The Salmonella typhimurium AhpC polypeptide is not essential for virulence in BALB/c mice but is recognized as an antigen during infection.

The OxyR regulon is known to mediate protection against oxidizing agents in Salmonella typhimurium. We reported previously that ahp, one of the OxyR-regulated loci, is induced during macrophage interaction (K. P. Francis, P. D. Taylor, C. J. Inchley, and M. P. Gallagher, J. Bacteriol. 179:4046-4048, 1997). We now report on the effects of disrupting ahp or oxyR on virulence in a BALB/c mouse model. Surprisingly, insertion of a Mudlux derivative within ahpC was found to result in attenuation, while irreversible inactivation of the locus through insertion of a cml cassette did not. An SL1344 derivative carrying an oxyR::kan disruption was also found to be as virulent as the parental strain. Moreover, both cell-mediated and humoral responses to AhpC were found to develop during the course of infection, probably through T-helper-cell (type I) activation. These results indicate that, although not essential for virulence, AhpC is expressed by S. typhimurium during infection of BALB/c mice and constitutes a target for the immune system.

Identification of the ahp operon of Salmonella typhimurium as a macrophage-induced locus.

Previously, we tagged a macrophage-induced Salmonella typhimurium locus with Mudlux (K. P. Francis and M. P. Gallagher, Infect. Immun. 61:640-649, 1993). The insertion lies within the OxyR-regulated ahpC locus and conveys alkyl peroxide sensitivity. Plasmid-encoded ahp reverses sensitivity but reduces luminescence. This suggests that OxyR is titrated by the multicopy ahp promoter.

Heligmosomoides polygyrus: influence of infection on lymphocyte subpopulations in mouse mesenteric lymph nodes.

The immune response to a primary infection of Heligmosomoides polygyrus (Nematospiroides dubius) was studied by flow cytometry in three strains of mice, BALB/c, CBA, and NIH. The chief feature of the response was a pronounced increase, during the first week, in the proportion of B lymphocytes in the mesenteric lymph nodes. All three strains also showed an increase in lymph node cellularity, although this was delayed in NIH and CBA mice. Total B cell numbers thus also increased, particularly in the BALB/c and NIH strains but only in the latter was this response maintained throughout the 4-week study. Although the early changes in B cell frequency were similar in all three strains, B cell responses were greatest in BALB/c mice, and most prolonged in NIH, when they persisted into the adult phase of the infection. These features distinguished them from CBA mice, and could be associated with known variations in resistance to challenge infections. An increase in T cell numbers was delayed in comparison with the changes in the B cell population, and the ratio of 'helper' to 'suppressor/cytotoxic' T cells remained more or less constant in all three strains. There was thus no evidence for an increase in the frequency of suppressor T cells in any strain of mouse.

Early lymphocytic responses to Heligmosomoides polygyrus infections in mice.

Responses to parasite antigens were studied in three strains of mice, BALB/c, CBA and NIH, during the initial phases of a primary infection with the intestinal nematode Heligmosomoides polygyrus. Changes in the rate of in vivo cell division were analysed in mesenteric lymph nodes and spleens during the phases of larval maturation and adult establishment, and related to changes in organ size and cellularity. The nature of the proliferating cell populations was also investigated by flow cytometry, carried out on cell suspensions prepared at the time when larval development was complete. The variation in the ability of the strains of mice to become resistant to a challenge infection was manifest as only slight differences in their initial responses to infection. All three strains showed an increase in 125I-iododeoxyuridine incorporation in their mesenteric lymph nodes and spleen, and an increase in B cell frequency over that of T cells in the draining lymph nodes. Although lymph node weight in NIH mice continued to rise over a 4 week period, the majority of responses measured were short lived, peaking 10 to 14 days after infection. The low responder status of CBA mice was thus reflected in a transient and relatively small enlargement of lymphoid tissues, but their early proliferative responses to antigen were similar in scale to those of responder strains.

The contribution of B-cell proliferation to spleen enlargement in Babesia microti-infected mice.

Flow cytofluorimetric analysis showed that B-cell proliferation makes a major contribution to the enlargement and increased cellularity of the spleen, which are characteristic of Babesia microti infections in mice. Expansion of the B-cell population was accompanied by modulation of the cell surface, which affected most B lymphocytes, and which was detected as a reduction in the density of surface immunoglobulin. This effect was noted as early as Day 7, shortly after the appearance of parasites in the circulation and the onset of gross spleen changes. In contrast to the results for B cells, the frequency of splenic T cells declined, and when the data were transformed into absolute numbers it became clear that only limited T-cell proliferation had occurred. There was no evidence to suggest that the balance of T-cell subsets was shifted in favour of suppressor T cells. The relationships of these results to reports of immunosuppression by this parasite are discussed.

Suppressive effects of Corynebacterium parvum on primary and secondary antibody responses in mice.

The splenic plaque forming cell (PFC) response of mice to an intraperitoneal injection of sheep erythrocytes was severely depressed by prior treatment with Corynebacterium parvum given four days beforehand by the same route. However, total antibody levels were less affected, and soon attained near normal titres. This implied that the effects of C. parvum were limited to the spleen, and that other tissues gave a substantial response during the period when the splenic response was suppressed. Equally, this apparently local immunosuppressive effect of C. parvum failed to inhibit the eventual development of a normal memory cell pool. It was also shown that primed cells in the spleen, challenged during the period when C. parvum inhibited primary IgG responses, were relatively refractory to its suppressive effects.

Age-related changes in cell localization and proliferation in lymph nodes and spleen after antigenic stimulation.

Antigen-dependent localization of 51Cr-labelled lymphocytes, and the subsequent uptake of IUdR into lymphoid organs has been studied as a function of age. Measures of cell localization indicated that while old age can alter the patterns of entry of lymphocytes into lymph nodes and spleen, these changes are variable and probably not sufficient alone to explain decreased primary antibody responses in old animals. Proliferation of cells, however, was consistently affected in both organs and this phenomenon is discussed in terms of abnormal T-cell function.

Changes in lymphocyte accumulation and proliferation in the lymph nodes draining the pregnant uterus.

Changes in weight, lymphocyte accumulation and cellular proliferation have been measured in the lymph nodes draining the uterus during inter- and intra-strain pregnancies and compared with similar effects after other antigenic stimuli. From the data obtained it was concluded that "paternal" antigenic stimulation from the conceptus initiated an immune response in these nodes. The mechanisms of the subsequent suppression of this response are discussed.

Influence of antigenic competition on the development of antibody-forming cell clones.

Isoelectric focusing in polyacrylamide gels was used to investigate the anti-sheep red blood cell antibody responses of mice subjected to antigenic competition. A reduction in the number and intensity of antibody bands was found, even in situations where the suppression of IgG antibody titres was minimal, while with large reductions in titre, antibody bands were rarely seen. It thus appeared that the output of individual B-cell clones was severely depressed during competition. It was concluded that inhibition of clonal expansion is an important feature of competition, and that this may reflect a normal regulatory activity which acts to limit cellular proliferation during immune responses. This conclusion was supported by observations on the level of DNA synthesis, following immunization with sheep red cells, in the spleens of normal and suppressed mice.

Age-related changes in localization of injected radiolabelled lymphocytes in the lymph nodes of antigen-stimulated mice.

The localization of i.v. injected syngeneic lymph node cells, radiolabelled with 51Cr or 75Se-L-selenomethionine, was studied in male CBA/H mice aged between 3 and 30 months. The following results were obtained. (1) Localization of cells from young adult donors was greater in the s.c. lymph nodes of old than of young recipients, the main increase being between 15 and 17 months of age. Increases in lymph node weight and DNA-synthesis were also seen at this time; but the rise in cell localization was significant even when calculated per unit of tissue weight. Splenic localization either declined slightly with age or, like the liver, showed no significant change. (2) Local antigenic stimulation by a single injection of sheep erythrocytes into one front footpad, 24 hr before lymph node cell injection, resulted in increased localization in the regional lymph nodes of 3-17 month old, but rarely of 24-30 month old mice. (3) No consistent differences in localization were observed between lymph node cells from 4-month and 25-month old donors. Both age-related and antigen-related increases in cell localization were at least partly attributable to an enhanced rate of entry of lymphocytes from the blood to the lymph nodes. Although the changes underlying the decline in antigen-related localization of cells in old recipients have still to be clarified, it is probable that the defective immune responses of old mice result partly from this decline.

Characteristics of immunological memory in mice. II. Resistance of nonrecirculating memory cells to antigen-mediated suppression of the secondary antibody response.

Mice were primed and subsequently challenged at various times with subcutaneous injections of sheep erythrocytes, and some characteristics of the secondary responses in the draining brachial and axillary lymph nodes were investigated. It was found that the secondary response within primed nodes was resistant to immunological preemption, a competition-like phenomenon which severely depresses primary responses. Since it was also shown that circulating memory cells could be inhibited by preempting injections of antigen, it was concluded that the resistance of primed nodes to preemption was due to the presence within them of a nonrecirculating subpopulation of memory cells. The size of this population was dependent both on the amount of priming antigen and the time after priming. The observation that the response given by these cells remained unaffected by doses of antigen which could depress a primary response does not favor the view that suppression of immune responses by preemption or antigenic competition is due to a factor which acts directly and indiscriminately on all immunologically competent cells.

Characteristics of immunological memory in mice. I. Separate early generation of cells mediating IgM and IgG memory to sheep erythrocytes.

The kinetics of the generation of primed IgM and IgG antibody-forming cell precursors, and of helper T-cell populations, were analyzed in mice whose primary responses to high and low doses of SRBC were arrested at intervals by the immunosuppressive agents cyclophosphamide monohydrate and specific antibody. The extent to which immunological memory was established in these animals before blockade of the primary response was assessed by the hemolytic plaque assay following challenge 12 wk after priming. The presence of IgG B-memory cells and T-memory cells in suppressed mice was further investigated by the transfer into these animals of syngeneic SRBC-stimulated thymocytes or anti-theta-treated spleen cells. It was found that the progenitors of secondary IgM-synthesizing cells were primed almost immediately after injection of antigen, and that early blockade of the primary response resulted in a raised IgM response after challenge. On the other hand, priming for a secondary IgG response took at least 4 days, and was dose-dependent, although helper T populations for a secondary IgG response appeared 3 days after antigen injection. It appeared that both IgM and IgG memory cells may be considered as Y cells in terms of the X-Y-Z scheme of lymphocyte activation, but that the two populations are generated at different times after exposure to antigen. The size of either Y-cell population at any given time is dependent upon the amount of antigen available to provoke differentiation to antibody-forming Z cells, and the IgM Y-cell population in particular is likely to be depleted during the course of a normal 1 degrees response. When IgM Y cells were maintained for long periods as a result of immunosuppression, their secondary antibody response was independent of the primed T cells necessary for a secondary IgG response.