The effect of long-term caloric restriction on function of T-cell subsets in old mice

The effect of caloric restriction (from weaning to old age) on CD3-stimulated CD4+ and CD8+ lymphocyte proliferation and calcium mobilization was examined. Young ad libitum (ad lib) fed, old ad lib fed, old calorically restricted, and old calorically restricted mice which were fed ad lib during the last 6 weeks of their life (restricted/refed) were compared in both BDF1 [(C57BL/6 x DBA/2)F1] and C57BL/6 mice. Proliferation of CD4+ cells was lower in old ad lib animals than in young animals; this difference was not seen in CD8+ cells. Those CD4+ cells which did proliferate in old ad lib animals underwent similar cell cycle progression as young cells. In calorically restricted and calorically restricted/refed animals, CD4+ cell proliferation was similar to the young animals, and CD8+ cells showed a higher proliferative capacity than cells from either young or old ad lib mice. Differences in proliferative capacity were not correlated with alterations in transmembrane signaling efficiency as peak [Ca2+]i was reduced in both T-cell subsets in all groups of old mice relative to young mice. Additionally, reduced [Ca2+]i was observed in the CD8+ subset for which there was no deficit in proliferation, and the enhanced proliferation seen in old restricted and old restricted/refed mice did not manifest as increased [Ca2+]i mobilization. The percentage of CD4+ cells from both mouse strains was reduced in all groups of old mice compared with young mice, while the percentage of CD8+ cells was generally similar in young and all groups of old mice. Our studies would suggest that lifelong caloric restriction of mice prevents the age-associated decrease in T-cell proliferative capacity but that the enhanced proliferation of these cells is not due to increased efficiency of transmembrane signaling.     

Intact antigen receptor-mediated generation of inositol phosphates and increased intracellular calcium in CD4 CD8 T lymphocytes from MRL lpr mice

The predominant T lymphocytes that accumulate in the peripheral lymphoid tissues of mice homozygous for the lpr gene bear the phenotype CD3+CD4-CD8-. By certain functional criteria these cells would appear to have impaired CD3-mediated signal transduction, in that they do not respond to alloantigen and produce little if any detectable IL-2 or other lymphokines. However, the signal pathway appears adequate for achieving other T cell functions, including induction of high affinity IL-2R, and thymic deletion. To clarify the basis of this seeming discrepancy, we examined transmembrane signal transduction in T cell subsets of lpr/lpr (lpr) and +/+ mice, as defined by increased [Ca2+]i and the generation of inositol phosphates (InsPs). Stimulation of lpr CD4-CD8- cells with anti-CD3 antibody produced prompt and sustained increases in the concentration of [C2+]i and in InsPs. Similar responses occurred in mature T cells from lpr and +/+ mice, except for the somewhat slower kinetics of their increased [Ca2+]i. In marked distinction to the anti-CD2-mediated response, Con A, even in high doses, could not stimulate any increase of [Ca2+]i in lpr CD4-CD8- cells, and only modest increases in InsPs. Mature T cells, whether of lpr or +/+ origin, yielded normal increased [Ca2+]i with Con A. The reason for the differences in signal transduction between anti-CD3 and Con A stimulation of lpr CD4-CD8- cells may relate to the absence of surface structures on these immature T cells that are required for activation by Con A but not by anti-CD3. The data demonstrate that the CD3 complex in lpr CD4-CD8- T cells can couple to phospholipase C to hydrolyze phosphoinositides. These activation properties of lpr CD4-CD8- T cells have interesting functional parallels to thymocytes at the time of thymic selection, as well as tolerance induction of mature T lymphocytes.     

Reduced proliferation in T lymphocytes in aged humans is predominantly in the CD8+ subset, and is unrelated to defects in transmembrane signaling which are predominantly in the CD4+ subset

Peripheral blood lymphocytes (PBL) from elderly donors have a reduced proliferative response to phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies (mAb) compared to those from young donors. To examine whether this is due to intrinsic deficiencies in proliferative potential of T-cell subsets, we compared the growth of unsorted PBL vs sorted CD4+ or CD8+ CD11- cells after anti-CD3 mAb or PHA stimulation. Unsorted PBL of elderly donors (greater than 65 years) showed a significant decrease in proliferation compared to young donors (20-30 years) when stimulated with anti-CD3 mAb or PHA. Sorted CD4+ and CD8+ cells were grown in culture in the absence of accessory cells under optimized growth conditions (CD28 mAb, interleukin 2 and beta-mercaptoethanol present). CD4+ cells from elderly donors showed no reduced growth after anti-CD3 mAb stimulation and only slightly decreased growth after stimulation with PHA. CD8+ CD11- cells from elderly donors, however, showed a 20-30% reduction in the proportion of cells proliferating in response to the mitogens and up to 40% reduction in the rate of cell-cycle progression of the responding cells. We examined whether this reduced proliferation is related to decreased efficiency of signal transduction by comparing this to the mobilization of intracellular free calcium ([Ca2+]i) and calcium channel activity after stimulation with anti-CD3 mAb or PHA. [Ca2+]i was measured in CD4 and CD8 subsets of young and elderly donors using a flow cytometric assay with the dye indo-1. Compared to cells from young donors, CD4+ cells from elderly donors showed a [Ca2+]i response which was up to 26% lower after stimulation with CD3 and 10% lower after stimulation with PHA. This appeared to be related to decreased calcium channel activity in elderly donors, rather than mobilization of intracellular Ca2+ stores. CD8+ cells from elderly donors, however, had a slightly, but significantly, greater [Ca2+]i response to CD3 mAb and PHA than did cells from young donors. Since the age-dependent defect in proliferation is mainly in CD8+ cells, but the [Ca2+]i decline is predominantly in the CD4+ subset, these results suggest that the reduced proliferation of T cells from older donors is not related to decreased efficiency of transmembrane signal transduction.     

Stress modulates calcium mobilization in immune cells

Both acute and chronic restraint stress modulated mitogen-induced increases in cytoplasmic free-calcium concentrations ([Ca2+]i) in mouse spleen cells. Dual-color analysis of lymphocyte subpopulations demonstrated that acute (2 hour) restraint stress suppressed mitogen-stimulated increases in [Ca2+]i in CD4+ T cells, but enhanced [Ca2+]i in CD8+ T cells. Chronic restraint stress (2 hours daily for up to 21 days) resulted in a significant suppression of mitogen-stimulated increases in [Ca2+]i in CD4+ T cells at 3 and 7 days, but not at 21 days. CD8+ T cells were unaffected by chronic stress. Chronic stress (for 7 days) had a modest suppressive effect on mitogen-induced Ca2+ responses in B cells. Within T lymphocyte subpopulations, both acute and chronic stress predominantly affected CD4+ T cells, which may induce a functional reversal of the CD4/CD8 ratios in vivo. Such a reversal could result in suppression of a variety of immune responses such as lymphocyte proliferation and antigen-specific antibody production. These findings indicate that the inhibitory effects of stress on calcium mobilization in lymphocytes may be an early event mediating stress-induced immunosuppression.     

Heterogeneity among T cells in intracellular free calcium responses after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry

Measurement of intracellular ionized calcium concentrations ([Ca2+]i) has been indispensable in elucidating the central role of [Ca2+]i as a trigger of cellular responses to activating stimuli. Such studies have employed the dye quin2, which has not been readily adapted to analysis of individual small cells. We show here that the calcium response of large numbers of single cells can be analyzed with the use of flow cytometry and the recently described dye, indo-1. Such analyses demonstrate for the first time the heterogeneous nature of the [Ca2+]i response to mitogenic stimuli within populations of peripheral blood lymphocytes (PBL). By simultaneous quantitation of one- or two-color surface immunofluorescence labels, some of this heterogeneity of [Ca2+]i response in PBL is shown to be related to cellular immunophenotype. Almost all T cells responded to anti-CD3 antibody; however, the response is greater among CD4+ than CD8+ cells, and within the CD4+ population the rate of response to stimulation by antibody to CD3 differed between subpopulations defined by expression of the common leukocyte marker p220. In contrast, not all T cells responded to phytohemagglutinin (PHA), even at very high doses. As with anti-CD3, after stimulation with PHA, CD4+ cells showed a larger proportion of responding cells than did CD8+ cells. In separate experiments, indo-1 was found not to impair reproductive viability of PBL, thereby providing the potential for analysis of functional activity after the separation of cells by sorting on the basis of the [Ca2+]i response to stimuli. Mixing experiments indicated that a response of a subpopulation representing as little as 0.3% of total cells could be readily detected. Thus, the flow cytometric assay with indo-1 is the first technique that allows the quantitative analysis of response differences of small subpopulations of cells and intercellular variation in [Ca2+]i.     

Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.     

The sphingosine 1-phosphate receptor agonist FTY720 differentially affects the sequestration of CD4+/CD25+ T-regulatory cells and enhances their functional activity

The sphingosine 1-phosphate (S1P) receptor agonist FTY720 is well known for its immunomodulatory activity, sequestering lymphocytes from blood and spleen into secondary lymphoid organs and thereby preventing their migration to sites of inflammation. Because inflammation is critically dependent on a balance between Ag-specific Th/effector cells and T-regulatory cells, we investigated the effect of FTY720 on T-regulatory cell trafficking and functional activity. An increased number of CD4+/CD25+ T cells was found in blood and spleens of FTY720-treated mice, and transfer of these cells resulted in a significantly more pronounced accumulation in spleens but not lymph nodes after treatment, suggesting that this compound differentially affects the homing properties of T-regulatory cells compared with other T cell subsets. Indeed, CD4+/CD25+ T cells express lower levels of S1P1 and S1P4 receptors and demonstrate a reduced chemotactic response to S1P. Moreover, analysis of the functional response of FTY720-treated CD4+/CD25+ T cells revealed an increased suppressive activity in an in vitro Ag-specific proliferation assay. This correlated with enhanced function in vivo, with T-regulatory cells obtained from FTY720-treated mice being able to suppress OVA-induced airway inflammation. Thus, FTY720 differentially affects the sequestration of T-regulatory cells and importantly, increases the functional activity of T-regulatory cells, suggesting that it may have disease-modifying potential in inflammatory disorders.     

Murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore.

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells. Interestingly, only CD4+ T cells but not CD8+ T cells or B cells responded to the calcium ionophore by proliferation. The inability of CD8+ T cells or B cells to respond was not related to decreased elevation in the intracellular ionized calcium [Ca2+]i concentration induced by the ionophore, because activated CD4+ T, CD8+ T and B cells all exhibited similar elevation in [Ca2+]i. The inability of CD8+ T cells to respond to calcium ionophore was probably due to insufficient production of autocrine growth factors, such as IL-2, inasmuch as the addition of exogenous IL-2 could completely restore the CD8+ T cell responsiveness. Also, exogenous rIL-1 could partially restore purified T cell response to calcium ionophore, whereas, rIL-6 failed to do so. IL-2, but not IL-4, acted as an autocrine growth factor for T cells responding to the calcium ionophore in the presence of SAC, since, antibodies against IL-2 or IL-2 receptor (IL-2R) but not against IL-4, could inhibit the T cell proliferation. Furthermore, exogenous rIL-2 but not rIL-4 supported the proliferation of T cells to calcium ionophore in the absence of accessory cells. Our results suggest that murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore and that this may not depend on the early activation signal such as the elevation in [Ca2+]i) induced by the ionophore but may depend on subsequent signals which regulate endogenous growth factor production.     

IL-7-dependent homeostatic proliferation in the presence of a large number of T cells in gld mice

In gld mice, CD4 and 8-double-negative (DN) T cells as well as naive and memory-phenotype T cells accumulate in the peripheral lymphoid organs. Although Fas ligand (L) defect accounts for the progressive accumulation of abnormal DN T cells, the existence of other mechanisms which may be involved in the defective homeostasis in gld mice has been unclear. In this study, we analyze T-cell homeostasis in gld mice using adoptive transfer systems. It was shown that a gld, but not C57BL/6 (B6), environment led to augmented proliferation of B6 T cells transferred without up-regulation of CD69. Thus, the augmented T-cell proliferation seemed to result from mal-homeostatic proliferation even in the presence of a large number of recipient T cells. T cells from lpr mice showed no significant proliferation in the B6 environment, suggesting that the absence of Fas-Fas L interaction was not responsible for the mal-homeostatic proliferation. Although similar levels of IL-7 mRNA were detected in gld and B6 spleens, the intensity of CD127 and the proportion of CD127+ cells in the T cells were significantly lower in gld mice than in B6 mice, suggesting that IL-7 excess in a gld environment is responsible for the abnormal proliferation of transferred T cells. The administration of anti-CD127 antibody inhibited the proliferation of transferred lymphocytes. Thus, IL-7-dependent proliferation seems to be involved in the abnormal proliferation of lymphocytes in gld recipients.     

Extrathymic T cell maturation. Phenotypic analysis of T cell subsets in nude mice as a function of age.

T cell maturation in an extrathymic environment has been studied using as a model the congenitally athymic nude mouse. Phenotypic analyses as a function of age were conducted on lymphocytes obtained from the spleens and lymph nodes of nude mice through use of mAb recognizing T cell surface markers and multiparameter flow cytometry. The data show that nude mice accumulate increasing numbers of lymphocytes bearing Thy-1, CD3, CD4, and CD8 with age characterized by a progression from heterogeneous dim to more homogeneous bright expression. In contrast, the expression of heat-stable Ag (HSA), a marker of immature thymocytes, decreases with age. By analogy to intrathymic maturation, spleens and lymph nodes in nude mice contain T cells defined as immature, transitional, and mature based on the expression of these markers. Although the proportion of CD4+ and CD8+ T cells associated with bright CD3 expression increases with age, at no age are significant numbers of CD4+8+ cells observed, in contrast to intrathymic T cell maturation. In addition to the frequently observed inversion in the ratio of CD4 to CD8, the CD8 T cell subpopulation in older nude mice contains mainly mature cells (CD8+, CD3+, HSA-) whereas only 50% of CD4+ T cells express the mature (CD4+, CD3+, HSA-) phenotype. At any age, the spectrum of phenotypes observed indicates that lymph nodes contain more mature T cells than spleen, suggesting a role for environmental Ag in driving extrathymic maturation, a process occurring most efficiently among CD8+ T cells. Because extrathymic maturation mirrors some but not all aspects of the intrathymic pathway, we propose that the nude mouse may be a useful model for further dissecting those interactions crucial to establishing the T cell repertoire in euthymic individuals as well as elucidating the contribution of extrathymically derived T cells to the peripheral immune system.     

Distribution of cycling T lymphocytes in blood and lymphoid organs during immune responses

Proliferation of murine T lymphocytes in blood, lymph nodes, and spleen was studied in four in vivo stimulation systems, using BrdU pulse-labeling of DNA-synthesizing cells. The T cell response to the superantigen Staphylococcus enterotoxin B (SEB) was studied in detail. Vbeta8+ T cells showed a peak of DNA synthesis 16-24 h after SEB injection, and the percentage of BrdU+ CD4 and CD8 T cells was higher in blood than in lymph nodes and spleen. DNA synthesis was preceded by massive migration of Vbeta8+ cells from blood to lymphoid organs, in which the early activation marker CD69 was first up-regulated. SEB-nonspecific Vbeta6+ cells showed minimal stimulation but, when cycling, also expressed a high level of CD69. The other systems studied were injection of the IFN-gamma inducer polyinosinic:polycytidylic acid, infection by the BM5 variants of murine leukemia virus (the causative agent of murine AIDS), and T cell expansion after transfer of normal bone marrow and lymph node cells into recombinase-activating gene-2-deficient mice. In each case, a peak of T cell proliferation was observed in blood. These data demonstrate the extensive redistribution of cycling T cells in the first few hours after activation. Kinetic studies of blood lymphocyte status appear crucial for understanding primary immune responses because cycling and redistributing T lymphocytes are enriched in the circulating compartment.     

Human T cells in the SCID-hu mouse are phenotypically normal and functionally competent

SCID-hu mice are heterochimeric animals that are constructed by transplanting human fetal thymus (Thy), liver (Liv), and/or lymph nodes into congenitally immunodeficient C.B-17 scid/scid (SCID) mice. Sensitive and specific two-color flow cytometric assays were used to evaluate human lymphocytes from peripheral blood of SCID-hu mice. Kinetic studies presented in this report show long term T lymphopoiesis in SCID-hu mice. Approximately one-half of SCID-hu mice constructed with Thy and Liv tissue develop detectable levels of circulating human T cells by 4 mo after transplantation. The average level of circulating human cells in SCID-hu mice is generally less than 2% of the total lymphoid cells in the peripheral blood of these mice. Some SCID-hu mice with as high as 13% human lymphocytes, however, have been detected. Nearly all human cells in the peripheral blood of SCID-hu mice are CD3+ cells that express TCR-alpha beta. The percentages of gamma delta+, CD4+, CD8+, CD25+, CD69+, and Leu-8+ cells among CD45+ cells in SCID-hu blood are similar to the levels found in adult peripheral blood. On average, 74% of SCID-hu T cells express CD45RA and 18% express CD29. Functional studies demonstrate that cells from SCID-hu Thy/Liv grafts or human T cells from SCID-hu peripheral blood are functionally competent to respond to mitogens or allogeneic human cells in vitro. They are similar to fetal thymocytes or adult T cells, respectively, in these responses. These studies demonstrate that the SCID-hu mouse is a useful model for the analysis of human immune differentiation and function in vivo.     

Changes in proliferation activity and relative distributions of lymphoid cell subpopulations in congenitally athymic nu/nu mice and lurcher mice with spontaneous olivopontocerebellar degeneration

Changes observed in mice with congenital damage of some part of the CNS-neuroendocrine-immune regulatory system are described. nu/nu mice with congenital absence of thymus and Lurcher mice with spontaneous olivopontocerebellar degeneration displayed changes in the histoarchitecture of adrenal gland, immune organs (thymus, spleen, axillar lymph nodes) and intestine. Changes were also observed in IgM+, IgG+, CD4+ and CD8+ lymphoid cell subpopulations in the main lymphoid organs--the spleen and axillar lymph nodes and in the proliferative ability of whole lymphoid cell populations. The extreme decrease of lymphoid T-cell subpopulations in athymic nu/nu mice is the consequence of the absence of thymus, the organ of their maturation. On the other hand, a relative increase of B-cell subpopulations was found in this mouse strain. A relative decrease of CD4+ lymphocytes and a different influence of immunization on B-cell subpopulations were found in the spleen in neurodeficient Lurcher mice. The high percentage of apoptotic cells, cells in the S-phase of cell cycle and increased proliferation index in nu/nu mice suggest that the turnover and renewal of lymphoid cells in the spleen in nu/nu mice is more rapid than in control immunocompetent BALB/c mice.     

Effect of cytochalasins on cytosolic-free calcium concentration and phosphoinositide metabolism in leukocytes

Cytochalasins are routinely used to stimulate a variety of functions in eukaryotic cells even though their precise mode of action remains to be elucidated. In the present work we used the fluorescent Ca2+ indicator quin2 to study the effect of various cytochalasins, cytochalasins A, B, C, D, E (CA, CB, CC, CD, CE) and dihydrocytochalasin B (dhCB) on the intracellular Ca2+ concentration ([Ca2+]i) in various types of leukocytes, viz, neutrophils and lymphocytes. In human neutrophils, cytochalasins increase [Ca2+]i mainly by releasing Ca2+ from membrane-bound, intracellular stores. Thus, in order to readily appreciate the effect of cytochalasins on [Ca2+ )i, these cells must be loaded with low intracellular quin2 concentrations. On the other hand, in peripheral blood lymphocytes, splenocytes and thymocytes, the increase in [Ca2+]i is predominantly due to an increased Ca2+ influx from the extracellular medium. In addition, we found that in neutrophils these drugs prolong the increase in [Ca2+]i induced by chemotactic peptides, probably by increasing the cell permeability to Ca2+. Finally, in thymocytes, cytochalasins potentiate the production of inositol phosphates induced by the polyclonal mitogen concanavalin A (conA).     

Ca2+ influx pathways mediated by swelling or stores depletion in mouse thymocytes

We used fura-2 video imaging to characterize two Ca2+ influx pathways in mouse thymocytes. Most thymocytes (77%) superfused with hypoosmotic media (60% of isoosmotic) exhibited a sharp, transient rise in the concentration of intracellular free Ca2+ ([Ca2+]i). After a delay of approximately 70 s, these swelling-activated [Ca2+]i (SWAC) transients reached approximately 650 nM from resting levels of approximately 100 nM and declined from a time constant of 20 s. Peak [Ca2+]i during transients correlated with maximum volume during swelling. Regulatory volume decrease (RVD) was enhanced in thymocytes exhibiting SWAC transients. Three lines of evidence indicate that Ca2+ influx, and not the release of Ca2+ from intracellular stores, underlies SWAC transients in thymocytes. First, thymocytes swollen in Ca2+-free media failed to respond. Second, Gd3+ and La3+ inhibited SWAC influx with Kd's of 3.8 and 2.4 microM, respectively. Finally, the depletion of Ca2+ stores with thapsigargin (TG) before swelling did not inhibit the generation, nor decrease the amplitude, of SWAC transients. Cell phenotyping demonstrated that SWAC transients are primarily associated with immature CD4-CD8- and CD4+CD8+ thymocytes. Mature peripheral lymphocytes (mouse or human) did not exhibit SWAC transients. SWAC influx could be distinguished from the calcium release-activated Ca2+ (CRAC) influx pathway stimulated by store depletion with TG. In TG- treated thymocytes, [Ca2+]i rose steadily for approximately 100 s, peaked at approximately 900 nM, and then declined slowly. Simultaneous activation of both pathways produced an additive [Ca2+]i profile. Gd3+ and La3+ blocked Ca2+ entry during CRAC activation more potently (Kd's of 28 and 58 nM, respectively) than Ca2+ influx during SWAC transients. SWAC transients could be elicited in the presence of 1 microM Gd3+, after the complete inhibition of CRAC influx. Finally, whereas SWAC transients were principally restricted to immature thymocytes. TG stimulated the CRAC influx pathway in all four thymic CD4/CD8 subsets and in mature T cells. We conclude that SWAC and CRAC represent separate pathways for Ca2+ entry in thymocytes.     

CD8 is needed for development of cytotoxic T cells but not helper T cells.

A mutant mouse strain without CD8 (Lyt-2 and Lyt-3) expression on the cell surface has been generated by disrupting the Lyt-2 gene using embryonic stem cell technology. In these mice, CD8+ T lymphocytes are not present in peripheral lymphoid organs, but the CD4+ T lymphocyte population seems to be unaltered. Cytotoxic response of T lymphocytes from these mice against alloantigens and viral antigens is dramatically decreased. Proliferative response against alloantigens and in vivo help to B lymphocytes, however, are not affected. These data suggest that CD8 is necessary for the maturation and positive selection of class I MHC restricted cytotoxic T lymphocytes but is not required on any of the intermediate thymocyte populations (CD8+CD4-TcR- or CD4+CD8+TcRlow) during the development of functional class II MHC restricted helper T cells.     

A bone marrow-derived APC in the gut-associated lymphoid tissue captures oral antigens and presents them to both CD4+ and CD8+ T cells

We have previously reported that feeding OVA to C57BL/6 mice can lead to a weak CTL response that is dependent on CD4+ T cell help and is capable of causing autoimmunity. In this study, we investigated the basis of the class I and class II-restricted Ag presentation required for such CTL induction. Two days after feeding OVA, Ag-specific CD4+ and CD8+ T cells were seen to proliferate in the Peyer's patches and mesenteric lymph nodes. Little proliferation was evident in other lymphoid tissues, except at high Ags doses, in which case some dividing CD4+ T cells were observed in the spleen and peripheral lymph nodes. Using chimeric mice, the APC responsible for presenting orally derived Ags was shown to be derived from the bone marrow. Examination of the Ag dose required to activate either CD4+ or CD8+ T cells indicated that a single dose of 6 mg OVA was the minimum dose that consistently stimulated either T cell subset. These data indicate that oral Ags can be transported from the gut into the gut-associated lymphoid tissue, where they are captured by a bone marrow-derived APC and presented to both CD4+ and CD8+ T cells.     

Antibody to a novel 95-kDa surface glycoprotein on human B cells induces calcium mobilization and B cell activation

These findings characterize a 95-kDa glycoprotein on the surface of B lymphocytes recognized by the mAb G28-8. This protein (designated Bgp95), previously classified as a CD39 molecule, is unique based on functional, cell distribution, and immunochemical criteria. Biochemical analyses revealed that Bgp95 is a 95-kDa glycoprotein with N-linked carbohydrate and is reduced to about 70-kDa after treatment with endoglycopeptidase F. In functional studies, stimulation by G28-8 mAb or its F(ab')2 fragments induced a G0 to G1 cell cycle transition and was synergistic with PMA, anti-mu, or anti-CDw40 in stimulating proliferation of resting B cells. G28-8 mAb also could induce increases of cytoplasmic free calcium concentration [Ca2+]i in a subpopulation of tonsillar or peripheral blood B cells. The G28-8 mAb alone induced a steady increase in [Ca2+]i detectable even 1 h after stimulation. Cross-linking the G28-8 mAb with a second mAb specific for murine kappa light chains induced a more rapid increase of [Ca2+]i which peaked at 10 to 20 min and then declined. At 1 h after stimulation, [Ca2+]i was higher in B cells stimulated with G28-8 alone than in B cells stimulated with G28-8 plus anti-kappa. The same conditions of cross-linking with the anti-kappa which increased the kinetics of the [Ca2+]i response decreased the proliferative response which otherwise followed co-incubation of the mAb with B cell growth factor or PMA. Thus, conditions leading to rapid but transient [Ca2+]i increase via Bgp95 may not be as effective at stimulating B cell proliferation as conditions favoring a slower prolonged [Ca2+]i response. Although the Bgp95 molecule is present on activated buoyant tonsillar B cells, mAb to Bgp95 did not trigger [Ca2+]i increases in these cells. These results suggest that the Bgp95 protein may function in early B cell activation and that its signal mechanisms are altered by the activation state of the cell.     

Rat peripheral CD4+CD8+ T lymphocytes are partially immunocompetent thymus-derived cells that undergo post-thymic maturation to become functionally mature CD4+ T lymphocytes

CD4+CD8+ double-positive (DP) T cells represent a minor subpopulation of T lymphocytes found in the periphery of adult rats. In this study, we show that peripheral DP T cells appear among the first T cells that colonize the peripheral lymphoid organs during fetal life, and represent approximately 40% of peripheral T cells during the perinatal period. Later their proportion decreases to reach the low values seen in adulthood. Most DP T cells are small size lymphocytes that do not exhibit an activated phenotype, and their proliferative rate is similar to that of the other peripheral T cell subpopulations. Only 30-40% of DP T cells expresses CD8beta chain, the remaining cells expressing CD8alphaalpha homodimers. However, both DP T cell subsets have an intrathymic origin since they appear in the recent thymic emigrant population after injection of FITC intrathymically. Functionally, although DP T cells are resistant to undergo apoptosis in response to glucocorticoids, they show poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2. A fraction of DP T cells are not actively synthesizing the CD8 coreceptor, and they gradually differentiate to the CD4 cell lineage in reaggregation cultures. Transfer of DP T lymphocytes into thymectomized SCID mice demonstrates that these cells undergo post-thymic maturation in the peripheral lymphoid organs and that their CD4 cell progeny is fully immunocompetent, as judged by its ability to survive and expand in peripheral lymphoid organs, to proliferate in response to CD3 ligation, and to produce IL-2 upon stimulation.