CD4(+) T-cell subsets that mediate immunological memory to Mycobacterium tuberculosis infection in mice

We have studied CD4(+) T cells that mediate immunological memory to an intravenous infection with Mycobacterium tuberculosis. The studies were conducted with a mouse model of memory immunity in which mice are rendered immune by a primary infection followed by antibiotic treatment and rest. Shortly after reinfection, tuberculosis-specific memory cells were recruited from the recirculating pool, leading to rapidly increasing precursor frequencies in the liver and a simultaneous decrease in the blood. A small subset of the infiltrating T cells was rapidly activated (<20 h) and expressed high levels of intracellular gamma interferon and the T-cell activation markers CD69 and CD25. These memory effector T cells expressed intermediate levels of CD45RB and were heterogeneous with regard to the L-selectin and CD44 markers. By adoptive transfer into nude mice, the highest level of resistance to a challenge with M. tuberculosis was mediated by CD45RB(high), L-selectin(high), CD44(low) cells. Taken together, these two lines of evidence support an important role for memory cells which have reverted to a naive phenotype in the long-term protection against M. tuberculosis.     

Anamnestic responses of mice following Mycobacterium tuberculosis infection

The anamnestic response is the property of the immune system that makes vaccine development possible. Although the development of a vaccine against Mycobacterium tuberculosis is an important global priority, there are many gaps in our understanding of how immunological memory develops following M. tuberculosis infection or after BCG vaccination. In experiments designed to compare the anamnestic response of susceptible and resistant mouse strains, major histocompatibility complex-matched memory-immune C3.SW-H2(b)/SnJ and C57BL/6 mice both demonstrated better control of bacterial replication following reinfection with M. tuberculosis than control mice. Nevertheless, this memory response did not appear to have any long-term protective effect for either mouse strain. A greater understanding of the immunological factors that govern the maintenance of immunological memory following exposure to M. tuberculosis will be required to develop an effective vaccine.     

Intracellular expression of interleukin-4 and interferon-gamma by a Mycobacterium tuberculosis antigen-stimulated CD4+ CD57+ T-cell subpopulation with memory phenotype in tuberculosis patients

In some chronic pathological conditions, antigen persistence activates and expands the CD4+ CD57+ T-cell subset. The host immune response against tuberculosis infection is maintained through the continuous presence of antigen-stimulated effector/memory helper T cells. To determine whether CD4+ CD57+ T cells were also expanded in human tuberculosis, we analysed (by flow cytometry) the phenotype of peripheral blood CD4+ T cells from 30 tuberculosis patients and 30 healthy controls. We observed a significant increase in the CD4+ CD57+ T-cell subset in tuberculosis patients in comparison to healthy controls (P < 0.001). Most CD4+ CD57+ T cells exhibited a CD28- CD45RO+ CD62L- phenotype, which is associated with memory cells. In vitro, a higher number of antigen-stimulated CD4+ CD57+ T cells produced intracellular interferon-gamma and interleukin-4 compared with antigen-stimulated CD4+ CD57- T cells (P < 0.001). These findings suggest that the majority of CD4+ CD57+ T cells correspond to a phenotype of activated memory T cells.     

Vaccination for Mycobacterium tuberculosis infection: reprogramming CD4 T-cell homing into the lung

Development of effective tuberculosis vaccines is hampered by insufficient understanding of protective immunity. Here, Woodworth et al.¹ show secondary effector CD4 T cells generated after Mtb challenge of H56/CAF01 vaccinated mice display superior lung homing compared with primary effectors. Vaccination generates large populations of parenchymal lung effector cells by inducing CXCR3⁺KLRG1⁻ cells that continuously migrate from lymph nodes to lung, and limiting the generation of non-protective CX3CR1⁺KLRG1⁺ intravascular effectors, providing insight vaccine-mediated protection against tuberculosis.     

T-cell cytokine responses in human infection with Mycobacterium tuberculosis.

Compared with healthy tuberculin reactors, Mycobacterium tuberculosis-stimulated peripheral blood mononuclear cells from tuberculosis patients had diminished production and mRNA expression of the Th1 cytokines gamma interferon and interleukin 2 (IL-2), with no change in production and mRNA expression for the Th2 cytokines IL-4, IL-10, and IL-13. These results were confirmed by evaluation of T cells and CD4+ cells. At the level of systemic T cells, development of tuberculosis is associated with diminished Th1 but not enhanced Th2 responses.     

Inflammatory response following intranasal infection with Mycobacterium avium complex: role of T-cell subsets and gamma interferon.

The role of CD4+ and CD8+ T cells in the response to intranasal infection with a Mycobacterium avium complex isolate (MAC) was investigated. Depletion of CD4+ T cells by injected antibody exacerbated infection in the lung, spleen, and liver. There were decreased numbers of inflammatory cells in the lungs of CD4-depleted mice and a significant decrease in lung cytotoxic activity. The neutrophil response was unaffected, and in CD4-depleted mice, unlike intact infected mice, these cells were found with large numbers of associated MAC. Purified CD4+ splenic T cells produced gamma interferon (IFN-gamma) in vitro in response to MAC antigen. IFN-gamma production by cultured spleen, lung, or mediastinal lymph node cells was markedly reduced in CD4-depleted mice. In contrast, CD8+ T cells did not produce IFN-gamma in vitro, and depletion of CD8+ T cells from infected mice had no effect on bacterial growth or lung cell activation. Depletion of IFN-gamma by injected monoclonal antibody had effects similar to those of CD4 depletion, namely, exacerbation of infection and decreased lung cell cytotoxicity. We conclude that CD4+ T cells are the main T cells involved in the lung response to MAC infection and that this response is at least partially dependent on the production of IFN-gamma.     

T-cell immune responses in Mycobacterium avium-infected mice.

Mycobacterium avium infection was substantially more severe in C57BL/6 (Bcgs) than in (C57BL/6 x DBA/2)F1 hybrid (Bcgr) mice both in terms of bacterial growth in the spleens and lungs and in host survival. Prior Mycobacterium bovis BCG vaccination resulted in increased resistance as well as enhanced tuberculin hypersensitivity to both PPD-S (Mycobacterium tuberculosis) and PPD-A (M. avium). Mice heavily infected with M. avium were used as T-cell donors in an adoptive transfer system. Substantial resistance was observed for both recipient hosts regardless of the genotype of the donor strain. Transfer of resistance was ablated by treatment of the immune spleen cells with anti-Thy 1.2 monoclonal antibody and complement or by cyclophosphamide treatment. Spleen cells which were monodepleted of L3T4+ or Lyt-2+ T cells did not lose their ability to transfer resistance against a subsequent challenge. However, when these cells were doubly deleted, all resistance was ablated in both the BCG-susceptible and -resistant mice. The recipient host expressed a detectable adoptive immune response although the donor had been unable to reduce the growth of the primary M. avium infection in vivo.     

T-cell responses to CD1-presented lipid antigens in humans with Mycobacterium tuberculosis infection

CD1-restricted presentation of lipid or glycolipid antigens derived from Mycobacterium tuberculosis has been demonstrated by in vitro experiments using cultured T-cell lines. In the present work, the frequency of T-cell responses to natural mycobacterial lipids was analyzed in ex vivo studies of peripheral blood lymphocytes from human patients with pulmonary tuberculosis, from asymptomatic individuals with known contact with M. tuberculosis documented by conversion of their tuberculin skin tests, and from healthy tuberculin skin test-negative individuals or individuals vaccinated with Mycobacterium bovis BCG. Proliferation and gamma interferon enzyme-linked immunospot assays using peripheral blood lymphocytes and autologous CD1(+) immature dendritic cells revealed that T cells from asymptomatic M. tuberculosis-infected donors responded with significantly greater magnitude and frequency to mycobacterial lipid antigen preparations than lymphocytes from uninfected healthy donors. By use of these methods, lipid-antigen-specific proliferative responses were minimally detectable or absent in blood samples from patients with active tuberculosis prior to chemotherapy but became detectable in blood samples drawn 2 weeks after the start of treatment. Lipid antigen-reactive T cells were detected predominantly in the CD4-enriched T-cell fractions of circulating lymphocytes, and anti-CD1 antibody blocking experiments confirmed the CD1 restriction of these T-cell responses. Our results provide further support for the hypothesis that lipid antigens serve as targets of the recall response to M. tuberculosis, and they indicate that CD1-restricted T cells responding to these antigens comprise a significant portion of the circulating pool of M. tuberculosis-reactive T cells in healthy individuals with previous exposure to M. tuberculosis.     

Protective immunity afforded by attenuated, PhoP-deficient Mycobacterium tuberculosis is associated with sustained generation of CD4+ T-cell memory

Definition of protective immunity induced by effective vaccines is important for the design of new pathogen control strategies. Inactivation of the PhoP response-regulator in Mycobacterium tuberculosis results in a highly attenuated strain that demonstrates impressive protective efficacy in pre-clinical models of tuberculosis. In this report we demonstrate that the protection afforded by the M. tuberculosis phoP mutant strain is associated with the long-term maintenance of CD4(+) T-cell memory. Immunization of mice with SO2 resulted in enhanced expansion of M. tuberculosis-specific CD4(+) T cells compared with vaccination with the BCG vaccine, with an increased frequency of these cells persisting at extended time-points after vaccination. Strikingly, vaccination with SO2 resulted in sustained generation of CD4(+) T cells displaying a central memory phenotype, a property not shared by BCG. Further, SO2 vaccination markedly improved the generation of polyfunctional cytokine-secreting CD4(+) T cells compared with BCG vaccination. The improved generation of functionally competent memory T cells by SO2 correlated with augmented recall responses in SO2-vaccinated animals after challenge with virulent M. tuberculosis. This study defines a mechanism for the protective effect of the SO2 vaccine and suggests that deletion of defined virulence networks may provide vaccine strains with potent immuno-stimulatory properties.     

Multifunctional CD4+ T cells correlate with active Mycobacterium tuberculosis infection

Th1 CD4⁺ T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN‐γ/IL‐2/TNF‐α triple expressors, IFN‐γ/IL‐2, IFN‐γ/TNF‐α or TNF‐α/IL‐2 double expressors or IFN‐γ, IL‐2 or TNF‐α single expressors) of CD4⁺ T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85–90%TB patients, were only present in 10–15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12‐ to 15‐fold) proportions of IL‐2/IFN‐γ double and IFN‐γ single expressors as compared with the other CD4⁺ T‐cell subsets. Proportions of the other double or single CD4⁺ T‐cell expressors did not differ between TB and LTBI subjects. These distinct IFN‐γ, IL‐2 and TNF‐α profiles of M. tuberculosis‐specific CD4⁺ T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB‐infected patients after completion of anti‐mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4⁺ T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti‐mycobacterial therapy.     

Maintenance of CD8(+) T-cell memory following infection with recombinant sindbis and vaccinia viruses

CD8(+) T-cell memory is critical for protection against pathogens poorly controlled by humoral immunity. To characterize two distinct vaccine vectors, the acute and memory CD8(+) T-cell responses to an HIV-1 epitope (p18) expressed by recombinant vaccinia (vp18) and Sindbis (SINp18) viruses were compared. Whereas 9 to 13% of CD8(+) splenocytes were p18 specific during the acute response to vp18, 4% were induced by SINp18 as revealed by class I tetramer staining. Increased T-cell activation by vp18 was confirmed by higher numbers of both p18-specific IFN-gamma-secreting splenocytes and activated CD8(+) and CD4(+) T cells. Although higher frequencies of p18-specific CD8(+) T cells during primary responses correlated with higher frequencies during memory, the overall decline was only two- to threefold during the transition to memory, demonstrating equally efficient maintenance of memory in SINp18- as in vp18-immune mice. Despite modest in vivo activation, SINp18-induced CD4(+) T cells secreted substantial amounts of IFN-gamma and IL-2, potentially contributing to sustained CD8(+) memory. Collectively the data indicate that Sindbis virus recombinants provide effective vaccines for inducing protective memory CD8(+) T cells in the absence of the extensive inflammation and replication associated with vaccinia virus.     

Immunization with a DNA vaccine cocktail protects mice lacking CD4 cells against an aerogenic infection with Mycobacterium tuberculosis

Tuberculosis (TB) is the most common opportunistic disease and a potentially fatal complication among immunocompromised individuals infected with human immunodeficiency virus (HIV). Effective vaccination against TB in persons with HIV has been considered unlikely because of the central role that CD4 cells play in controlling tuberculous infections. Here we show that the vaccination of CD8^−/− mice with a TB DNA vaccine cocktail did not significantly enhance protective responses to a Mycobacterium tuberculosis infection. In contrast, immunization with a DNA vaccine cocktail or with the current TB vaccine, Mycobacterium bovis BCG, induced considerable antituberculosis protective immunity in immune-deficient mice lacking CD4 cells. In vaccinated CD4^−/− animals, substantially reduced bacterial burdens in organs and much improved lung pathology were seen 1 month after an aerogenic M. tuberculosis challenge. Importantly, the postchallenge mean times to death of vaccinated CD4^−/− mice were significantly extended (mean with DNA cocktail, 172 ± 7 days; mean with BCG, 156 ± 22 days) compared to that of naïve CD4^−/− mice (33 ± 6 days). Furthermore, the treatment of DNA-vaccinated CD4^−/− mice with an anti-CD8 or anti-gamma interferon (IFN-γ) antibody significantly reduced the effect of immunization, and neither IFN-γ^−/− nor tumor necrosis factor receptor-deficient mice were protected by DNA immunization; therefore, the primary vaccine-induced protective mechanism in these immune-deficient mice likely involves the secretion of cytokines from activated CD8 cells. The substantial CD8-mediated protective immunity that was generated in the absence of CD4 cells suggests that it may be possible to develop effective TB vaccines for use in HIV-infected populations.Tuberculosis (TB) remains a significant global threat to public health, with two million people dying from Mycobacterium tuberculosis infections each year and eight million cases of TB developing annually (6). The increasing linkage of TB with the human immunodeficiency virus (HIV) pandemic has magnified this tragedy in the past decade. The World Health Organization estimates that at least six million people worldwide are coinfected with M. tuberculosis and HIV (18). The HIV-M. tuberculosis coinfection rates exceed 5% in eight African countries, and in South Africa alone two million adults are coinfected (9). In these developing countries, TB is the most prevalent cause of morbidity and mortality for HIV-positive adults. In contrast to immunocompetent individuals, who have a 10% lifetime risk of disease following TB infection, persons coinfected with M. tuberculosis and HIV have a nearly 10% annual risk of developing disease.A primary reason for the continued failure to curb the global TB epidemic is the absence of a highly effective vaccine. Although the current TB vaccine, Mycobacterium bovis BCG, has been widely used for decades, its efficacy in controlled clinical trials has been extremely variable and its value in protecting against the most prevalent form of the disease, adult pulmonary TB, is doubtful (7). Moreover, the effectiveness of BCG in preventing TB in HIV-infected individuals is uncertain. Since BCG is a live vaccine (attenuated but not avirulent) and since clinical cases of reactivated BCG have been reported for HIV-infected persons, BCG vaccination has not been indicated for immunocompromised individuals (33).The development of a new vaccine against TB for use in HIV-positive persons has been considered unlikely because of the presumed essential roles that CD4 cells play in controlling TB infections. Mice that lack CD4 cells or that are aberrant in major histocompatibility complex class II presentation are extremely sensitive to a TB challenge and cannot effectively control acute TB infections (3, 13). The greatly enhanced susceptibility of HIV patients to both primary and reactivated disease argues that CD4 cells also play a prominent role in protective immune responses against human TB. The primary antituberculosis effector function of CD4⁺ T cells involves the production and secretion of cytokines which activate macrophages to control or eliminate the intracellular bacilli (13). In addition to this more direct role of CD4 cells in limiting tuberculous infections, CD4 cells also assist in the development of primary CD8 T-cell responses (23). CD4 cells stimulate professional antigen-presenting cells (APCs) primarily via CD40-CD40 ligand interactions; these activated APCs efficiently costimulate antigen-specific naïve CD8 T cells. Additionally, cytokine production from CD4 cells enhances the proliferation of primed CD8 cells.Despite the critical immune functions of CD4 cells, recent studies have demonstrated that protective immunity to pathogens can be generated in the absence of CD4 cells. For instance, substantial CD8 T-cell responses to the influenza virus, lymphocytic choriomeningitis virus (LCMV), and pathogenic fungi can occur in mice lacking CD4 cells (17, 26, 34, 35). In a Listeria monocytogenes model, similar levels of activated CD8 T cells were detected in CD4^−/− and wild-type (WT) C57BL/6 mice after an infection. Importantly, the epitope-specific CD8 T cells that were generated established long-term memory in CD4^−/− mice and were capable of producing an effective recall response (21). Under circumstances in which CD4 help is not required for the generation of effective CD8 T-cell responses, alternate pathways for the activation of APCs exist. For influenza virus, a direct infection of dendritic cells results in the upregulation of costimulatory molecules. For bacteria, dendritic cells can be activated through recognition by Toll-like receptors (TLRs) of bacterial products such as peptidoglycan, glycolipids, and lipoproteins (2, 29, 30).Based on these findings in other immune-deficient disease models, we evaluated whether cell-mediated antituberculosis protective immunity could be induced in mice lacking CD4 cells by immunization with a DNA vaccine cocktail or with BCG. Previously, members of our laboratory generated a DNA cocktail that expressed mycobacterial proteins fused at the N terminus to ubiquitin (UB), a eukaryotic intracellular targeting sequence (10, 11). These UB-conjugated proteins were designed to enhance major histocompatibility complex class I presentation. In studies using WT C57BL/6 mice, it was shown that vaccination with this combination was more effective than immunization with the individual single components and that the sustained protective immunity induced by the plasmid mixture was equivalent to the level of protection elicited by the BCG vaccine (10, 11). We report here that substantial antituberculosis protective immunity can be induced in the absence of CD4 cells. Immunization of CD4^−/− mice with either the DNA vaccine cocktail or BCG leads to significantly decreased lung and spleen bacterial burdens and much improved lung pathology, relative to naïve controls, after an aerogenic M. tuberculosis infection. Most importantly, the survival of the vaccinated animals was substantially extended compared to nonimmunized CD4^−/− controls.     

Evolution of cell types and T-cell subsets in the spleens of Mycobacterium bovis BCG-resistant and M. bovis BCG-susceptible strains of mice after infection with M. bovis BCG.

In mice, the early host response to intravenous infection with small doses of dispersed Mycobacterium bovis BCG is controlled by the Bcg gene. After infection with a low dose of M. bovis BCG, Lyt-1+ cells were generated in the spleens of BCG-susceptible mice (Bcgs) in parallel with an increase in the proportion of phagocytic cells. Very few changes occurred in the splenic cell types of BCG-resistant mice (Bcgr).     

Gamma interferon responses of CD4 and CD8 T-cell subsets are quantitatively different and independent of each other during pulmonary Mycobacterium bovis BCG infection

Gamma interferon (IFN-gamma) is a key cytokine in host defense against intracellular mycobacterial infection. It has been believed that both CD4 and CD8 T cells are the primary sources of IFN-gamma. However, the relative contributions of CD4 and CD8 T-cell subsets to IFN-gamma production and the relationship between CD4 and CD8 T-cell activation have not been examined. By using a model of pulmonary mycobacterial infection and various immunodetection assays, we found that CD4 T cells mounted a much stronger IFN-gamma response than CD8 T cells at various times after mycobacterial infection, and this pronounced IFN-gamma production by CD4 T cells was attributed to both greater numbers of antigen-specific CD4 T cells and a greater IFN-gamma secretion capacity of these cells. By using major histocompatibility complex class II-deficient or CD4-deficient mice, we found that the lack of CD4 T cells did not negatively affect primary or secondary CD8 T-cell IFN-gamma responses. The CD8 T cells activated in the absence of CD4 T cells were capable of immune protection against secondary mycobacterial challenge. Our results suggest that, whereas both CD4 and CD8 T cells are capable of IFN-gamma production, the former represent a much greater cellular source of IFN-gamma. Moreover, during mycobacterial infection, CD8 T-cell IFN-gamma responses and activation are independent of CD4 T-cell activation.     

Dispensability of surfactant proteins A and D in immune control of Mycobacterium tuberculosis infection following aerosol challenge of mice

Surfactant proteins A and D (SP-A and -D) play a role in many acute bacterial, viral, and fungal infections and in acute allergic responses. In vitro, human SPs bind Mycobacterium tuberculosis and alter human and rat macrophage-mediated functions. Here we report the roles of SP-A and SP-D in M. tuberculosis infection following aerosol challenge of SP-A-, SP-D-, and SP-A/-D-deficient mice. These studies surprisingly identified no gross defects in uptake or immune control of M. tuberculosis in SP-A-, SP-D-, and SP-A/-D-deficient mice. While both SP-A- and SP-D-deficient mice exhibited evidence of immunopathologic defects, the CD11b(high) CD11c(high) dendritic cell populations and the gamma interferon (IFN-γ)-dependent CD4(+) T cell response to M. tuberculosis were unaltered in all genotypes tested. Together, these data indicate that SP-A and SP-D are dispensable for immune control of M. tuberculosis in a low-dose, aerosol challenge, murine model of tuberculosis (TB).     

HIV-1 infection alters CD4+ memory T-cell phenotype at the site of disease in extrapulmonary tuberculosis

HIV-1-infected people have an increased risk of developing extrapulmonary tuberculosis (TB), the immunopathogenesis of which is poorly understood. Here, we conducted a detailed immunological analysis of human pericardial TB, to determine the effect of HIV-1 co-infection on the phenotype of Mycobacterium tuberculosis (MTB)-specific memory T cells and the role of polyfunctional T cells at the disease site, using cells from pericardial fluid and blood of 74 patients with (n = 50) and without (n = 24) HIV-1 co-infection. The MTB antigen-induced IFN-γ response was elevated at the disease site, irrespective of HIV-1 status or antigenic stimulant. However, the IFN-γ ELISpot showed no clear evidence of increased numbers of antigen-specific cells at the disease site except for ESAT-6 in HIV-1 uninfected individuals (p = 0.009). Flow cytometric analysis showed that CD4+ memory T cells in the pericardial fluid of HIV-1-infected patients were of a less differentiated phenotype, with the presence of polyfunctional CD4+ T cells expressing TNF, IL-2 and IFN-γ. These results indicate that HIV-1 infection results in altered phenotype and function of MTB-specific CD4+ T cells at the disease site, which may contribute to the increased risk of developing TB at all stages of HIV-1 infection.     

CD8(+) T cells participate in the memory immune response to Mycobacterium tuberculosis

The contribution of CD8(+) T cells to the control of tuberculosis has been studied primarily during acute infection in mouse models. Memory or recall responses in tuberculosis are less well characterized, particularly with respect to the CD8 T-cell subset. In fact, there are published reports that CD8(+) T cells do not participate in the memory immune response to Mycobacterium tuberculosis. We examined the CD8(+) T-cell memory and local recall response to M. tuberculosis. To establish a memory immunity model, C57BL/6 mice were infected with M. tuberculosis, followed by treatment with anti-mycobacterial drugs and prolonged rest. The lungs of memory immune mice contained CD4(+) and CD8(+) T cells with the cell surface phenotype characteristic of memory cells (CD69(low) CD25(low) CD44(high)). At 1 week postchallenge with M. tuberculosis via aerosol, > or =30% of both CD4(+) and CD8(+) T cells in the lungs of immune mice expressed the activation marker CD69 and could be restimulated to produce gamma interferon (IFN-gamma). In contrast, <6% of T cells in the lungs of naive challenged mice were CD69(+) at 1 week postchallenge, and IFN-gamma production was not observed at this time point. CD8(+) T cells from the lungs of both naive and memory mice after challenge were cytotoxic toward M. tuberculosis-infected macrophages. Our data indicate that memory and recall immunity to M. tuberculosis is comprised of both CD4(+) and CD8(+) T lymphocytes and that there is a rapid response of both subsets in the lungs following challenge.     

Specificity of a protective memory immune response against Mycobacterium tuberculosis.

We have investigated the memory T-cell immune response to Mycobacterium tuberculosis infection. C57BL/6J mice infected with M. tuberculosis were found to generate long-lived memory immunity which provided a heightened state of acquired resistance to a secondary infection. The T-cell response of memory immune mice was directed to all parts of the bacilli, i.e., both secreted and somatic proteins. Major parts of the memory T-cell repertoire were maintained in a highly responsive state by cross-reactive restimulation with antigens present in the normal microbiological environment of the animals. A resting non-cross-reactive part of the memory repertoire was restimulated early during a secondary infection to expand and produce large amounts of gamma interferon. The molecular target of these T cells was identified as a secreted mycobacterial protein with a molecular mass of 3 to 9 kDa.