Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene.

The virulence of the mycobacteria that cause tuberculosis depends on their ability to multiply in mammalian hosts. Disruption of the bacterial erp gene, which encodes the exported repetitive protein, impaired multiplication of M. tuberculosis and M. bovis Bacille Calmette-Guérin in cultured macrophages and mice. Reintroduction of erp into the mutants restored their ability to multiply. These results indicate that erp contributes to the virulence of M. tuberculosis.

An example of idiotypic mimicry.

We have evaluated the impact of transgenic immunoglobulin (TGIg) expression on endogenous antibody repertoires. The transgenic system was chosen as to allow for normal recombination of endogenous Ig genes, secretion of TGIg from early development on, and distinguishing the TGIg from endogenous Ig by several serological markers on the C and V regions of the molecules. The transgenic construct encodes a complete anti-(4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NP) antibody molecule carrying a well-defined idiotype, bearing a lambda 1 light chain and a chimeric heavy chain encoded by a human alpha 2 C region devoid of its membrane exon, and the murine B1.8 VDJ-region. Endogenous antibody repertoires were analyzed in mitogen-driven limiting dilution cultures, in single-cell assays for naturally activated Ig-secreting cells, and in hybridomas derived by direct fusion of spleen cells from unmanipulated animals. The results show that a very high frequency of splenic resting B cells and plasma cells in transgenic animals produce IgM with B1.8-cross-reactive idiotypes. This was confirmed by hybridoma analysis which also established that the levels of transgene expression and of idiotype-positive IgM production by the same cell are not correlated. The affinities of idiotype-positive endogenous Ig varied, but were generally several orders of magnitude lower than the transgene-encoded idiotype. V regions from idiotype-cross-reactive IgM heavy chains showed marked diversity in sequences that were all different from the transgenic B1.8. These results are compatible with idiotypic mimicry resulting from intercellular selection based on degenerate, whole V region reactivities.

Mycobacterium tuberculosis Des protein: an immunodominant target for the humoral response of tuberculous patients.

The phoA gene fusion methodology permitted the identification of a new Mycobacterium tuberculosis exported protein, Des. This protein has significant sequence similarities to plant acyl-acyl carrier protein desaturases, which are enzymes involved in general fatty acid biosynthesis as well as in mycolic acid biosynthesis in mycobacteria. As shown by Western blot and enzyme-linked immunosorbent assay experiments, the Des protein is a major B-cell antigen that was recognized by all the tuberculous M. tuberculosis- and M. bovis-infected human patients tested.

Characterization of the Mycobacterium tuberculosis erp gene encoding a potential cell surface protein with repetitive structures.

Using the phoA gene fusion methodology adapted to mycobacteria, several Mycobacterium tuberculosis DNA fragments encoding exported proteins were recently identified. In this paper, the molecular cloning, genomic positioning, nucleotide sequence determination and transcriptional start site mapping of a new M. tuberculosis gene, identified by this methodology, are reported. This gene was called erp (for exported repetitive protein) and has a sequence similar to that of the Mycobacterium leprae 28 kDa antigen irg gene M. tuberculosis erp gene contains a putative iron box close to the mapped transcriptional start site. The predicted Erp protein displays a typical N-terminal signal sequence, a hydrophobic domain at the C-terminus and harbours repeated amino acid motifs. These structural features are reminiscent of cell-wall-associated surface proteins from Gram-positive bacteria. We found that these repeats are conserved among M. tuberculosis isolates, and are absent from the published M. leprae irg gene sequence. In addition to being present in M. leprae, erp sequences were found in other members of the M. tuberculosis complex, but not in other mycobacteria tested. These results suggest that erp might encode a cell surface component shared by major pathogenic mycobacteria.

Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.

Murine AIDS protects mice against experimental cerebral malaria: down-regulation by interleukin 10 of a T-helper type 1 CD4+ cell-mediated pathology.

The retrovirus LP-BM5 murine leukemia virus induces murine AIDS in C57BL/6 mice that has many similarities with human AIDS; Plasmodium berghei ANKA causes experimental cerebral malaria in the same strain of mice. The outcome of malaria infection was studied in mice concurrently infected with the two pathogens. The retrovirus significantly reduced the gravity of the neurological manifestations associated with Plasmodium berghei ANKA infection. The protection against experimental cerebral malaria induced by murine AIDS increased with duration of viral infection and, hence, with the severity of the immunodeficiency. Interleukin 10, principally from splenic T cells, was shown to play a crucial role in this protection.

Abnormal selection of antibody repertoires in retrovirus-induced murine AIDS.

The mature B cell repertoire in the course of murine AIDS (MAIDS) was investigated. The polymerase chain reaction (PCR) was used to amplify a large diversity of rearranged Ig H chain genes in normal or infected mice, 2 and 8 wk after virus inoculation. Libraries were constructed from the polymerase chain reaction products. By sequencing V-D-J clones in these libraries and analyzing the respective complementary determining region 3 (CDR3), we have shown at 8 wk the emergence of a population of B cells with significantly less N diversity, some sequences lacking any N addition, a typical feature of fetal repertoires known for degeneracy, and autoreactivities. This decreased N diversity was not present 2 wk after inoculation and could not be related to a defect in terminal deoxytransferase expression because the steady-state levels of terminal deoxytransferase mRNA were found normal in MAIDS bone marrow 8 wk after inoculation. FACS analyses revealed a decreased number of bone marrow B cells (B220+, sIgM+) in MAIDS already present at 2 wk, suggesting an alteration in the pathway of B cell differentiation and resulting in a decrease of peripheral B cells renewal. A relative enrichment of spleen cells in long lived B cells as a consequence of this blockade may participate in the abnormal antibody repertoire selection occurring in MAIDS. These data suggest in the MAIDS pathogeny the relationship between an abnormal repertoire selection and the pathologic process.

Peripheral lymphoid hyperplasia and central lymphoid depletion in mice treated with a bacterial B-cell mitogen (F3'EP-Si/p90).

In order to further understand the mechanism mediating the mitogenic and immunosuppressor effects of p90, a protein produced by Streptococcus intermedius, flow cytometric studies were performed on peripheral and central lymphoid organs of mice treated with this protein. p90 induced a strong blastogenic B-cell response in the spleen and lymph nodes, followed by a slight but significant polyclonal T-cell activation. B-cell repertoire analysis indicated that polyclonal B-cell responses affected similarly both CD5+ and conventional (CD5-) B cells in the spleen. Repertoire analysis of T cells failed to reveal any preferential stimulation of the V beta T-cell receptor (V beta-TcR) families studied. Peripheral lymphoid hyperplasia was observed concomitantly with central lymphoid depletion. In the bone marrow, pre-B and B cells were profoundly depleted, with a more pronounced effect on small pre-B cells. In the thymus, double-positive (CD4+CD8+) thymocytes were preferentially eliminated, with a relative enrichment of single positive (either CD4+ or CD8+) and double-negative (CD4-CD8-) thymocytes.

Protective effect of a T-cell-dependent immunosuppressive, B-cell-mitogenic protein (F3'EP-Si, or P90) produced by Streptococcus intermedius.

The role of a previously described bacterial protein (F3'EP-Si), now designated P90, in the survival of Streptococcus intermedius in the host was investigated, and the immunosuppressive and B-cell-mitogenic effects of this protein were further characterized. C57BL6 mice treated with P90 were about 50 times more susceptible to infection with this bacterium than untreated mice. One of seven splenocytes of C57BL/6 mice were activated by P90. Marked splenomegaly was observed in mice treated with P90, with increased numbers of splenic mononuclear cells and polyclonal immunoglobulin-secreting plaque-forming cells. Peak responses were seen on day 3 for immunoglobulin M (IgM) and on day 5 for IgG, with an isotypic pattern consisting predominantly of IgG2a and IgG2b. When mice were treated with P90 before being primed with sheep erythrocytes, polyclonal immunoglobulin synthesis was accompanied by an ephemeral stimulation of the specific immune response against sheep erythrocytes that was quickly replaced by a dramatic immunosuppression. In contrast, when mice were treated with P90 after being primed, the polyclonal activation was comparatively much less evident and there was no suppression of the specific immune response. Immunosuppression was considerably reduced in mice thymectomized as adults or depleted of CD8+ cells. Adoptive transfer experiments showed that B cells obtained from P90-treated mice were less able to respond to an antigenic challenge, even in the presence of normal T cells, and that T cells obtained from P90-treated mice could actively suppress the specific immune response of normal B cells.

Normal serum immunoglobulins influence the numbers of bone marrow pre-B and B cells.

The homeostatic mechanisms controlling B lymphocyte output from bone marrow are not well understood. The present experiments evaluated putative influences of circulating immunoglobulins (Ig) on bone marrow (BM) pre-B and B cell populations. Injections into normal mice of Ig isolated from normal mouse serum, resulted in a dose-dependent and reversible reduction in numbers of BM B lineage cells, in particular of small B220+ surface IgM- cells. Maximal effects were observed upon injection of isologous polyclonal Ig and were independent of mature T cells. These results suggest a feedback modulation of peripheral Ig on cellular activities in BM B lineage compartments, mediated by mechanisms that seem to involve the variable regions of the Ig molecule.

Stimulation of B and T cells by in vivo high dose immunoglobulin administration in normal mice.

Adult BALB/c mice were injected intravenously with a preparation of pooled normal murine IgG (400 mg/kg/day, on five consecutive days) and studied 8, 15, and 60 days later. High dose IgG administration increased the total numbers of splenic activated B and CD4+ (but not CD8+) T cells, as well as the numbers of splenic Ig-secreting cells, particularly in the IgG isotypes. Reactivities to some autoantigens, but not to bacterial or other heteroantigens, were selectively amplified amongst IgM-secreting cells. IgG administration did not alter the specific primary immune response to heterologous erythrocytes or bacterial dextran. No cellular alterations were detected in the lymph nodes or peritoneal cavity of treated animals. Most of these effects subsided with time, but some autoantibody reactivities remained elevated 60 days later. The present results suggest that the therapeutic effects of high dose IgG administration which have been reported in human diseases might be associated with the immunostimulatory activities of such treatment.

Zidovudine (azido dideoxythymidine) inhibits characteristic early alterations of lymphoid cell populations in retrovirus-induced murine AIDS.

Using flow cytometry technology and multiparameter analyses, we report early and characteristic alterations in lymphoid cell profile in spleen and lymph nodes due to LP-BM5 retrovirus disease (murine AIDS (MAIDS)) and the effect of azido dideoxythymidine, a nucleoside inhibitor, on these changes. MAIDS has been characterized by rapid and profound lymphoproliferation accompanied by hypergammaglobulinemia and immunosuppression. As early as 2 wk postinfection, there is a selective depletion of CD8+ cells whereas the total number of CD4+ cells increases throughout the first 8 wk of infection although the frequency is relatively stable. These population changes were partially delayed by oral AZT therapy for 6 wk postinfection. Ly-6C (AL-21) is expressed on roughly 50% of CD4+ and CD8+ cells in C57BL/6 mice. In MAIDS, the residual population of CD8+ cells is primarily Ly-6C+. The CD4+ cells have a transient increase in ratio of Ly-6C+/Ly-6C- cells at 2 wk postinfection but by 6 wk are primarily Ly-6C-. There was an increase in both the total number and percentage of Mac 1+ cells and a selective depletion of certain splenic B cell subpopulations. Azido dideoxythymidine delays these early population changes.

Major histocompatibility complex-linked and T cell-dependent selection of antibody repertoires. Quantitation of I-E-related specificities in normal mice.

Autoreactive B cell repertoires with major histocompatibility complex (MHC) class II (I-E)-related specificities were investigated by quantitating frequencies of specific B lymphocyte clonal precursors in unmanipulated normal and athymic BALB/c mice and in I-E-negative, MHC-congenic BALB.B10 mice. Clonal culture supernatants containing anti-I-E antibodies were identified by their selective binding to I-Ek alpha Ed beta-transfected fibroblasts, and those containing anti-anti-I-E antibodies were detected by their selective binding to anti-I-E monoclonal antibodies. Analysis of splenic B lymphocytes from BALB/c mice revealed high frequencies of both specificities in the compartment of large, naturally activated cells, but not among small, resting lymphocytes. The selection of such clones was found to be MHC linked because of their absence in BALB.B10 mice, and T cell dependent because of their reduced frequency in athymic BALB/c mice. The positive selection of V regions representing complementarities and mimicries of self-class II antigens may suggest a set of mechanisms participating in the maintenance of natural tolerance.

Ontogenic development of autoantibody repertoires in spleen and peritoneal cavity of normal mice: examples of T cell-dependent and -independent reactivities.

The ontogenic development of B cell clonal precursors (BCP) reactive to bromelain-treated, syngeneic erythrocytes (BrMRC) and to single-stranded DNA has been studied by limiting dilution of both spleen and peritoneal cells. It was found that the frequency of anti-BrMRC BCP in the spleen is very low up to 4 weeks of age and slowly increases thereafter, to reach adult levels by 6-10 weeks. In the peritoneal cavity, no such BCP can be found before 2 weeks, but they occur at a very high frequency already by 3 weeks of age. Injection of adult, normal syngeneic T cells at birth has no apparent effect on the representation of anti-BrMRC BCP in the peritoneal cavity, but brings these to adult levels or even higher in the spleen already at 3 weeks of age. Accordingly, adult athymic (nude) mice contain normal frequencies of BrMRC-specific BCP in the peritoneal cavity but are devoid of such clones in the spleen. In contrast, the frequency of anti-DNA BCP is very high throughout postnatal development in both spleen and peritoneal cavity, of normal and athymic mice, in both resting and naturally activated splenic B cell compartments, and it is independent of T cell transfers into nude animals. These results indicate the role of T cells in the establishment of some clonal specificities in the adult, splenic autoreactive B cell repertoire.

The immune response to bacterial dextrans. VI. No correlation between the frequency of cells expressing a major anti-dextran idiotype and the idiotype profiles of specific antibody responses.

C57BL/6 mice respond to Dextran B512 (Dex) with a predominant idiotype (Id) (17-9) while no such Id-positive antibodies are identified in the specific antibody response of BALB/c mice. We used limiting dilution systems to determine the absolute frequencies of clonal B-cell precursors producing the 17-9 Id in these two mouse strains and analysed the correlation between Id-expression and antibody activity at the clonal level. The results show very similar frequencies of anti-Dex and Id-positive B cells in both strains, but C57BL/6 mice contained fourfold higher frequencies of Dex-specific clonal precursors which are Id-positive. This kind of clone, although not used in the specific response of BALB/c mice, constitute roughly 20% of their anti-Dex repertoire and they are readily induced in this strain. Thus, immunization of both strains with anti-idiotypic antibodies results in the production of Id-positive anti-Dex antibodies with serum titres that directly correlate with precursor frequencies. The results show, therefore, that the section of clonal repertoires utilized in a primary immune response varies with the immunogen, even if thymus-independent. These observations are discussed in the context of the genetic controls of anti-Dex antibody responses and on the general question of the utilization of available antibody repertoires in immune responses.

Idiotype-specific regulation might contribute to specific unresponsiveness in dextran-primed mice.

Priming of adult responder mice with optimal immunogenic doses of dextran alpha, 1-6 results in reduced antibody responses to secondary antigenic challenge. We have now quantitated dextran-specific clonal precursors in the large and small B-lymphocyte compartments within several days or several months after priming with either dextran or antiidiotypic antibodies directed to a dominant idiotype of C57BL/6 mice which accounts for more than 50% of the antibody response. The results show that "secondary unresponsiveness" correlates with idiotype-directed depletion of the appropriate specificities from the immunocompetent resting B-cell pool.

Lymphoproliferative syndrome associated with c-myc expression driven by a class I gene promoter in transgenic mice.

We have produced transgenic mice carrying an H-2K/human c-myc fusion gene. In this construct, the human c-myc proto-oncogene expression is driven by the 5' flanking sequences (including promoter) of the class I H-2Kb gene, which have previously been shown to direct the expression of a marker gene, the human growth hormone (hGH), in most tissues of H-2K/hGH transgenic mice. Comparative analysis, by S1 nuclease mapping, of the H-2K and human c-myc gene expression in different organs of the H-2K/myc mice shows that exogenous c-myc and endogenous H-2K expression is found in most organs examined. However, the liver is a notable exception, for here c-myc expression is very weak. The exogenous c-myc expression is maximal in lymphoid organs of all H-2K/myc transgenic strains. One strain, H-2K/myc 27, hereditarily develops a lymphoproliferative syndrome which eventually leads to death. The H-2K/myc 27 lymphoid tissues are profoundly abnormal: pre-B cells as well as mature B cells are underrepresented in the bone marrow but the thymus as well as lymph nodes are largely infiltrated by B cells. Moreover, in the thymus, the proportions of the different thymic cell populations are altered. However, in the other H-2K/myc transgenic strains, even in those expressing a comparable or even higher level of myc, no pathology has been observed over a period of 20 months. Our results, therefore, demonstrate that constitutive enforced c-myc expression might disturb lymphocyte development, but does not directly lead to malignancy.

T cell dependence of the "natural" autoreactive B cell activation in the spleen of normal mice.

A high frequency of clonal precursor B cells producing lytic antibodies to syngeneic erythrocytes treated with bromelain (BMRC) is revealed in normal mouse spleen cells by lipopolysaccharide-driven limiting dilution analysis. All such specificities are recovered as activated blasts after density gradient fractionation, the "small lymphocyte" pool being depleted of anti-BMRC reactivities. In contrast, the spleens of athymic (nude) mice contain undetectably low frequencies of these specificities in either lymphocyte compartment. Transfer of relatively low numbers of normal syngeneic splenic T lymphocytes to adult nude mice restores the high frequency of anti-BMRC clonal precursor B cells, again in the activated, but not in the resting spleen cell fractions. Large total T cells are more than tenfold better than resting T cells in reconstitution potential, as are enriched CD4+ as compared to CD8+ cells which are practically devoid of activity in this respect. These results apply exclusively to B cells at a differentiative stage that allows for extensive clonal expansion, since there is a marked difference between the frequency of clonal precursors determined by limiting dilution analysis and the frequency of Ig-secreting plaque-forming cells of the same specificity, and induced by the same mitogen in short-term cultures. The implications of these findings for the physiology of autoreactivity and repertoire selection in the compartment of perinatal B cells are discussed.

On the validity of using lipopolysaccharide-driven limiting dilution systems for clonable B-cells to analyse functional antibody repertoires.

Analyses of B-cell repertoires by mitogen-driven limiting dilution systems (LDA) followed by specific antibody detection on immobilized antigens (ELISA), while constituting the only method available to determine absolute frequencies of any given specificity, provide no indications as to the functional ability of clonal precursors scored as positive to actually respond to antigen in vivo. We have now addressed this question by quantitating dextran alpha 1----6-specific clonal precursors among small, resting, and large activated B cells in the spleens of mice, before and after priming with optimal doses of antigen. The results demonstrate that virtually all B cells scored as antigen-specific in LDA/ELISA systems do respond to antigen and undergo blast transformation after priming in vivo.