Phenotype and distribution of T lymphocytes in Peyer's patches of athymic mice

The distribution and phenotype of T (Thy-1.2+) cells was examined in Peyer's patches of 8 and 16 week old athymic mice by peroxidase and two-color-fluorescence immunohistochemistry. Despite the generally recognized T cell deficiency of nude mice, some T cells consistently occurred in Peyer's patch domes in all mice. However, many Thy-1.2+ lymphocytes lacked cell surface markers for either helper T cells (L3T4) or cytotoxic/suppressor T cells (Lyt-2), indicating that these cells may be an immature subset of T cells. These cells may represent a population of resident T cell precursors delayed in maturation or T cells newly immigrated to Peyer's patches.     

Phenotype and distribution of T lymphocytes in Peyer's patches of athymic mice

Summary The distribution and phenotype of T (Thy-1.2^–) cells was examined in Peyer's patches of 8 and 16 week old athymic mice by peroxidase and two-color-fluorescence immunohistochemistry. Despite the generally recognized T cell deficiency of nude mice, some T cells consistently occurred in Peyer's patch domes in all mice. However, many Thy-1.2⁺ lymphocytes lacked cell surface markers for either helper T cells (L3T4) or cytotoxic/suppressor T cells (Lyt-2), indicating that these cells may be an immature subset of T cells. These cells may represent a population of resident T cell precursors delayed in maturation or T cells newly immigrated to Peyer's patches.     

L3T4+ T cells regulate Abelson virus-induced lymphomagenesis

To evaluate the role of T cells in regulation of lymphomagenesis, experiments were performed using Abelson murine leukemia virus (AMuLV). In vitro transformation of bone marrow target cells by this B lymphotropic retrovirus was inhibited by peripheral lymph node cells from naive mice. The inhibitory activity depended on Thy-1+ L3T4+ cells but did not require Lyt-2+ cells. In vivo depletion of L3T4+ T cells with a mAb (GK1.5) altered the course of AMuLV-induced lymphoma. L3T4 depletion of naturally resistant C57BL/6 mice resulted in dramatic susceptibility to lymphoma induction. Lymphoma cells from anti-L3T4-treated C57BL/6 mice infected with AMuLV displayed the B lineage transformation marker P1606C3. These studies reveal an important immunologic component of Abelson disease resistance involving L3T4+ T cells.     

T lymphocytes that express CD4 and the alpha beta-T cell receptor but lack Thy-1. Preferential localization in Peyer's patches

Thy-1- T cells expressing CD4 and the alpha beta-TCR have been identified in murine lymphoid tissues. These cells are particularly prevalent in Peyer's patches (PP), representing 17 +/- 3% of PP CD4 T cells, whereas they are much less prevalent in spleen, lymph nodes, lamina propria, or peritoneum. Phenotypic studies of fresh-isolated PP T cells demonstrate that all PP CD4 T cells (both Thy-1- and Thy-1+) express CD3, alpha beta-TCR, and CD5 (Lyt-1), whereas none coexpress CD8 (Lyt-2). Thy-1- and Thy-1+ CD4 T cell lines generated from PP also coexpress CD3 and alpha beta-TCR, but are heterogeneous in expression of CD5 and again do not coexpress CD8. Further studies revealed that Thy-1- CD4+ T cells were not present in nude mice. Short term stimulation of Thy-1+ CD4+ PP T cells with anti-CD3 resulted in loss of Thy-1 in a substantial fraction of these cells. Functional studies of Thy-1- and Thy-1+ CD4+ PP T cells indicate that fresh-isolated Thy-1- CD4+ cells do not proliferate in response to insoluble anti-CD3 but do proliferate when stimulated with soluble anti-CD3 in the presence of feeder cells. In contrast, Thy-1+ CD4+ cells proliferate well to both stimuli. However, Thy-1- CD4+ PP T cells adapted to in vitro culture exhibit vigorous proliferative responses when stimulated with either form of anti-CD3. Evaluation of lymphokine secretion by fresh-isolated Thy-1- and Thy-1+ CD4+ PP T cells revealed that both make substantial amounts of IL-2; however, Thy-1- T cells made less IL-4 than their Thy-1+ counterparts. Neither population made IL-5 or IFN-gamma. Similarly, Thy-1- and Thy-1+ CD4 T cell lines made similar amounts of IL-2; again Thy-1- T cells made less IL-4; and in this case Thy-1- T cells made IL-5 albeit significantly less than the Thy-1+ cells. Finally, immunohistochemical studies suggested that many of the CD4+ T cells in PP germinal centers were Thy-1-, indicating that Thy-1- and Thy-1+ CD4 T cells differ in their distribution within the PP. These studies thus define a phenotypically and functionally distinct T cell population which is most prevalent in murine Peyer's patches.     

Correlation of extracellular matrix components with the cytoarchitecture of mouse Peyer's patches

Summary The distribution patterns of extracellular matrix elements were determined to ascertain whether they play a role in the localization of lymphocytes in discrete T-cell, B-cell and dome antigen-processing domains within Peyer's patches. Antibodies against collagen types I, III and IV, laminin and fibronectin were applied to cryosections of mouse Peyer's patches and localized by direct or indirect immunoperoxidase methods. T-cell domains were identified with a monoclonal antibody against Thy-1.2. Labeled reticular fibers in distinctive patterns were more numerous in parafollicular and dome areas than within follicles. Germinal centers contained few such fibers. In parafollicular areas, fibers were oriented predominantly toward follicle domes; their distribution corresponded to T-cell zones and lymphocyte traffic areas, with their orientation being parallel to the migration pathways of lymphocytes from high endothelial venules to the antigen-processing domes. Subepithelial and subendothelial basal laminae were immunopositive for type-IV collagen, laminin and fibronectin. The dome subepithelial basal lamina had pore-like discontinuities through which lymphocytes migrated to and from the epithelium. The correspondence of the distribution patterns of extracellular matrix to specific functional domains of Peyer's patches suggests that this matrix provides a structural framework for lymphocyte migration and localization.     

Origin of autoreactive T helper cells. I. Characterization of Thy-1+, Lyt-, L3T4- precursors in the spleen of normal mice

A new population of dull Thy-1+, Ly-1-, Lyt-2-, L3T4- PNA- cells, resistant to a double cytotoxic treatment by monoclonal antibodies to these T cell markers plus complement, has been isolated from the spleen of normal adult BALB/c and DBA/2 mice (Tkr cells). These cells exhibit no spontaneous autoreactivity or alloreactivity but can be activated with concanavalin A (Con A). Once activated, they differentiate into bright Thy-1+, Ly-1+, Lyt-2-, L3T4+ PNA- T lymphocytes. Con A-activated Tkr cells also strongly proliferate in the presence of allogeneic or syngeneic dendritic cells in secondary cultures. Moreover, contrary to other Con A-stimulated T cell populations, they induce B lymphocytes to proliferate and to differentiate into Ig-secreting cells at a very high level. Con A-activated Tkr cells are therefore very potent polyclonal B cell activators. Restimulated of Tkr cells by syngeneic dendritic cells can be inhibited by anti-L3T4 or anti-class II monoclonal antibodies. The results suggest that Tkr cells are the precursors of class II-specific autoreactive T helper cells. Tkr cells are absent in the spleen of B6 animals. This indicates that their expression might be genetically controlled. It also suggests that Tkr cells may not be the unique splenic precursors of autoreactive T cells. Con A activation of Tkr cells in Click's medium is 2-mercaptoethanol dependent and highly sensitive to pCO2, like the response of thymocytes. Tkr cells are also absent in the spleen of nude mice. We conclude that Tkr cells represent splenic precursors of autoreactive T helper cells equivalent to Thy-1+, Ly-2-, L3T4- PNA- cortical thymocytes.     

Regulation of the anti-alpha (1,3) dextran response: two populations of dextran-reactive B cells that differ in their T cell requirements for induction to antibody synthesis

The in vitro antibody response to dextran B1355S, a thymus-independent Type 2 antigen, requires T cell-derived lymphokines but is not thought to require an activation signal from an antigen-specific T helper cell. The present study demonstrates that there are two dextran-reactive B cell populations in BALB/c mice with respect to the T cell requirements for the generation of antibody-forming cells. One population found among dextran-reactive spleen B cells from 12- to 14-mo-old BALB/c mice generated anti-dextran PFC in the presence of B cell growth factor (BCGF II) and IL 2 or the combination of BCGF II, IL 2, and IFN-gamma. A second population of dextran-reactive B cells found in spleen and Peyer's patches of 2-mo-old unprimed mice did not respond to these same lymphokines, but did generate anti-dextran plaque-forming cells in the presence of Thy-1.2+, L3T4+ T cells from Peyer's patches. However, splenic B cells obtained from 2-mo-old mice that had been primed with dextran 2 to 3 days after birth were shown to be responsive to the same lymphokines as dextran-reactive B cells from 12- to 14-mo-old mice. These results suggest that previous priming with dextran B1355S induces a dextran-specific B cell population that can be activated to antibody-forming cells in the presence of antigen and T cell-derived lymphokines, whereas a second, unprimed population requires an additional activation signal from L3T4+ T cells.     

Differentiation of thymocytes in fetal organ culture: analysis of phenotypic changes accompanying the appearance of cytolytic and interleukin 2-producing cells

At the 14th day of gestation, embryonic thymocytes+ are large, functionally incompetent cells with H-2K+ Thy-1+ B14- Ly-2- L3T4- phenotype, some of which express TL antigen. Differentiation of these cells in organ culture is characterized by: 1) appearance of cells expressing Ly-2 and L3T4 molecules, first among the population of large cells, after 2 days of culture; 2) appearance of small H-2K- Thy-1+TL+B14+Ly-2+L3T4+ and H-2K-Thy-1+TL+B14-Ly-2+ L3T4+ cells between days 2 and 4; 3) accumulation of small H-2K- Thy-1+ TL+ B14- Ly-2+ L3T4+ (but not H-2K- Thy-1+ TL+ B14+ Ly-2+ L3T4+) cells until day 5 of culture, and their subsequent gradual disappearance which is paralleled by an increase of the proportion of medium-sized H-2K+ Thy-1+ TL- B14- cells with various Ly-2 L3T4 phenotypes; 4) appearance and subsequent accumulation of cytolytic and IL 2-producing cells between days 4 and 6. Comparison of these results with the data from similar in vivo studies shows that differentiation of organ-cultured thymocytes rather closely follows the in vivo development only during the first week of culture and shows significant deviations thereafter. Precursors of cytolytic cells and cytolytic effector cells as well as IL 2-producing cells are found among both Ly-2+ and Ly-2- populations of thymocytes, indicating that there is no clear association between Ly-2 phenotype and the ability to kill or to secrete IL 2.     

In vivo dynamics of pulmonary lymphoid cell subpopulations generated against pulmonary metastasis: evaluation by bronchoalveolar lavage fluid.

The dynamics of lymphoid cell subpopulations in bronchoalveolar lavage fluid (BALF) and the systemic lymphoid organs of mice after intravenous injection of B16 melanoma cells were examined with a fluorescence-activated cell sorter. The lymphoid cell subpopulations of BALF and mediastinal lymph nodes showed significant changes in numbers and proportions, while those of other lymphoid organs including inguinal lymph nodes, thymus and spleen, showed little change. In week 1, the cells with a Thy-1.2+, Lyt-1+, L3T4-, Lyt-2- phenotype and asialo-Gm1+ cells in BALF significantly increased and L3T4+ cells slightly increased in number. By week 3, the numbers of Lyt-2+ cells in BALF markedly increased in number (by about 90 times) compared with controls. The number of Thy-1.2+ cells in mediastinal lymph nodes also increased significantly by week 3. Mice that had been pretreated with an immunosuppressive dose of cyclophosphamide were also inoculated intravenously with B16 melanoma cells. In these mice, a significantly increased number of pleural tumors developed and the number of Thy-1.2+ cells in BALF was markedly reduced from week 1 to 3. The results indicate that L3T4 and Lyt-2 double negative T-cells and natural killer (NK) cells may be generated and/or mobilized to the lung in an early phase of experimental metastasis of B16 melanoma cells and that at a later stage, when multiple metastases develop, T-cells with a Lyt-2+ phenotype markedly increase, probably as an expression of a host reaction against proliferating metastatic tumor cells.     

The cells responsible for murine natural cytotoxic (NC) activity: a multi-lineage system

Previous studies on the surface phenotype of natural cytotoxic (NC) cells defined by negative selection with antibodies and complement showed that most if not all NC activity is the property of "null" cells that did not express a variety of lymphoid markers, including some expressed by natural killer (NK) cells. In the present study we show that when murine C57BL/6 spleen cells were sorted by flow cytometry into fractions positive or negative for Qa-5, Ly-2.2, Thy-1.2, L3T4, or surface immunoglobulin (sIg) and for high or low expression of H-2Kb, the pattern of NC activities was quite different from the negative selection experiments with antibody and complement. Enrichment of NC activity tested against WEHI-164 targets was observed in the H-2Kb high, Qa-5+, Thy-1.2+, and Ly-2.2- fractions, and to a lesser extent in the L3T4+ and sIg- fractions. However, significant NC activity, although lower than in the unseparated cells, was also found in the H-2Kb low, Qa-5-, Thy-1.2-, L3T4-, Ly-2.2+, and sIg+. With the exception of the anti-Ig, all the reagents were monoclonal antibodies. By comparison, NK activity tested against YAC-1 targets was clearly enriched in the H-2Kb high, Ly-2.2-, sIg-, and to a lesser extent, Thy-1.2+ sorted fractions, whereas most of the NK activity was in the L3T4- fractions. These results indicate that NC activity against WEHI-164 targets is mediated by an heterogeneous population of effector cells, which includes cells with markers of both the T and the B lineages, as well as of NK cells. These studies also show that negative selection with antibodies and complement is not always a reliable method for defining the surface phenotype of effector cells.     

Identification of early stages of T lymphocyte development in the thymus cortex and medulla

Thymocyte subpopulations with a phenotype suggesting they are early stages of T cell development in the adult mouse thymus were characterized and isolated by using multiparameter flow cytometry and sorting, in conjunction with selective killing with antibody and complement (C). The intrathymic localization of these subpopulations was assessed by dipping the thymus in fluorescent dyes to selectively label outer-cortical cells. The main phenotypic markers used were sensitivity to C-mediated lysis by the monoclonal antibody B2A2 (which spares most prothymocytes but kills most thymocytes), the expression of the T cell lineage specific markers Ly-2 and L3T4, and the levels of the common T cell antigens Ly-1 and Thy-1. A preliminary selection for cells lacking Ly-2 and L3T4, or resistant to B2A2 and C, produced a population of large cells, only 5% of all thymocytes and distinct from the typical cortical blast cells. This population of putative early thymocytes was itself heterogeneous, consisting of eight subpopulations separable by phenotype and intrathymic localization. One group of two subpopulations (B2A2-, Ly-1++, Thy-1+ and either Ly-2+ L3T4- or Ly-2- L3T4+) appeared to be of medullary location, and their phenotype suggested they could have been early members of the medullary lineages. Another group of two subpopulations (B2A2-, Ly-1++, Thy-1-, Ly-2-, L3T4- and B2A2-, Ly-1++, Thy-1+, Ly-2- L3T4-) did not show a clear localization pattern and may have represented cells in an earlier stage of transition to medullary phenotype and location. A quite different group of three subpopulations (B2A2++, Ly-1-, Thy-1-, Ly-2- L3T4-; B2A2++, Ly-1-, Thy-1+, Ly-2-, L3T4-; and B2A2++, Ly-1+, Thy-1++, Ly-2- L3T4-) was concentrated in the outer cortex and seemed to represent a series of stages of a cortical pathway, before the typical cortical blast cells. Finally, a very minor subset (0.2% of thymocytes), lacking all these markers, was concentrated in the outer cortex; this fifth group had the phenotype expected of the earliest intrathymic precursor cells. The results suggest that the separate developmental streams of cortical and medullary thymocytes may be traced back, via these minor early blast subpopulations, to common precursor cells in the outer cortex.     

Immunohistochemical examination of Peyer's patches in autoimmune mice

Summary The distribution of T-cells and B-cells in Peyer's patches was examined in three autoimmune model mice, MRL/Mp-lpr/lpr, BXSB, NZBWF1/J mice and normal BALB/c mice, between one and ten months old. A multiple layering technique was used for immunohistochemical detection of lymphocyte surface antigens of T-cells (Thy1.2, Lyt1, Lyt2) and B-cells (surface IgM) and peanut agglutinin receptor for germinal center cells. The T-cell population of female MRL/Mp-lpr/lpr mice increased markedly with age, and the B-cell population of the male BXSB mouse tended to increase. However, little change was observed with age in the NZBWF1/J mice. The immunohistochemical properties of the Peyer's patches in the three autoimmune model mice were different.     

Immunohistochemical examination of Peyer's patches in autoimmune mice

The distribution of T-cells and B-cells in Peyer's patches was examined in three autoimmune model mice, MRL/Mp-lpr/lpr, BXSB, NZBWF1/J mice and normal BALB/c mice, between one and ten months old. A multiple layering technique was used for immunohistochemical detection of lymphocyte surface antigens of T-cells (Thy1.2, Lyt1, Lyt2) and B-cells (surface IgM) and peanut agglutinin receptor for germinal center cells. The T-cell population of female MRL/Mp-lpr/lpr mice increased markedly with age, and the B-cell population of the male BXSB mouse tended to increase. However, little change was observed with age in the NZBWF1/J mice. The immunohistochemical properties of the Peyer's patches in the three autoimmune model mice were different.     

L3T4+ cytotoxic T lymphocytes specific for class I H-2 antigens are activated in primary mixed lymphocyte reactions

Thy-1+, L3T4+, Ly-2- cytotoxic lymphocytes (CTL) are generated in a primary anti-H-2d mixed lymphocyte reaction, by using responders depleted of Ly-2+ cells. In addition to expressing the L3T4 marker, as detected by anti-L3T4 antibody and complement-mediated elimination, the L3T4+ CTL are inhibited by L3T4 antibody. The observation of these L3T4+ CTL in cells recovered from primary mixed lymphocyte reactions confirms the previous reports. However it is demonstrated for the first time that a subpopulation of these are class I-specific by their specific inhibition with an antiserum to class I antigens. The class I specificity of the CTL was further shown by their ability to kill class II antigen negative P815 tumor cells. The lysis of this target cell by L3T4+ CTL was also specifically blocked by the class I antiserum. The data is consistent with the presence also of a class II-specific population of L3T4+ cytotoxic cells. The fact that a level of L3T4+ cell-mediated cytotoxic activity comparable to Ly-2+ cytolytic activity is generated in a primary mixed lymphocyte response, even though the precursor frequency of L3T4+ killer cells is 10 times lower than for Ly-2+ killers, is suggestive of their physiologic significance. It was also shown that the activation of these cells is not dependent on the presence of xenogeneic serum components or exogenous helper or mitogenic factors in the culture medium. The findings provide further evidence against both the phenotype-function and phenotype-major histocompatibility complex antigen specificity models of T cell diversity.     

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Administration of monoclonal anti-T cell antibodies retards murine lupus in BXSB mice

Murine lupus in BXSB mice is associated with B cell hyperactivity, monocyte proliferation, and impaired T cell function. However, the significance of these abnormalities, and the relationship among them, has not been clearly established. To examine the role of T cells in the pathogenesis of autoimmune disease in BXSB mice, we depleted specific T cell subsets from BXSB males by using rat IgG2b monoclonal antibodies (MAb) to either Thy-1.2 (on all T cells) or L3T4 (on "helper/inducer" T cells). A single injection of anti-Thy-1.2 (6 mg i.v.) at age 3 mo produced a sustained 40 to 50% reduction in circulating T cells for 6 mo. Treatment prevented monocytosis, reduced anti-DNA antibody concentration, and retarded renal disease, but it did not prolong life. Repeated injections of rat MAb to Thy-1.2 were precluded by the development of a host immune response to rat immunoglobulin (Ig) that can cause anaphylaxis in BXSB mice. In contrast, rat MAb to L3T4 stimulated little or no immune response to rat Ig. We therefore were able to treat BXSB mice weekly with anti-L3T4 (2 mg i.p.) from age 3 to 12 mo. Treatment reduced circulating L3T4+ cells beneath the level of detection by fluorescence analysis. It also significantly reduced monocytosis, anti-DNA antibody production, renal disease, and mortality. These findings establish that monocytosis and autoimmunity in BXSB mice are promoted by T cells. They extend our previous observation that MAb to L3T4 retard autoimmunity in NZB/NZW F1 mice. Our finding that treatment with MAb to L3T4 is effective in two strains of lupus-prone mice suggests that treatment with MAb to Leu-3/T4, the human homologue for L3T4, may be effective in people with systemic lupus erythematosus.     

Subpopulations of early thymocytes. A cross-correlation flow cytometric analysis of adult mouse Ly-2-L3T4-(CD8-CD4-) thymocytes using eight different surface markers

Putative early thymocytes, the Ly-2-L3T4-(CD8-CD4-) cells representing 3 to 4% of adult CBA mouse thymic lymphocytes, were isolated in high purity (99.5%). They were then stained by using mAb and analyzed by flow cytometry for the expression of six additional surface antigenic markers. Cross-correlation of the data obtained from a complete series of successive two-parameter analyses revealed the existence of about 11 discrete subsets, falling into four-main groups, within the Ly-2-L3T4- population. All subsets consisted of relatively large lymphoid cells. The most numerous group of Ly-2-L3T4- cells was Ly-1 low B2A2-M1/69 high Thy-1 high Pgp-1 low and by these markers resembled Ly-2+L3T4+ cortical blasts. Many of the cells in this group were positive for the IL-2R and/or for MEL-14. A second major group of Ly-2-L3T4- cells was Ly-1 high B2A2-M1/69 low Pgp-1 high, and resembled in some respects activated mature T cells. This group had previously been shown to be absent from the embryonic thymus. The group could be divided into Thy-1 high and Thy-1 low subsets. None of the cells in this group were positive for the IL-2R and very few expressed MEL-14. A third group, 13% of the Ly-2-L3T4- population, was Ly-1 low B2A2-M1/69 low Pgp-1 high, and could also be divided into Thy-1 high and Thy-1 low subsets. A final minor group, 9% of the Ly-2-L3T4- population, was Ly-1 high B2A2-M1/69 high Pgp-1 low Thy-1 high. The particular pattern of markers on these subsets, combined with subsequent information on their properties, makes it unlikely that they all represent sequential steps in one continuous developmental stream, and indicates that complex developmental steps have occurred, even at this supposedly early stage of T cell differentiation.     

The membrane-bound chemokine CXCL16 expressed on follicle-associated epithelium and M cells mediates lympho-epithelial interaction in GALT

The recently identified CXCL16 has dual functions as a transmembrane adhesion molecule and a soluble chemokine. In this study we found that CXCL16 mRNA and protein were expressed constitutively on the follicle-associated epithelium covering Peyer's patches (PPs), isolated lymphoid follicles, and cecal patches, but minimally on the villous epithelium in the murine gastrointestinal tract. The CXCL16 receptor CXCR6/Bonzo was constitutively expressed on subpopulations of CD4+ and CD8+ T cells isolated from PPs. The expression of CXCR6/Bonzo on the PP T cells was up-regulated after stimulation with anti-CD3 and anti-CD28 mAbs. The activated PP T cells showed chemotactic migration in response to the soluble N-terminal chemokine domain of CXCL16. Furthermore, the activated PP T cells selectively adhered to cells expressing murine CXCL16. To determine the physiological role of CXCL16 in GALT, we first carefully analyzed T cell distribution in PPs. T cells localized not only in the interfollicular region but also at a lesser frequency in the subepithelial dome (SED) and in the germinal center of lymphoid follicles. Consistently, the majority of the adoptive transferred activated T cells migrated into the SED and the interfollicular region. However, the neutralization of CXCL16 specifically reduced the migration of the adoptive, transferred, activated T cells into the SED of PPs. These data suggest that CXCL16 expressed on the follicle-associated epithelium plays an important role in the recruitment and retention of activated T cells in the SED and should, at least partially, be responsible for lymphocyte compartmentalization in GALT.     

Characterization of the inflammatory response in the central nervous system of mice susceptible or resistant to demyelination by Theiler's virus

Intracerebral inoculation of mice with Theiler's murine encephalomyelitis virus results in an intense inflammatory response of mononuclear leukocytes which infiltrate into the central nervous system. Resistant strains of mice have the ability to clear virus whereas susceptible strains become infected persistently and are associated with chronic demyelination which is proposed to be immune-mediated. In an attempt to better understand the role of the immune response during demyelination, mononuclear leukocytes were isolated from the central nervous system of infected mice and stained by an immunoperoxidase technique with anti-Thy-1.2, anti-L3T4, anti-Lyt-2 and anti-MAC-1 mAb. Infection of susceptible SJL/J mice resulted in a biphasic immune response which peaked on days 7 and 27 post-infection. In contrast, a single peak (day 7) was observed in resistant C57BL/10SNJ mice. The presence of Thy-1.2, L3T4, and MAC-1+ cells was similar between the two strains. However, although the number of Lyt-2+ cells peaked on day 7 in C57BL/10SNJ mice, they were not detected in SJL/J mice until 14 days post-infection and gradually increased in number over the course of infection. To further study the role of T cells in demyelination, serial frozen sections of brain and spinal cord were stained for the presence of Lyt-2 and L3T4+ cells in the lesions of chronically infected SJL/J mice. L3T4+ cells were observed predominantly in perivascular regions while Lyt-2+ cells were observed infiltrating the parenchyma. These results provide further evidence that Lyt-2+ lymphocytes are important in the mechanism of susceptibility/resistance to Theiler's murine encephalomyelitis virus-induced demyelination.     

T cell subsets and IFN-gamma production in resistance to systemic candidosis in immunized mice

In addition to previous evidence for the roles of T cell-dependent immunity and delayed-type hypersensitivity in acquired resistance to systemic candidosis in mice, in the present study we have investigated the relative contributions of L3T4+ and Lyt-2+ lymphocytes in the protective immunity induced by vaccination with low virulence Candida albicans cells. We have also addressed the issue of the mode of Candida Ag recognition by specific T cells leading to cytokine release. Spleen cells from immunized mice produced high levels of IFN-gamma in vitro in response to Candida Ag, and this activity was abolished only by the combined treatment of the responder population with anti-L3T4 and anti-Lyt-2.2 mAb plus C. Positively selected L3T4+ and Lyt-2+ cells also produced IFN-gamma in vitro, provided accessory cells (plastic-adherent and Thy-1- Ia- splenocytes, respectively) were added to the lymphocyte-yeast cell cocultures. The production of IFN-gamma by purified L3T4+ and Lyt-2+ cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to the cultures. In vivo, administration of anti-L3T4, anti-Lyt-2.2 mAb or a combination of both significantly impaired the resistance of immunized mice to challenge with virulent C. albicans, as manifested by increased recovery of the yeast from the mouse kidneys. A similar effect was observed upon neutralization of endogenous IFN-gamma by treatment with rat mAb. These results suggest that both L3T4+ and Lyt-2+ T cells play a role in acquired immunity to systemic C. albicans infection, and that their activity may involve IFN-gamma-mediated stimulation of candidacidal mechanisms.