Thymic differentiation of TCRαβ+ CD8αα+ IELs

Summary:  Intraepithelial lymphocytes (IELs) contain several subsets, but the origin of the T-cell receptor (TCR)αβ⁺ CD8αα⁺ IELs has been particularly controversial. Here we provide a synthesis, based on recent work, that attempts to unify the divergent views. The intestine has a primordial function in lymphopoiesis, and precursors with the potential to differentiate into T cells are found both in the epithelium and underlying lamina propria. Moreover, the thymus has been reported to export cells to the intestine that are not fully differentiated. TCRαβ⁺ CD8αα⁺ IELs can differentiate in the intestine from each of these sources, but in normal euthymic mice, the thymus appears to be the major source for TCRαβ⁺ CD8αα⁺ IELs. This unique IEL subset is a self-reactive population that requires exposure to self-agonists for selection in the thymus, similar to other regulatory T-cell populations. IELs transition through a double-positive (DP) intermediate in the thymus, but they originate from a subset of the DP cells that can be identified by its expression of CD8αα homodimers. The agonist-selected cells in the thymus are TCRβ⁺ but CD4 and CD8 double negative. The evidence suggests that reacquired expression of CD8αα and downregulation of CD5 occur after thymus export, perhaps in the intestine under the influence of interleukin-15. As a result of agonist exposure, a new gene expression program is activated. Therefore, the increased understanding of the developmental origin of TCRαβ⁺ CD8αα⁺ IELs may help us to understand how they participate in immune regulation and protection in the intestine.     

Curriculum vitae of intestinal intraepithelial T cells: their developmental and behavioral characteristics

Summary:  The alimentary tract has an epithelial layer, consisting mainly of intestinal epithelial cells (IECs), that is exposed to the exterior world through the intestinal lumen. The IEC layer contains many intestinal intraepithelial T cells (IELs), and the total number of IELs constitutes the largest population in the peripheral T-cell pool. Virtually all γδ-IELs and many αβ-IELs in the mouse small intestine are known to express CD8αα homodimers. A wide range of evidence that supports extrathymic development of these CD8αα⁺ IELs has been collected. In addition, while several studies identified cells with precursor T-cell phenotypes within the gut epithelium, how these precursors, which are dispersed along the length of the intestine, develop into γδ-IELs and/or αβ-IELs has not been clarified. The identification of lymphoid cell aggregations named 'cryptopatches' (CPs) in the intestinal crypt lamina propria of mice as sites rich in T-cell precursors in 1996 by our research group, however, provided evidence for a central site, whereby precursor IELs could give rise to T-cell receptor-bearing IELs. In this review, we discuss the development of IELs in the intestinal mucosa and examine the possibility that CPs serve as a production site of extrathymic IELs.     

Crucial function of the pre-T-cell receptor (TCR) in TCRP selection, TCRβ allelic exclusion and αβ versus γδ lineage commitment

Summary: The analysis of T-cell receptor (TCR) βselection, TCRβ allelic exclusion and TCRβ rearrangement in γδ T cells from normal and pre-TCR-deficient mice has shown that the pre-TCR has a crucial role in T-lyinpbocyte development:

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  The pre-TCR is by far the most effective receptor that generates large numbers of CD4⁺8⁺ T cells with productive TCRβ rearrangements.
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  In the absence of the pre-TCR, TCRβ rearrangement proceeds in developing cells irrespective of whether they already contain a productive TCRβ gene.
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  The pre-TCR directs developing T cells to the αβ lineage because y5 T cells from pTα^-/- mice proceed much further in TCRβ rearrangement than γδ T cells from wild-type mice. It is argued that the pre-TCR commits developing T cells to the αβ lineage by an instructive mechanism, which has largely replaced an evolutionarily more ancient mechanism that involves stochastic αβ lineage commitment.

     

CD8alpha+beta(low) effector T cells in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by the loss of self-tolerance to nuclear antigens. Aberrant T-cell function plays a central role in lupus pathogenesis. We and others previously demonstrated that peripheral TCRαβ⁺CD3⁺ T cells express CD8β either at a high (CD8β^high) or low density (CD8β^low), thereby defining two functionally distinct subsets. CD8β^low T cells express predominantly CD8αα and less CD8αβ as a coreceptor, display a differentiated phenotype and exert effector function. CD8β^high T cells appear to be the precursors expressing predominantly the heterodimeric efficient CD8αβ coreceptor, exhibiting a naïve phenotype and high proliferative capacity. In the present study, the distribution and functional properties of CD8β^high and CD8β^low T cells of SLE patients were compared ( n  = 20) with those of healthy subjects ( n  = 16). It was found that expansion of CD8β^low T-cell subset correlated with disease activity indicating chronic antigenic stimulation leading to a major lack of naïve CD8β^high precursor T cells in SLE. Functional characteristics of CD8β^low T cells including production of cytokines and cytotoxic granules were not significantly different between patients with SLE and healthy individuals. We speculate that unbalanced CD8β^high/CD8β^low T-cell relation reflects a skewed homeostasis within the CD8⁺ T-cell compartment towards fully differentiated effector T cells possibly due to persistent antigen stimulation in SLE.     

Generation of gut-homing T cells and their localization to the small intestinal mucosa

Summary:  The intestinal mucosa represents the largest body surface toward the external environment and harbors numerous T lymphocytes that take up resident within the intestinal epithelium or in the underlying lamina propria (LP). The intraepithelial lymphocytes include subsets of 'unconventional' T cells with unclear ontogeny and reactivity that localize to this site independently of antigen-specific activation in secondary lymphoid organs. In contrast, the majority of the 'conventional' gut T cells are recruited into the intestinal mucosa subsequent to their activation in intestinal inductive sites, including Peyer's patches (PPs) and mesenteric lymph nodes (MLNs). T cells homing to the small intestine express a distinct pattern of homing molecules, allowing them to interact with and transmigrate across intestinal postcapillary endothelium. At least some of these homing molecules, including the integrin α₄β₇ and the chemokine receptor CCR9, are induced on T cells during their activation in PPs or MLNs. Mucosal dendritic cells (DCs) play a key role in this process, but not all intestinal DCs possess the ability to confer a gut-homing capacity to T cells. Instead, functionally specialized CD103⁺ DCs derived from the small intestinal LP appear to selectively regulate T-cell homing to the small intestine.     

Lamina Propria T Cell Subsets in the Small and Large Intestine of Euthymic and Athymic Mice

We investigated lamina propria T cells from the small intestine (jejunum/ileum) and the large intestine (colon) of euthymic (BALB/c, C. B-17, C57BL/6) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/ xid) mice. CD3⁺ T cells represented about 40% of the lamina propria lymphocytes (LPL) from the small or the large intestine of euthymic mice, and 20–30% of the LPL populations from the small or large intestine of athymic mice. In the lamina propria T cell population of the small intestine, 85% were of the αβ lineage in euthymic mice, but only 40% were of the αβ lineage in athymic mice. T cells of the αβ lineage were thus more frequent than T cells of the αβ lineage in the intestinal lamina propria T cells of extrathymic origin. CD4⁺ T cells represented 40% of the lamina propria T cells in the small as well as in the large intestine of euthymic mice, and 20–30% of the T cells in the lamina propria of the nude mouse gut. In euthymic mice, 40% of the T cells in the small intestine lamina propria, and 30% of the T cells in the colonic lamina propria were CD8⁺, In intestinal lamina propria T cell populations of athymic mice, the CD8⁺ T cell population was expanded. Most (60–70%) CD8+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice expressed the homodimeric CD8α⁺β^? form of the CD8 coreceptor. A fraction of 15–20% of all CD3+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice were ‘double negative’ CD4^? CD8^?. A large fraction of the TCRαβ⁺ T cells in the colonic lamina propria (but not in the small intestine lamina propria) of euthymic mice expressed the CD2 and the CD28 costimulator molecules, the adhesion molecule LECAM-1 (CD62 L), and could be activated in vitro by CD3 ligation. These data reveal a considerable heterogeneity in the surface phenotype and the functional phenotype of murine lamina propria T cells.     

γδ T cells: firefighters or fire boosters in the front lines of inflammatory responses

Summary:  Intradermal inoculation of cloned self-reactive αβ T cells into the footpads of mice induced cutaneous graft-versus-host disease (GVHD), and after recovery from GVHD, the epidermis became resistant to subsequent attempts to induce GVHD. Resistance to GVHD was not induced in the epidermis of T-cell receptor δ-deficient (TCRδ^−/−) mice that lacked γδ T cells bearing canonical Vγ5/Vδ1⁺γδTCRs, known as dendritic epidermal T cells (DETCs), and resistance was restored by reconstitution of these mutant mice with precursors of Vγ5⁺ DETCs. Pulmonary infection by Cryptococcus neoformans induced an increase of γδ T cells in the lung, and in comparison with wildtype mice, TCRδ^−/− mice eliminated C. neoformans more rapidly and synthesized more interferon-γ in the lung. In the mouse small intestine, the absence of γδ T cells is associated with a reduction in epithelial cell turnover and downregulation of the expression of major histocompatibility complex class II molecules. The protective role of γδ T cells was verified in a dextran sodium sulfate-induced inflammatory bowel disease (IBD) model, whereas in a spontaneous model of IBD, γδ T cells were involved in the exacerbation of colitis in TCRα^−/− mice. Taken together, in addition to the homeostatic regulation of epithelial tissues, γδ T cells appear to play a pivotal role in the modification of inflammatory responses induced in many organs containing epithelia.     

Expression of α5 (CD49e) and α6 (CD49f) Integrin Subunits on T Cells in the Circulation and the Lamina Propria of Normal and Inflammatory Bowel Disease Colonic Mucosa

Intestinal lamina propria T cells are believed to be derived, via the systemic circulation, from gut-associated lymphoid tissue. After migration into the lamina propria, T cells are capable of luminally directed migration following the loss of surface epithelial cells. For adhesion and migration within the extracellular matrix, T cells are likely to utilize the integrin family of adhesion molecules. The aim of this study was to quantitatively and qualitatively investigate the expression of α₅ and α₆ integrin subunits on the surface of human T cells that: (a) migrated out of the lamina propria, (b) remained resident within the matrix and (c) were present in the circulation. In both subpopulations of CD4 and CD8-positive T cells, from both normal and inflamed (inflammatory bowel disease) colonic mucosa, there were significantly fewer α₅ and α₆-positive cells than in the peripheral blood. In addition, there were significantly fewer α₆ integrin molecules on the surface of CD4 and CD8-positive lamina propria T-cell subpopulations, compared with those in the circulation. Our studies suggest that, following migration into the lamina propria, there is down-regulation of α₅ and α₆ integrin-subunit expression on the surface of T cells. Molecules other than members of very late activation antigen-5 (VLA-5) (α₅β₁) and VLA-6 (α₆β₁) families of adhesion molecules are likely to be important in interactions with extracellular components in the lamina propria of normal and inflamed human colonic mucosa.     

Variation in Gut-Homing CD27-Negative Lymphocytes in Inflammatory Colon Disease

Prolonged antigenic stimulation results in lymphocyte shedding of CD27, a member of the tumour necrosis factor receptor (TNFR) family, and transformation to a stable phenotype capable of synthesizing interleukin-4 (IL-4). Co-expression of α4β7 identifies those cells with gut-homing potential. We have investigated these cell populations in patients with inflammatory colonic disease. Circulating and lamina propria mononuclear cells were isolated from patients with Crohn's disease (CD), ulcerative colitis (UC), non-inflammatory bowel disease (non-IBD) colonic inflammation and healthy controls. Double and triple colour flow cytometry for CD3, CD4, CD27, α4β7 and intracellular cytokines was performed. Circulating CD4+CD27– populations were increased in patients with CD (8.8 ± 0.8%, P  < 0.001), UC (12.2 ± 1.9%, P  < 0.001) and non-IBD colitis (10.5 ± 1.3%, P  < 0.01) as compared with controls (6.1 ± 0.5%). CD4⁺CD27⁻α4β7⁺ cells were increased in CD ( P  < 0.01). Lamina propria CD4⁺CD27⁻ populations were depressed significantly in CD ( P  < 0.05), UC ( P  < 0.02) and non-IBD colitis ( P  < 0.03). Mucosal CD4⁺CD27⁻ cells synthesized IL-4 in preference to interferon-γ. Thus, colonic inflammation is associated with alterations in gut-tropic circulating and mucosal populations of differentiated memory T cells with the phenotype of predominantly IL-4-synthesizing cells.     

Vδ1+ Gamma/Delta T Lymphocytes Infiltrating Human Lung Cancer Express the CD8α/α Homodimer

Murine γ/δ T lymphocytes localize to different epithelial tissues and are phenotypically distinct from peripheral γ/δ T cell-populations in that they show limited TCR diversity, express the CD8 α/α homodimer and lack the CD8β chain. In humans, a compartmentalization of γ/δ cells sharing similar phenotypic features has been documented to date only in the case of intestinal epithelium. In the present study we show that about half of Vδ1⁺ (as well as Vδ1⁻Vδ2⁻) γ/δ lymphocytes, which can be selectively expanded from human lung cancers, coexpress the CD8α/α homodimer. The accumulation of intraepithelial CD8⁺γ/δ⁺ lymphocytes might then be a more general phenomenon, possibly as a result of common mechanisms operating at those sites.     

Regulatory T cells in spontaneous autoimmune encephalomyelitis

Summary: Spontaneous experimental autoimmune encephalomyelitis (EAE) develops in 100% of mice harboring a monoclonal myelin basic protein (MBP)-specific CD4⁺αβ T-cell repertoire. Monoclonality of the αβ T-cell repertoire can be achieved by crossing MBP-specific T-cell receptor (TCR) transgenic mice with either RAG^−/− mice or TCR α^−/−/TCR β^−/− double knockout mice. Spontaneous EAE can be prevented by a single administration of purified CD4⁺ splenocytes or thymocytes obtained from wild-type syngeneic mice. The regulatory T cells (T-reg) that protect from spontaneous EAE need not express the CD25 marker, as effective protection can be attained with populations depleted of CD25⁺ T cells. Although the specificity of the regulatory T cells is important for their generation or regulatory function, T cells that protect from spontaneous EAE can have a diverse TCR α and β chain composition. T-reg cells expand poorly in vivo , and appear to be long lived. Finally, precursors for T-reg are present in fetal liver as well as in the bone marrow of aging mice. We propose that protection of healthy individuals from autoimmune diseases involves several layers of regulation, which consist of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁻ T-reg cells, and anti-TCR T cells, with each layer potentially operating at different stages of T-helper cell-mediated immune responses.     

γδ T-Cell Precursor-Derived CD4− CD8−αβ T Cells Retain γδ Cell Function

We have previously shown that some of the DNαβ⁺ T cells arising in TcRα-chain transgenic mice are of γδ T cell origin, based on phenotypic data and on their status of TcR gene rearrangements. In the present report we investigated the impact of αβ TcR expression on the functional programme of the mature γδ precursor-derived DNαβ⁺ T cells. Our results demonstrate that both their proliferative capacity and their cytokine production profile are similar to that of γδ T cells. Furthermore, both transgenic DNαβ⁺ T cells and DNγδ⁺ T cells up-regulate CD8α expression after activation, but, in contrast to CD4⁺αβ T cells, are unable to induce proliferation of naive B cells. Thus, our results suggest that the effector functions of mature T cells are determined independently of the TcR isotype, probably at an early stage of differentiation, and thereby have important implications for current models of T-cell lineage commitment.     

Human Intestinal B-Cell Blasts and Plasma Cells Express the Mucosal Homing Receptor Integrin α4β7

Interactions between homing receptors on circulating leucocytes and endothelial addressins regulate tissue-specific cellular extravasation. Although integrin á4β7 appears to be the main receptor for guthoming T lymphocytes, less is known about molecules mediating mucosal B cell homing. Expression of integrin α4β7 on B lymphocytes, B cell blasts, and plasma cells in human gut-associated lymphoid tissue (GALT; the Peyer's patches and appendix) and lamina propria was studied by multi-colour immunofluorescence applied on cryosections. Isolated mononuclear cells from the same tissue compartments were examined by flow cytometry and compared with peripheral blood B cells. Integrin α4β7 was expressed by IgA⁺ B cell blasts and plasma cells (CD38^high) in the lamina propria, B cell blasts in GALT, and sIgD⁺ B lymphocytes in peripheral blood. In contrast, GALT sIgD⁺ B lymphocytes were negative or only weakly positive for α4β7. These results suggested that B lymphocytes down-regulate αAβ7 upon extravasation in GALT but up-regulate this integrin after antigen-priming. Thus, α4β7 may be a homing receptor also for B cell blasts extravasating in the gut lamina propria, where this integrin is maintained on plasma cells, perhaps as a local retention factor.     

γδ intraepithelial lymphocytes drive tumor necrosis factor-α responsiveness to intestinal iron challenge: relevance to hemochromatosis

Summary: The dependence of intestinal epithelial cell (IEC) growth and differentiation on intraepithelial lymphocytes (IELs) expressing the gamma/delta (γδ) T-cell receptor (TCR), suggested a potential role for γδ⁺ IELs in the regulation of iron absorption. We therefore examined the levels of hepatic iron and the IEL cytokine responses in C57BL/6J control and class I and TCR knockout lines (placed on a C57BL/6J genetic background) following the administration of supplemental dietary iron. The highest level of liver iron was found in the β2-microglobulin knockout (β2m^-/-) mice followed by the TCR-δ knockout (TCRδ^-/-) animals. TCR-α knockout (TCRα^-/-) and control animals did not differ in their iron levels. Liver iron loading correlated inversely with rhe ability of the mice to generate an IEL tumor necrosis factor (TNE)-α response. These observations suggest a model in which IEC iron loading is communicated to IELs via the HFE class I protein. The result of this communication is the initiation of TNE-α release by γδ⁺ IELs (sustained by macrophages and dendritic cells) contributing to the upregulation of ferritin expression and possibly to the normal maintenance of the IEC apoptotic pathway.     

Vβ Usage and T Regulatory Cells in Children Following Partial or Total Thymectomy after Open Heart Surgery in Infancy

During open heart surgery in infants the thymus was usually removed, partly or completely. Our previous studies on 16 such children indicated reduced T-cell output later in life with signs of extrathymic maturation of the T cells, but no reduction in T regulatory cells (CD4⁺CD25⁺). The diversity of the T-cell repertoire in these children was examined to test if the extrathymic microenvironment could alter Vβ usage. The expression of Foxp3 and CD127 in CD4⁺CD25^high T cells was measured in order to determine whether the T regulatory cells had the phenotype of natural T regulatory cells. There was a wide distribution of Vβ usage in both study and control groups. Significant variability was found in Vβ usage for CD4⁺ and CD8⁺ T cells when the distribution of the percentage of T cells expressing each Vβ family was analysed between individuals within each group ( P  < 0.001; Kruskal–Wallis). Significant difference was also found in average usage of Vβ2, Vβ5.1 and Vβ14 chains within CD4⁺ T cells and Vβ2, Vβ8 and Vβ21.3 chains within CD8⁺ cells between the groups ( P  < 0.05; Student's t -test). There was no difference between the two groups with regard to the proportion of CD4⁺CD25^high T cells and no difference in the average expression of Foxp3 or CD127 within the CD4⁺CD25^high population. Our data provide evidence that cardiothoracic surgery in infants and total or partial thymectomy alters Vβ usage, suggesting more limited selection in such children than in the control group. The frequency of natural T regulatory cells seems to be unimpaired.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Rheumatoid Inflammatory T-Cell Clones express mostly Th1 but also Th2 and Mixed (Th0-Like) Cytokine Patterns

This study was performed in order to characterize whether T cells from rheumatoid synovial inflammation belong to the Th1- or Th2-like functional subsets. Cytokine production was studied in 26 CD4⁺αβ⁺ and 2 CD8⁺αβ T-cell clones from the synovial fluid, the synovial membrane and peripheral blood of 5 patients. Fifteen of the CD4⁺ clones were raised against various mycobacterial antigens and 11 CD4⁺ clones and 2 CD8⁺ clones were raised unspecifically using PHA and/or IL-2. The specificities of these clones are not known. In the mycobacterial antigen-specific group, all CD4⁺'αβ T-cell clones produced IFN-γ at high levels, while the production of IL-4 was generally absent or low (< 1 ng/ml), consistent with a Thl-like profile. Some of these clones, however, also produced various amounts of IL-10 which has been regarded as a Th2 product but can be produced also in lower amounts by Thi cells. One HSP-65-specific clone produced levels of IL-4 and IL-10 in the same order as that of IFN-γ, thus appearing to be Th0-like. Among the 11 unspecific CD4⁺ clones, 7 showed a Thl-like pattern but with lower levels of IFN-γ than the antigen-specific clones. However, three clones did not produce any IFN-γ activity but produced IL-4 and one of them also produced distinct amounts of IL-10, compatible with a Th2-like pattern. In addition, one of the clones also showed an almost equally strong IFN-γ and IL-4 production, thus most likely representing a Th0-like clone.