Cell-mediated immune responses to chlamydial antigens in guinea pigs injected with inactivated chlamydiae

Cell-mediated immunity (CMI) to chlamydial antigens was readily induced in guinea pigs by a single injection of Betaprone-inactivated chlamydiae in complete Freund adjuvant. The CMI was measured in vivo by delayed hypersensitivity skin tests, and in vitro by inhibition of migration of peritoneal exudate cells and by proliferation of lymph node lymphocytes. There was an overall correlation between in vivo and in vitro responses. Of the in vitro assays, migration inhibition reflected the state of sensitization, as judged by skin tests, more uniformly than lymphocyte stimulation. Extensive inter- and intra-species cross-reactivity was noted between LB-1, a strain ofC. trachomatis, and three strains ofC. psittaci, 6BC, GPIC, and 562F. Cross-reactivity between LB-1 and 6BC was one-way only, by all three parameters: LB-1 elicited strong cross-reactions in 6BC-immunized animals but not vice versa. Antichlamydial antibodies could not be demonstrated in any of the animals by microimmunofluorescence.     

Analysis of the humoral immune response to chlamydial genital infection in guinea pigs.

Studies using the guinea pig model of chlamydial genital infection with the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) have shown that serum and local antibodies play a role both in the resolution of infection and in protection against reinfection. Thus, this model is suited for further exploration of immune mechanisms and for vaccine studies with chlamydial macromolecules. We have further characterized the model by assessing the antigen-specific antibody response to experimental genital infection by using immunoblotting to assay both genital secretions and serum. The GPIC agent was characterized by analysis of outer membrane proteins, which indicated that the GPIC agent possessed a major outer membrane protein (MOMP), with a molecular mass of 39 kilodaltons (kDa), and a 61-kDa protein, analogous to cysteine-rich 60-kDa proteins or doublets of Chlamydia trachomatis strains. As indicated by immunoblotting, most infected animals produced serum immunoglobulin G antibodies to MOMP, the 61-kDa proteins, an 84-kDa outer membrane protein, and lipopolysaccharide. Such serum antibodies persisted for at least 813 days after primary genital infection. Immunoglobulin A antibodies against the 61-kDa proteins, lipopolysaccharide, and MOMP, but not the 84-kDa protein, were detected in secretions. Animals challenged with GPIC 825 days after primary infection became infected again despite the presence of serum antibodies, but the period of chlamydial shedding was significantly shorter and less intense than in primary infections. Although the specific mechanism is not known, these data suggest that a long-lasting immune effect is capable of altering the course of infection late after primary infection. Correlation of the antigen-specific antibody response and other immune parameters with the duration and degree of protective immunity induced by infection or vaccination may be helpful in further understanding the nature of such protective immunity.     

Effect of estradiol on chlamydial genital infection of female guinea pigs.

Female guinea pigs were treated daily with 1 mg of beta-estradiol-3-benzoate intramuscularly beginning 14 days before intravaginal inoculation with the chlamydial agent of guinea pig inclusion conjunctivitis and continuing during the course of the infection. Treatment with estradiol was found to markedly influence the course of genital infection with the chlamydial agent of guinea pig inclusion conjunctivitis, producing infections of greater intensity and longer duration than those in control animals. Moreover, pathogenesis was altered in that ascending infection was observed, resulting in endometritis, cystic salpingitis, and cystitis. Infection in the controls was limited to the cervix and vagina. Estradiol treatment increased the apparent number of infected cells in the cervix and vagina as detected by histopathology and immunofluorescent staining. Humoral and cell-mediated immune responses to the chlamydial agent of guinea pig inclusion conjunctivitis were comparable in estradiol-treated and untreated animals. These data indicate that hormonal manipulation may have profound effects on the course of chlamydial genital infections.     

Immunity to vaginal reinfection in female guinea pigs infected sexually with Chlamydia of guinea pig inclusion conjunctivitis.

Guinea pig boars were inoculated intraurethrally with the chlamydial agent of guinea pig inclusion conjunctivitis (GPIC). At the heights of their urethral infections, they were caged with sows in estrus. Whereas some of the sows had not been previously exposed to GPIC agent, others had received an intravaginal inoculation 5 to 8 weeks earlier. Those sows for which infected boars provided the first exposure were challenged by intravaginal inoculation 5 to 8 weeks later. Vaginal and conjunctival scrapings were taken regularly and stained for chlamydial inclusions. Titers of serum anti-GPIC antibodies and of vaginal secretory IgA anti-GPIC antibodies were determined by immunofluorescence. Our results show for the first time that a sexually acquired vaginal GPIC infection induces immunity to manual reinfection of the vagina. Because of the high incidence of secondary conjunctival infections among the vaginally infected sows, we could not provide a sound statistical basis for our tentative conclusion that manual infection of the vagina induces immunity to sexual reinfection. The results of our antibody titrations confirm previous work showing that vaginal GPIC infection induces formation of both serum antibody and vaginal secretory immunoglobulin A antibody.     

Characterization of chlamydial genital infection resulting from sexual transmission from male to female guinea pigs and determination of infectious dose

A major problem in the study of chlamydial genital infections in animal models has been the use of varied doses of chlamydiae for infection in different laboratories. It is clearly desirable to use a dose which approximates that of natural sexual infection, but that dose to date has not been determined because of the inability of researchers to quantify chlamydiae in semen. Fortunately, sexual transmission of chlamydiae has been described for the guinea pig model of infection with the chlamydial agent of guinea pig inclusion conjunctivitis (GPIC). In this study, we undertook to determine the approximate infection dose in actual sexual transmission by comparing the kinetics of infection in female guinea pigs acquired via sexual contact to those of genital infections induced artificially with known quantities of chlamydiae. Groups of guinea pigs were infected intravaginally with 10(4), 10(3), 10(2), and 10(1) inclusion-forming units (IFU) of GPIC, and the kinetics of the infection were determined. Infection with 10(2) IFU produced infections with lower peak levels than those in animals receiving 10(4) or 10(3) IFU. Seventy percent of animals receiving 10(2) IFU became infected, while 100 and 79% of animals receiving 10(4) and 10(3) IFU, respectively, became infected. Animals receiving 10(2) IFU also had a longer incubation period. Of 19 animals that mated with infected males, 63.2% became infected, with an infection course which was not significantly different than that of the 10(2)-IFU-infected group. The data suggest that female guinea pigs received approximately 10(2) IFU by sexual transmission. Of interest was the observation that the guinea pigs infected by sexual transmission shed organisms for a significantly shorter time period than that of any group that was artificially infected. This result suggests that there may be factors associated with semen which passively transfer antimicrobial activity to the female or enhance the innate host response in the female. Immunization of females with an inactivated vaccine was also found to elicit a protective immune response against sexual challenge, demonstrating that the model can be used in the evaluation of possible vaccine candidates and/or methodologies. There is currently no other animal model available for any sexually transmitted disease in which the disease or the ability to prevent the disease may be studied in animals infected by the natural means.     

Pathogenesis of endometritis and salpingitis in a guinea pig model of chlamydial genital infection.

The development of tubal obstruction and subsequent infertility is a major sequelum of upper genital tract infection with Chlamydia trachomatis; however, little is known about the pathogenesis of the infection. In this investigation, the authors present a detailed study of the progression of ascending chlamydial infection in female guinea pigs resulting from intravaginal inoculation of the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC). Isolation of chlamydiae from different tissues of the genital tract revealed definitive evidence for ascending infection that was not dose-related. By 7 days after infection, GPIC was isolated from the endometrium and oviducts of 78% of the animals. Pathologic changes analogous to those seen in human chlamydial disease, including polymorphonuclear, mononuclear, and plasma cell infiltration, were seen in the endometrium and oviducts, although not all isolation positive animals developed overt tubal disease. Long-term fibrosis, often in combination with hydrosalpinx, was noted in the mesosalpingeal tissue in 20% of the animals. Thus, the guinea pig:GPIC system represents a model for ascending chlamydial infection resulting from vaginal inoculation of normal guinea pigs that closely approximates the disease as seen in humans and can be used to study the pathogenesis of chlamydial genital infection.     

Susceptibility to reinfection after a primary chlamydial genital infection is associated with a decrease of antigen-specific T cells in the genital tract.

We tested the hypothesis that the intensity of specific antichlamydial T cell-mediated immunity in the genital tract of female guinea pigs infected intravaginally with the chlamydial agent of guinea pig inclusion conjunctivitis would determine the resistance or susceptibility to reinfection after a primary chlamydial infection. T cell-enriched lymphocytes were isolated by collagenase treatment of genital tract tissues from either infected or control uninfected female guinea pigs at various times after infection. The nylon wool-enriched T lymphocytes were evaluated for expression of antigen-specific T cell-mediated immunity in vitro by using a blast transformation assay. Both uninfected and infected genital tracts contained T cells, as evidenced by reactivity to concanavalin A, although a greater number of T lymphocytes was detected in the genital tracts of infected animals compared with that in controls. Significant antigen-specific T-cell activity could be detected in the genital tract tissue by 7 days after a primary genital tract infection with the chlamydial agent of guinea pig inclusion conjunctivitis. When antigen-specific activity was assessed at different times after infection, the intensity of the response of genital tract-associated T lymphocytes was directly proportional to the degree of resistance of the animals to genital challenge. Thus, susceptibility of animals to reinfection by chlamydiae appears to be associated with the intensity of the local T cell-mediated immune responses in the genital tract of infected animals.     

Protein antigens of Chlamydia psittaci present in infected cells but not detected in the infectious elementary body.

Ocular infection of guinea pigs with the guinea pig inclusion conjunctivitis (GPIC) strain of Chlamydia psittaci produces a clinical condition representative of acute chlamydial conjunctivitis in humans. Guinea pigs which had recovered from two challenges with GPIC were used as a source of sera for the identification of antigens present in GPIC-infected tissue culture cells but absent in the infectious elementary body (EB). Immunoblots of lysates of infected HeLa cells probed with the convalescent-phase sera identified protein antigens of 22, 34, and 52 kDa (p22, p34, and p52, respectively) that were not detected in lysates of purified EB or in uninfected HeLa cells. Protein p22 was also not detected in lysates of purified reticulate bodies. Immunoblotting of lysates of HeLa cells infected with other chlamydiae demonstrated that the antigenicity of p22 and p34 was subspecies specific. Immunoblotting was also used to detect p22 and p34 in lysates of the conjunctivae of infected guinea pigs. Adsorption of convalescent-phase sera with GPIC EB produced a reagent with dominant reactivity toward p22, p34, and a 28-kDa EB protein. Immunofluorescent staining of GPIC-infected HeLa cells demonstrated that these adsorbed sera labeled the inclusion and inclusion membrane, with no apparent reactivity toward EB or reticulate bodies. Collectively, these data identify non-EB chlamydial components which may be released into the inclusion during intracellular growth.     

Dissociation of immune determinants of outer membrane proteins of Chlamydia psittaci strain guinea pig inclusion conjunctivitis.

Chlamydia trachomatis is an important human pathogen. Research to develop a Chlamydia vaccine has focused on the major outer membrane protein (MOMP). Determinants of this protein elicit serovar-specific neutralizing antibodies which are thought to play a critical role in protective immunity. MOMP-specific antibody responses are highly variable in the polymorphic population. Genetic factors which might influence the MOMP-specific immune response are consequently of particular interest. The C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) is a natural pathogen of the guinea pig that causes both ocular and genital tract infections that closely resemble those caused by C. trachomatis in humans. As such, it provides an excellent model for disease. In this report, we explore the influence of major histocompatibility complex-linked genes on the MOMP-specific antibody response in mice immunized with either whole GPIC elementary bodies or recombinant GPIC MOMP. Our results indicate that the MOMP-specific antibody response is major histocompatibility complex linked such that mice of the H-2d haplotype are high responders while mice of the H-2k haplotype are low responders. We demonstrate that MOMP-specific B cells are present in H-2k strains which are, however, deficient in MOMP-specific helper T cells. Although immunization of low-MOMP-responder strains with whole chlamydial elementary bodies induces high levels of immunoglobulin G antibody specific for Omp2, the cysteine-rich outer membrane protein, MOMP-specific B cells are unable to receive help from Omp2-specific T cells. The failure of intermolecular help from Omp2-specific T cells and related observations raise important issues regarding the processing and presentation of chlamydial antigens and the design of optimal subunit vaccines.     

Subjects recovering from human ocular chlamydial infection have enhanced lymphoproliferative responses to chlamydial antigens compared with those of persistently diseased controls.

Cell-mediated immune responses to chlamydial and common recall antigens were measured in 26 subjects whose clinical signs of trachoma persisted over 6 months of follow-up and in 21 subjects whose clinical signs resolved spontaneously over the same period. Seven-day lymphocyte proliferative responses to chlamydial but not common recall antigens were significantly greater in subjects whose disease resolved spontaneously. There was, however, no detectable difference between the two groups in gamma interferon levels in supernatants from lymphocyte cultures stimulated with these antigens. These results are consistent with the hypothesis that cell-mediated immune responses play an important role in the clearance of ocular chlamydial infection in humans.     

Effect of antithymocyte serum on the course of chlamydial genital infection in female guinea pigs

The treatment of female guinea pigs, infected in the genital tract with the chlamydial agent of guinea pig inclusion conjunctivitis, with rabbit anti-guinea pig thymocyte serum extended the course of the infection by 20 to 30 days. The rabbit anti-guinea pig thymocyte serum was shown to suppress delayed hypersensitivity responses to the guinea pig inclusion conjunctivitis agent and the contact allergen oxazolone. The appearance of antibody in genital secretions was delayed, but the infection persisted at low levels even when normal serum and secretory antibody titers were attained, indicating that cell-mediated immunity may play a role in the resolution of chlamydial genital infections.     

Cell-mediated immune responses of guinea pigs to an inactivated phase I Coxiella burnetii vaccine.

The ability of a killed phase I Coxiella burnetii vaccine to induce cell-mediated immune responses in guinea pigs was studied. Cell-mediated immune responses were assessed by the inhibition of macrophage migration and lymphocyte transformation assays. The macrophage migration response occurred rapidly and was detected at high levels, but was relatively short-lived. In contrast, the lymphocyte transformation response developed more slowly, and persisted for a longer period. The vaccine, given in a single dose or in two doses 1 week apart, protected guinea pigs from a subsequent virulent challenge.     

Local Host Response to Chlamydial Urethral Infection in Male Guinea Pigs

Very little is known about the host response to chlamydial genital infection in the male, particularly about the nature of the local response in the urethra. In this study, the pathological and immunologic responses to urethral infection of the male guinea pig with Chlamydia caviae (Chlamydophila caviae) were characterized both during a primary infection and following a challenge infection. A dose-response experiment found that the 50% infectious dose for male urethral infection was 78 inclusion-forming units. The histopathologic response was similar to that of the female, with an initial acute inflammatory response followed by a chronic inflammatory response and plasma cell infiltration. Production of IgG and IgA antibodies in local urethral secretions developed following infection, and levels of both increased in a typical anamnestic response following a challenge infection. CD4 and CD8 T cells, as well as B cells, were observed in the local site by flow cytometry, with a slightly increased number of CD8 cells. Following challenge infection, the dominant anamnestic response was solely in the B-cell compartment, with only a minimal number of T cells. The T-cell response was clearly a Th1 response, as judged by increased levels of gamma interferon (IFN-γ), interleukin-12 p40 (IL-12p40), and IL-2. The proinflammatory cytokines and chemokines IL-8, IL-1β, tumor necrosis factor alpha (TNF-α), CCL2 (monocyte chemoattractant protein 1 [MCP-1]), and CCL5 (RANTES) were elicited in the urethra following primary infection, but only CCL5 showed increased levels upon challenge. This study represents the first comprehensive analysis of the local immune response in the male urethra to a chlamydial genital infection.Chlamydia trachomatis is the most common bacterial cause of sexually transmitted infections in the United States and worldwide. The vast majority of research on Chlamydia has been devoted to infections of females, particularly because of the severe sequelae to infection, such as ectopic pregnancy and tubal infertility. However, Chlamydia is the most common bacterial agent of sexually transmitted infections in males as well, causing urethritis and, in some cases, epididymitis and prostatitis, and has been associated with male infertility. A recent epidemiological study reported an increasingly high prevalence rate of 161.1 cases per 100,000 men (29). As in females, the majority of chlamydial infections in males are asymptomatic; thus, the individual may unknowingly transmit the infection to his partner. Quinn and colleagues estimated the probability of transmission for both men and women at 68% (15).Despite the high prevalence of chlamydial infection among males, very little is actually known about the immunobiology and pathogenesis of the infection in males. The human penile urethra is known to possess all of the necessary immune components for antigen presentation and cellular and humoral immune responses, suggesting that this region is clearly able to launch immune responses against chlamydial infection (14). Human male genital tract-derived epithelial cells have been shown to support the growth of C. trachomatis in vitro and to secrete the cytokines interleukin-1 (IL-1), IL-6, and IL-8 upon infection, cytokines that are associated with the inflammatory and innate host responses (1). In the only report of a study of the local immune response of human males, Pate and colleagues reported higher levels of chlamydia-specific IgG and IgA antibodies in urethral swabs from C. trachomatis-infected males than in those from uninfected controls (12). Interestingly, the only cytokine whose levels they found to be elevated in urethral secretions was IL-8. Nevertheless, it is almost a “mission impossible” to define the relation between the infection course and the development of the host response in the local urethral site in infected human males, making the use of animal models imperative.To date, only limited studies on male genitourinary tract infection with chlamydiae have been performed with nonhuman primate (3, 4, 9), mouse (11), and rat (5, 6) models. Although the nonhuman primates closely parallel human disease, basic research studies on the host response are impractical because of the expense. The mouse is a difficult model because of the inability to obtain repeated urethral specimens from individual animals and the very small size of the target tissue for cytokine studies. While it is likely that rats can be infected in the urethra, this has not been reported, and studies with male rats have used direct injection into the epididymis, clearly an unnatural route.However, male guinea pigs can be infected intraurethrally with Chlamydia caviae (Chlamydophila caviae), the agent of guinea pig inclusion conjunctivitis (GPIC) (10, 26). In addition, investigators can collect urethral swabs repeatedly from the same animal, and the guinea pig affords the only animal model in which sexual transmission of chlamydial infection to female guinea pigs can be reliably accomplished (10, 24). In the male guinea pig, the infection resolves about 3 weeks after urethral inoculation, and animals are immune to reinfection. Resolution of the infection appears to be dependent on the humoral immune response, although the role of the cell-mediated response has yet to be explored (26). Interestingly, Patterson and Rank reported that male guinea pigs had a high level of resistance to reinfection with GPIC for as long as 150 days, in contrast to female guinea pigs, which had comparable protection against reinfection for only 30 days after the primary infection (13). Since there is virtually no information on the nature of the local immune response in the male urethra to chlamydial infection, in the present study, we used the model of male guinea pigs inoculated intraurethrally with the GPIC agent to comprehensively characterize the local host response, including the histopathologic response and parameters of innate, humoral, and cell-mediated immunity in both primary infection and reinfection.     

Humoral and cellular immune response to a Microsporum canis recombinant keratinolytic metalloprotease (r-MEP3) in experimentally infected guinea pigs.

In order to better understand the host-fungus relationship in Microsporum canis dermatophytosis and to identify major fungal antigens, the immune response to a crude exoantigen preparation and to a purified recombinant keratinolytic metalloprotease (r-MEP3) was evaluated in guinea pigs experimentally infected with M. canis. Humoral and cellular immune responses were assessed from day 0 to day 57 post-infection (PI), the former by enzyme-linked immunosorbent assay (ELISA) and the latter via a lymphocyte proliferation assay. Infected guinea pigs developed humoral and cellular responses to both M. canis exoantigen and r-MEP3, while no specific immune response to these antigens was observed in control animals. This is the first report on the development of both humoral and cell-mediated immune responses to a purified keratinase in M. canis dermatophytosis.     

Humoral immunity in the resolution of genital infection in female guinea pigs infected with the agent of guinea pig inclusion conjunctivitis.

Female guinea pigs infected in the genital tract with the chlamydial agent of guinea pig inclusion conjunctivitis were selectively immunosuppressed by varying regimens of cyclophosphamide (Cy) treatment. Temporary suppression of both humoral and cell-mediated immunity by daily treatment of Cy (25 mg/kg) for 13 days resulted in a prolonged infection, whereas daily treatment for the duration of the experiment totally prevented the development of humoral and cell-mediated responses and produced an intense and prolonged infection which did not resolve. When humoral immunity alone was suppressed by treatment with Cy (250 and 150 mg/kg) at 9-day intervals, the infection again did not resolve. Treatment with 100 mg of Cy per kg at 9-day intervals resulted in an extended infection which resolved concomitantly with the development of antibody to guinea pig inclusion conjunctivitis. These data indicate that humoral immunity is essential for the recovery of female guinea pigs from guinea pig inclusion conjunctivitis genital infection. A market weight loss was observed which could not be attributed to Cy treatment alone. Edematous and ulcerative changes of the external genitalia were also noted.     

Characterization of lymphocyte response in the female genital tract during ascending Chlamydial genital infection in the guinea pig model

It is well known that pathology caused by chlamydial infection is associated closely with the host response to the organism and that both innate and adaptive host responses contribute to tissue damage. While it is likely that the organism itself initiates the acute inflammatory response by eliciting cytokine and chemokine production from the host cell, the adaptive response is the result of activation of the cell-mediated immune response. While there are several studies describing the nature of the pathologic response in primate, guinea pig, and murine models, there is less information on the kinetics of the CD4 and CD8 response following primary and challenge infections. In this study, we have quantified by flow cytometry the mononuclear cell response to genital infection with the agent of guinea pig inclusion conjunctivitis in the cervix, endometrium, and oviducts at various times following a primary intravaginal infection and after a challenge infection. Tissues from individual animals were assessed for cells expressing CD4, CD8, or Mac-1 and for B cells. Peak responses of each subset occurred 10 to 14 days after a primary infection. The number of Mac-1-expressing cells in each tissue site was found to be dependent on the size of the inoculating dose of chlamydiae. The responses of each cell type were generally stronger in the cervix than in the upper genital tract. In contrast to the murine model but consistent with the primate models, there were equal numbers of CD4 and CD8 cells present in the infiltrates. Twenty-one days after challenge infection, which was performed 50 days after the primary infection, there was a significant increase in the number of CD4, CD8, and B cells in the oviduct compared to the number of these cells at the same time after a primary infection, providing clear cellular evidence for a cell-mediated immune pathologic response.     

An enzyme-linked immunosorbent assay (ELISA) for anti-chlamydial secretory immunoglobulin A in guinea pig tears

A method is described which permits the assay of specific secretory immunoglobulin A (sIgA) antibodies produced by guinea pigs in response to ocular infection with the guinea pig inclusion conjunctivitis strain of Chlamydia psittaci (GPIC agent). The enzyme-linked immunosorbent assay (ELISA) developed was shown to be more sensitive and less subjective than the micro-immunofluorescence assay as a means of assaying specific antibody.     

Lack of allelic polymorphism for the major outer membrane protein gene of the agent of guinea pig inclusion conjunctivitis (Chlamydia psittaci).

The major outer membrane protein gene (omp1) was sequenced for each of six Chlamydia psittaci (guinea pig inclusion conjunctivitis [GPIC]) strains isolated from guinea pigs. Five of the isolates were obtained in the United States during the 1960s and 1970s, including the prototype strain isolated by Murray in 1962. The other isolate was obtained from a guinea pig in England. The nucleotide sequence of the omp1 gene for each strain was identical. The lack of omp1 allelic polymorphism among GPIC isolates suggests that, unlike C. trachomatis, the GPIC agent lacks antigenic variation in the major outer membrane protein.     

Electron microscopic observations concerning the in vivo uptake and release of the agent of guinea-pig inclusion conjunctivitis (Chlamydia psittaci) in guinea-pig exocervix

This report details electron-microscopical observations concerning C. psittaci infection in vivo. The model employed was that of the guinea-pig infected at the exocervical region with the agent of guinea-pig inclusion conjunctivitis (GPIC). Our observations indicate that chlamydial particles gain access to their target cells by the mechanism of endocytosis. Single GPIC elementary bodies were seen to be positioned within individual endosomes. The observations reported here provide evidence that chlamydial particles that had undergone their developmental cycle within the exocervical epithelial cells may leave the epithelium in 2 ways; within entire infected cells that had been shed into the lumen of the cervix and by means of the liberation of chlamydial particles from disrupted cells. The mechanism of cell disruption and shedding is thought to involve the large number of PMNs observed to be present within the enlarged intercellular spaces of the infected epithelium.     

Studies on HLA antigens and cellular and humoral autoimmune phenomena in patients with myasthenia gravis.

The HLA phenotype of fifty-four patients with myasthenia gravis (MG) was compared to that of 600 controls. When male and female patients were compared separately with the control group, HLA-B12 was increased in MG males (P less than 0-023) and HLA-A1, B8 in MG females (P less than 0-023). In addition, HLA-A1, B8 was correlated with the early onset type of MG and with a more severe clinical course (Osserman scale IIb-III) in female patients. Cell-mediated immune responses were studied in a twenty-one hospitalized MG patients. Specific cell-mediated immunity to highly purified muscle proteins was investigated using the two-step-leucocyte migration agarose test and general cell-mediated immunity was studied by determining the cutaneous response to four extrinsic antigens. Cellular immune activity to muscle antigens occurred in fourteen out of twenty-one patients (67%) with MG and with one exception, in none of the controls. There was no significant difference in the LIF inducing ability of the different muscle antigens. A statistically significant correlation between HLA-A1 and B8 and either cell-mediated immune reactivity to muscle antigens or humoral autoimmune phenomena or clinical parameters was not found. Only a trend toward an association between HLA-A1 and B8 and a cell-mediated immune response to muscle antigens could be demonstrated. Positive delayed skin reactions to extrinsic antigens were observed with the same frequency as in healthy blood donors.