice in which transgenic TCR-expressing cells are the only lymphocytes present (T/R⁻ mice). In this study, crosses of T/R⁺ mice with mice deficient for B cells, CD8⁺ T cells, NK1.1 CD4⁺ T (NKT) cells, γ/δ T cells, or α/β T cells indicated that α/β CD4⁺ T cells were the only cell population capable of controlling the self-reactive T cells. To confirm the protective role of CD4⁺ T cells, we performed adoptive transfer experiments. CD4⁺ T cells purified from thymi or lymph nodes of normal mice prevented the occurrence of spontaneous EAE in T/R⁻ mice. To achieve full protection, the cells had to be transferred before the recipient mice manifested any symptoms of the disease. Transfer of CD4⁺ T cells after the appearance of symptoms of EAE had no protective effect. These results indicate that at least some CD4⁺ T cells have a regulatory function that prevent the activation of self-reactive T cells.     

Type I Interferon-mediated Stimulation of T Cells by CpG DNA

Immunostimulatory DNA and oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNA) are strongly stimulatory for B cells and antigen-presenting cells (APCs). We report here that, as manifested by CD69 and B7-2 upregulation, CpG DNA also induces partial activation of T cells, including naive-phenotype T cells, both in vivo and in vitro. Under in vitro conditions, CpG DNA caused activation of T cells in spleen cell suspensions but failed to stimulate highly purified T cells unless these cells were supplemented with APCs. Three lines of evidence suggested that APC-dependent stimulation of T cells by CpG DNA was mediated by type I interferons (IFN-I). First, T cell activation by CpG DNA was undetectable in IFN-IR^−/− mice. Second, in contrast to normal T cells, the failure of purified IFN-IR^−/− T cells to respond to CpG DNA could not be overcome by adding normal IFN-IR⁺ APCs. Third, IFN-I (but not IFN-γ) caused the same pattern of partial T cell activation as CpG DNA. Significantly, T cell activation by IFN-I was APC independent. Thus, CpG DNA appeared to stimulate T cells by inducing APCs to synthesize IFN-I, which then acted directly on T cells via IFN-IR. Functional studies suggested that activation of T cells by IFN-I was inhibitory. Thus, exposing normal (but not IFN-IR^−/−) T cells to CpG DNA in vivo led to reduced T proliferative responses after TCR ligation in vitro.     

Antagonist Peptide Selects Thymocytes Expressing a Class II Major Histocompatibility Complex–restricted T Cell Receptor into the CD8 Lineage

CD4/CD8 lineage decision is an important event during T cell maturation in the thymus. CD8 T cell differentiation usually requires corecognition of major histocompatibility complex (MHC) class I by the T cell receptor (TCR) and CD8, whereas CD4 T cells differentiate as a consequence of MHC class II recognition by the TCR and CD4. The involvement of specific peptides in the selection of T cells expressing a particular TCR could be demonstrated so far for the CD8 lineage only. We used mice transgenic for an MHC class II-restricted TCR to investigate the role of antagonistic peptides in CD4 T cell differentiation. Interestingly, antagonists blocked the development of CD4⁺ cells that normally differentiate in thymus organ culture from those mice, and they induced the generation of CD8⁺ cells in thymus organ culture from mice impaired in CD4⁺ cell development (invariant chain–deficient mice). These results are in line with recent observations that antagonistic signals direct differentiation into the CD8 lineage, regardless of MHC specificity.     

CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production

Peripheral tolerance may be maintained by a population of regulatory/suppressor T cells that prevent the activation of autoreactive T cells recognizing tissue-specific antigens. We have previously shown that CD4⁺CD25⁺ T cells represent a unique population of suppressor T cells that can prevent both the initiation of organ-specific autoimmune disease after day 3 thymectomy and the effector function of cloned autoantigen-specific CD4⁺ T cells. To analyze the mechanism of action of these cells, we established an in vitro model system that mimics the function of these cells in vivo. Purified CD4⁺CD25⁺ cells failed to proliferate after stimulation with interleukin (IL)-2 alone or stimulation through the T cell receptor (TCR). When cocultured with CD4⁺CD25⁻ cells, the CD4⁺CD25⁺ cells markedly suppressed proliferation by specifically inhibiting the production of IL-2. The inhibition was not cytokine mediated, was dependent on cell contact between the regulatory cells and the responders, and required activation of the suppressors via the TCR. Inhibition could be overcome by the addition to the cultures of IL-2 or anti-CD28, suggesting that the CD4⁺CD25⁺ cells may function by blocking the delivery of a costimulatory signal. Induction of CD25 expression on CD25⁻ T cells in vitro or in vivo did not result in the generation of suppressor activity. Collectively, these data support the concept that the CD4⁺CD25⁺ T cells in normal mice may represent a distinct lineage of “professional” suppressor cells.     

Major Histocompatibility Complex Class I–restricted Cross-presentation Is Biased towards High Dose Antigens and Those Released during Cellular Destruction

Naive T cells recirculate mainly within the secondary lymphoid compartment, but once activated they can enter peripheral tissues and perform effector functions. To activate naive T cells, foreign antigens must traffic from the site of infection to the draining lymph nodes, where they can be presented by professional antigen presenting cells. For major histocompatibility complex class I–restricted presentation to CD8⁺ T cells, this can occur via the cross-presentation pathway. Here, we investigated the conditions allowing antigen access to this pathway. We show that the level of antigen expressed by peripheral tissues must be relatively high to facilitate cross-presentation to naive CD8⁺ T cells. Below this level, peripheral antigens did not stimulate by cross-presentation and were ignored by naive CD8⁺ T cells, although they could sensitize tissue cells for destruction by activated cytotoxic T lymphocytes (CTLs). Interestingly, CTL-mediated tissue destruction facilitated cross-presentation of low dose antigens for activation of naive CD8⁺ T cells. This represents the first in vivo evidence that cellular destruction can enhance access of exogenous antigens to the cross-presentation pathway. These data indicate that the cross-presentation pathway focuses on high dose antigens and those released during tissue destruction.     

Tyrosine Phosphorylation of Pyk2 Is Selectively Regulated by Fyn During TCR Signaling

The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyrosine phosphorylation of the TCR-ζ chain and the ZAP-70 PTK. In an effort to identify the targets that are specifically regulated by Fyn, we have studied the tyrosine phosphorylation of Pyk2, a recently discovered new member of the focal adhesion kinase family PTK. We demonstrated that Pyk2 was rapidly tyrosine phosphorylated following TCR stimulation. TCR-induced tyrosine phosphorylation of Pyk2 was selectively dependent on Fyn but not Lck. Moreover, in heterologous COS-7 cells, coexpression of Pyk2 with Fyn but not Lck resulted in substantial increases in Pyk2 tyrosine phosphorylation. The selective regulation of Pyk2 tyrosine phosphorylation by Fyn in vivo correlated with the preferential phosphorylation of Pyk2 by Fyn in vitro. Our results demonstrate that Pyk2 is a specific target regulated by Fyn during TCR signaling.Engagement of the TCR evokes a series of signal transduction events critical for the functional activation of T cells (reviewed in reference 1). Signal transduction through the TCR is also important for T cell development (1). The earliest detectable signaling event after TCR stimulation is the activation of protein tyrosine kinases (PTKs)¹, resulting in the tyrosine phosphorylation of cellular proteins (1). Lck and Fyn, two cytoplasmic PTKs of the Src family, have been implicated as the initiating PTKs for TCR signaling. Lck is critical for TCR signaling. Mutant T cell lines lacking functional Lck or T cells from lck ^−/− mice respond to TCR stimulation with very limited tyrosine phosphorylation of cellular proteins, greatly decreased calcium mobilization, and reduced proliferation (2–4). Lck also plays a critical role in T cell development, as lck ^−/− mice have a pronounced reduction in thymocyte numbers and a block in thymocyte development at the early CD4⁺CD8⁺ stage (2). The residual progression of thymocytes from CD4⁻CD8⁻ to CD4⁺CD8⁺ stage in lck ^−/− mice depends upon the redundant function of Fyn. Combined disruption of both Lck and Fyn (lck ^−/−/fyn ^−/−) completely arrests thymocyte development at the CD4⁻CD8⁻ stage (5, 6). Fyn is also important for TCR signaling. Mature CD4⁺ and CD8⁺ thymocytes from fyn ^−/− mice are severely impaired in TCR signaling as measured by calcium mobilization, protein tyrosine phosphorylation, IL-2 production, and proliferation (7, 8). Peripheral T cells from fyn ^−/− mice are also impaired in TCR signaling, albeit to a lesser degree (7, 8).The mechanisms by which Lck or Fyn regulates proximal TCR signaling are still not completely understood. One important issue is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have studied the TCR signaling pathway in T cells from lck ^−/− or fyn ^−/− mice in an attempt to identify targets for Lck and Fyn. We have shown previously that Lck is the primary PTK that regulates the tyrosine phosphorylation of the TCR subunits and of ZAP-70 (9), a Syk family PTK critical for TCR signaling (reviewed in reference 10). The identity of the downstream target(s) for Fyn has not been identified. In the present study, we have focused our efforts in identifying target(s) whose phosphorylation is specifically regulated by Fyn. Previous studies have shown that Fyn interacts with a number of proteins in T cells (11–16), including several proteins migrating from 110 to 130 kD. These proteins are potential targets for Fyn, though their identities have not been fully elucidated. A recently discovered cytoplasmic PTK Pyk2 has a molecular mass of 112 kD (17–19). Pyk2 is a member of the focal adhesion kinase (FAK) family and has been shown to play important roles in signal transduction of neuronal cells (17, 20, 21). Because Pyk2 is also expressed in T cells (18, 19), we examined whether it might be a target for Fyn during TCR signaling. Our data demonstrate that Pyk2 is a novel Fyndependent tyrosine-phosphorylated substrate during TCR signaling.     

Uncovering a novel role of PLCβ4 in selectively mediating TCR signaling in CD8+ but not CD4+ T cells

Because of their common signaling molecules, the main T cell receptor (TCR) signaling cascades in CD4⁺ and CD8⁺ T cells are considered qualitatively identical. Herein, we show that TCR signaling in CD8⁺ T cells is qualitatively different from that in CD4⁺ T cells, since CD8α ignites another cardinal signaling cascade involving phospholipase C β4 (PLCβ4). TCR-mediated responses were severely impaired in PLCβ4-deficient CD8⁺ T cells, whereas those in CD4⁺ T cells were intact. PLCβ4-deficient CD8⁺ T cells showed perturbed activation of peripheral TCR signaling pathways downstream of IP₃ generation. Binding of PLCβ4 to the cytoplasmic tail of CD8α was important for CD8⁺ T cell activation. Furthermore, GNAQ interacted with PLCβ4, mediated double phosphorylation on threonine 886 and serine 890 positions of PLCβ4, and activated CD8⁺ T cells in a PLCβ4-dependent fashion. PLCβ4-deficient mice exhibited defective antiparasitic host defense and antitumor immune responses. Altogether, PLCβ4 differentiates TCR signaling in CD4⁺ and CD8⁺ T cells and selectively promotes CD8⁺ T cell–dependent adaptive immunity.     

CRTAM controls residency of gut CD4+CD8+ T cells in the steady state and maintenance of gut CD4+ Th17 during parasitic infection

Retention of lymphocytes in the intestinal mucosa requires specialized chemokine receptors and adhesion molecules. We find that both CD4⁺CD8⁺ and CD4⁺ T cells in the intestinal epithelium, as well as CD8⁺ T cells in the intestinal mucosa and mesenteric lymph nodes, express the cell adhesion molecule class I–restricted T cell–associated molecule (Crtam) upon activation, whereas the ligand of Crtam, cell adhesion molecule 1 (Cadm1), is expressed on gut CD103⁺DCs. Lack of Crtam–Cadm1 interactions in Crtam^−/− and Cadm1^−/− mice results in loss of CD4⁺CD8⁺ T cells, which arise from mucosal CD4⁺ T cells that acquire a CD8 lineage expression profile. After acute oral infection with Toxoplasma gondii, both WT and Crtam^−/− mice mounted a robust TH1 response, but markedly fewer TH17 cells were present in the intestinal mucosa of Crtam^−/− mice. The almost exclusive TH1 response in Crtam^−/− mice resulted in more efficient control of intestinal T. gondii infection. Thus, Crtam–Cadm1 interactions have a major impact on the residency and maintenance of CD4⁺CD8⁺ T cells in the gut mucosa in the steady state. During pathogenic infection, Crtam–Cadm1 interactions regulate the dynamic equilibrium between newly formed CD4⁺ T cells and their retention in the gut, thereby shaping representation of disparate CD4⁺ T cell subsets and the overall quality of the CD4⁺ T cell response.Class I–restricted T cell–associated molecule (Crtam) is an Ig-like cell surface protein that was originally found on activated NKT cells (Kennedy et al., 2000), NK cells, and CD8⁺ T cells (Arase et al., 2005; Boles et al., 2005; Galibert et al., 2005) and shown to bind the cell adhesion molecule 1 (Cadm1, also known as Nectin like [Necl] 2; Arase et al., 2005; Boles et al., 2005; Galibert et al., 2005). Cadm1 is a cell surface molecule of the nectin and Necl families that is expressed on CD8α DCs (Galibert et al., 2005; Poulin et al., 2010), epithelial cells, neurons, and tumor cells (Sakisaka and Takai, 2004; Mizutani et al., 2011). Crtam–Cadm1 interactions strengthen NK cell and CD8⁺ T cell effector functions (Arase et al., 2005; Boles et al., 2005; Galibert et al., 2005; Murakami, 2005) and promote the retention of virus-specific CD8⁺ T cells within LNs (Takeuchi et al., 2009). One report proposed that Crtam is essential for the establishment of CD4⁺ T cell polarization after TCR engagement, a process which blocks CD4⁺ T cell division and induces the capacity to secrete IFN-γ, IL-17, and IL-22 (Yeh et al., 2008).The immune system associated with the gastrointestinal mucosa comprises large numbers of dispersed lymphoid cells that reside in the epithelium and the underlying lamina propria. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) include antigen-experienced CD8⁺ and CD4⁺ T cells, γδ T cells, various subsets of innate lymphoid cells (ILCs), and IgA-secreting plasma cells (Jabri and Ebert, 2007; Cerutti, 2008; Cheroutre et al., 2011; Sheridan and Lefrançois, 2011; Spits et al., 2013). Homing and residency of IELs and LPLs in the mucosa requires specialized chemokine receptors, such as CCR9, CCR6, and CXCR6, which detect chemokines released by gut epithelial cells (CCL25, CCL20, and CXCL16, respectively; Johansson-Lindbom and Agace, 2007). Integrins, like CD103 (α_E) and α₄β₇, also play an essential role in promoting homing and retention of IELs and LPLs in the mucosa by binding E-cadherin and MAdCAM-1 on epithelial cells and vascular endothelial cells, respectively (Johansson-Lindbom and Agace, 2007).T cell acquisition of homing and adhesion molecules is induced by T cell interaction with DCs (Mora et al., 2008; Villablanca et al., 2011). Among the disparate subsets of DC in the intestinal lamina propria and mesenteric LNs (mLN), the CD103⁺ DC subset produces retinoic acid (RA), which induces the gut homing receptors CCR9 and α₄β₇ on lymphocytes (Coombes et al., 2007; Mora et al., 2008; Villablanca et al., 2011). Gut-associated CD103⁺ DCs also produce TGF-β, which induces the expression of CD103 on T cells (Coombes et al., 2007; Mora et al., 2008; Villablanca et al., 2011). In addition to imprinting gut-homing capacity on T cells, gut CD103⁺ DCs control the differentiation of CD4⁺ T cells by priming regulatory CD4⁺ T cells during the steady state (Mucida et al., 2007) and TH1 and TH17 cells during inflammation (DePaolo et al., 2011; Hall et al., 2011).Here, we investigated the impact of Crtam–Cadm1 interaction in the intestinal immune system. We find that Crtam is expressed upon activation on all CD8⁺ T cells of the intestinal mucosa and mLN, intraepithelial CD4⁺ T cells, and intraepithelial CD4⁺CD8⁺ T cells, whereas Cadm1 is expressed on gut CD103⁺ DCs. Crtam–Cadm1 interactions have a major impact on the maintenance of intraepithelial CD4⁺CD8⁺ T cells and a limited influence on the presence of mucosal CD4⁺ and CD8⁺ T cells. Crtam^−/− and Cadm1^−/− mice almost completely lacked CD4⁺CD8⁺ T cells in the intestinal epithelium under steady-state conditions and had fewer CD4⁺ and CD8⁺ T cells in the intestinal mucosa than WT mice. CD4⁺CD8⁺ T cells arise from CD4⁺ T cells that acquire a CD8 T cell lineage gene expression profile upon reaching the intestinal mucosa (Mucida et al., 2013; Reis et al., 2013). Therefore, we further investigated the role of Crtam–Cadm1 interactions in governing CD4⁺ T cell homing and maintenance in the intestinal mucosa, and found that Crtam^−/− CD4⁺ T cells reconstituted the intestinal CD4⁺ T cell and CD4⁺CD8⁺ T cell subsets less effectively than did WT CD4⁺ T cells after transfer into Rag1^−/− mice. Moreover, fewer intestinal CD4⁺ T cells in Crtam^−/− mice expressed gut homing, adhesion, and retention molecules typical of their counterparts in WT mice (including CCR9, CD103, and CD69), and hence adhered less firmly to the intestinal mucosa.Crtam deficiency did not affect the intrinsic capacity of naive CD4⁺ T cells to differentiate into IFN-γ– or IL-17–producing CD4⁺ T cells in vitro. After acute oral infection with Toxoplasma gondii, both WT and Crtam^−/− mice mounted a robust TH1 response and cleared the intestinal infection. However, a preferential reduction of TH17 cells was evident within the intestinal mucosa CD4⁺ T cells in the Crtam^−/− mice. The almost exclusive TH1 response in Crtam^−/− mice resulted in more efficient clearance of intestinal infection. Antibody blockade of IL-17 in WT mice orally infected with T. gondii recapitulated the enhanced host response of Crtam^−/− mice. Thus, the defects in T cell gut homing and maintenance in Crtam^−/− mice preferentially affected TH17, whereas TH1 were relatively unaffected. Because the differentiation of CD4⁺CD8⁺ T cells from CD4⁺ T cells skews CD4⁺ T cell cytokine production toward IFN-γ at the expenses of IL-17 production (Mucida et al., 2013; Reis et al., 2013), the need for continuous replacement of lost CD4⁺CD8⁺ T cells in Crtam^−/− mice may result in a relative depletion of TH17 cells. Together, these results demonstrate that Crtam–Cadm1 interactions have a major impact on the residency and maintenance of CD4⁺CD8⁺ T cells in the gut mucosa in the steady-state. During mucosal responses against pathogenic infection, Crtam–Cadm1 interactions may be required to retain a balanced representation of disparate CD4⁺ T cell subsets, thereby influencing the overall quality of the CD4⁺ T cell response.     

Essential and Partially Overlapping Role of CD3γ and CD3δ for Development of αβ and γδ T Lymphocytes

CD3γ and CD3δ are two highly related components of the T cell receptor (TCR)–CD3 complex which is essential for the assembly and signal transduction of the T cell receptor on mature T cells. In gene knockout mice deficient in either CD3δ or CD3γ, early thymic development mediated by pre-TCR was either undisturbed or severely blocked, respectively, and small numbers of TCR-αβ⁺ T cells were detected in the periphery of both mice. γδ T cell development was either normal in CD3δ^−/− mice or partially blocked in CD3γ^−/− mice. To examine the collective role of CD3γ and CD3δ in the assembly and function of pre-TCR and in the development of γδ T cells, we generated a mouse strain with a disruption in both CD3γ and CD3δ genes (CD3γδ^−/−). In contrast to mice deficient in either CD3γ or CD3δ chains, early thymic development mediated by pre-TCR is completely blocked, and TCR-αβ⁺ or TCR-γδ⁺ T cells were absent in the CD3γδ^−/− mice. Taken together, these studies demonstrated that CD3γ and CD3δ play an essential, yet partially overlapping, role in the development of both αβ and γδ T cell lineages.     

An Alternate Pathway for T Cell Development Supported by the Bone Marrow Microenvironment: Recapitulation of Thymic Maturation

In the principal pathway of α/β T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4⁺ and CD8⁺ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4⁺ and CD8⁺ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4⁺CD8⁺ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.     

B7-1 Engagement of Cytotoxic T Lymphocyte Antigen 4 Inhibits T Cell Activation in the Absence of CD28

Ligation of cytotoxic T lymphocyte antigen 4 (CTLA4) appears to inhibit T cell responses. Four mechanisms have been proposed to explain the inhibitory activity of CTLA4: competition for B7-1 and B7-2 binding by CD28; sequestration of signaling molecules away from CD28 via endocytosis; delivery of a signal that antagonizes a CD28 signal; and delivery of a signal that antagonizes a T cell receptor (TCR) signal. As three of these potential mechanisms involve functional antagonism of CD28, an experimental model was designed to determine whether CTLA4 could inhibit T cell function in the absence of CD28. TCR transgenic/recombinase activating gene 2–deficient/CD28–wild-type or CD28-deficient mice were generated and immunized with an antigen-expressing tumor. Primed T cells from both types of mice produced cytokines and proliferated in response to stimulator cells lacking B7 expression. However, whereas the response of CD28^+/+ T cells was augmented by costimulation with B7-1, the response of the CD28^−/− T cells was strongly inhibited. This inhibition was reversed by monoclonal antibody against B7-1 or CTLA4. Thus, CTLA4 can potently inhibit T cell activation in the absence of CD28, indicating that antagonism of a TCR-mediated signal is sufficient to explain the inhibitory effect of CTLA4.     

CD5 Expression Is Developmentally Regulated By T Cell Receptor (TCR) Signals and TCR Avidity

Recent data indicate that the cell surface glycoprotein CD5 functions as a negative regulator of T cell receptor (TCR)-mediated signaling. In this study, we examined the regulation of CD5 surface expression during normal thymocyte ontogeny and in mice with developmental and/or signal transduction defects. The results demonstrate that low level expression of CD5 on CD4⁻CD8⁻ (double negative, DN) thymocytes is independent of TCR gene rearrangement; however, induction of CD5 surface expression on DN thymocytes requires engagement of the pre-TCR and is dependent upon the activity of p56^lck. At the CD4⁺CD8⁺ (double positive, DP) stage, intermediate CD5 levels are maintained by low affinity TCR–major histocompatibility complex (MHC) interactions, and CD5 surface expression is proportional to both the surface level and signaling capacity of the TCR. High-level expression of CD5 on DP and CD4⁺ or CD8⁺ (single positive, SP) thymocytes is induced by engagement of the α/β-TCR by (positively or negatively) selecting ligands. Significantly, CD5 surface expression on mature SP thymocytes and T cells was found to directly parallel the avidity or signaling intensity of the positively selecting TCR–MHC-ligand interaction. Taken together, these observations suggest that the developmental regulation of CD5 in response to TCR signaling and TCR avidity represents a mechanism for fine tuning of the TCR signaling response.     

The JNK Pathway Regulates the In Vivo Deletion of Immature CD4+CD8+ Thymocytes

The extracellular signal-regulated kinase (ERK), the c-Jun NH₂-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4⁻CD8⁻ into CD4⁺CD8⁺ double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4⁺ cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment– mediated signals.     

CD8β Increases CD8 Coreceptor Function and Participation in TCR–Ligand Binding

To study the role of CD8β in T cell function, we derived a CD8α/β⁻ (CD8^−/−) T cell hybridoma of the H-2K^d–restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8α alone or together with CD8β. All three hybridomas released interleukin 2 upon incubation with L cells expressing K^d–peptide derivative complexes, though CD8α/β cells did so more efficiently than CD8α/α and especially CD8^−/− cells. More strikingly, only CD8α/β cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab′ fragments of the anti-K^d α3 monoclonal antibody SF11.1.1 or substitution of K^d D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR–ligand binding on CD8α/β cells was ~5- and 20-fold more avid than on CD8α/a and CD8^−/− cells, respectively. SF1-1.1.1 Fab′ or K^d mutation D227K reduced the TCR photoaffinity labeling on CD8α/β cells to approximately the same low levels observed on CD8^−/− cells. These results indicate that CD8α/β is a more efficient coreceptor than CD8α/α, because it more avidly strengthens TCR–ligand binding.     

Thromboxane A2 acts as tonic immunoregulator by preferential disruption of low-avidity CD4+ T cell–dendritic cell interactions

Interactions between dendritic cells (DCs) and T cells control the decision between activation and tolerance induction. Thromboxane A2 (TXA2) and its receptor TP have been suggested to regulate adaptive immune responses through control of T cell–DC interactions. Here, we show that this control is achieved by selectively reducing expansion of low-avidity CD4⁺ T cells. During inflammation, weak tetramer-binding TP-deficient CD4⁺ T cells were preferentially expanded compared with TP-proficient CD4⁺ T cells. Using intravital imaging of cellular interactions in reactive peripheral lymph nodes (PLNs), we found that TXA2 led to disruption of low- but not high-avidity interactions between DCs and CD4⁺ T cells. Lack of TP correlated with higher expression of activation markers on stimulated CD4⁺ T cells and with augmented accumulation of follicular helper T cells (T_FH), which correlated with increased low-avidity IgG responses. In sum, our data suggest that tonic suppression of weak CD4⁺ T cell–DC interactions by TXA2–TP signaling improves the overall quality of adaptive immune responses.T cells have evolved to quickly react to potentially dangerous microbes by recognizing pathogen-derived peptide (p)-MHC complexes displayed on antigen-presenting cells, in particular DCs. Because T cells are selected in the thymus for their ability to recognize self-pMHC complexes (Morris and Allen, 2012) and numerous self-reactive T cells are released into the periphery (Su et al., 2013), peripheral tolerance education is critical to avoid activation of autoreactive T cells. Studies using intravital two-photon microscopy (2PM) of reactive PLNs have shed light on the dynamic T cell–DC interactions and their correlation with full versus curtailed T cell activation and tolerance induction. The amount of cognate pMHC complexes on activated DCs is critical in determining the transition of a highly motile scanning-mode T cell to an immotile, stably interacting one (Cahalan and Parker, 2006; Henrickson and von Andrian, 2007; Bajénoff and Germain, 2007). Such stable T cell–DC interactions (>8h) are a prerequisite for full effector T cell differentiation (Rachmilewitz and Lanzavecchia, 2002). Thus, in presence of high amounts of cognate pMHC on activated DCs, T cells decelerate rapidly, whereas T cells show a motile DC sampling behavior when cognate pMHC levels are low. Altered peptide ligands (APLs) with reduced affinity for a given TCR also decrease the length of T cell–DC interactions, limiting T cell activation. Under tolerogenic conditions (i.e., in the absence of co-stimulation), 2PM studies uncovered shortened T cell–DC interactions (Hugues et al., 2004) although this is still controversial (Shakhar et al., 2005). Similarly, the presence of regulatory T (T reg) cells reduces T cell–DC interactions and subsequent T cell activation (Tadokoro et al., 2006; Tang et al., 2006).A perhaps counterintuitive recent finding has revealed a significant increase in CD8⁺ T cell immune response avidity in presence of T reg cells (Pace et al., 2012). This is due to T reg cell–mediated suppression of excessive interactions between DCs and CD8⁺ T cells bearing TCRs with low avidity for pMHC complexes. In the absence of T reg cells, uncontrolled CCR5 ligand secretion by activated DCs induces attraction of bystander TCR clones with low affinity for pMHC complexes, which decreases overall avidity and memory T cell generation of the resulting immune response. Whether a comparable mechanism also exists to selectively support activation of high avidity CD4⁺ T cells by immunoregulatory factors is currently unknown.The short-lived arachidonic acid–derived lipid thromboxane A2 (TXA2) has been suggested to regulate adaptive immune responses (Kabashima et al., 2003). Activated DCs and other cell types produce TXA2, which binds its G-protein coupled receptor TP expressed in thymocytes and naive but not effector/memory CD4⁺ and CD8⁺ T cells. Addition of high amounts of the TP agonist I-BOP induces chemokinesis in naive T cells and decreases in vitro aggregate formation between T cells and DCs, causing reduced T cell activation (Kabashima et al., 2003). Combined with the observation that TXA2 levels rapidly rise in reactive PLN during immune responses (Moore et al., 1989), these data suggest a model where TXA2 may act as a general suppressor of T cell–DC interactions. In line with this hypothesis, aged TP-deficient T cells develop lymphoid hyperplasia and high antibody titers (Kabashima et al., 2003). Yet, it has remained unknown how TXA2 signaling affects dynamic CD4⁺ T cell interactions with DC displaying varying pMHC abundance and affinity in vivo, and how this impacts avidity patterns of responding T cells.Here, we show that during sterile and microbial inflammation, absence of TP resulted in increased expansion of low-avidity CD4⁺ T cells. Using 2PM imaging of cellular interactions in reactive PLNs, we report that paracrine TXA2 signaling preferentially disrupted low-avidity interactions between DCs and OT-II CD4⁺ T cells induced by low cognate pMHC levels or low-affinity peptide. As a consequence, TP^−/− OT-II CD4⁺ T cells show increased expression of early activation markers, as well as augmented accumulation of follicular helper T cells (T_FH) compared with WT OT-II CD4⁺ cells. High numbers of TP^−/− T_FH correlated with increased low-avidity IgG production, thus thwarting the overall quality of the adaptive immune response. In sum, our data uncover a previously unappreciated contribution of a tolerance-inducing mechanism for preferential activation of high avidity CD4⁺ T cells.     

Cytotoxic T Lymphocyte Antigen 4 Is Induced in the Thymus upon In Vivo Activation and Its Blockade Prevents Anti-CD3–mediated Depletion of ?Thymocytes

The development of a normal T cell repertoire in the thymus is dependent on the interplay between signals mediating cell survival (positive selection) and cell death (negative selection or death by neglect). Although the CD28 costimulatory molecule has been implicated in this process, it has been difficult to establish a role for the other major costimulatory molecule, cytotoxic T lymphocyte antigen (CTLA)-4. Here we report that in vivo stimulation through the T cell receptor (TCR)–CD3 complex induces expression of CTLA-4 in thymocytes and leads to the association of CTLA-4 with the SH2 domain–containing phosphatase (SHP)-2 tyrosine phosphatase. Moreover, intrathymic CTLA-4 blockade dramatically inhibits anti-CD3–mediated depletion of CD4⁺CD8⁺ double positive immature thymocytes. Similarly, anti-CD3–mediated depletion of CD4⁺CD8⁺ double positive cells in fetal thymic organ cultures could also be inhibited by anti–CTLA-4 antibodies. Thus, our data provide evidence for a role of CTLA-4 in thymic selection and suggest a novel mechanism contributing to the regulation of TCR-mediated selection of T cell repertoires.