Low Relative Frequencies of CD26+ CD4+ Cells in Long-Term Nonprogressing Human Immunodeficiency Virus Type 1-Infected Subjects

A broad antibody panel was used for immunophenotyping of human immunodeficiency virus type 1 (HIV-1)-infected patients who were long-term nonprogressors (LTNP). The LTNP were compared with patients in the early phase of infection and patients who had progressed to advanced immunodeficiency. Changes in CD8⁺ subset distribution were observed mainly at acquisition of HIV-1 infection, whereas CD4⁺ subset changes appeared during progression of HIV-1 infection. The decreasing levels of CD4⁺ cells were characterized by an increasing frequency of cells expressing the activation markers HLA-Dr and CD45RO but not the CD28 surface antigen. The LTNP exhibited significant changes compared to HIV-negative patients in almost all markers. Compared to patients in the early phase of infection, the only difference was a relatively lower frequency of CD4⁺ cells expressing CD26 among the LTNP. The results show that HIV-1-infected persons who have no signs of immunodeficiency despite many years of infection have an immunophenotypic pattern that is substantially different from that of noninfected persons. Despite the long duration of infection, the LTNP exhibit a pattern similar to that of newly infected persons, with the exception of lower expression of CD26 on CD4⁺ cells.     

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.     

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.     

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.     

Evolution of Vaccinia Virus-Specific CD8+ Cytotoxic T-Lymphocyte Responses in Primary Vaccinees and Revaccinees

Determination of successful vaccination with vaccinia virus is based on visual confirmation of a dermal response (take). Some revaccinees do not manifest a take, which may be due to a preexisting immunity rather than to poor technique or inadequate virus. Cytotoxic T-lymphocyte (CTL) response appears to be the most important immune defense in limiting response to vaccination. We evaluated vaccinia virus-specific CTL responses in revaccinees. Subjects with and without takes displayed comparable CTL responses. Vaccinia virus-specific CD8⁺ CTL responses may be useful in interpreting the response to vaccination, particularly in individuals who are revaccinated and have difficult-to-interpret visual takes.     

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.     

Expansion of a Novel Pulmonary CD3− CD4+ CD8+ Cell Population in Mice during Chlamydia pneumoniae Infection

A new pulmonary T-cell-like lymphocyte population with the phenotype CD3⁻ CD4⁺ CD8⁺ was discovered in mice. CD4⁺ CD8⁺ but CD3⁺ cells among murine intestinal intraepithelial lymphocytes have previously been described. We describe herein a dramatic expansion of the CD3⁻ CD4⁺ CD8⁺ cell population in response to experimental respiratory infection. After intranasal Chlamydia pneumoniae infection, CD4⁺ CD8⁺ cells became transiently the dominant lymphocyte type (maximum of 87% of all lymphocytes) in the lungs of NIH/S mice but remained virtually undetectable in spleen and blood. The enrichment of these cells was not a C. pneumoniae-specific event, since infection of NIH/S mice with influenza A virus also resulted in an increase in the number of CD4⁺ CD8⁺ cells (maximum of 42% of all lymphocytes). In addition to outbred NIH/S mice, two other mouse strains were studied: BALB/c (H-2^d) and C57BL/6 (H-2^b). C. pneumoniae-infected BALB/c mice responded with an intermediate increase in the number of CD4⁺ CD8⁺ cells in lungs, whereas C57BL/6 mice did not respond. The double-positive CD4⁺ CD8⁺ cells lacked a major part of the T-cell receptor complex, being both CD3⁻ and TCR αβ⁻. However, when they were stimulated in vitro with a T-cell mitogen, they responded by proliferation but did not secrete gamma interferon. The dramatic expansion of this cell population at the infection site suggests an active role for them in respiratory infection, but the specification of this requires further study.     

Immune CD8+ T Cells Prevent Reactivation of Toxoplasma gondii Infection in the Immunocompromised Host

Toxoplasma gondii remains a serious cause of morbidity and mortality in individuals that are immunosuppressed, patients with AIDS in particular. The cellular immune response, especially by gamma interferon (IFN-gamma)-producing CD8(+) T cells, is an essential component of protective immunity against the parasite. In the present study the role of CD8(+) T cells during the reactivation of Toxoplasma infection in an immunocompromised murine model was evaluated. Chronically infected mice were challenged with LP-BM5 virus, and the kinetics of CD8(+) T-cell function was studied. At 10 weeks after viral infection, mice showed obvious signs of systemic illness and began to die. At this stage, CD8(+) T cells were unresponsive to antigenic stimulation and unable to kill Toxoplasma-infected targets. IFN-gamma production by the CD8(+) T cells from dual-infected animals reached background levels, and a dramatic fall in the frequency of precursor cytotoxic T lymphocytes was observed. Histopathological analysis of the tissues demonstrated signs of disseminated toxoplasmosis as a result of reactivation of infection. However, treatment of the dual-infected animals with immune CD8(+) T cells at 5 weeks post-LP-BM5 challenge prevented the reactivation of toxoplasmosis, and mice continued to live. Our study for the first time demonstrates a therapeutic role for CD8(+) T cells against an opportunistic infection in an immunocompromised state.     

Antigen-Specific CD8+ T Cells Protect against Lethal Toxoplasmosis in Mice Infected with Neospora caninum

Neospora caninum is a coccidial protozoan parasite that appears morphologically indistinguishable from Toxoplasma gondii and that infects a large range of mammals. Both inbred and outbred strains of mice exhibit a high degree of resistance to infection with N. caninum. Three inbred strains of mice (A/J, BALB/c, and C57BL/6) that were infected intraperitoneally with N. caninum were protected against a lethal challenge from T. gondii. Vaccine-induced protection was Neospora dose dependent. A rise in the CD8⁺ T-cell population in mice that had been vaccinated with N. caninum and challenged with T. gondii was observed. Adoptive transfer of CD8⁺ T-cell splenocytes from N. caninum-infected mice was protective against challenge with Toxoplasma. The CD8⁺ T cells from Neospora-infected mice proliferate to both Neospora and Toxoplasma antigens in vitro and secrete substantial quantities of gamma interferon when pulsed with the parasite antigen. These observations demonstrate that N. caninum protects against lethal T. gondii infection by the induction of CD8⁺ T cells that are immunoreactive to both parasites.     

CD4+ CD25+ T-Cell Production in Healthy Humans and in Patients with Thymic Hypoplasia

Regulatory T cells are found primarily in the CD4⁺ CD25⁺ fraction of T cells and play an important role in the prevention of autoimmunity. We examined CD4⁺ CD25⁺ T cells in 33 healthy children and adults and compared them to a population with an inherited form of thymic hypoplasia and a predisposition to autoimmune disease. Absolute numbers of CD4⁺ CD25⁺ T cells were markedly higher in healthy infants than in infants with chromosome 22q11.2 deletion syndrome.     

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.     

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.     

Processing of Mycobacterium tuberculosis Bacilli by Human Monocytes for CD4+ αβ and γδ T Cells: Role of Particulate Antigen

Mycobacterium tuberculosis readily activates both CD4⁺ and Vδ2⁺ γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and γδ T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2⁺ γδ T cells. For γδ T cells, recognition of M. tuberculosis-infected monocytes was dependent on Vδ2⁺ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4⁺ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vδ2⁺ γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4⁺ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4⁺ and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4⁺ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4⁺ and γδ T cells.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is spread readily from person to person by inhalation of aerosolized mycobacteria (8). A hallmark of M. tuberculosis infection is the ability of most healthy individuals to control the infection by mounting an acquired immune response, in which antigen-specific T cells and mononuclear phagocytes arrest the growth of M. tuberculosis bacilli and maintain control over dormant bacilli within granulomas (reviewed in reference 25). This protective cellular immune response results in conversion of the tuberculin skin test from negative to positive and probably in increased resistance to reinfection with tubercle bacilli.CD4⁺ αβ-T-cell-receptor (αβ TCR)-bearing T cells (CD4⁺ T cells) are readily activated by mycobacterial antigens and have a dominant role in the protective immune response to M. tuberculosis in humans (2, 34). These CD4⁺ T cells not only secrete cytokines but also serve directly as cytotoxic effector cells against M. tuberculosis-infected macrophages (6). In addition to CD4⁺ T cells, M. tuberculosis antigens activate other human T-cell subsets such as γδ TCR⁺ T cells (γδ T cells) (15, 16, 18). Vδ2⁺ and Vγ9⁺ γδ T cells are particularly responsive to live M. tuberculosis (15). A role for both γδ and CD4⁺ T cells in protective immunity to acute M. tuberculosis infection has been demonstrated in murine models (20, 21, 26, 27). A recent study of humans suggests that Vγ9⁺ and Vδ2⁺ γδ T-cell numbers and function are reduced in tuberculosis patients (23).Functional comparisons of human CD4⁺ and γδ T-cell responses of healthy tuberculin-positive persons demonstrate that both T-cell subsets have similar cytotoxic effector functions for M. tuberculosis-infected monocytes and produce large amounts of gamma interferon (IFN-γ), with γδ T cells being slightly more efficient producers of IFN-γ than CD4⁺ T cells (37). Despite similarities in function, these two T-cell subsets differ in the mycobacterial antigens recognized by their TCRs and the manners in which antigens are presented to them by M. tuberculosis-infected mononuclear phagocytes. CD4⁺ T cells recognize a wide diversity of mycobacterial peptides in the context of class II major histocompatibility complex (MHC) molecules, which include secreted as well as somatic antigens (6, 13, 33, 37). In contrast, Vγ9⁺ and Vδ2⁺ γδ T cells, the dominant γδ TCR subsets activated by M. tuberculosis, recognize mycobacterial antigens in a non-MHC-restricted manner and the repertoire of antigens includes small phosphate-containing antigens such as TUBag’s (5, 9, 19, 22, 29, 36).Both blood monocytes and alveolar macrophages infected with M. tuberculosis are efficient antigen-presenting cells for mycobacterial antigen-specific CD4⁺ and γδ T cells (1, 5). However, little is known about how M. tuberculosis-infected mononuclear phagocytes process antigens for these two T-cell subsets. M. tuberculosis bacilli are taken up by mononuclear phagocytes through a variety of surface receptors, including complement receptor 4, mannose receptor, and complement receptor 3 (17, 31, 32). Within mononuclear phagocytes, the mycobacteria reside within phagosomes and modulate the phagosome by preventing fusion with acidic lysosomal compartments (7). Although the vacuolar membranes surrounding the phagosome acquire endosomal markers, the vesicular proton ATPase is actively excluded, resulting in an elevated pH of 6.3 to 6.5 compared to the normal lysosomal pH of 4.5 (7, 35). The elevated pH in the phagosome does not appear to inhibit the ability of mycobacterial antigens to be processed and presented to CD4⁺ and Vδ2⁺ γδ T cells. This study was undertaken to gain insight into the mechanisms used by monocytes infected with live M. tuberculosis bacilli to process mycobacterial antigens for presentation to both CD4⁺ and γδ T cells.     

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.     

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.     

Most CD56+ Intestinal Lymphomas Are CD8+CD5− T-Cell Lymphomas of Monomorphic Small to Medium Size Histology

The expression of the natural killer (NK) cell marker CD56 has been reported to occur in NK cell lymphomas/leukemias and a small group of peripheral T-cell lymphomas but has not been studied extensively in primary intestinal non-B-cell lymphomas. Normal human jejunal intraepithelial lymphocytes (IELs) are mainly T-cell receptor (TCR)-αβ⁺CD3⁺CD8⁺CD5^low and include an ~15% fraction of CD56⁺ cells that could be the cells of origin for CD56⁺ intestinal T-cell lymphoma (ITL). To test this hypothesis, 70 cases diagnosed as ITL were immunophenotyped, and 15 CD56⁺ cases (21%) were identified. The majority of the CD56⁺ lymphomas was of monomorphic small to medium-sized histology, shared the common phenotype βF1^±CD3ε/cyt⁺CD8⁺CD4⁻CD5⁻CD57⁻TIA-1⁺ and had clonally rearranged TCR γ-chain genes. In contrast, the CD56⁻ lymphomas were mainly composed of pleomorphic medium and large cells or had a morphology most consistent with anaplastic large-cell lymphoma and were mostly CD8⁻. These findings suggest that the majority of CD56⁺ intestinal lymphomas are morphologically and phenotypically distinct T-cell lymphomas most likely derived from activated cytotoxic CD56⁺CD8⁺ IELs. Some overlapping histological and clinical features between CD56⁺ and CD56⁻ ITLs indicate that the former belong to the clinicopathological entity of ITL. The consistent expression of cytotoxic-granule-associated proteins introduces ITL (both CD56⁺ and CD56⁻) into the growing family of usually aggressive extranodal lymphomas of cytotoxic T-cell and NK-cell derivation. In contrast to putative NK-cell lymphoma of the sinonasal region, intestinal NK-cell lymphoma seems to be very rare.     

γδ+ T Cells Preferentially Respond to Live Rather than Killed Malaria Parasites

We have compared the in vitro responses of peripheral blood T cells from malaria-unexposed donors to live Plasmodium falciparum schizonts, freeze-thawed schizont extracts (P. falciparum schizont extracts [PfSE]), and parasite culture supernatants. We show that the cells responding to PfSE and parasite culture supernatants are predominantly CD4⁺ TCRαβ⁺ while in the presence of live schizonts there is an additional activation of TCRγδ⁺ cells. Activation of TCRγδ⁺ cells in response to PfSE was seen only when irradiated autologous feeder cells or recombinant interleukin-2 (IL-2) was added to the cultures. Live schizonts but not PfSE induced significant IL-2 production in vitro in the first 5 days after stimulation, suggesting that induction of early IL-2 by live parasites may contribute to the marked activation of the TCRγδ⁺ population.     

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.