CD8 Alpha Research Articles

Practical aspects of immunocytochemistry in canine lymphomas

The aim of this study was to evaluate the utility of immunocytochemistry in a standard veterinary practice and to determine the immunophenotype of tumor cells in cases of multicentric lymphoma in dogs by immunocytochemical analysis of fine-needle biopsy specimens. The study was performed on cytological samples collected from 54 dogs, in which multicentric lymphoma was recognised based on clinical data, cytology or cytology and histology, and follow-up information. Diagnosis of lymphoma was established according to the updated Kiel classification. Immunocytochemical assays were conducted using commercially available antibodies to the pan T-lymphocyte marker CD3 and B cell antigen receptor complex CD79 alpha. Among all animals examined B cell lymphoma was recognized in 42/54 (77.8%) of cases, while in the remaining 12/54 (22.2%) of dogs T cell lymphoma was recognized. In 11 animals with lymphoma recognized cytologically, in which an entire lymph node was obtained for histology, the results of routine cytology and immunocytochemistry fully corresponded with findings revealed by histology and immunohistochemistry. Immunocytochemistry can be successfully conducted in smears stored at room temperature for 24 hours without changes of staining results. It can be stated that application of standard cytopathological assessment in connection with immunocytochemistry of lymph nodes samples collected from dogs with lymphoma is a method of choice for establishing final diagnosis, and avoids the need for reexamination or collection of tissue samples for histopathology and immunohistochemistry during surgical procedures in ambiguous cases.

Subsets of migrating intestinal dendritic cells

Dendritic cells (DCs) in the intestine are heterogeneous. Phenotypically different populations of conventional DCs have been identified in the intestinal lamina propria, Peyer's patches, and in the draining mesenteric lymph nodes, to which these DCs constitutively migrate. Markers used to identify these populations include major histocompatibility complex class II, CD11c, CD8 alpha, CD11b, and CD103. Extensive studies in rats, summarized here, which involved collection of migrating DCs by thoracic duct cannulation after mesenteric lymphadenectomy, have clearly demonstrated that the subsets of migrating intestinal lymph DCs have different functional properties. The subsets might play different roles in the induction of oral tolerance and in driving systemic immune responses after vaccination or intestinal stimulation with Toll-like receptor ligands. The use of these surgical techniques allows investigation of the functions of purified subsets of migrating DCs. However, in the rat, these studies are limited by the range of available reagents and are difficult to compare with data from other species in this fast-moving field. Recent refinements have enabled the collection of migrating intestinal DCs from mice; our initial results are described here. We believe that these studies will generate exciting data and have the potential to resolve important questions about the functions of migrating intestinal DC subsets.

Langerin‐expressing dendritic cells in gut‐associated lymphoid tissues

Dendritic cells (DCs) are key regulators of the immune system. They act as professional antigen-presenting cells and are capable of activating naive T cells and stimulating the growth and differentiation of B cells. According to their molecular expression, DCs can be divided into several subsets with different functions. We focus on DC subsets expressing langerin, a C-type lectin. Langerin expression is predominant in skin DCs, but langerin-expressing DCs also exist in mucosal tissue and can be induced by immunization and sometimes by nutrient deficiency. Topical transcutaneous immunization induces langerin(+)CD8 alpha(-) DCs in mesenteric lymph nodes (MLNs), which mediate the production of antigen-specific immunoglobulin A antibody in the intestine. Yet, in one recent study, langerin(+) DCs were generated in gut-associated lymphoid tissue and contributed to the suppressive intestinal immune environment in the absence of retinoic acid. In this review, we focus on the phenotypic and functional characteristics of langerin(+) DCs in the mucosal tissues, especially MLNs.

The CD8+ dendritic cell subset

Mouse lymphoid tissues contain a subset of dendritic cells (DCs) expressing CD8 alpha together with a pattern of other surface molecules that distinguishes them from other DCs. These molecules include particular Toll-like receptor and C-type lectin pattern recognition receptors. A similar DC subset, although lacking CD8 expression, exists in humans. The mouse CD8(+) DCs are non-migrating resident DCs derived from a precursor, distinct from monocytes, that continuously seeds the lymphoid organs from bone marrow. They differ in several key functions from their CD8(-) DC neighbors. They efficiently cross-present exogenous cell-bound and soluble antigens on major histocompatibility complex class I. On activation, they are major producers of interleukin-12 and stimulate inflammatory responses. In steady state, they have immune regulatory properties and help maintain tolerance to self-tissues. During infection with intracellular pathogens, they become major presenters of pathogen antigens, promoting CD8(+) T-cell responses to the invading pathogens. Targeting vaccine antigens to the CD8(+) DCs has proved an effective way to induce cytotoxic T lymphocytes and antibody responses.

Single Cell Profiling of B Cell Receptor Signaling and Apoptosis Networks in Chronic Lymphocytic Leukemia: Association with in Vitro Sensitivity to Fludarabine Monophosphate (F-ara-A).

Abstract 1263Poster Board I-285 BackgroundIn conjunction with antigen-driven responses, ligand-independent signaling (termed tonic signaling) through both the pre-B cell receptor and B-cell receptor has an important role in B cell development, maturation and survival. In addition to the recognized role of CD79 alpha and CD79 beta BCR signaling, tyrosine phosphatases can impact tonic BCR signaling (Wienands et al. PNAS, 93 p.7865 (1996), Monroe Nat. Rev. Immunol. 6 p.283 (2006)). We previously subjected chronic lymphocytic leukemia (CLL) cells with modulators of BCR signaling and monitored their responses using flow cytometry-based Single Cell Network Profiling (SCNP). Of the many signaling modulators studied, hydrogen peroxide treatment (a general inhibitor of tyrosine phosphatase activity) augmented BCR signaling in a subset of CLL patient samples evaluated. In the remaining samples there was an apparent lack of response to hydrogen peroxide. These data suggested that differential phosphatase activity proximal to BCR signaling was driving the biology of these two patient groups. ObjectivesStudies were designed to evaluate whether there were any associations between tonic and/or ligand-dependent BCR signaling and in vitro sensitivity to fludarabine, as well as whether such response profiles showed a relationship to the hydrogen peroxide-dependent signaling we observed previously. Methods23 CLL samples and 7 healthy PBMCs were treated with anti-m alone, hydrogen peroxide alone or the combination for 10 minutes. Separate aliquots of the same sample were exposed to F-ara-A for 48 hours. SCNP was carried out on gated B cells with quantitation of single cell measures of intracellular phosphorylated kinases and adaptor proteins downstream of the BCR. Additionally, the relative activation status of several protein markers of the apoptotic cascade (cytoplasmic cytochrome C, cleaved caspase 3, and cleaved PARP) was measured. ResultsAs previously observed, CLL samples could be segregated into one of two groups exhibiting either responsive or refractory signaling after exposure to hydrogen peroxide alone. Moreover, responsive signaling in CLL cells was correlated in that all the measured components of the canonical B cell receptor network (p-Lyn, p-Syk, p-BLNK, p-PLC-gamma-2, p-Erk and p-Akt) showed the same phosphorylation response: either augmented in unison, or not activated at all. In vitro F-ara-A treatment (48 hours in the presence of 1mM F-ara-A) of parallel samples from these same CLL patients identified distinct populations of apoptosis responsive and refractory cells. Surprisingly, the capacity of patient samples to show augmented BCR signaling in response to hydrogen peroxide was associated prominently with the ability of cells in these patients to exhibit apoptotic proficiency to F-ara-A in vitro. This implies a link between mechanisms governing apoptosis in these CLL cells, survival pathways, and cell states that govern the role of phosphatase activity and BCR signaling potential. ConclusionsThis study reveals a link between tonic BCR signaling and regulation of apoptosis pathways. This suggests that the subgroup of CLL patients with active phosphatase activity (which suppresses BCR responses) have cell populations that are responsive to F-ara-A, a standard drug in CLL therapy. Conversely, the presence of CLL cells in a patient sample that remain unresponsive to hydrogen peroxide repression of phosphatase activity appear to identify patient samples which cannot undergo apoptosis in response to in vitro F-Ara-A exposure. The clinical implications of this work will be the focus of future translational studies. DisclosuresEvensen:Nodality Inc.: Employment, Equity Ownership. Palazzo:Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership. Fantl:Nodality, Inc.: Employment, Equity Ownership.

T Helper 17 Cells Promote Cytotoxic T Cell Activation in Tumor Immunity

Although T helper 17 (Th17) cells have been found in tumor tissues, their function in cancer immunity is unclear. We found that interleukin-17A (IL-17A)-deficient mice were more susceptible to developing lung melanoma. Conversely, adoptive T cell therapy with tumor-specific Th17 cells prevented tumor development. Importantly, the Th17 cells retained their cytokine signature and exhibited stronger therapeutic efficacy than Th1 cells. Unexpectedly, therapy using Th17 cells elicited a remarkable activation of tumor-specific CD8(+) T cells, which were necessary for the antitumor effect. Th17 cells promoted dendritic cell recruitment into the tumor tissues and in draining lymph nodes increased CD8 alpha(+) dendritic cells containing tumor material. Moreover, Th17 cells promoted CCL20 chemokine production by tumor tissues, and tumor-bearing CCR6-deficient mice did not respond to Th17 cell therapy. Thus, Th17 cells elicited a protective inflammation that promotes the activation of tumor-specific CD8(+) T cells. These findings have important implications in antitumor immunotherapies.

Flt3-L Increases CD4+CD25+Foxp3+ICOS+Cells in the Lungs of Cockroach-Sensitized and -Challenged Mice

We previously reported in an ovalbumin-induced model of allergic asthma that Fms-like tyrosine kinase 3 ligand (Flt3-L) reversed airway hyperresponsiveness (AHR) and airway inflammation, and increased the number of regulatory CD11c(high)CD8 alpha(high)CD11b(low) dendritic cells in the lung. In this study, we investigated the effect of Flt3-L in a clinically relevant aeroallergen-induced asthma on the phenotypic expression of lung T cells. Balb/c mice were sensitized and challenged with cockroach antigen (CRA), and AHR to methacholine was established. These mice received three intraperitoneal injections of anti-CD25 antibody (PC61; 250 microg) and Flt3-L (3 microg) daily for 10 days. Cytokines and Ig levels in the serum were measured and differential bronchoalveolar lavage fluid (BALF) cell counts were examined. Flt3-L reversed AHR to methacholine to the control level. Flt3-L significantly decreased levels of BALF IL-5, IFN-gamma, eosinophilia and substantially increased IL-10 and the number of CD4(+)CD25(+) Forkhead winged helix transcription factor box P3 (Foxp3(+)) IL-10(+) T cells in the lung. Administration of PC61 antibody blocked the effect of Flt3-L and substantially increased AHR, eosinophilia, and BALF IL-5 and IFN-gamma levels, and decreased BALF IL-10 levels and the number of CD4(+)CD25(+)Foxp3(+)IL-10(+) T cells. Flt3-L significantly decreased CD62-L, but increased inducible costimulatory molecule and Foxp3 mRNA expression in the CD4(+)CD25(+) T cells isolated from lungs of Flt3-L-treated, CRA-sensitized mice compared to CRA-sensitized mice without Flt3-L treatment and PBS control group. Flt3-L significantly inhibited the effect of CRA sensitization and challenge to increase GATA3 expression in lung CD4(+)CD25(+) T cells. Collectively, these data suggest that the therapeutic effect of Flt3-L is mediated by increased density of naturally occurring CD4(+)CD25(+)Foxp3(+)IL-10(+)ICOS(+) T-regulatory cells in the lung. Flt3-L could be a therapeutic strategy for the management and prevention of allergic asthma.

Anti-CD28 antibody- and IL-4-induced human T cell proliferation is sensitive to rapamycin.

Rapamycin (RAPA) is a potent immunosuppressant. In this study we investigated the effect of RAPA on T cell proliferation triggered by various stimuli in an in vitro human model. The proliferation of T cells stimulated via an alternative pathway using phorbol myristate acetate (PMA) and anti-CD28 antibody (alpha CD28) in the absence of antigen-presenting cells (APC) was strongly inhibited by RAPA. T cell proliferation provoked via a combination of CD3/TCR and CD28 pathways using anti-CD3 antibody (alpha CD3) plus alpha CD28 was also inhibited by RAPA in the presence of APC. The mitogen (phytohaemagglutinin (PHA) or alpha CD3)-induced up-regulation of expression of the IL-2 receptor alpha chain (IL-2R alpha) and the IL-4 receptor (IL-4R) was sensitive to RAPA. This suggests that RAPA's interference with the IL-2 and IL-4 autocrine loops during T cell activation might contribute to RAPA's overall immunosuppressive effect. We have further demonstrated in a two-stage culture system that RAPA strongly inhibited IL-4-stimulated proliferation of T cells, the latter being either pretreated with alpha CD3 in the presence of APC, or with PMA plus alpha CD28 in the absence of APC. The result suggests that the Ca++ influx during the pretreatment is not obligatory for T cells to achieve IL-4 responsiveness. The results also indicate that RAPA's antiproliferative effect on IL-4-stimulated T cells is not contingent on the various mechanisms of cell priming. Therefore, RAPA's major target is probably at the second stage after the priming. Our study has extended current knowledge about the effect of RAPA on human T cells.

Trans‐presentation of IL‐15 by intestinal epithelial cells drives development of CD8 alpha alpha T cells

IL‐15, which is trans‐presented by IL‐15Rα+ cells to neighboring IL‐2Rβ/γC+ cells, is crucial for the development of intestinal intraepithelial lymphocytes (IEL). Parenchymal cells have a major role in the trans‐presentation of IL‐15 to IELs; however, the specific identity of this cell type is unknown. Since trans‐presentation requires cell‐cell contact, we hypothesize that intestinal epithelial cells (IEC) are the parenchymal cell trans‐presenting IL‐15 to IELs. To determine whether IECs trans‐present IL‐15 to developing IELs, we developed a mouse model with IL‐15Rα expressed exclusively by the IECs. This model was generated by driving IL‐15Rα under the Villin promoter and crossing to the IL‐15Rα−/− background. Analysis of these transgenic mice detected high levels of IL‐15Rα by the IECs. Among the IELs, Thy1‐ CD8αα TCRαβ and TCRγδ subsets were restored to normal or higher levels as compared to control and IL‐15Rα−/− mice. The increases in cell number did not appear to be the result of cell expansion as no differences in proliferation rate was detected among transgenic, control, or IL‐15Rα−/− mice. Collectively, this study demonstrates that IL‐15Rα expression by IECs alone is completely sufficient to direct all the IL‐15‐mediated events driving the development of CD8αα IELs. This research is supported by NIH grant AI070910 and the MDACC Trust Fellowship.

In vivo transformation of mouse conventional CD8α+ dendritic cells leads to progressive multisystem histiocytosis

Division and proliferation of dendritic cells (DCs) have been proposed to contribute to homeostasis and to prolonged antigen presentation. Whether abnormal proliferation of dendritic cells causes Langerhans cell histiocytosis (LCH) is a highly debated topic. Transgenic expression of simian virus 40 (SV40) T antigens in mature DCs allowed their transformation in vivo while maintaining their phenotype, function, and maturation capacity. The transformed cells were differentiated splenic CD8 alpha-positive conventional dendritic cells with increased Langerin expression. Their selective transformation was correlated with higher steady-state cycling compared with CD8 alpha-negative DCs in wild-type and transgenic mice. Mice developed a DC disease involving the spleen, liver, bone marrow, thymus, and mesenteric lymph node. Surprisingly, lesions displayed key immunohistologic features of Langerhans cell histiocytosis, including expression of Langerin and absence of the abnormal mitoses observed in Langerhans cell sarcomas. Our results demonstrate that a transgenic mouse model with striking similarities to aggressive forms of multisystem histiocytosis, such as the Letterer-Siwe syndrome, can be obtained by transformation of conventional DCs. These findings suggest that conventional DCs may cause some human multisystem LCH. They can reveal shared molecular pathways for human histiocytosis between humans and mice.

Changes in thymus size, cellularity and relation between thymocyte subpopulations in young adult rats induced by Somatostatin-14

The role of somatostatin on inhibition of both normal and tumor cell cycle, secretion of endocrine and exocrine cells, as well as induction apoptosis is well documented. However, its effect on T cell development and thymic structure is not fully clarified. In order to investigate the influence of somatostatin in vivo on the thymus structure and T cell development, the young adult Albino Oxford male rats were intracerebroventriculary treated with somatostatin-14. We examined the thymus compartments and its cellularity, through assessment of morphometric parameters by stereological method, and the relation between thymocytes subpopulations, over expression of CD4, CD8 and T-cell receptor (TCR) alpha beta by flow cytometry. Additionally, we also determined the body and thymus weight of the rats, during the first three months of life, to define the time of SRIH-14 application. A decrease of relative thymus weight from the fourth weeks of postnatal life, and an unchanged relative thymus weight obtained in treated group indicates that SRIH-14 in young adult rats inhibits growth of whole organism, not only thymus. The changes in the absolute number and numerical density of cortical thymocytes indicate that SRIH-14 alters the true lymphoid tissue. SRIH-14 changes relation between thymocyte subsets, increase number of CD4(-)CD8(-)TCR alpha beta(-) and CD4(-)CD8(+)TCR alpha beta(hi) thymocyte subsets as well as the CD4(-)CD8(-)TCR alpha beta(low/hi) thymocytes, while decrease number of CD4(+)CD8(+) TCR alpha beta(-/low/hi) thymocyte subsets. These results indicate that somatostatin-14 is not involved in the control of the physiologic involution of the thymus, although induces thymic weight loss through the reduction of true lymphoid tissue. In addition, changes in frequency of thymocyte subpopulations, especially immature cells, indicate that SRIH-14 modulates thymocytes development and maturation.

Ethanol Consumption Modifies Dendritic Cell Antigen Presentation in Mice

Alcohol consumption impairs type 1 cell-mediated adaptive immune responses both in vivo and in vitro. The present study investigated the effect of alcohol consumption on antigen-presenting cell (APC) populations and cytokine production. BALB/c were fed ethanol-containing, pair-fed isocaloric liquid control, or solid diets for 11 days. Macrophage and dendritic cell (DC) populations were isolated by paramagenetic bead separation and used to present ovalbumin (OVA) to highly purified syngeneic CD4+ T cells derived from DO11.10 T cell receptor transgenic mice in coculture. DC isolated from diet-fed mice were also used to present OVA to highly purified CD4+ T cells derived from antigen-naïve DO11.10Rag2-/- mice that are devoid of memory T cells. In vitro cytokine responses, interleukin (IL) -2, IL-6, IL-12, IL-13, IL-17A, and interferon-gamma (IFN-gamma) were measured by enzyme-linked immunosorbent assay. Flow cytometry measured cell surface molecule expression. Alcohol consumption impairs delayed hypersensitivity responses (type 1) and enhances serum IgE levels (type 2). CD11c+ DC, but not F4/80+ macrophages, support cytokine responses by purified CD4+ T cells. CD11c+ DC derived from ethanol consuming BALB/c mice show diminished ability to support IFN-gamma responses by purified CD4+ T cells derived from DO11.10 or DO11.10Rag2-/- mice. Subset analysis indicates that of the 3 "conventional" DC subsets found in mouse spleens, CD11c+CD8(alpha)+ DCs are both responsible for OVA presentation and susceptible to the effects of ethanol. Ethanol consumption does not overtly alter the percent of splenic DC, but does increase the surface density of CD11c on these cells. Data show that cocultures containing purified CD4+ T DO11.10 cells and APC derived from alcohol-consuming mice show decreased IL-6, IL-12, IL-17A, and IFN-gamma and increased IL-13 cytokine production in response to OVA stimulation. Ethanol alters CD11c+CD8(alpha)+ DC function, affecting cytokines responsible for adaptive immune responses. A unifying hypothesis for the underlying mechanism(s) of ethanol's effect upon adaptive immune function is proposed.

Leptin-Dependent Toll-Like Receptor Expression and Responsiveness in Preadipocytes and Adipocytes

Leptin, an adipokine mainly produced by adipocytes, has been well characterized with regard to its regulatory function on immune cells. Thus the question occurred of how adipocytes and preadipocytes interact with the immune system and whether or not this communication is regulated by leptin. With the present study we evaluated the Toll-like receptor (TLR) expression and TLR ligand-specific activation of murine preadipocytes and adipocytes in the presence [wild type (WT), 3T3L1] or absence of leptin (ob/ob) or leptin signaling (db/db). The ob/ob as well as db/db adipocytes and preadipocytes were characterized by a significant up-regulation of TLR1 to -9 expression when compared with WT cells. In WT preadipocytes the TLR responsiveness increased during maturation to adipocytes; however, stimulation of ob/ob and db/db cells resulted in a 10- to 20-fold higher interleukin-6 production. Signaling studies revealed, in addition to the increased TLR expression, alterations in the phosphoinositide 3 kinase signaling cascade in ob/ob and db/db cells as an explanation for this increased responsiveness. In conclusion, the present study indicates the expression and responsiveness of TLR1 to -9 in murine preadipocytes as well as adipocytes, both of which are strongly regulated by the adipokine leptin. In summary, these data further emphasize the role of fat tissue in the immune system.

Histopathological findings in the gastrointestinal tract of primate recipients of porcine renal xenografts following different immunosuppressive regimens

Cyclophosphamide (CYP) and methotrexate (MTX) have been used as immunosuppressants in induction or maintenance protocols in a large variety of xenotransplantation models. Combining the use of transgenic porcine organs expressing human decay-accelerating factor (hDAF) with immunosuppressive therapy that included the use of CYP or MTX, survival of primate recipients of life-supporting renal xenografts has been prolonged. However, both drugs can cause significant systemic toxicity and, in particular, gastrointestinal (GI) toxicity. To date only limited data have been reported on the histopathological features deriving from the use of such agents in non-human primates. Cyclophosphamide or MTX was used as part of the immunosuppressive regimen in 15 bilaterally nephrectomized non-human primate (Macaca fascicularis) recipients of a life-supporting hDAF porcine kidney. At post-mortem, a detailed analysis of the GI tract in animals receiving either CYP or MTX was performed. Paraffin-embedded sections of each portion of the GI tract were prepared and stained with hematoxylin and eosin (H&E). In some animals, additional investigations by immunohistochemistry (CD3, CD5, CD20, CD79 alpha cy, lambda, and kappa light chains) and by in situ hybridization for EBV encoded RNA (EBER) were undertaken. The xenografted animals from the CYP group had a mean survival of 31 days (range: 0 to 90 days); animals from the MTX group survived a median of 14 days (range: 0 to 39 days). GI complications were the most frequent cause of euthanasia after renal failure. In CYP-treated animals GI-tract lesions were primarily characterized by diffuse, severe lymphoplasmocytic mucosal inflammatory infiltrate. Variable degrees of villi atrophy and fusion, gut-associated lymphoid tissue (GALT) and goblet cell hyperplasia were also observed. In MTX-treated primates, findings were consistent with severe villi atrophy associated with mild-to-moderate disseminated lymphoplasmocytic infiltration. In conclusion, GI tract lesions are an early and consistent finding when CYP or MTX are used as induction agents in this model. The two compounds induce different types of GI tract damage, however, in agreement with their different mechanisms of action. Whilst CYP primarily determines inflammatory lesions, MTX leads to a degenerative type of damage. This study indicates that immunosuppressive drugs can cause severe GI tract damage in primate recipients of renal xenografts and may be responsible for life-threatening lesions.

Human CD8+ intraepithelial lymphocytes: a unique model to study the regulation of effector cytotoxic T lymphocytes in tissue

The epithelium of the human small intestine contains a large population of intraepithelial cytolytic alphabeta T-cell receptor (TCR) CD8 alpha beta T lymphocytes (IE-CTLs), whose main role is to sustain epithelial integrity by rapidly eliminating infected and damaged cells. In mouse, the recognition of inducible/modified self-molecules, i.e. non-classical major histocompatibility complex (MHC) class I molecules, is mediated by the TCR and natural killer receptors (NKRs) co-expressed on the cell surface of a non-conventional autoreactive CD8 alpha alpha alpha beta TCR cell subset. In contrast, in humans, the recognition of non-classical MHC class I molecules induced by stress and inflammation on intestinal epithelial cells (IECs) is principally mediated by NKRs expressed on conventional CD8 alpha beta alpha beta TCR cells. By sensing microenvironmental signals of inflammation and stress through NKRs, IE-CTLs fine tune their TCR activation threshold. Furthermore, IE-CTLs under particular conditions, involving interleukin-15 upregulation, acquire the capacity to kill distressed intestinal epithelial cells in an antigen non-specific manner. Adaptive IE-CTLs appear hence to have autoreactive properties and modulate their immune response based on innate signals, reflecting the fitness of the tissue.

Thymic differentiation of TCRαβ+ CD8αα+ IELs

Intraepithelial lymphocytes (IELs) contain several subsets, but the origin of the T-cell receptor (TCR)alphabeta(+) CD8 alpha alpha(+) 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. TCR alpha beta(+) CD8 alpha alpha(+) 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 TCR alpha beta(+) CD8 alpha alpha(+) 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 CD8 alpha alpha homodimers. The agonist-selected cells in the thymus are TCRbeta(+) but CD4 and CD8 double negative. The evidence suggests that reacquired expression of CD8 alpha alpha 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 TCR alpha beta(+) CD8 alpha alpha(+) IELs may help us to understand how they participate in immune regulation and protection in the intestine.

The extrathymic T‐cell differentiation in the murine gut

The gut epithelial border is in continuous contact with exogenous antigens and harbors a distinctive and very abundant CD8 alpha alpha intraepithelial T-lymphocyte effector population. We describe here the characteristics of these cells that distinguish them from all other T-cell types in the body as well as their functions in local protection. We also describe how these cells differentiate from local precursors present in the gut cryptopatches (CPs) following a pathway of T-cell differentiation unique to the gut wall. Finally, we describe the origin of the precursors of CD8 alpha alpha T cells, which come from the bone marrow in athymic mice but are first imprinted in the thymus in euthymic mice. Indeed, CD3(-)CD4(-)CD8(-) T-cell-committed precursors can leave the thymus before T-cell receptor rearrangements and then colonize the gut CPs, proceeding with their differentiation within the gut wall.

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

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 gammadelta-IELs and many alphabeta-IELs in the mouse small intestine are known to express CD8 alpha alpha homodimers. A wide range of evidence that supports extrathymic development of these CD8 alpha alpha(+) 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 gammadelta-IELs and/or alphabeta-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.

Cytokine and antibody responses of reactivated murine toxoplasmosis upon administration of dexamethasone

Toxoplasma gondii has been shown to result in life-threatening encephalitis in immunocompromised patients after reactivation of dormant parasites. In order to obtain information on immune responses related to this phenomenon, BALB/c mice were infected with 25 cysts of the 76K strain of T. gondii, then, treated orally with dexamethasone (Toxo/Dexa-treated group) in order to reactivate the chronic toxoplasmosis. None of the T. gondii-infected mice died during the experimental periods, whereas the Toxo/Dexa-treated mice evidenced a significant attenuation of survival periods. Toxoplasma-specific IgG2a, IgA and IgM titers in sera were significantly depressed in the Toxo/Dexa-treated mice; however, the IgG1 sera titers were similar to those seen in the Toxoplasma-infected mice. The percentages of CD4+ and CD8 alpha + T cells in the Toxo/Dexa-treated mice were significantly reduced 2 weeks after dexamethasone treatment. IFN-gamma and IL-10 production levels in the Toxo/Dexa-treated mice were depressed significantly, whereas IL-4 production was increased temporarily. The expression levels of the Toxoplasma-specific P30 and B1 genes were found to have been increased in the Toxo/Dexa-treated mice in comparison with the Toxoplasmainfected mice. Collectively, the findings of this study demonstrate that reactivation of murine toxoplasmosis as the result of dexamethasone treatment induced a depression in Th1 immune responses, whereas Th2 immune responses were not significantly influenced.

Redirection of T Cells by Delivering a Transgenic Mouse-Derived MDM2 Tumor Antigen-Specific TCR and its Humanized Derviative Is Governed by the CD8 Coreceptor and Affects Natural Human TCR Expression

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.