Mutations in the tyrosine phosphatase CD45 gene in a child with severe combined immunodeficiency disease.

The hematopoietic-specific transmembrane protein tyrosine phosphatase CD45 functions to regulate Src kinases required for T- and B-cell antigen receptor signal transduction. So far, there have been no reports to our knowledge of a human deficiency in a tyrosine-specific phosphatase. Here, we identified a male patient with a deficiency in CD45 due to a large deletion at one allele and a point mutation at the other. The point mutation resulted in the alteration of intervening sequence 13 donor splice site. The patient presented at 2 months of age with severe combined immunodeficiency disease. The population of peripheral blood T lymphocytes was greatly diminished and unresponsive to mitogen stimulation. Despite normal B-lymphocyte numbers, serum immunoglobulin levels decreased with age. Thus, CD45 deficiency in humans results in T- and B-lymphocyte dysfunction.

Depletion of CD8+ cells abolishes memory in acquired immunity against Chlamydia pneumoniae in BALB/c mice.

The importance of T cells in Chlamydia pneumoniae infection in mice was assessed by comparing wild-type BALB/c mice with nude mice and mice depleted in vivo of either CD4+ or CD8+ T cells. Whereas wild-type mice cleared the primary infection in 3 weeks, nude mice were only able to restrict the infection and could not clear it during the observation period of 56 days. Nude mice exhibited a greater number of macrophages in their lungs and the pulmonary cells secreted a higher level of tumour necrosis factor-alpha (TNF-alpha) than wild-type mice. Depletion of CD4+ cells did not change the overall infection kinetics of the primary infection. However, depletion of CD8+ cells resulted in a slightly impaired clearance of the bacteria in the late stages of primary infection. To assess the role of the two T-cell subsets in the acquired immunity that develops during primary infection in wild-type BALB/c mice, in vivo depletions were performed during reinfection. Prior to reinfection, immunocompetent wild-type mice were infected and natural immunity was allowed to form. During reinfection, depletion of CD4+ cells did not have any effect on infection kinetics, whereas depletion of CD8+ cells abolished the protection, reverting the infection kinetics and bacterial load to the same levels found in wild-type mice during primary infection. These results show that T cells are necessary for clearing C. pneumoniae infection in mice. Furthermore, whereas neither of the two main T-cell subsets, separately, were essential for clearance of primary infection, the induced protective immunity was strongly CD8 dependent.

Leishmania major infection in interleukin-4 and interferon-gamma depleted mice.

The outcome of experimental Leishmania major infection in mice is closely correlated with the type of CD4+ helper T cell (Th) response. Whereas a Th1 response is host protective, a Th2 response leads to a disseminated, fatal course of disease. Previous studies in this murine model have shown, that the two prominent Th1 and Th2 cytokines, interferon (IFN)-gamma and interleukin (IL)-4, themselves play a major role in the determination of the resulting Th response. Treatment of susceptible mouse strains (BALB/c) with anti-IL-4 induces a Th1 response, allowing the animals to cure the infection. Treatment of resistant strains (e.g. C3H/HeN) with anti-IFN-gamma induces a Th2 response with dissemination of the disease. In this report, we investigated the course of infection and Th response in susceptible and resistant mice treated with anti-IL-4 and anti-IFN-gamma simultaneously. Both mouse strains showed an initial exacerbation of the disease and an overall reduced cytokine response early after infection. Later during infection both strains had a strong Th1 response that was resulting in cure of disease in C3H/HeN mice. BALB/c mice however, could not control the spread of infection despite the strong Th1 response.

Lactobacilli and streptococci induce interleukin-12 (IL-12), IL-18, and gamma interferon production in human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells (PBMC) were stimulated with three nonpathogenic Lactobacillus strains and with one pathogenic Streptococcus pyogenes strain, and cytokine gene expression and protein production were analyzed. All bacteria strongly induced interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha mRNA expression and protein production. S. pyogenes was the most potent inducer of secretion of IL-12 and gamma interferon (IFN-gamma), and two of three Lactobacillus strains induced IL-12 and IFN-gamma production. All strains induced IL-18 protein production. IL-10 and IL-4 production was induced weakly and not at all, respectively. Our data show that nonpathogenic lactobacilli and pathogenic streptococci can induce Th1 type cytokines IL-12, IL-18, and IFN-gamma in human PBMC.

Local immune responses to Chlamydia pneumoniae in the lungs of BALB/c mice during primary infection and reinfection.

Cell-mediated immune (CMI) responses play a major role in protection as well as pathogenesis of many intracellular bacterial infections. In this study, we evaluated the infection kinetics and assessed histologically the lymphoid reactions and local, in vitro-restimulated CMI responses in lungs of BALB/c mice, during both primary infection and reinfection with Chlamydia pneumoniae. The primary challenge resulted in a self-restricted infection with elimination of culturable bacteria by day 27 after challenge. A mild lymphoid reaction characterized the pathology in the lungs. In vitro CMI responses consisted of a weak proliferative response and no secretion of gamma interferon (IFN-gamma). The number of lung-derived mononuclear cells increased substantially during the primary infection; the largest relative increase was observed in B cells (B220(+)). After reinfection, the number of lung-derived mononuclear cells increased further, and the response consisted mainly of T cells. The reinfection was characterized in vivo by significant protection from infection (fewer cultivable bacteria in the lungs for a shorter period of time) but increased local lymphoid reaction at the infection site. In vitro, as opposed to the response in naive mice, acquired immunity was characterized by a strongly Th1-biased (IFN-gamma) CMI response. These results suggest that repeated infections with C. pneumoniae may induce Th1-type responses with similar associated tissue reactions, as shown in C. trachomatis infection models.

Production of human tumor necrosis factor alpha, interleukin-6, and interleukin-10 is induced by lactic acid bacteria.

To investigate the role of cytokines in interactions between lactic acid bacteria and the immune system, we measured production of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-10 from human peripheral blood mononuclear cells after stimulation with live or glutaraldehyde-fixed bacteria. Production of tumor necrosis factor alpha, IL-6, and, in some cases, IL-10 was induced in amounts even greater than those obtained with lipopolysaccharide as a stimulant. Our results suggest that lactic acid bacteria can stimulate nonspecific immunity.

Biological characterization of Campylobacter fetus lipopolysaccharides.

Lipopolysaccharides (LPS) of three strains of Campylobacter fetus (subspp. fetus and venerealis, and serotypes A and B), a bacterium of veterinary importance but also a cause of various infections in humans, were assessed for their ability to induce mitogenicity, gelation of Limulus amebocyte lysate, lethal toxicity in mice, and pyrogenicity in rabbits. All C. fetus LPS exhibited activities lower than those of Salmonella typhimurium LPS. LPS of C. fetus subsp. fetus serotype A had the lowest activity in all assays. Since the majority of C. fetus subsp. fetus isolates from humans are serotype A, the lower biological activities of this LPS may aid the pathogenesis of such strains. The lower activities of C. fetus LPS compared with those of S. typhimurium LPS may reflect the presence of longer fatty acid chains in the lipid A of C. fetus LPS, whereas interstrain differences in C. fetus LPS bioactivities may be related to some property influenced by composition of the saccharide moiety.

Effect of interleukin-10 on NF-kB and AP-1 activities in interleukin-2 dependent CD8 T lymphoblasts.

Interleukin-10 is a multifunctional cytokine, which regulates the function of various cell types of the immune system. In CD8 T cells it is known to accelerate the interleukin-2 dependent proliferation and to induce the differentiation of these cells to active cytolytic cells. Now we have studied interleukin-10 induced intracellular signaling mechanisms in human interleukin-2 dependent CD8 T lymphoblasts. The data obtained demonstrate that interleukin-10 alone can activate the AP-1 transcription factor and potentiate the interleukin-2 induced NF-kappa B activity. Moreover, interleukin-10 induced a rapid tyrosine phosphorylation of several proteins. The pattern of proteins phosphorylated was very similar to that induced by interleukin-2. Together, these findings suggest that tyrosine kinase dependent activation of NF-kappa B and AP-1 transcription factors are involved in the signaling mechanism of interleukin-10. This activation pathway resembles that of interleukin-2 in the same cell type.

Reconstitution of C.B-17 scid mice with BALB/c T cells initiates a T helper type-1 response and renders them capable of healing Leishmania major infection.

C.B-17 scid mice, which were found to be very susceptible to infection with Leishmania major, were reconstituted with various doses of T cells, T plus B cells or unfractionated spleen cells from nonhealer BALB/c mice. All reconstitution protocols, except for the transfer of very high numbers of BALB/c spleen cells, led to a spontaneously healing infection and resistance to reinfection, rather than the lethal, nonhealing infection typical of BALB/c mice. These healing responses were associated with a strong T helper 1 (Th1)-like response characterized by delayed-type hypersensitivity (DTH) responsiveness, but no elevation of serum IgE, and by the production of high levels of interferon-gamma (IFN-gamma), but no interleukin-4 (IL-4) by lymph node and spleen cells after restimulation with antigen in vitro. The development of this Th1 response from BALB/c Th cells requires IFN-gamma during the initial infection period. Treatment of scid mice with a single injection of neutralizing anti-IFN-gamma antibody prior to infection and reconstitution prevented healing and permitted the development of a Th-2 like response as indicated by elevated serum IgE, but no DTH, and by the production of IL-4, but very little IFN-gamma, after antigen stimulation in vitro. As few as 10(4) transferred T cells led to a Th1-like response, suggesting that the IFN-gamma is of host rather than donor origin. The transfer of very high numbers (7.5 x 10(7)) of BALB/c spleen cells overcame the effects of the IFN-gamma and led to the nonhealing infection and cytokine pattern characteristic of BALB/c mice. The enrichment or depletion of B cells from the transferred T cells had no measurable effect upon the development of a healing response in reconstituted scid mice.

IL-4 induces a Th2 response in Leishmania major-infected mice.

The infection of mice with Leishmania major can cause either a fatal disseminated disease or a localized healing disease, depending on the genetic background of the mice. A strong correlation has been shown between disease outcome and the nature of the T cell response, with healer strains developing a Th1-like response and nonhealer strains a Th2-like response. The treatment of nonhealer BALB/c mice with a single dose of an anti-IL-4 antibody, given at the time of infection with L. major, allowed these mice to develop healing Th1-like responses, suggesting that IL-4 is required in BALB/c mice for the differentiation of Th cells into Th2 cells. Anti-IL-4 had to be present during the first 2 wk of infection to have this effect. Anti-IL-4 caused a marked shift from a Th2 to a Th1 pattern of cytokine expression within 4 days, in vivo, and injections of IL-4 had the opposite effect on the early response in healer C3H/HeN mice. These findings demonstrate that IL-4 can induce the development of Th2 response to L. major infection in vivo.

Depletion of asialo-GM1+ cells from the F1 recipient mice prior to irradiation and transfusion of parental spleen cells prevents mortality to acute graft-versus-host disease and induction of anti-host specific cytotoxic T cells.

Graft-versus-host disease (GVHD) was induced in irradiated (750 rad) (CBA x C57BL/6)F1 hybrid mice by an intravenous injection of 30 x 10(6) CBA spleen cells and 5 x 10(6) syngeneic F1 bone marrow cells. The GVHD resulted in the death of 80% of recipients within 9 days. However, when radioresistant Asialo-GM1+ cells were depleted from the recipients with a single injection of anti-Asialo-GM1 antibody 2 days before irradiation and transplantation, mortality decreased significantly (to 11%). During the GVHD, anti-host specific cytotoxic T cell (CTL) activity could be shown in vitro in the spleens of mice suffering from the GVHD if suppressor activity was first abolished by in vitro culture procedures. This CTL activity, however, was not detectable in the spleens of anti-ASGM1 antibody pretreated hosts. The results indicate that radioresistant ASGM1+ cells of host origin are necessary for the induction of both anti-host CTL and lethal GVHD.

Asialo GM1+ NK cells have opposite roles in the activation of alloreactive cytotoxic T lymphocyte (CTL) response in vitro and in vivo.

The immuno-regulatory role of AsialoGM1+ murine NK cells in the induction of allogeneic cytotoxic T cell responses was studied in vivo and in vitro. Depletion of ASGM1+ cells from the (CBAxC57BL/6)F1 cells used for the foot-pad immunization of CBA mice greatly reduced the formation of allospecific CTLs. However, highly purified ASGM1+ cells were not efficient stimulators of alloCTLs in vivo by themselves. When the same genetic combination was used in alloCTL stimulation culture in vitro, ASGM1+ cells were seen to suppress the activation of the CTL response. The opposite roles of ASGM1+ cells in the alloCTL activation in vivo and in vitro were seen when both lymphoid cells (spleen cells or peripheral blood cells) and non-lymphoid cells (epidermal cells) were used as stimulators. The data presented in this paper suggest that during the alloCTL activation, ASGM1+ cells have an important enhancing role in vivo; but in vitro these cells suppress the antigen-presenting cells.

Induction of TNP-specific cytotoxic T lymphocyte memory in vivo in the absence of T helper cell activity.

The question of whether TH cells are required for the priming of CTL precursors (CTLp) in vivo was studied by using Txbm mice (Thymectomized, irradiated, and stem cell-reconstituted mice). In these mice, TNP-specific CTL could be induced in vitro with TNP-coupled spleen cells only if the cultures were supplemented with an IL 2-containing supernatant (ConAsup). In contrast to normal mice, TNP-specific Lyt-2-TH cells could not be induced by skin painting with trinitrochlorobenzene (TNCB) (as tested by the ability to help CTL formation from thymocyte or normal spleen precursors). These data confirm previous findings that Txbm mice possess CTLp but that their TH compartment is deficient. TNCB skin painting had, however, a clear priming effect on the CTLp population: spleen cells from TNCB-painted mice could give rise to specific CTL with a lower amount of ConAsup than spleen cells from unprimed mice. In addition to this, priming changed the CTLp so that stimulation with lightly coupled cells (0.1 mM trinitrobenzene sulfonic acid [TNBS] instead of 10 mM TNBS) became effective. These changes took place without a significant increase in the frequency of TNP-specific CTL precursors. The data obtained are consistent with the concept that at least with some antigens, CTLp proliferation (clonal expansion), which is probably caused by activated TH cells, is not required for the induction of immunologic memory in vivo.

Natural killer (NK) cells and graft-versus-host disease (GVHD): no correlation between the NK cell levels and GVHD in the murine P----F1 model.

Graft-versus-host disease (GVHD) was induced in (CBA X C57BL/6) F1 mice by i.v. injection of 50 X 10(6) parental spleen cells. The GVHD induced an enhanced NK (anti-YAC-1) cytotoxicity during the first 2 weeks after the spleen cell transfusion. This cytotoxic activity was shown to be mediated by asialo GM1-positive, partially Thy-1-positive and nylon-wool (NW) non-adherent cells, thus being classical NK cells. Depletion of NK-cell activity from donor and/or recipient mice with anti-asialo GM1 antibody prior to the spleen cell transfer did not prevent the GVHD as judged by the splenomegaly assay. Also, when NK activity was potentiated with polyinosinic-polycytidylic acid (pIC), no effect on the GVHD was seen. These data suggest that NK cells are not crucial for the development of GVHD in this model.

The effect of cyclophosphamide on cytotoxic T-lymphocyte responses: inhibition of helper T-cell induction in vitro.

The effects of cyclophosphamide (Cy) on the different cell populations participating in the cytotoxic T lymphocyte (CTL) response against haptenated (trinitrophenyl, TNP) syngeneic cells were studied. Pretreatment of responder cell donor mice with 150 mg/kg Cy decreased the cytotoxicity against TNP-modified syngeneic target cells almost to the background level. When TH cells were added to the culture the cytotoxicity increased significantly. Helper T cells were generated in vivo by priming the mice with TNP-modified syngeneic spleen cells or sensitizing the mice with a reactive hapten (TNCB). However, if the TH cell donor mice were treated with Cy before in vivo priming, the cytotoxicity reached the normal level, which indicated that TH precursors were not destroyed by Cy treatment and TH induction was even more effective after Cy. These data indicate that the decrease of the response by this Cy dose is not due to the sensitivity of CTL or TH precursors. Mice could be primed with male-specific (HY) antigen in spite of Cy pretreatment. However, Cy pretreatment caused a latent period of 2 weeks when effective CTL could not be generated in vitro, but after that the capacity for CTL generation was restored. These experiments confirm that pretreatment of responder cell donor mice with Cy does not destroy CTL or TH precursors, but rather affects their in vitro restimulation probably by destroying a short lived 'inducer' cell that is needed.