Protection against Mycobacterium tuberculosis Infection by CD8+ T Cells Requires the Production of Gamma Interferon

The role of CD8 T cells in controlling Mycobacterium tuberculosis infections in mice was confirmed by comparing the levels of growth of the organism in control, major histocompatibility complex class II knockout, and athymic mice and by transferring T-cell populations into athymic mice. By using donor mice which were incapable of making gamma interferon (IFN-γ), it was shown that IFN-γ production was essential for CD8 cell mediation of protective immunity against M. tuberculosis.

Cell-mediated immunity is crucial for the control of mycobacterial infections. Athymic mice (4) and mice whose T cells have been depleted (22, 23) are much more susceptible to infection with mycobacteria than euthymic or unmanipulated mice. However, the contributions of the different components of the T-cell response are unclear. CD4 T cells are thought to play a major role in controlling infections with the primary human tubercle bacillus, Mycobacterium tuberculosis; individuals with reduced CD4 counts, from infection with human immunodeficiency virus, for example, are known to be more susceptible to M. tuberculosis infections (12). Activation of CD4 cells by antigen in association with major histocompatibility complex (MHC) class II molecules results in clonal expansion and the production of cytokines, most notably gamma interferon (IFN-γ), which activate macrophages so that they become mycobactericidal. Mice with deletions of the IFN-γ gene are much more susceptible to M. tuberculosis infection than wild-type mice (5, 9). However, in addition to CD4 cells, other components of the cell-mediated response are thought to play roles in controlling infection with M. tuberculosis. For example, CD8 T cells have been shown to be involved (20, 24): β2 microglobulin-deficient knockout mice, which lack an effective CD8 response, show increased susceptibility to M. tuberculosis infection (10). Other cell types, such as T cells bearing the γ/δ T-cell receptor (19) and NK cells (1), are also thought to have roles in protection against intracellular bacteria, while a number of T cells with novel phenotypes and unknown functions have been shown to recognize mycobacterial antigens (2, 28).CD8 T cells are known to contribute to the protective response against M. tuberculosis, but the mechanism(s) by which they exert this protective effect is unknown. CD8 T cells produce a range of cytokines, including IFN-γ (11, 17, 25, 26), but their primary role is thought to be cytotoxic. However, it has recently been shown that mice with a targeted disruption in either the perforin gene or the granzyme gene and mice which are Fas receptor defective are no more susceptible to infection with M. tuberculosis than are wild-type mice (6, 16). Since perforin (13, 18) and Fas-Fas ligand interactions (21, 27, 31) are thought to be the primary mechanisms of cytotoxicity mediated by CD8 T cells, such cells may contribute their antimycobacterial activity through noncytotoxic pathways.In this study, we have used MHC class II-deficient mice and athymic mice to confirm the role of non-CD4 T-cell-mediated mechanisms in protection against M. tuberculosis infection. Using transfer of purified CD4 and CD8 cells into athymic mice, we have demonstrated that these cells contribute equally to protective immunity in this system. However, by using mice with deletions of the IFN-γ gene as T-cell donors, we have shown that production of IFN-γ is required in order for CD8 T cells to exert their antimycobacterial effect.In preliminary experiments, the levels of growth of M. tuberculosis in MHC class II knockout, athymic, and normal mice were compared. MHC class II knockout (Aβ^−/−) mice were obtained as a breeding nucleus (kindly provided by D. Gray, Hammersmith Hospital, London, United Kingdom, with permission from D. Mathis, Institut National de la Santé et de la Recherche Médicale). These mice were bred from heterozygous (Aβ^+/−) parents and genotyped as described previously (7). Heterozygous littermates were used as controls. Stock cultures of M. tuberculosis H₃₇Rv were grown in Dubos 7H9 broth for 14 days, and then they were aliquoted and stored in liquid nitrogen. For infection, aliquots were thawed, diluted in phosphate-buffered saline, and inoculated intraperitoneally into mice. The infection was monitored by removing the lungs and spleens of infected mice at various intervals; the baseline level of infection of each tissue was estimated by harvesting organs from the mice 18 h after infection and determining viable counts. The tissues were weighed and homogenized by shaking with 2-mm-diameter glass beads in chilled saline with a Mini-Bead Beater (Biospec Products, Bartlesville, Okla.), and 10-fold dilutions of the suspension were plated onto Dubos 7H11 agar with Dubos oleic albumic complex supplement (Difco Laboratories, Surrey, United Kingdom). Numbers of CFU were determined after the plates had been incubated at 37°C for approximately 20 days. The results are shown in Fig. ​Fig.1A1A and B. In control mice, there was a transient increase in bacterial counts in the spleen, followed by a steady decline over 60 days and then by a levelling out of the infection at approximately 10⁴ CFU per g of tissue. In MHC class II knockout mice, there was an initial growth of the infection over the first 60 days, followed by a plateau phase during which the infection appeared to be controlled but was significantly more severe than in wild-type mice (Fig. ​(Fig.1A).1A). In lung tissue (Fig. ​(Fig.1B),1B), a similar pattern emerged, except that in the MHC class II knockout mice, control of the infection broke down in some of the mice after about 60 days, when there was a sudden increase in bacterial counts. By day 80, counts had reached approximately 10⁷ CFU per g of tissue, a 10,000-fold increase over the counts seen in wild-type mice. Open in a separate windowFIG. 1Growth of M. tuberculosis in the tissues of MHC class II knockout, control, and athymic mice. (A and B) Growth in spleens and lungs, respectively, of MHC class II knockout mice (•) and their wild-type littermates (▪). (C and D) Growth in spleens and lungs, respectively, of MHC class II knockout (•) and athymic (▴) mice. Data are the geometric means ± the standard errors of the means for three to five mice. An asterisk indicates a significant difference between values for MHC class II knockout and control mice (P < 0.05 by Students’ t test). A double asterisk denotes that at the indicated time, all remaining mice in the group were killed because of the widely disseminated nature of the infection.These results emphasize the importance of the MHC class II-CD4 T-cell pathway in controlling M. tuberculosis infection. However, in spite of the fact that after the first few days of infection there was always a highly significant difference between the level of viable M. tuberculosis organisms in MHC class II knockout mice and the level in control mice, some control of bacterial multiplication did appear to occur in the MHC class II knockout mice. In order to demonstrate that this apparent partial control of the infection in MHC class II knockout mice was mediated by T cells, we compared growth in these mice with growth in athymic mice. Athymic (nude) BALB/c mice were obtained from a breeding colony at the National Institute for Medical Research. Athymic and MHC class II knockout mice were infected intraperitoneally, and the infections were monitored as described above. Whereas the MHC class II knockout mice were again able to control the infection to some degree, growth in athymic mice was unchecked and the mice had to be killed at 40 days because of overwhelming infection (Fig. ​(Fig.1C1C and D).These results confirm the importance of CD4 cells in controlling M. tuberculosis infections but also suggest that a contribution is made by non-CD4-mediated mechanisms. It has previously been shown that depletion of CD8 cell populations in mice with anti-CD8 antibodies (20) or abolition of a CD8 response by disruption of the β2 microglobulin gene (10) renders mice highly susceptible to infection with M. tuberculosis. CD8 T cells have also been implicated in human tuberculosis; CD8⁺ T cells with specificity for mycobacterium-pulsed target cells have been described (14, 32), and an individual with recurrent tuberculosis was found to have a specific reduction in CD8 T cells (3).In order to investigate the contribution of CD8 T cells to the control of M. tuberculosis infections in mice, total spleen cells, CD4 T cells, and CD8 T cells were transferred from control BALB/c mice into infected athymic BALB/c mice. Splenocytes were incubated in hypotonic medium to lyse erythrocytes and washed twice. To obtain highly purified populations of CD4 and CD8 cells, cell suspensions were enriched by negative selection with T-cell-subset columns (R & D Systems Inc., Minneapolis, Minn.) according to the manufacturer’s instructions. The resulting populations were >90% CD4 or CD8 T cells, as determined by flow cytometric analysis. The cells were washed, resuspended in sterile saline, and injected intravenously such that recipient mice received 5 × 10⁶ cells. The mice were then infected with M. tuberculosis, and organs were harvested 21 days later for CFU counts. The results of a typical experiment are shown in Fig. ​Fig.2.2. In athymic mice which had not received any transferred cells, the infection reached approximately 10⁷ CFU per g in the lung (Fig. ​(Fig.2A)2A) and 10⁸ CFU per g in the spleen (Fig. ​(Fig.2B).2B). Transfer of total spleen cells from naive BALB/c mice reduced the number of CFU 100- to 1,000-fold in both tissues. It appeared that CD4 and CD8 T cells contributed approximately equally to the observed protection. Open in a separate windowFIG. 2Infection of athymic mice with M. tuberculosis following transfer of splenocytes from euthymic mice. (A and B) Results for the lungs and spleen, respectively, of mice infected intravenously with approximately 10⁶ CFU 21 days prior to harvest. Transfer of cells was carried out 24 h before infection. Data are the means ± the standard errors of the means for three to five mice. Mice received either no cells, total spleen cells, CD4 cells, or CD8 cells. All three groups of mice which received cells showed significantly reduced CFU counts compared to controls (P < 0.05 by Student’s t test).The mechanism by which CD8 T cells exert this antimycobacterial response is not understood. It has been suggested that the cytotoxicity of mycobacterium-laden target cells could be involved, perhaps through the release of M. tuberculosis bacilli from ineffective macrophages to cells with greater antimycobacterial potential (15). However, perforin or granzyme knockout mice and Fas receptor-defective mice, when infected, did not display any increased susceptibility to infection, compared to wild-type controls (6, 16). Interestingly, both the perforin knockout mice and the Fas receptor-defective mice had elevated levels of cytokines, including IFN-γ, in the absence of infection, and levels in infected mice were similar to those seen in wild-type mice (16). Thus, neither perforin-, granzyme-, nor Fas-mediated cytotoxicity appeared to be involved in the control of these experimental infections (6, 16). Conversely, however, Silva and colleagues (29) produced CD8⁺ T-cell clones which were capable of conferring protection against M. tuberculosis in recipient mice, and the level of protection correlated with the level of cytotoxic activity rather than with the level of IFN-γ secretion.In a recent study of human cytotoxic cells with mycobacterial specificity, it was found that CD4⁻ CD8⁻ T cells lysed macrophages through a Fas-Fas ligand interaction but the lysis was not associated with mycobacterial killing, whereas CD8⁺ T-cells lysed macrophages by a Fas-independent pathway and the lysis resulted in the killing of mycobacteria (30). The human T-cell lines used for these experiments were unusual in that they were CD1 restricted.Since CD8 T cells were clearly able to confer significant levels of protection against M. tuberculosis in our cell transfer model, we next investigated the role of IFN-γ in this protection. Again athymic mice were recipients of either total spleen cells or CD8 cells. This time, however, donor mice were either normal BALB/c mice or IFN-γ knockout mice (8) and recipient mice received 3 × 10⁶ cells. The results (Fig. ​(Fig.3)3) clearly demonstrate the requirement for IFN-γ. Transfer of total spleen cells or CD8 T cells from normal mice gave protection, although the level of protection was slightly lower than that seen in the previous experiment (Fig. ​(Fig.2).2). This was probably because the number of cells transferred was lower (3 × 10⁶ rather than 5 × 10⁶). However, the protection seen in both organs was significant (P < 0.05). Importantly, transfer of cells from IFN-γ knockout mice gave no protection. Open in a separate windowFIG. 3Infection of athymic mice with M. tuberculosis following transfer of splenocytes from control BALB/c and IFN-γ knockout (IFN-γ −VE) BALB/c mice. (A and B) Results for the lungs and spleen, respectively. The experimental design was identical to that for Fig. ​Fig.2.2. Mice received either no cells, total spleen cells from wild-type BALB/c mice, CD8 cells from BALB/c mice, total spleen cells from IFN-γ knockout mice, or CD8 cells from IFN-γ knockout mice. Mice which received cells from control BALB/c mice (total spleen or CD8 cells) showed significantly reduced CFU counts compared to naive athymic mice (P < 0.05); there were no significant differences between values for naive athymic mice and mice which received either total spleen cells or CD8 cells from IFN-γ knockout BALB/c mice.Thus, the results reported in this study confirm the role of CD8 T cells in the control of M. tuberculosis infections in mice. We have also demonstrated that this control requires the ability of the CD8 cells to produce IFN-γ, suggesting that such cells may exert their effects through classical cytokine-mediated macrophage activation rather than through a cytotoxic mechanism. The recent demonstration that human CD1-restricted CD8 T cells were able to kill mycobacteria in vitro through a cytotoxicity-mediated pathway (30) suggests that different subpopulations of CD8 cells may have different effector mechanisms; since no murine equivalent of the CD1-restricted CD8 T cell has been described, this mechanism may be absent in mice. Alternatively, the results reported earlier for murine CD8 T-cell lines (29) or human CD8, CD1-restricted T-cell lines (30) may reflect the activity of primed or memory T cells, whereas the results reported in the present study reflect the activity of unprimed cells. Primed CD8 T cells have been shown to be hyperreactive to antigenic challenge in vitro and may employ different effector mechanisms. That production of IFN-γ by CD8 T cells is required in order to control infection has also been reported for viral infections (11, 26), where cytotoxicity has long been thought to be the major mechanism of CD8-mediated antiviral activity. IFN-γ and other cytokines have been shown to be major components of the mechanism by which hepatitis B virus is controlled in mice by CD8 cells without the killing of hepatocytes (11). The results reported in this study demonstrate that IFN-γ is essential for CD8-mediated protection against M. tuberculosis infection in mice.     

Identification and Characterization of TspA, a Major CD4+ T-Cell- and B-Cell-Stimulating Neisseria-Specific Antigen

In search for novel T-cell immunogens involved in protection against invasive meningococcal disease, we screened fractionated proteins of Neisseria meningitidis (strain SD, B:15:P1.16) by using peripheral blood mononuclear cells (PBMCs) and specific T-cell lines obtained from normal individuals and patients convalescing from N. meningitidis infection. Proteins of iron-depleted meningococci produced higher PBMC proliferation indices than proteins of iron-replete organisms, indicating that iron-regulated proteins are T-cell immunogens. Insoluble proteins of the iron-depleted cells, which produced better T-cell stimulation than soluble ones, were fractionated by using sodium dodecyl sulfate-polyacrylamide gels and recovered as five fractions (F1 to F5) corresponding to decreasing molecular weight ranges. The proteins were purified (by elution and precipitation) or electroblotted onto nitrocellulose membranes (dissolved and precipitated) before use in further T-cell proliferation assays. One of the fractions (F1), containing high-molecular-mass proteins (>130 kDa), consistently showed the strongest T-cell proliferation responses in all of the T-cell lines examined. F1 proteins were subdivided into four smaller fractions (F1A to F1D) which were reexamined in T-cell proliferation assays, and F1C induced the strongest responses in patients' T-cell lines. Rabbit polyclonal antibodies to F1C components were used to screen a genomic expression library of N. meningitidis. Two major clones (C1 and C24) of recombinant meningococcal DNA were identified and fully sequenced. Sequence analysis showed that C24 (1,874 bp) consisted of a single open reading frame (ORF), which was included in clone C1 (2, 778 bp). The strong CD4(+) T-cell-stimulating effect of the polypeptide product of this ORF (named TspA) was confirmed, using a patient T-cell line. Immunogenicity for B cells was confirmed by showing that convalescent patients' serum antibodies recognized TspA on Western blots. Additional genetic sequence downstream of C24 was obtained from the meningococcal genomic sequence database (Sanger Centre), enabling the whole gene of 2,761 bp to be reconstructed. The DNA and deduced amino acid sequence data for tspA failed to show significant homology to any known gene, except for a corresponding (uncharacterized) gene in Neisseria gonorrhoeae genome sequences, suggesting that tspA is unique to the genus Neisseria. The DNA and deduced amino acid sequence of the second ORF of clone C1 showed significant homology to gloA, encoding glyoxalase I enzyme, of Salmonella typhimurium and Escherichia coli. Thus, we have identified a novel neisserial protein (TspA) which proved to be a strong CD4(+) T-cell- and B-cell-stimulating immunogen with potential as a possible vaccine candidate.     

Murine γδ T Lymphocytes Elicited during Plasmodium yoelii Infection Respond to Plasmodium Heat Shock Proteins

γδ T cells accumulate during Plasmodium infections in both murine and human malarias. The biological role of these cells and the antigens that they recognize are not clearly understood, although recent findings indicate that γδ T cells in general influence both innate and antigen-specific adaptive host responses. We examined the accumulation of γδ T cells elicited during infection with virulent and avirulent Plasmodium yoelii parasites in relatively susceptible and resistant strains of mice. Our results indicated that in nonlethal malaria infections, γδ T cells comprise a larger proportion of splenic T cells than in lethal infections and that only a live infection is capable of inducing an increase in the percentage of γδ T cells in vivo. Furthermore, we demonstrate that γδ T cells elicited during a P. yoelii infection respond by proliferation in vitro to P. falciparum heat shock proteins (HSPs) of 60 and 70 kDa, suggesting a possible immunological involvement of parasite HSPs in this arm of the cellular immune response during malarial infection in mice.     

Association of CD4+ CD25+ T Cells with Prevention of Severe Destructive Arthritis in Borrelia burgdorferi-Vaccinated and Challenged Gamma Interferon-Deficient Mice Treated with Anti-Interleukin-17 Antibody

CD4⁺ CD25⁺ T cells are a population of regulatory T cells responsible for active suppression of autoimmunity. Specifically, CD4⁺ CD25⁺ T cells have been shown to prevent insulin-dependent diabetes mellitus, inflammatory bowel disease, and pancreatitis. Here, we present evidence that CD4⁺ CD25⁺ T cells also play a major role in controlling the severity of arthritis detected in Borrelia burgdorferi-vaccinated gamma interferon-deficient (IFN-γ°) C57BL/6 mice challenged with the Lyme spirochete. When B. burgdorferi-vaccinated and challenged IFN-γ° mice were treated with anti-interleukin-17 (IL-17) antibody, the number of CD4⁺ CD25⁺ T cells increased in the local lymph nodes. Furthermore, histopathologic examination showed the mice to be free of destructive arthritis. When these anti-IL-17-treated B. burgdorferi-vaccinated and challenged mice were also administered anti-CD25 antibody, the number of CD4⁺ CD25⁺ T cells in the local lymph nodes decreased. More importantly, severe destructive arthropathy was induced. In addition, delayed administration of anti-CD25 antibody decreased the severity of the arthritis. These results suggest that CD4⁺ CD25⁺ T cells are involved in regulation of a severe destructive arthritis induced with an experimental model of vaccination and challenge with B. burgdorferi.     

Immunization of Mice with a TolA-Like Surface Protein of Trypanosoma cruzi Generates CD4+ T-Cell-Dependent Parasiticidal Activity

The gene family encoding a trypomastigote-specific protein restricted to the part of the flagellum in contact with the cell body of the trypomastigote form of Trypanosoma cruzi has been isolated, characterized, and expressed in a baculovirus expression system. The gene family contains three tandemly repeated members that have 97 to 100% sequence identity. The predicted protein encoded by the gene family has both significant amino acid sequence identity and other physical and biological features in common with the TolA proteins of Escherichia coli and Pseudomonas aeruginosa. Based on these similarities, we have designated this gene family tolT. Immunization of mice with recombinant TolT generates a population of CD4(+) T lymphocytes that recognize T. cruzi-infected macrophages, resulting in the production of gamma interferon (IFN-gamma), which leads to NO production and a 50 to 60% reduction in parasite numbers compared to that seen with infected macrophages incubated with naive T cells. This population of T cells also produces both IFN-gamma and interleukin 2 (IL-2) but not IL-4 or IL-5 when incubated with spleen cells stimulated with TolT antigen, indicating that they are of the T-helper 1 type. T cells from mice chronically infected with T. cruzi also produce significant levels of IFN-gamma when cocultured with macrophages and either TolT protein or paraflagellar rod protein, indicating that both of these flagellar proteins produce positive T-cell responses in mice chronically infected with T. cruzi.     

Control of Leishmania infantum Infection Is Associated with CD8+ and Gamma Interferon- and Interleukin-5-Producing CD4+ Antigen-Specific T Cells

Visceral leishmaniasis is a severe and lethal disease caused by the protozoan parasites of the genus Leishmania. In areas where leishmaniasis is endemic, most infected individuals control the infection and remain asymptomatic; chemotherapy of visceral leishmaniasis restores some immunity which protects against relapses. In the present study, Leishmania-specific T-cell clones were established from six asymptomatic and five cured patients. Cytokines production by these clones was analyzed. A large fraction of the parasite-specific T-cell clones from asymptomatic patients were CD8(+) and produced high amounts of gamma interferon (IFN-gamma). Most CD4(+) T-cell clones from two asymptomatic subjects exhibited an unusual phenotype: production of high levels of IFN-gamma low levels of interleukin-4, (IL-4), but high levels of IL-5. In contrast, only few parasite-specific CD8(+) T-cell clones were obtained from cured patients after chemotherapy; moreover, CD4(+) T-cell clones from these patients exhibited an heterogeneous profile of cytokines from Th1-like to Th2-like phenotypes. These results point to CD8(+) T cells and to IL-5- and IFN-gamma-producing CD4(+) T cells as possible contributors to human resistance to Leishmania infection. They should stimulate new immunological approaches in the control of this disease.     

Increased Foxp3+ CD4+ Regulatory T Cells with Intact Suppressive Activity but Altered Cellular Localization in Murine Lupus

Foxp3⁺ CD4⁺ regulatory T (T_reg) cells play a pivotal role in the maintenance of dominant self tolerance. Understanding how the failures of immune control by T_reg cells are involved in autoimmune diseases is important for the development of effective immunotherapies. In the present study, we analyzed the characteristics of endogenous T_reg cells in (NZB × NZW) F1 (BWF1) mice, a murine model of systemic lupus erythematosus. Unexpectedly, T_reg number and frequency in aged BWF1 mice with developing lupus nephritis were increased, not decreased, and in vitro suppressive activity in lymphoid organs was intact. In addition, T_reg cells trafficked to target organs because cells were present in the kidney and lung. T_reg cells of aged BWF1 mice exhibited altered localization within lymph organs, however, and an altered phenotype, with higher expression levels of chemokine receptors and activation markers, suggesting a highly activated cellular state. Notably, the expression levels of co-stimulatory molecules were also markedly enhanced in the T_reg cells of aged BWF1 mice. Furthermore, T_reg cells of BWF1 mice did not show any suppressive effects on antibody production in vitro. Taken together, we conclude that T_reg cells in BWF1 mice are not predisposed to functional incompetence but rather are present in a highly activated state.     

Identification of Promiscuous Epitopes from the Mycobacterial 65-Kilodalton Heat Shock Protein Recognized by Human CD4+ T Cells of the Mycobacterium leprae Memory Repertoire

By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4(+) T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killed M. leprae and then tested for proliferative reactivity against overlapping peptides comprising both the M. leprae and Mycobacterium tuberculosis HSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between the M. tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) represented M. leprae HSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences completely identical between the M. leprae and M. tuberculosis HSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4(+) T cells which belong to the human memory T-cell repertoire against M. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.     

Roles of CD4+ T Cells and Gamma Interferon in Protective Immunity against Babesia microti Infection in Mice

Babesia microti produces a self-limiting infection in mice, and recovered mice are resistant to reinfection. In the present study, the role of T cells in protective immunity against challenge infection was examined. BALB/c mice which recovered from primary infection showed strong protective immunity against challenge infection. In contrast, nude mice which failed to control the primary infection and were cured with an antibabesial drug did not show protection against challenge infection. Treatment of immune mice with anti-CD4 monoclonal antibody (MAb) diminished the protective immunity against challenge infection, but treatment with anti-CD8 MAb had no effect on the protection. Transfer of CD4(+) T-cell-depleted spleen cells resulted in higher parasitemia than transfer of CD8(+) T-cell-depleted spleen cells. A high level of gamma interferon (IFN-gamma), which was produced by CD4(+) T cells, was observed for the culture supernatant of spleen cells from immune mice, and treatment of immune mice with anti-IFN-gamma MAb partially reduced the protection. Moreover, no protection against challenge infection was found in IFN-gamma-deficient mice. On the other hand, treatment of immune mice with MAbs against interleukin-2 (IL-2), IL-4, or tumor necrosis factor alpha did not affect protective immunity. These results suggest essential requirements for CD4(+) T cells and IFN-gamma in protective immunity against challenge infection with B. microti.     

Increase in Gamma Interferon-Secreting CD8+, as Well as CD4+, T Cells in Lungs following Aerosol Infection with Mycobacterium tuberculosis

Although it is well established that CD4(+) T cells are required for the protective immune response against tuberculosis (TB), there is some evidence that CD8(+) T cells are also involved in the host response to Mycobacterium tuberculosis. There is, however, a paucity of information on the pulmonary CD8(+) T-cell response during infection. We therefore have compared the changes in both CD8(+) and CD4(+) T cells following aerosol infection with M. tuberculosis. There was an observed delay between the peak of infection and the activated T-cell response in the lung. The kinetics of CD8(+) and CD4(+) T-cell responses in the lung were identical, both peaking at week 8, 4 weeks later than the peak of cellular response in draining lymph nodes. Similar changes in activation/memory phenotypes occurred on the pulmonary CD8(+) and CD4(+) T cells. Following in vitro restimulation, both subsets synthesized gamma interferon, a cytokine essential for controlling M. tuberculosis infection. Since lung CD8(+) T cells are actively expanded during aerosol M. tuberculosis infection, it is important that both CD8(+) and CD4(+) T cells be targeted in the design of future TB vaccines.     

CD8+-T-Cell Immunity against Toxoplasma gondii Can Be Induced but Not Maintained in Mice Lacking Conventional CD4+ T Cells

T-cell immunity is critical for survival of hosts infected with Toxoplasma gondii. Among the cells in the T-cell population, CD8(+) T cells are considered the major effector cells against this parasite. It is believed that CD4(+) T cells may be crucial for induction of the CD8(+)-T-cell response against T. gondii. In the present study, CD4(-/-) mice were used to evaluate the role of conventional CD4(+) T cells in the immune response against T. gondii infection. CD4(-/-) mice infected with T. gondii exhibited lower gamma interferon (IFN-gamma) messages in the majority of their tissues. As a result, mortality due to a hyperinflammatory response was prevented in these animals. Interestingly, T. gondii infection induced a normal antigen-specific CD8(+)-T-cell immune response in CD4(-/-) mice. No difference in generation of precursor cytotoxic T lymphocytes (pCTL) or in IFN-gamma production by the CD8(+)-T-cell populations from the knockout and wild-type animals was observed. However, the mutant mice were not able to sustain CD8(+)-T-cell immunity. At 180 days after infection, the CD8(+)-T-cell response in the knockout mice was depressed, as determined by pCTL and IFN-gamma assays. Loss of CD8(+)-T-cell immunity at this time was confirmed by adoptive transfer experiments. Purified CD8(+) T cells from CD4(-/-) donors that had been immunized 180 days earlier failed to protect the recipient mice against a lethal infection. Our study demonstrated that although CD8(+)-T-cell immunity can be induced in the absence of conventional CD4(+) T cells, it cannot be maintained without such cells.     

In Vitro Expansion of T-Cell-Receptor Vα2.3+ CD4+ T Lymphocytes in HLA-DR17(3), DQ2+ Individuals upon Stimulation with Mycobacterium tuberculosis

The T-cell receptor (TCR) Valpha/beta gene product expression upon in vitro stimulation with mycobacteria was investigated to assess whether T-cell proliferation was associated with any specific TCR V gene usage. T-cell-enriched populations from peripheral blood of Mycobacterium bovis BCG-vaccinated healthy blood donors were stimulated in vitro with live or killed M. tuberculosis or with a soluble extract thereof. TCR Valpha/beta repertoire analysis of reactive CD4(+) and CD8(+) T cells revealed a selective HLA-DR17(3), DQ2-restricted expansion of Valpha2.3(+) CD4(+) T cells upon stimulation with live M. tuberculosis or its soluble extract. Third-complementarity-determining-region (CDR3) length analysis of the expanded Valpha2.3(+) T cells indicated an oligoclonal pattern with short CDR3 lengths in six of seven HLA-DR17(3), DQ2(+) individuals tested. In addition, Valpha/Vbeta repertoire analysis of T lymphocytes from a DR17(3), DQ2(+) donor before and after BCG vaccination revealed that positivity of skin test reactivity was associated with expansion of Valpha2.3(+) CD4(+) T lymphocytes with preferential use of a short CDR3 peak length after in vitro stimulation. Separation of M. tuberculosis soluble extract by fast protein liquid chromatography (FPLC) purification indicated that fractions corresponding to molecular masses of 60 to 70 and 15 to 25 kDa were particularly effective in eliciting Valpha2.3(+) CD4(+) T-cell expansion.     

Clearance of Virulent but Not Avirulent Rhodococcus equi from the Lungs of Adult Horses Is Associated with Intracytoplasmic Gamma Interferon Production by CD4+ and CD8+ T Lymphocytes

Rhodococcus equi is a gram-positive bacterium that infects alveolar macrophages and causes rhodococcal pneumonia in horses and humans. The virulence plasmid of R. equi appears to be required for both pathogenicity in the horse and the induction of protective immunity. An understanding of the mechanisms by which virulent R. equi circumvents protective host responses and by which bacteria are ultimately cleared is important for development of an effective vaccine. Six adult horses were challenged with either virulent R. equi or an avirulent, plasmid-cured derivative. By using a flow cytometric method for intracytoplasmic detection of gamma interferon (IFN-γ) in equine bronchoalveolar lavage fluid (BALF) cells, clearance of the virulent strain was shown to be associated with increased numbers of pulmonary CD4⁺ and CD8⁺ T lymphocytes producing IFN-γ. There was no change in IFN-γ-positive cells in peripheral blood, suggesting that a type 1 recall response at the site of challenge was protective. The plasmid-cured strain of R. equi was cleared in horses without a significant increase in IFN-γ-producing T lymphocytes in BALF. In contrast to these data, a previous report in foals suggested an immunomodulating role for R. equi virulence plasmid-encoded products in downregulating IFN-γ expression by equine CD4⁺ T lymphocytes. Intracytoplasmic detection of IFN-γ provides a method to better determine whether modulation of macrophage-activating cytokines by virulent strains occurs uniquely in neonates and contributes to their susceptibility to rhodococcal pneumonia.     

Primary Role for CD4+ T Lymphocytes in Recovery from Oropharyngeal Candidiasis

Oropharyngeal candidiasis is associated with defects in cell-mediated immunity and is commonly seen in human immunodeficiency virus positive individuals and AIDS patients. A model for oral candidiasis in T-cell-deficient BALB/c and CBA/CaH nu/nu mice was established. After inoculation with 10(8) Candida albicans yeasts, these mice displayed increased levels of oral colonization compared to euthymic control mice and developed a chronic oropharyngeal infection. Histopathological examination of nu/nu oral tissues revealed extensive hyphae penetrating the epithelium, with polymorphonuclear leukocyte microabscess formation. Adoptive transfer of either naive or immune lymphocytes into immunodeficient mice resulted in the recovery of these animals from the oral infection. Reconstitution of immunodeficient mice with naive CD4(+) but not CD8(+) T cells significantly decreased oral colonization compared to controls. Interleukin-12 and gamma interferon were detected in the draining lymph nodes of immunodeficient mice following reconstitution with naive lymphocytes. This study demonstrates the direct requirement for T lymphocytes in recovery from oral candidiasis and suggests that this is associated with the production of cytokines by CD4(+) T helper cells.     

Change of Mouse CD5+ B1 Cells to a Macrophage-Like Morphology Induced by Gamma Interferon and Inhibited by Interleukin-4

The in vitro effects of gamma interferon (IFN-γ) on the mouse CD5⁺ B1-cell line, TH2.52, a hybridoma between mouse B lymphoma and mouse splenic B cells that expresses a series of B1 markers, were investigated. A significant number of macrophage-like cells appeared in the cultures of TH2.52 cells exposed to IFN-γ, these adhering to plastic dishes and exhibiting phagocytic activity. Positive for esterase staining, the macrophage-like cells returned to the original TH2.52 morphology upon removal of IFN-γ. The change was prevented by treatment with SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase and by transfection of a p38 MAP kinase dominant-negative mutant. Further, interleukin-4 (IL-4) inhibited IFN-γ-induced phosphorylation of p38 MAP kinase and the appearance of macrophage-like cells. IFN-γ and IL-4 exhibited contradictory actions on morphological change of CD5⁺ B1 cells into macrophage-like cells. Differential regulation of CD5⁺ B1 cells by IFN-γ, a Th1 cytokine, and IL-4, a Th2 cytokine, may have clear immunological significance.     

CD69 Expression on CD4+ T Lymphocytes after In Vitro Stimulation with Tuberculin Is an Indicator of Immune Sensitization against Mycobacterium tuberculosis Antigens

The expression of the CD69 antigen on CD4 T lymphocytes after in vitro stimulation with purified protein derivative (2 tuberculin units) was used to evaluate the tuberculin reactivities of 52 individuals from four experimental groups: Mycobacterium bovis BCG-vaccinated healthy individuals with a negative tuberculin skin test (TST) result (group A), BCG-vaccinated healthy individuals with a positive TST result (group B), patients with active tuberculosis (TB) before treatment (group C), and individuals with clinically inactive TB who had previously completed a prescribed course of chemotherapy (group D). The expression of CD69 on CD4 T lymphocytes was significantly higher in patients with active TB (16.2% ± 7.3%), individuals with clinically inactive TB (10.5% ± 7.4%), and healthy individuals with a positive TST result (15.5% ± 7.2%) than in healthy individuals with a negative TST result (3.8% ± 4.3%) (P < 0.005). We confirmed the correlation between CD69 antigen expression on T lymphocytes after stimulation with tuberculin and the TST induration diameter (Spearman rho = 0.783; P < 0.001), an assay for gamma interferon (the Quantiferon-TB assay; Spearman rho = 0.613; P < 0.001), and the lymphocyte BLAST transformation test (Spearman rho = 0.537; P < 0.001). Our results demonstrate the usefulness of the determination of CD69 on CD4 T lymphocytes after in vitro stimulation with tuberculin as a rapid indicator of immune sensitization against Mycobacterium tuberculosis.     

CD4+ T-Lymphocyte and Immunoglobulin G2 Responses in Calves Immunized with Anaplasma marginale Outer Membranes and Protected against Homologous Challenge

Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4⁺ T cells, and CD4⁺ T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.     

Increase of γδ T Lymphocytes in Murine Lungs Occurs during Recovery from Pulmonary Infection by Nocardia asteroides

Previous studies have demonstrated that gammadelta T lymphocytes are important for host resistance to pulmonary infection of the murine lung by log-phase cells of Nocardia asteroides. To study the role of gammadelta T cells in nocardial interactions in the murine lung, C57BL/6J wild type and C57BL/6J-Tcrd (gammadelta T-cell knockout mice) were infected intranasally with log-phase cells of N. asteroides GUH-2. At 3, 5, and 7 days after infection, the gammadelta T cells were quantified by multiparameter flow cytometry. At the same time, Gram and hematoxylin-eosin stains of paraffin sections were performed to monitor the host responses. The data showed that gammadelta T lymphocytes increased significantly within the lungs after intranasal infection, and the peak of this cellular increase occurred at 5 days. Furthermore, at this time, greater than 50% of the CD3 T-cell receptor (TCR)-positive (CD3+) cells were gammadelta TCR positive. Histological examination clearly showed divergent inflammatory responses in the lungs of wild-type mice compared to gammadelta T-cell knockout mice. The C57BL/6J-Tcrd mice were less capable of clearing the organism, and the polymorphonuclear leukocyte response lasted longer than in wild-type C57BL/6J mice. These results showed that gammadelta T cells were actively involved in modulating the innate host responses to murine pulmonary infection by N. asteroides.     

Fas Ligand-Expressing B-1a Lymphocytes Mediate CD4+-T-Cell Apoptosis during Schistosomal Infection: Induction by Interleukin 4 (IL-4) and IL-10

A previous study of the murine model of Schistosoma mansoni infection has implicated splenic CD19(+) B lymphocytes as Fas ligand (FasL)-bearing mediators of CD4(+) T-lymphocyte apoptosis. The present study shows that B-cell deficiency leads to decreased CD4(+) T-cell apoptosis during infection and compares FasL expression and killer function of B-1a- and CD5(-) B-lymphocyte subsets. B-1a cells from uninfected mice displayed constitutive expression of FasL compared with that of CD5(-) B cells. FasL expression was enhanced following worm egg deposition and antigenic stimulation on both subsets of B cells. Purified B-1a cells from uninfected mice were potent effectors of CD4(+) T-cell apoptosis, and the killing effect was enhanced during schistosome infection. FasL expression by splenic B cells required CD4(+)-T-cell help that was replaced by addition of culture supernatants from antigen-stimulated splenocytes of infected mice. The culture-supernatant-stimulated FasL expression was inhibited by anti-interleukin 10 (IL-10) and anti-IL-4 antibodies. Culture of purified B cells with recombinant IL-4 (rIL-4), rIL-10, and soluble egg antigens (SEA) led to increased expression of FasL on B-1a cells. These results suggest that FasL-expressing, splenic B-1a cells are important mediators of SEA-stimulated CD4(+)-T-cell apoptosis and that maximal FasL expression on B-1a cells is dependent on antigenic stimulation and the presence of IL-4 and IL-10.     

Correlation of CD4+ T-Cell Counts Estimated by an Immunocapture Technique (Capcellia) with Viral Loads in Human Immunodeficiency Virus-Seropositive Individuals

As antiretroviral therapy becomes more affordable, valid, reliable, and inexpensive laboratory tests are also needed to monitor the progression of disease in people with human immunodeficiency virus (HIV) infection. The CD4⁺ T-cell counts estimated by Capcellia, an immunocapture method, and flow cytometry were compared and were correlated with HIV type 1 (HIV-1) load. There was a significant negative correlation between the HIV-1 load and CD4⁺ T-cell counts estimated by flow cytometry (r = −0.63, P = <0.001) as well as between the HIV-1 load and CD4⁺ T-cell counts estimated by Capcellia (r = −0.61, P = <0.001). Capcellia is a cost-effective, user-friendly assay that correlated well with HIV-1 load determinations for individuals both with and without treatment.