Cyclosporine A Prevents the Generation of Single Positive (Lyt2+ L3T4−, Lyt2− L3T4+) Mature T Cells, but not Single Positive (Lyt2+ T3−) Immature Thymocytes, in Newborn Mice

The influence of cyclosporine A (CsA) on T-cell maturation was investigated in newborn mice. CsA treatment during the pre- and postnatal periods resulted in a hypoplasia of peripheral lymphatic organs, and absence of mature T3+ T cells in lymph nodes and spleens; no functional T-cell reactivity was observed. In thymuses of CsA-treated mice, no T3+ single positive Lyt2+ or T3+L3T4+ thymocytes could be found, but double positive (DP) cells were readily detected. A thymocyte subset with the phenotype Lyt2+L3T4-T3- was still discernible; this population was non-functional in vitro. The data show that the maturation of single positive (SP) T cells is critically influenced by CsA; under the conditions used here we found no evidence that 'leaky' autoreactive SP T cells develop in CsA-treated newborn mice.     

Functional double-negative T cells in the periphery express T cell receptor Vβ gene products that cause deletion of single-positive T cells

A proportion of peripheral T cells lack surface expression of the CD4 or CD8 coreceptor molecules and hence are designated as “double negative” (DN). Most DN T lymphocytes express the Γ/β T cell receptor (TcR), but a minor fraction of them, in both humans and mice, express the α/β TcR. Whereas α/β⁺ DN T lymphocytes are infrequent (< 1%) in conventional lymphoid organs (spleen, blood, lymph node), they account for two-thirds of the T cells residing in adult bone marrow. Analysis of the TcR Vβ repertoire expressed by peripheral DN T cells revealed a high frequency of cells bearing autoreactive TcR that cause deletion of “single-positive” (SP) (CD4⁺CD8^? or CD4^?CD8⁺) T cells. Peripheral DN cells thus represent a cell type that is relatively resistant to clonal deletion. Furthermore, such cells have not been inactivated (anergized) in vivo since they proliferate and secrete interleukins in response to cross-linking by monoclonal antibodies specific for these Vβ gene products that are deleted in SP T cells. These results might help to understand the association of peripheral expansion of DN cells and development of autoimmune diseases.     

Exogenous Thy-1.1 expression as a marker for thymocyte maturation in transgenic mice.

We established a line of transgenic mice carrying the exogenous mouse Thy-1.1 gene (8.2 Kb Eco RI genomic DNA fragment). In these mice, Thy-1.1 was expressed on thymocytes but not on peripheral T cells, presumably due to the lack of cis-acting element(s) on the microinjected genomic DNA. Even in the thymus, however, while most of the CD3/TCR- thymocytes were positive for the transgenic Thy-1.1 gene expression, the CD4+ or CD8+ single positive (SP) thymocytes were composed of two groups, one Thy-1.1+ and one Thy-1.1-, suggesting that the former belongs to cells at premature stages of terminal T cell differentiation. There was no difference in the amount of CD3/TCR complexes expressed on two such SP thymocyte subsets. In the double negative (DN) thymocytes, all the CD3/TCR+ cells were Thy-1.1-. These results suggest that the maturational process of CD3+ DN thymocytes differs from that of SP thymocytes. The unique distribution of Thy-1.1+ population among CD3+ thymocytes suggests that the transgenic Thy-1.1 gene expression can serve as a useful marker to examine terminal maturation processes of CD3+ thymocytes.     

Mouse hepatitis viral infection induces an extrathymic differentiation of the specific intrahepatic alpha beta-TCRintermediate LFA-1high T-cell population.

Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent model for the study of thymic and extrathymic T-cell subpopulation disorders induced during viral hepatitis. It was recently reported that, in addition to the intrathymic T-cell differentiation pathway, an extrathymic differentiation pathway of alpha beta-T-cell receptor (TCR) T lymphocytes exists in the liver, and becomes important under pathological situations such as autoimmune diseases, malignancies or hepatic bacterial infections. In the present study, we compared the phenotypes of resident hepatic, splenic or thymic T-cell subpopulations during the acute viral hepatitis induced by HMV3 in susceptible C57BL/6 mice. The number of liver-resident mononuclear cells (MNC) increased during the viral infection, while cellularity decreased. Single positive (SP) CD4+ cells strongly increased in both the liver and thymus, while double positive (DP) (CD4+ CD8+) cells, present in the liver and thymus of mock-infected mice, decreased in C57BL/6 mice during the viral infection. A shift of alpha beta-TCRintermediate T cells toward alpha beta-TCRhigh was evidenced in the liver and thymus of infected mice, but not in the spleen. The few alpha beta-TCRint double negative (DN) (CD4-CD8-) cells also decreased following viral infection. alpha beta-TCRint or high lymphocytes expressing high levels of leucocyte function antigen-1 (LFA-1) increased in the liver of MHV3-infected mice. In addition, liver-resident T cells expressed strongly the CD44 (Pgp-1) activation marker, suggesting that they were either activated or antigen experienced during the viral infection. No significant change in T-cell subpopulations was detected in the spleen, suggesting that MHV3 infection could induce an early in situ differentiation of resident hepatic T cells rather than a recruitment of lymphocytes from peripheral lymphoid organs.     

A step-wise expansion of intestinal intraepithelial T lymphocytes in association with microbial colonization is defined by sensitivity to cyclosporin A.

Murine intestinal intraepithelial lymphocytes (IELs) consist of T cells bearing alpha beta-antigen receptor (alpha beta-IELs) and those bearing gamma delta-IELs). Although gamma delta-IELs outnumber alpha beta-IELs in germ-free (GF) mice, oral inoculation of fecal suspension from conventional (CV) mice into GF mice induced the increase in number of alpha beta-IELs, leaving the number of gamma delta-IELs unchanged, and the number of alpha beta-IELs reached the level of CV mice by 3 weeks after conventionalization. Expansion of alpha beta-IELs and increase in their CD44+ subset in conventionalized mice were not affected until 2 weeks after beginning of daily injection of cyclosporin A (CsA). However, further expansion of alpha beta-IELs during 2-3 weeks after conventionalization was blocked by injection of CsA. Although the relative constitution of CD4- 8-, CD4+ 8-, CD4- 8 alpha alpha+, CD4- 8 alpha beta+ and CD4+ 8+ subsets among alpha beta-IELs was comparable between control and CsA-treated groups, CsA injection resulted in the decrease in ratio of high-density fraction cells to low density fraction cells in IELs. CsA completely abrogated the expansion of T cells in peripheral lymph nodes stimulated by alloantigens in vivo, and proliferation of IELs from GF mice induced by immobilized anti-alpha beta-T-cell receptor (TCR) monoclonal antibodies (mAb) in vitro was also eliminated by CsA. These results indicate that microbial colonization-induced expansion of alpha beta-IELs is subdivided into two steps: the early phase of expansion takes place via TCR-non-mediated pathway and the late phase of expansion requires TCR-mediated signal transduction.     

CD8+ T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+ TCRβ− Thymocytes in SCID Mice

During T-cell development the transition in the thymus of CD4-CD8- double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T-cell receptor (TCR)-beta-chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B-17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.     

Effect of cyclosporin A on T cell immunity. II. Defective thymic education of CD4 T helper cell function in cyclosporin A-treated mice

Cyclosporin A (CsA) is an immunosuppressive agent that is widely used in transplantation. Recent animal studies indicate that CsA can affect the development of immunity so that autoreactive T lymphocytes are generated. In this study, mice were treated with CsA prior to irradiation and transplantation of syngeneic bone marrow to determine whether CsA pretreatment would affect the ability of the bone marrow recipients to develop normal T cell function. Our results indicate that (a) thymuses of CsA-treated mice do not contain single-positive thymocytes (i.e. L3T4+Ly-2- or L3T4-Ly-2+) during i.p. treatment with 15 mg/kg/day of CsA; (b) both populations of single-positive thymocytes reappear within 2 weeks of termination of CsA and (c) irradiation and bone marrow reconstitution of these CsA-treated mice results in reconstitution of normal numbers of L3T4+ and Ly-2+ cells, but the L3T4+ T cells to not provide T helper function, as determined by interleukin 2 production and cytotoxic T lymphocytes generation. These findings indicate that CsA can affect thymic microenvironment and may be important as a model for investigating intrathymic T cell maturation. Our results may also have clinical implications for T lymphocyte development in transplant patients receiving CsA.     

Changes in sensitivity of peripheral lymphocytes of autoimmune gld mice to FasL-mediated apoptosis reveal a mechanism for the preferential deletion of CD4-CD8-B220+ T cells

During thymic selection 'mis-selected' CD8(+) T cells exit to the periphery where they are deleted by a Fas/FasL-mediated mechanism, presumably as a result of activation by self-antigens. In the absence of functional FasL, as is the case in autoimmune gld mice, these 'mis-selected' T cells develop into unique Thy1(+)CD4(-)CD8(-) TCRalphabeta(+)B220(+) lymphocytes [abnormal double negative T (DN T) cells]. Using bioactive FasL-bearing vesicles [FasL vesicle preparation (FasL VP)], we were able to induce acute apoptosis in freshly isolated lymphocytes and to demonstrate that peripheral lymphocytes of gld mice become more sensitive to the FasL-mediated apoptosis as they age. Furthermore, flow cytometric analyses indicated that within this peripheral lymphocyte population, the abnormal DN T cells were preferentially eliminated. The exquisite sensitivity of these abnormal DN T cells is attributed to their increased membrane Fas expression with a concomitant reduction of cytosolic FLIP(L). Our data support the hypothesis that specific components of the Fas-mediated apoptotic pathway are modulated in favor of the elimination of auto-reactive T cells as well as those CD8(+) T cells that are 'mis-selected' in the thymus and escape to the periphery.     

角质细胞生长因子对老龄鼠胸腺细胞增殖作用的研究

目的观察角质细胞生长因子（KGF）通过恢复胸腺上皮细胞（TEC）数量促进老龄鼠胸腺细胞增殖的作用。方法①体内实验：BALB／C小鼠10只，随机分为KGF处理组和对照组，每组各5只，连续7d分别给予皮下注射重组人KGF（5mg·k^-1·d^-1）和生理盐水，4周后检测小鼠胸腺和脾组织的细胞总数，流式细胞仪分别检测胸腺组织中CD4单阳性（CD4SP）、CD8单阳性（CD8SP）、CD4CD8双阳性（DP）、CD4CD8双阴性（DN）以及脾组织中CD4SP、CD8SP各T细胞亚群的比例；②体外实验：鼠胸腺上皮来源的1C6细胞，分别经含不同浓度（25、50、100、200、400、800ng／ml）的重组人KGF和不含重组人KGF的完全培养液（对照组）作用24h，采用噻唑蓝（MTT）法测定细胞增殖率。结果细胞计数和流式细胞仪检测显示，与对照组相比，KGF处理组平均胸腺细胞总数和CD4SP、CD8SP、DP、DN各T细胞亚群的平均细胞数以及KGF处理组平均脾细胞总数和CD4SP、CD8SP各T细胞亚群的平均细胞数均有明显增加（均P〈0．05）。MTT法检测显示，在低浓度（〈200ng／ml）时，1C6细胞增殖率随KGF浓度的增加不断升高；当KGF浓度为200ng／ml时，1C6细胞增殖率达到最高值；而在高浓度（〉200ng／ml）时，1C6细胞增殖率随KGF浓度的增加又逐渐下降；与对照组比较，浓度为100、200、400ng／ml的重组人KGF对1C6细胞具有明显的促增殖作用（均P〈0．05）。结论KGF可通过恢复老龄鼠TEC数量以及促进胸腺细胞生成和增加成熟T细胞向外周输出而增强机体的免疫力。     

Galactoside-specific plant lectin,Viscum album agglutinin-I induces enhanced proliferation and apoptosis of murine thymocytes in vivo

Galactoside-specific plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to act as a biomodulator with proinflammatory and apoptosis-inducing effects, however its cellular targets and mechanism of immunobiological action in vivo are less well understood. Therefore, in the present work the short- and long-term in vivo effects of VAA-I on thymocyte subpopulations and peripheral T cells were tested using a murine (Balb/c) model. Cell surface CD4/CD8 staining and flow cytometry allowed us to follow the changes of thymocyte subpopulations: CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), CD4+ or CD8+ single positive (SP) and mature peripheral T cells after single or repeated injections with low doses of VAA-I. The apoptosis of the cells was detected by flow cytometry using propidium iodide (PI) and Annexin V staining. To detect the short-term effects of the lectin, the animals were investigated 24 h after a single injection of 1 or 30 ng/kg body weight (BW) VAA-I+/-1 mg/kg Dexamethasone (DX). The total number of mature CD8+ SP thymocytes increased significantly with an enhancement of the ratio of apoptotic cells. In contrast, in the blood samples an elevated CD4/CD8 ratio was found. In the next trial, Balb/c mice were treated twice weekly with 1 or 30 ng/kg VAA-I+/-1 mg/kg DX for 3 weeks. The total cell count of thymocytes showed significant increases after both doses of VAA-I, but an elevated percentage of apoptotic cells was found only after treatment with 30 ng/kg VAA-I. SP thymocytes revealed higher increases in lectin-induced apoptosis than DN or DP cells. In addition, both lectin doses significantly inhibited the DX-induced reduction of all thymocyte subpopulations investigated. In conclusion, our data suggest that VAA-I is able to modulate the maturation of thymocytes in vivo.     

Alterations in the thymocyte phenotype of EphB-deficient mice largely affect the double negative cell compartment

In the present study, we have analysed the phenotype of EphB2 and/or EphB3 deficient thymocytes confirming and extending previous studies on the role of this family of molecules in T-cell differentiation. In all mutant thymuses statistically significant reduced cell contents were observed. This reduction of thymic cellularity correlated with increased proportions of apoptotic cells, largely both double negative (DN; CD4- CD8-) and double positive (CD4+ CD8+) cells, and decreased proportions of DN cycling cells. Adult deficient thymuses also showed increased proportions of DN cells but not significant variations in the percentages of other thymocyte subsets. In absolute terms, the thymocyte number decreased significantly in all thymocyte compartments from the DN3 (CD44- CD25+) cell stage onward, without variations in the numbers of both DN1 (CD44+ CD25-) and DN2 (CD44+ CD25+) cells. Remarkably, all these changes also occurred from the 15-day fetal EphB2 and/or EphB3 deficient mice, suggesting that adult phenotype results from the gradual accumulations of defects appearing early in the thymus ontogeny. As a reflection of thymus condition, a reduction in the number of T lymphocytes occurred in the peripheral blood and mesenteric lymph nodes, but not in spleen, maintaining the proportions of T-cell subsets defined by CD4/CD8 marker expression, in all cases.     

Proliferation of CD3+ B220- single-positive normal T cells was suppressed in B-cell-deficient lpr mice.

It is known that lpr mice develop systemic lymphadenopathy and lupus erythematosus-like autoimmune disease that are associated with the accumulation of CD4- CD8- (double-negative; DN) CD3+ B220+ abnormal T cells as well as normal mature CD4+ or CD8+ single-positive (SP) CD3+ T cells. In order to clarify the role of B cells in the lymphoproliferation and autoimmunity of lpr mice, we created B-cell-deficient C57BL/6 (B6) lpr mice (B6lpr/lpr microMT/microMT) by crossing B6lpr/lpr mice with B6 microMT/microMT mice in which the B-cell development was arrested at pre-B stage owing to a targeted disruption of the immunoglobulin mu heavy-chain gene locus. In the B-cell-deficient B6-lpr mice, both lymphadenopathy and splenomegaly were markedly suppressed. Although the accumulation of both CD3+ B220- SP normal T cells and CD3+ B220+ DN abnormal T cells was inhibited in the B-cell-deficient lpr mice, the decrease in numbers of CD3+ B220- SP normal T cells occurred more strikingly than that of the CD3+ B220+ DN abnormal T cells. Glomerulonephritis did not develop in the B-cell-deficient lpr mice over 40 weeks. The present results indicate that the B cells thus play a crucial role in the extensive proliferation of normal CD3+ B220- mature SP T cells rather than the accumulation of abnormal DN T cells.     

CD3 expression, modulation and signalling in T-cell subpopulations from MRL/Mp-lpr/lpr mice.

The expanded T-cell population of MRL/Mp-lpr2lpr mice is abnormal from a variety of standpoints. We have already shown that T-cell receptor expression and modulation are aberrant in the predominant CD4- CD8 (DN) T cell population. To investigate these abnormalities further, we examined CD3 expression and modulation in subpopulations of +/+ and lpr T cells and measured mitogen-induced Ca++ mobilization in DN lpr T cells. We found that expression and modulation of CD3 in CD4hi and CD8hi lpr single positive (SP) T cells are similar to that in +/+ T cells. We have, however, identified additional lpr cell subsets that are CD4lo or CD8lo. Their expression and modulation of CD3 are intermediate, between that of SP and DN lpr T cells. These subpopulations may thus represent a transitional stage between the SP and DN populations. The rapid modulation of CD3 in the DN population does not appear to be merely related to the lack of expression of CD4 or CD8, and may in fact cause (rather than result from) low CD3 expression. In addition, we observed impairment of CA++ mobilization in DN lpr T cells in response to concanavalin A or anti-CD3 antibody. These findings further define the abnormalities of T cells from lpr mice.     

Depletion of pro-inflammatory CD161(+) double negative (CD3(+)CD4(-)CD8(-)) T cells in AIDS patients is ameliorated by expansion of the γδ T cell population

In this present investigation, flow cytometry was utilized to evaluate 13 healthy controls and 31 HIV-1 infected patients who had advanced to the AIDS stage of infection (CD4 count below 200 cells/mm(3)), for the expression of CD161 on CD3(+) double negative (DN) (CD3(+)CD4(-)CD8(-)) T cells, CD4(+) T cells, CD8(+) T cells and γδ T cells. The observed depletion of CD161(+) T cells from peripheral circulation was due primarily to the loss of CD4(+)CD161(+) T cells; as these cells represented 8.67±0.74% of the total healthy control peripheral T cell population, while the CD4(+)CD161(+) T cells of the AIDS group represented only 3.35±0.41% (p=<0.0001) of the total peripheral T cell population. We have also shown here that the DN T cell population was more than doubled in the AIDS group, with the DN T cell population expanding from 3.29±0.45% of the healthy control peripheral T cell population to 8.64±1.16% (p=0.0001) of the AIDS group peripheral T cell population. By evaluating the expression of CD161 on the surface of the DN T cells we showed that within the healthy control group, 47.4±4.99% of the DN T cells were positive for the expression of CD161, while only 26.4±3.54% (p=0.002) of the AIDS group's DN T cells expressed CD161. Despite CD161 expression being halved on the DN T cells of the AIDS group, when we compared the total peripheral T cell percentage of CD161(+) DN T cells between the healthy control group and the AIDS group, there was no statistical difference. Even though only 26.4% DN T cells within the AIDS group were positive for CD161(+), the overall DN T cell population had expanded to such an extent that there was no statistical difference between the groups with regard to CD161(+) DN T cells as a percentage of the total peripheral T cell population. Furthermore, we showed that within the DN T cell population, there was an approximate 2:1 ratio of γδ to αβ T cells, and this ratio was maintained in both the healthy control group and the AIDS group. While evaluating γδ T cells we also discovered that CD8(+) γδ T cells were expanded from 0.62±.09% of the healthy control peripheral T cell population to 5.01±.88% (p=<0.0001) of the peripheral T cell population of the AIDS group; and that this population of CD8(+) γδ T cells underwent the same reduction in percentage of cells expressing CD161(+), further demonstrated that the phenomenon of CD161(+) percentage reduction and compensatory increase in total cell population was affecting the entire circulating γδ T cell population.     

Thymic and lymphoid changes and serum immunoglobulin abnormalities in mice receiving cyclosporine.

The authors recently demonstrated that cyclosporine (CsA) enhanced the development of murine thymic lymphomas by its tumor promoter-like action. To clarify the underlying mechanism, they investigated the morphologic alterations in the thymus and other lymphoid tissues and the serum immunoglobulin abnormalities in mice receiving CsA. Feeding male Swiss Webster mice with diets containing 0.015% and 0.027% CsA for 1, 2, 4, and 8 weeks led to a marked reduction of the thymic medulla. Thymocytes remaining after CsA treatment were completely destroyed by a single injection of cortisone acetate (8 mg/mice), and cortisone-resistant cells were markedly decreased. Immunoperoxidase staining using anti-keratin antibodies showed a decrease in the number of keratin-positive cells, presumably epithelial cells, in the thymus of CsA-treated mice. These changes were completely reversible within 4 weeks after withdrawing CsA from the diet. The lymph nodes and the gut-associated lymphoid tissues of CsA-treated mice showed blurring of the germinal centers, diminished Thy-1-positive lymphocytes, and proliferation of lymphocytes carrying IgG, IgM, and IgA on their surface. Similar changes were observed in the spleens of CsA-treated mice. There was a slight increase in serum IgG, a twofold increase in serum IgM, and a six- to tenfold increase in serum IgA. The results suggest that CsA interferes with the functions of both T and B lymphocytes in vivo and that disturbance of T-cell maturation in the thymus may be the mechanism by which CsA promotes the induction of thymic lymphomas in mice.     

Augmentation of delayed-type hypersensitivity to high dose sheep erythrocytes by cyclosporin A in the mouse: influence of drug dosage and route of administration and analysis of spleen cell populations.

When administered by various routes 48 h before a high systemic dose (10 degrees) of sheep red blood cells (SRBC), Cyclosporin A (CsA) prevented the suppression of delayed-type hypersensitivity (DTH) reactions elicited 4 days later. Augmentation of DTH was observed over a wide range (5-200 mg/kg) and with circulating CsA levels ranging below 45 ng/ml at the time of immunization or antigen challenge. Splenic lymphocytes from vehicle- and CsA-treated mice exhibited good proliferative responses to mitogen in vitro, but only those from CsA-treated animals responded to antigen. Expression of DTH was associated with a progressive, 2-fold increase in the absolute numbers of splenic L3T4+ cells, whereas no significant alteration in the number of Lyt-2+ lymphocytes was recorded. B cell and macrophage numbers in the spleen were unaffected by CsA. In contrast to its potentiating effects on cell-mediated immunity, CsA caused profound (up to 100%) suppression of the concomitant production of splenic anti-SRBC IgM-secreting plasma cells. Circulating anti-SRBC antibody levels were also markedly reduced. These data show that CsA can permit induction of TDTH, whilst suppressing T-dependent humoral immunity and without significant change in absolute numbers of Lyt-2+ cells.     

Anti-schistosomal activity of cyclosporin A: studies on murine spleen cells and the influence of a cyclosporin antagonist on resistance to infection.

Mice were treated with 5-day courses of cyclosporin A (CsA) around the time of infection with Schistosoma mansoni. Recovery of lung-stage worms 4-8 days post-infection (p.i.) was substantially reduced (80%) and no sexually mature adults were recovered from the hepatic portal system at 7 weeks p.i. Flow cytometric analysis of spleen cells from CsA-treated animals during the period of maximal parasite attrition revealed transient reductions in CD3+ and CD4+ cells and in the CD4+: CD8+ ratio compared with drug vehicle-treated, infected controls. No significant numerical changes in B cells, macrophages or eosinophils were detected relative to vehicle-treated infected mice. Transfer of spleen cells from CsA-treated donors 8 days after infection failed to confer increased resistance to S. mansoni infection on untreated recipients. Moreover, concomitant administration of CsA and an inducer of interleukin-2 production (ADA-202-718) did not interfere with the anti-schistosomal effect of CsA. Despite our incomplete understanding of the in vivo properties of CsA and reports of its paradoxical effects on immune responses, these new data indicate that the influence of CsA in schistosomiasis is unlikely to be mediated by modulation of host cell mediated immunity. This contrasts with certain other anti-parasitic effects of CsA which appear to be mediated by an action on T cells.     

Adhesion of peripheral blood lymphocytes of children with arthritis to human umbilical vein endothelial cells.

To determine whether adhesion of peripheral blood lymphocytes (PBL) of patients with juvenile rheumatoid arthritis (JRA) may be enhanced, adhesion of PBL of children with JRA, children with seronegative spondyloarthropathies (SSA), age-appropriate and adult controls, to human umbilical vein endothelial cells (HUVEC) was assessed in vitro. B and CD4 T lymphocytes in initial, adherent, and non-adherent cell fraction were identified by flow cytometry. B lymphocytes of all the younger subjects combined had a higher adherence to activated HUVEC compared with B lymphocytes of the adult donors. Except for greater adherence of HLA-DR+ CD4 T cells, lymphocytes of children with JRA showed no enhanced adhesion to either unactivated or activated HUVEC. The percentage of B cells adherent to activated HUVEC in each of the subject groups was 1.5-3.6-fold higher than adherent CD4 T lymphocytes. Surface analyses indicated higher percentages of CD49d (alpha 4)+ and CD29 (beta 1)+ CD4 T lymphocytes in adherent cells, but less of a differential in CD49 (alpha 4)+ and no difference in CD29 (beta 1)+ B lymphocytes. There were fewer Leu-8 (L-selectin)+ B and Leu-8+ CD4 T cells among adherent cells. The data suggest a greater adhesive capacity of B lymphocytes compared with CD4 T lymphocytes which is unrelated to disease, and the possibility that B lymphocytes may utilize adhesion molecules distinct from those of CD4 T lymphocytes. Only a small subset of T cells of patients with JRA may have an enhanced capacity for adhesion to endothelium.     

A cortisone sensitive CD3low subset of CD4+CD8- thymocytes represents an intermediate stage in intrathymic repertoire selection.

Two populations of CD4 single positive (SP) thymocytes were found in transgenic mice bearing class I-restricted Mls-1a reactive (V beta 8.1) TCR genes in the absence of the restriction element. CD3high CD4 SP cells were deleted in the presence of Mls-1a and were cortisone resistant, whereas CD3low CD4 SP cells were not deleted in the presence of Mls-1a and were cortisone sensitive. Intravenous transfer of CD3low CD4 SP cells into nude mice resulted in significant peripheral expansion of these cells with apparent upregulation of CD3. These data indicate that CD3low CD4 SP thymocytes represent an intermediate stage in the transition from CD3low double positive (DP) to CD3high SP thymocytes and raise the possibility that these cells may hve undergone positive but not negative selection events (at least to Mls-1a). Furthermore the fact that CD3high DP thymocytes were also deleted by Mls-1a in these mice suggests strongly that sensitivity to Mls-1a deletion is dependent upon stage of thymic maturation (as revealed by TCR density) rather than CD4/CD8 phenotype.     

Induction of Apoptosis in Mouse Thymocytes by Cyclosporin A: An In Vitro Study

The effect of cyclosporin A (CsA) on mouse thymocytes was investigated in vitro. Ultrastructural examination and DNA electrophoresis of 24hr-CsA-treated thymocytes showed chromatin condensation, cellular shrinkage and nuclear fragmentation in oligonucleosomal fragments. DNA labeling of CsA-treated-thymocytes with propidium iodide (PI) showed an increase in the number of apoptotic nuclei compared to untreated thymocytes. Furthermore, flow cytometric analysis using monoclonal antibodies (mAbs) specific to particular thymocyte subsets showed that CsA induces a decrease in the relative number of immature double positive (DP) CD4+CD8+ thymocytes in direct proportion to the concentration of CsA. No significant changes were observed in mature single positive (SP) CD4+CD8-and CD8+CD4-cells. Moreover, the cell viability of CsA-treated thymocytes was decreased. These results suggest that CsA induces the programmed cell death of thymocytes in vitro. Taken together with our previous study on the in vivo effect of CsA on mouse thymus and thymocytes, the present study confirms that, in addition to its effect on the thymic epithelium, CsA acts directly on thymocytes by inducing their programmed cell death. We postulate that immature DP thymocytes are the most likely targets of CsA.