Development of autoreactive L3T4+ T cells from double-negative (L3T4-/Ly-2-) Thy-1+ spleen cells of normal mice

Thy-1+/L3T4-/Ly-2- spleen cells were purified from normal C57BL/6 (B6) and C,B-17 mice. Cells within this subset expressed the T cell receptor (TcR) for antigen: the majority of cells in this subset were CD3+; a fraction of the cells was stained with the monoclonal antibody (mAb) F23.1; and the TcR molecule was immunoprecipitable with mAb F23.1 from cells within this subset. In limiting dilution analyses, about 1/30 cells within this subset were growth inducible in vitro by stimulation with phorbol myristate acetate (PMA) plus ionomycin; conditioned media containing interleukin (IL) 1, IL2, IL3 or IL4 activity neither triggered nor promoted in vitro growth of these cells. The in vitro generated T cells displayed the Thy-1+/L3T4+/Ly-2- surface phenotype and were self-reactive, i.e., proliferated preferentially in response to syngeneic stimulator cells, and secreted IL2 and IL3 only in response to syngeneic but not allogeneic stimulator cells. The proliferative response of these cells to syngeneic stimulator cells was blocked by anti-self Ia mAb. This autoreactive helper T cell subset was not inducible in purified Thy-1+ spleen cell subsets from athymic nude mice or scid mice. Autoreactive helper T cells did not express detectable levels of the IL2 receptor (IL2R), and their proliferative response was not blocked by anti-IL2R mAb. From PMA plus ionomycin-stimulated double-negative Thy-1+ spleen cells, 14 T cell clones were established in long-term culture which displayed the CD3+CD4+CD8- surface phenotype and were self-reactive.     

Genetics and strain distribution of concanavalin A-reactive Ly-2-, L3T4- peripheral precursors of autoreactive T cells

Cytotoxic treatment of BALB/c cells from different peripheral lymphoid tissues by a cocktail of monoclonal antibodies against Thy-1, Ly-1, L3T4 and Ly-2 differentiation markers (anti-T cocktail) plus complement eliminates all mature T lymphocytes. Yet a population of dull Thy-1+, Ly-1-, L3T4-, Ly-2-, corresponding to about 1% of the initial population, can be detected by flow cytometry which proliferate under concanavalin A stimulation. These anti-T killing-resistant cells (TKR) were previously shown to be capable of differentiating in culture into class II-restricted autoreactive T helper cells. We demonstrate here that such cells can be detected in mice of BALB/c and DBA/2 genetic background but are absent in C57BL/6 and B10 animals. The presence of TKR cells is dominant in (BALB/c x C57BL/6)F1 hybrids and genetically controlled by two genes which are neither H-2 nor Igh linked. TKR cells are also detected in young NZB mice but disappear with the development of the systemic autoimmune disease in old animals. Thy-1+, L3T4-, Ly-2- cells from MRL lpr/lpr mice also respond to concanavalin A but are removed by the anti-T treatment. Altogether, arguments are presented suggesting that TKR cells represent a particular subset of double-negative peripheral T cells which may correspond to autoreactive T cell recursors that would escape the thymic selection. We postulate that these cells are present in all mouse strains but their susceptibility to killing by anti-Thy-1 antibodies differs depending on background genes.     

Spontaneous production of interleukin 3 by T lymphocytes from autoimmune MRL/MP-lpr/lpr mice

MRL/MP-lpr/lpr (MRL/lpr) mice develop a lupus-like autoimmune disease and a massive generalized lymphadenopathy associated with proliferation of nonmalignant Thy-1⁺ Lyt-1⁺ cells. The mechanism(s) leading to outgrowth of these cells is unknown. We report here that Thy-1⁺, Lyt-1⁺, Lyt-2^? lymphocytes from spleens of MRL/lpr mice, but not from several strains of normal mice, spontaneously secrete IL3. The presence of IL3 is shown by: (a) the ability of the supernatants from unstimulated spleen cells of MRL/lpr (MRL/lpr SUP) to support growth of IL3 but not IL2 addicted cells and (b) the growth-promoting activity in MRL/lpr SUP was absorbed with IL3-dependent cells but not with IL2-dependent cells. Spontaneous release of IL3 was detected in supernatants from spleen cells of 6-week-old MRL/lpr mice and the titers of IL3 activity increased with age. Nylon wool-enriched cells from spleens of MRL/lpr mice proliferated in response to purified IL3 and IL3 secreted by MRL/lpr T cells, in a manner similar to nylon wool-passed cells from normal mice. The cells responding to both sources of IL3 were Thy-1⁺, Lyt-1⁺, Lyt-2^?. Thus, Thy-1⁺, Lyt-1⁺,2^? cells from spleen of MRL/lpr mice spontaneously secrete IL3 and respond normally to this lymphokine. Four Thy-1⁺, Lyt-1⁺,2^? cell lines derived from unstimulated spleen cells of MRL/lpr mice were established in culture with IL3. These IL3-sensitive T cell lines help syngeneic and H-2-compatible normal small “resting” B cells to mature into plasma cells secreting predominantly IgG₁, IgG₂ and IgA. Taken together, these data and previous findings that T cells from MRL/lpr mice have an impaired production of and response to IL2, strongly suggest that abnormal production of IL3 may account for the outgrowth of Thy-1⁺, Lyt-1⁺,2^? cells in the MRL/lpr mouse. Finally, a mechanism linking abnormal production of IL3 and B cell hyperactivity in these animals is proposed.     

The role of L3T4+ cells in the pathogenesis of lupus in lpr-bearing mice. I. Defects in the production of interleukins 2 and 3

Mice which bear the lpr gene spontaneously develop autoimmune syndromes characterized by massive expansion of an unusual T cell subset which is phenotypically Thy-1+, L3T4-, Lyt-2-, B220+. The mutant T cells are refractory to stimulation with mitogenic lectins and, by implication, are thought to be solely responsible for the defects in lymphokine production manifested by lpr mice. The contribution of the remaining L3T4+ T cell subset to the latter derangements has not been previously examined and is the focus of this study. We found that abnormalities in concanavalin A-induced interleukin 2 and 3 production in the spleens of MRL-lpr/lpr and C57BL/6.lpr mice occurred in the presence of limited infiltration with B220+, L3T4- T cells. Mixing experiments indicated that B220+ T cells were not suppressive. Furthermore, lpr spleen cells enriched for L3T4+ cells and depleted of sIg+, B220+ and Lyt-2+ cells demonstrated reductions in lymphokine production which were comparable to those seen in unfractionated preparations. Spleen cells from C57BL/6.lpr mice, enriched for L3T4+ cells, were also markedly impaired in a mixed leukocyte reaction in response to stimulator cells from the class II major histocompatibility complex mutant bm12. The results indicate that the aberrations in lymphokine production and proliferation in the spleen cells of lpr mice involve not only B220+ T cells but also L3T4+ cells and suggest a potential role for the L3T4+ subset in the pathogenesis of lupus in lpr-bearing mice.     

Characterization of functional T-cell lines derived from MRL mice

In an effort to characterize further the role of T cells in the autoimmune disease of MRL/Mp-lpr/lpr (lpr) mice, continuous cell lines were established from spleen and lymph nodes using EL-4 lymphoma supernatants as a source of T-cell growth factor(s). Five lines were derived from lpr spleen and lymph nodes, and an equal number from MRL/Mp- +/+ (+/+). All of the lines lost their alloreactivity after a short time in culture. Surprisingly, every line manifested marked proliferation in response to autologous irradiated spleen cells. This response was restricted to I-Ak, as it was blocked with monoclonal anti-I-Ak antibodies, and as B10.A(4R) accessory cells were stimulatory while B10.A(3R) were not. There was no difference in the degree of stimulation from lpr accessory cells compared to that in those from +/+ or other H-2k mice. The T-cell lines bore Thy-1, Ly-1, L3T4, and 7D4 (interleukin 2 (IL-2) receptor), but lacked Ly-2 and surface Ig. They proliferated in response to both conventional and recombinant DNA-derived IL-2. When cocultured with Ia-identical B cells, the T-cell lines provoked B-cell division and antibody production. The cells also caused intense proliferation when cultured with freshly isolated lpr (but not +/+) lymph node cells. The results indicate that lpr lymphoid tissue contains functional T cells reactive to autologous Ia molecules and capable of inducing both B-cell activation and the proliferation of lpr lymphocytes. Such cells may be of importance in inducing hypergammaglobulinemia, autoantibody production, and lymphoproliferation in these SLE mice.     

Auto-MHC class II-reactive T cell line obtained from MRL/+mice suffering from "lpr-GVHD". I. Characterization of surface phenotypes, specificities and functions in vitro

When MRL/Mp-(+)/+ (MRL/+) mice are lethally irradiated and then reconstituted with bone marrow or spleen cells from MRL/Mp-lpr/lpr (MRL/lpr) mice, they develop a graft-versus-host disease (GVHD)-like syndrome, colloquially known as "lpr-GVHD". To analyze the roles of the MRL/lpr T cells in the development of "lpr-GVHD" and autoimmune diseases, several T cell lines were established from the spleen cells of MRL/+ mice suffering from "lpr-GVHD". The surface phenotypes, specificities, and functions of a representative clone (l/+T1) of the cloned T cell lines were characterized. The l/+T1 cells showed Thy-1.2+, L3T4+ and T3+, but Lyt-2- and B220- phenotypes. Proliferative response was observed by co-culturing the cells with spleen cells from MRL/+, MRL/lpr, AKR/J, and C3H/HeN mice, but not from BALB/c or C57BL/6 mice. Furthermore, the l/+ T1 cells responded to spleen cells of B10.BR and B10.A but not B10.D2 mice. The proliferative response of l/+ T1 cells to MRL/+ spleen cells was inhibited by anti-I-Ek (but not anti-I-Ak or anti-Kk) antibodies, suggesting that the specificity of l/+T1 cell culture enhanced the proliferative response only in the presence of appropriate stimulators. Treatment of stimulator cells with J11d.2 + C (but not anti-Thy-1.2 + C or 33D1 + C) abolished the stimulatory effect, indicating that B cells are effective stimulator cells for auto-MHC class II-reactive l/+T1 cells. When MRL/+ splenic B cells were co-cultured with l/+T1 cells, both B cell proliferation and IgM production were observed. In addition, IgM-class rheumatoid factor and anti-ssDNA antibody activities were found in the supernatants of MRL/+ splenic B cells co-cultured with l/+T1 cells. These results are discussed in relation to "lpr-GVHD" and autoimmunity in MRL/lpr mice.     

Analysis of the function of L3T4+ T cells by in vivo treatment with monoclonal antibody to L3T4

We have used MAb to L3T4 to examine the function of L3T4+ T cells in normal and autoimmune mice. Treatment of mice with MAb to L3T4 profoundly depleted L3T4+ cells from the blood, spleen, and lymph nodes, but not the thymus. In BALB/c and C57BL/6 mice, selective depletion of L3T4+ cells blocked both primary and secondary humoral immune responses and inhibited, but did not prevent, cellular immune responses. In lupus-prone B/W and BXSB mice, depletion of L3T4+ cells significantly retarded autoimmune disease. Because the L3T4 antigen in mice is homologous to the CD4 antigen in humans, these findings have implications regarding the function of CD4+ T cells and the prospects for using MAb to CD4 as therapeutic agents.     

Elevated production of interferon-gamma and interleukin 4 by mature T cells from autoimmune lpr mice correlates with Pgp-1 (CD44) expression

The cell surface glycoprotein, Pgp-1 (CD44), has been shown to be a marker of murine memory T lymphocytes. When activated, Pgp-1hi memory T cells produce strikingly higher amounts of interferon-gamma (IFN-gamma) than naive Pgp-1lo T cells, yet both subsets make similar levels of interleukin (IL)2. Whereas Pgp-1hi cells represent only 20%-25% of peripheral T cells from most strains, this marker is expressed by the vast majority (greater than 90%) of T cells from autoimmune MRL mice homozygous for the lymphoproliferation (lpr) gene. The massive lymphadenopathy that develops in lpr/lpr mice is composed of both non-mature (CD4-CD8-) T cells as well as a greatly expanded number (up to 300-fold) of mature (CD4+CD8-,CD4-CD8+) T cells. Paralleling the expression of high levels of Pgp-1, we find that compared to normal mouse T cells, the lpr mature T lymphocyte subsets are also very high producers on a per cell basis of IFN-gamma and, for the CD4+ subset, IL 4. Increased concentrations of IFN-gamma and IL 4 produced by large numbers of lpr Pgp-1hi mature T cells could contribute to the autoimmune syndrome in MRL lpr/lpr mice through the effects of these cytokines on augmenting MHC class II expression and production of certain classes of antibodies.     

Prevention of B220+ T cell expansion and prolongation of lifespan induced by Lactobacillus casei in MRL/lpr mice.

We examined the therapeutic effect of heat-killed Lactobacillus casei (LC) on MRL/lpr mice. Ingestion of a diet containing 0.05% (w/w) LC from the weaning period prolonged the lifespan and tended to reduce the proportion of B220+ T cells in the spleen and mesenteric lymph nodes (MLN) of MRL/lpr mice. When LC was intraperitoneally injected once a week after the age of 8 weeks, I-A- macrophages accumulated in the spleen as well as the peritoneum and macrophage progenitors increased in the bone marrow. Moreover, the amount of IL-6 mRNA in peritoneal macrophages was reduced by LC injection. Splenocytes from LC-injected MRL/lpr mice exhibited lower proliferative responses to mitogens than those from control MRL/lpr mice and the increase in number of B220+ T cells in the spleen and MLN was prevented by LC injection. However, LC injection affected neither expression of interferon-gamma (IFN-gamma) and IL-4 mRNAs nor proliferative capacities of splenic T cells. Our findings demonstrate that LC injection accelerates macrophage recruitment and prevents the expansion of B220+ T cells without affecting the functions of T cells in MRL/lpr mice. These immunological modulations induced by LC may lead to prolongation of the lifespan of MRL/lpr mice.     

CD4-/CD8-T cells: amplification in spleens of mice following in vivo treatment with monoclonal antibody anti-L3T4

Amplification of L3T4-/Ly-2-(CD 4-/CD 8-) T cells following in vivo administration of anti-L3T4 monoclonal antibody was examined in the spleen of mice by flow cytometry and immunohistochemistry. BALB/c mice were given 4-7 weekly injections of either anti-L3T4 (1 mg/week) or phosphate-buffered saline (control group), and dispersed spleen cells and tissue sections analyzed for the presence of Thy-1.2+, L3T4+, or Ly-2+ cells, and for the absence of both L3T4 and Ly-2 on Thy-1.2+ cells. Prior to treatment, L3T4+ and Ly-2+ cells accounted for virtually all Thy-1.2+ cells in approximately a 2:1 ratio. Following anti-L3T4 treatment, L3T4+ cells were depleted, and Ly-2+ cells accounted for about 2/3 of the Thy-1.2+ cells. A population of L3T4-/Ly-2- T cells was generated that accounted for 20-30% of the Thy-1.2+ cells, accounted for most of the Thy-1.2+ cells in red pulp, and was also present among the predominant Ly-2+ cells in periarteriolar sheaths.     

Generation of L3T4-/Ly-2- T cells in Peyer's patches of mice treated with monoclonal antibody against helper T cells

The effect of in vivo administration of anti-L3T4 monoclonal antibody on the generation of an L3T4-/Ly-2- (CD4-/CD8-) population of Thy-1.2+ cells was examined in Peyer's patches of mice by two-color flow cytometry. Female BALB/c mice aged 8 wk were given 4-6 weekly injections of either anti-L3T4 (1 mg/wk) or phosphate-buffered saline (control group), and dispersed Peyer's patch cells analyzed for the presence and absence of L3T4 and Ly-2 on Thy-1.2+ cells. In anti-L3T4 treated mice, L3T4-/Ly-2- T cells accounted for 25-30% of the Thy-1.2+ cells, whereas in control mice these cells represented only 3-4% of the T cells. The remaining 70-75% of the Thy-1.2+ cells after anti-L3T4 treatment were Ly-2+ and not L3T4+. The L3T4-/Ly-2- population of Thy-1.2+ cells is a novel subset which has not been previously found in Peyer's patches and is amplified when helper T cells are depleted.     

Intestinal intraepithelial lymphocyte T cells are resistant to lpr gene-induced T cell abnormalities.

The mucosal immune system of the gastrointestinal (GI) tract consists of Peyer's patches (PP), which are IgA inductive sites, and more diffuse effector regions which include cells in the intraepithelial lymphocyte (IEL) compartment. Since autoimmune MRL lpr/lpr (MRL/lpr) mice develop a proliferating CD3+, CD4-, CD8- (double negative; DN), B220+ T cell subset in systemic lymphoid tissue, we have initiated studies to determine the distribution of CD3+, DN, B220+ T cells (B220+ T cells or lpr/lpr T cells) in the GI immune system. Specifically, we examined T cell subsets separated according to expression of CD4, CD8, Thy-1, B220, alpha/beta T cell receptor (TcR) and gamma/delta TcR in PP and IEL of MRL/lpr mice at 6, 12 and 21 weeks of age. Increased numbers of CD3+ T cells were noted in both PP and spleen of 12- and 21-week-old mice in which the development of autoimmune disorders were also evident. However, normal numbers of CD3+ IEL T cells were seen in MRL/lpr mice in all three age groups tested. When the presence of T cell lymphadenopathy was examined in both IgA inductive and effector tissues, the PP followed the B220+ T cell pattern seen in the spleen, where approximately 30%-50% of CD3+ T cells in the PP of 12- and 21-week-old MRL/lpr mice expressed the phenotype of lpr/lpr T cells and greater than 90% were alpha/beta TcR+. On the other hand, B220+ T cells had not developed in PP or spleen of 6-week-old MRL/lpr mice. Of interest was the finding that IEL from lpr/lpr homozygous mice did not contain B220+ T cells in any age group tested. In this regard, the IEL of MRL/lpr mice comprised an identical pattern and frequency of CD4-/CD8+, CD4+/CD8-, DN and CD4+/CD8+ (double positive, DP) T cell subsets as their normal counterparts (i.e. MRL +/+, BALB/c and C3H/HeN mice) which consisted of approximately 75%, approximately 7.5%, approximately 7.5% and approximately 10%, respectively. Further, Thy-1, gamma/delta TcR and alpha/beta TcR expression in these four subsets of MRL/lpr IEL were very similar to normal mice. These results suggest that the intestinal IEL compartment is minimally affected by the lpr/lpr mutation which induces T cell abnormalities and indicate that B220+ T cells do not preferentially home to IEL. Further, our results support the concept that IEL T cells develop as a separate T cell lineage from thymus-derived cells.     

Features of renal vasculitis in autoimmune MRL lpr/lpr mice: phenotypes and functional properties of infiltrating cells.

MRL lpr/lpr (MRL/1) mice spontaneously develop a widespread renal vasculitis. The majority of the cells in vasculitic lesions are bright Ly-1, L3T4 and la-positive in contrast to the cells found in lymph nodes and spleens of the old MRL/1 mice. However, despite differences in phenotypical patterns, B and T cells from arteritic lesions do not differ from mononuclear cells (MNC) eluted from MRL/1 lymph nodes with regard to the frequency of IgG secreting cells and the proliferative responses to Concanavalin A (Con A). Co-culture experiments with congeneic MRL+/+ (MRL/n) spleen cells indicate that the poor response to Con A of the MNC eluted from vasculitic lesions is, unlike the case of lymph node MNC, due to suppressive action of vasculitic cells on the indicator cell population. Further support for the activation status of infiltrating MHC in kidney vasculitic lesions, expressed by high in vivo uptake of 3H-thymidine, was obtained by autoradiography performed on frozen sections. The observed differences in phenotypic patterns and functional features between lymph node MNC and infiltrating vasculitic MNC indicate that different immune mechanisms may be responsible for the development of lymphadenopathy and vasculopathy, respectively in MRL/1 mouse.     

Auto-MHC class II-reactive T cell line obtained from MRL/+ mice suffering from lpr-GVHD. II. Analyses of functional characteristics of T cell line by in vivo administration.

Functional characteristics of an autoreactive (I-Ek-restricted) T cell line (l/+ T1), previously established from MRL/M(p-)+/+(MRL/+) mice with lpr-GVHD, were analyzed in vivo. Intravenous injection of l/+ T1 cells to non-irradiated H-2k (MRL/+ or AKR) mice (but not H-2d mice) induced enhanced spontaneous proliferation of recipient spleen cells; this was also I-Ek self-restricted. This augmented self-reactivity seemed to be mediated by recipient L3T4+ T cells, since few l/+ T1 cells were detected in the spleen cells of l/+ T1-injected AKR mice by cell surface marker analyses, and the treatment of the spleen cells with anti-Thy-1.1 antibody (Ab) or anti-L3T4 Ab plus complement abolished this enhanced spontaneous proliferation. The production of IgM rheumatoid factor (RF) in AKR mice and IgG RF in MRL/+ mice increased, although no enhancement of anti-ssDNA Ab production was observed. Judging from both spleen B cell proportion and serum Ig levels, autoantibody induction by the injection of l/+ T1 cells was not associated with polyclonal B cell activation. When lethally irradiated B10 congenic mice were used as recipients, B10. BR mice showed elevated levels of IgM anti-ssDNA and IgM RF 1 wk after l/+ T1 cell injection; it is likely that lethal irradiation causes autoantigens, particularly DNA, to be exposed. These findings suggest that the autoreactivity of l/+ T1 cells can be transferred to recipient L3T4+ T cells via T-T interaction or the immunological network, and that increased autoreactivity induces autoantibody production in the presence of autoantigen stimulation. In contrast to the stimulatory effects observed in AKR and MRL/+ mice, MRL/Mp-lpr/lpr(MRL/lpr) mice showed a different response to the injection of l/+ T1 cells; spontaneous proliferation of spleen cells and autoantibody production were not enhanced, and suppression of the mitogen responses was observed. It is discussed that lpr-GVHD may be due to these unusual features of MRL/lpr mice.     

Phenotypic and functional analyses on T-cell subsets in lymph nodes of MRL/Mp-lpr/lpr mice

By means of killing and/or FACS sorting the double-negative (DN) Lyt2-, L3T4- cells, Lyt2+ or L3T4+ cells and B220- cell populations were separated from T-cell-enriched lymph node (LN) cells of 4- to 5-month-old MRL/Mp-lpr/lpr mice. These highly purified cell populations were examined for their proliferative responses, interleukin 2 (IL2) production and expression of IL2 receptor (IL2R) in response to phorbol myristate acetate (PMA) and the calcium ionophore A23187 (A2) or PMA plus concanavalin A. The DNT-cell population was unable to respond to the stimuli and did not express IL2R. Thus the DN T cells, the major population responsible for the lymphadenopathy, possess fundamental defects in signal transduction as well as in the IL2-IL2R-mediated function. On the other hand, Lyt2+ or L3T4+ T cells obtained by sorting or B220- cells purified by the sorting after killing B220+ cells, exhibited proliferative responses indistinguishable from that of LN cells of the congenic MRL/Mp-+/+(+/+) mice. These cells also expressed IL2R after stimulation, however, the amount of IL2 produced was significantly lower than that produced by congenic +/+ cells. This suggested that phenotypically normal Lyt2+ or L3T4+ T cells of lpr LNs also possess a partial defect in the signal transduction system for IL2 production under the influence of the lpr gene.     

Treatment of murine lupus with monoclonal antibody to L3T4. II. Effects on immunohistopathology of thymus, spleen, and lymph node

Monoclonal antibody to L3T4 has been used successfully to suppress autoimmunity in the New Zealand black/New Zealand white F1 (B/W) mouse model for systemic lupus erythematosus. To clarify the immunopathology of murine lupus and determine the effects of anti-L3T4 treatment on the cellular composition and histopathology of lymphoid organs, we examined the distribution of lymphocyte subsets in cryostat sections of the thymus, spleen, and lymph nodes of B/W mice. Immunohistologic specimens were obtained from female B/W mice that had received weekly intraperitoneal injections of either rat monoclonal antibody to L3T4 (2 mg/mouse/week) or phosphate buffered saline (200 microliters/mouse/week) from age 5 months until euthanasia at 8 months. B and T cell domains in each organ were identified on serial sections with monoclonal antibody directed against B220 (all B cells), Thy-1.2 (all T cells), L3T4 (helper T cells), and Ly-2 (cytotoxic/suppressor T cells). In control mice, striking cytoarchitectural abnormalities were identified in the thymuses, and the spleen and lymph nodes were hypertrophied relative to anti-L3T4 treated mice. Thymic abnormalities included amplification of medulla, formation of thymomas, and cortical atrophy. Amplified medullary regions and thymomas in B/W mice contained numerous B cells and L3T4+ T cells but few Ly-2+ T cells. The enlarged spleens and lymph nodes of control mice consisted of numerous secondary follicles with germinal centers containing an unusual subpopulation of T cells that expressed L3T4 but not Thy-1.2. In contrast, mice treated with anti-L3T4 did not develop histopathologic changes characteristic of systemic lupus erythematosus in any organ. However, treatment depleted L3T4+ cells from the spleen and lymph nodes, and it modulated the expression of L3T4 by thymocytes. These observations demonstrate that treatment with anti-L3T4 not only interferes with L3T4-dependent T cell functions, but it also prevents progressive abnormalities in lymphoid tissue in lupus-prone B/W mice. This preservation of normal lymphoid structure may contribute to the beneficial effects of anti-L3T4 on autoimmunity.     

CD95 (Fas) may control the expansion of activated T cells after elimination of bacteria in murine listeriosis.

CD95 (Fas) is known to mediate activation-induced T-cell death by apoptosis. To understand the role of CD95 during the course of bacterial infection, we examined the kinetics of alphabeta and gammadelta T cells in the peritoneal cavities and livers of 5-week-old CD95-defective MRL/lpr mice after an intraperitoneal infection with Listeria monocytogenes. The number of bacteria in the spleen decreased to an undetectable level by day 10 after infection with 7 x 10(3) Listeria cells similar to the number in MRL/+/+ mice. The number of alphabeta T cells expressing CD44 and CD95 reached a maximum in the peritoneal cavity on day 6 after listerial infection and thereafter decreased gradually in MRL/+/+ mice, whereas CD44+ alphabeta T cells without CD95 expression continued to increase throughout the course of listerial infection in MRL/lpr mice. Freshly isolated T cells from MRL/+/+ mice infected with L. monocytogenes 10 days previously showed DNA fragmentation with apoptosis, whereas such fragmentation was not prominent in T cells from infected MRL/lpr mice. In correlation with the increased number of CD44+ alphabeta T cells, Listeria-specific T-cell proliferation of peritoneal exudate cells was significantly greater in MRL/lpr mice than in MRL/+/+ mice on day 10 after listerial infection. In contrast to alphabeta T cells, gammadelta T cells increased in number only transiently in the peritoneal cavity and liver after listerial infection in both MRL/lpr mice and MRL/+/+ mice. These results suggest that CD95-mediated cell death with apoptosis may be involved in termination of the alphabeta-T-cell-mediated immune response after the battle against L. monocytogenes has been won, whereas gammadelta T cells may undergo apoptosis independently of CD95 during the course of listerial infection.     

Determination of frequency of T cells expressing the T cell-specific serine proteinase 1 (TSP-1) reveals two types of L3T4+ T lymphocytes

TSP-1 is a murine T cell-specific serine proteinase which is exclusively expressed in activated but not in resting T lymphocytes. Among T lymphocyte clones tested so far the enzyme was found to be associated with all Ly-2+ but only with a fraction of L3T4+ lines. Here we have applied a limiting dilution system to determine the frequency of precursor cells of resting L3T4+ and Ly-2+ lymphocytes which can be induced in vitro by antigen/lectin to express TSP-1. T cell subsets were either positively enriched by flow cytofluorometry cell sorting or by negative selection using monoclonal antibodies and complement. Following stimulation of lymphocytes in vitro, individual microwells were tested for growth by visual examination and for the TSP-1 protein/enzyme by analyzing cell lysates using either a specific rabbit anti-TSP-1 antiserum and/or the chromogenic model peptide substrate H-D-Pro-Phe-Arg-p-nitroanilide. Moreover, a large panel of L3T4+ and Ly-2+ T lymphocyte clones generated from primary cultures were similarly investigated. In some cell cultures the presence of TSP-1 was also tested on the mRNA level using a TSP-1-specific oligonucleotide probe. The data show that the majority, if not all, of antigen/lectin-induced-Ly-2+ T cells expressed TSP-1. In contrast, only 12%-27% of the growing lectin or antigen-reactive L3T4+ lymphocytes were positive for the enzyme. Studies performed in parallel with L3T4+ and Ly-2+ lymphocyte populations sensitized in bulk culture showed that under these conditions similar levels of TSP-1-specific mRNA and enzyme activity are detected in both subsets. The finding of primary L3T4+ T lymphocyte clones with distinct patterns of TSP-1 production provides evidence for the existence of two types of L3T4+ effector cells with different functional capacities. The data also suggest a cooperation between distinct L3T4+ lymphocytes for induction of optimal TSP-1 activity in L3T4+ T cells.     

Macrophage activation by interferon-gamma from host-protective T cells is inhibited by interleukin (IL)3 and IL4 produced by disease-promoting T cells in leishmaniasis

BALB/c mice are highly susceptible to Leishmania major infection. They develop a progressive fatal disseminating disease even with a minimum infecting dose. However, these mice are able to contain the disease if they are exposed to sublethal gamma-irradiation shortly before infection. Earlier studies demonstrated that CD4+ T cells from mice which had recovered from infection (Tr) can adoptively transfer resistance. In contrast, CD4+ cells from mice with progressive disease (Ts) not only failed to protect, but can reverse the protective effect of the Tr cells. Spleen cells from BALB/c mice which had recovered from L. major infection or which had progressive disease were cultured with leishmanial antigens in vitro. The culture supernatant from spleen cells of recovered mice (TrSN) contains high levels of macrophage-activating factor (MAF) activity which can activate peritoneal macrophages to kill 51Cr-labeled P815 cells and to eliminate intracellular parasites as measured by the reduction in [3H]thymidine uptake by residual parasites released from macrophages following sodium dodecyl sulfate treatment. The MAF activity of TrSN parallels that of recombinant interferon-gamma (IFN-gamma). In contrast, culture supernatant of spleen cells from mice with progressive disease (TsSN) contains no detectable MAF but it is able to neutralize the MAF activity of TrSN. The MAF-inhibiting function of TsSN appears to be mediated by interleukin (IL)3 and IL4, since the MAF activity of TrSN and rIFN-gamma also can be inhibited by the addition of rIL3 and rIL4 but not by rIL1 or rIL2. Furthermore, the MAF-inhibiting activity of TsSN can be partially reversed by the addition of specific anti-IL3 or anti-IL4, but completely reversed by the combination of the two antibodies in vitro. These findings provide a mechanism for the immune regulation in leishmaniasis and a means by which the two subsets of CD4+ T cells influence each other through their modulation of macrophage function.     

L3T4+ T cells promoting susceptibility to murine cutaneous leishmaniasis express the surface marker Ly-24 (Pgp-1)

In a murine model of cutaneous leishmaniasis, the importance of T cell-dependent immunity has been documented by the susceptibility to parasite infection of athymic nude mice of both genetically resistant and genetically susceptible strains. T lymphocytes from uninfected mice have the capacity to promote resistance to Leishmania major infection in nude recipients, whereas T cells from mice chronically infected with L. major not only fail to mediate protection, but totally abrogate the host-protective effect of normal mouse lymphocytes. Both these effects are mediated by T cells which have the phenotype L3T4+Ly-2-. To discriminate between the two activities, we have tried to separate the L3T4+ population on the basis of additional cell surface markers and we have found that peripheral L3T4+ lymphocytes could be subdivided according to their expression of the Ly-24 (Pgp-1) surface marker. In adoptive transfer experiments, both the Ly-24+ and the Ly-24- subset of L3T4+ cells from uninfected mice had the capacity to mediate resistance to infection with L. major. However, disease-promoting activity was only found in the L3T4+Ly-24+ and not in the L3T4+Ly-24- subset of cells from mice with chronic cutaneous disease. Moreover, Ly-24 expression was strongly increased in lymphocytes from chronically infected mice and in vitro limiting dilution analysis confirmed that the vast majority of L. major-reactive T cells was L3T4+Ly-24+. In genetically susceptible mice with chronic cutaneous leishmaniasis, Ly-24 therefore appears to be a marker for lymphocytes with the capacity to abrogate resistance to disease, these cells being activated and expanding in the course of progressive L. major infection. Ly-24 expression is a useful tool for phenotypic identification and selective enrichment of antigen-activated and possibly memory T cells. It may facilitate the isolation of L. major-specific T cell clones with defined activities.