Recombinant BCG coexpressing Ag85B,ESAT-6 and Rv3620c elicits specific Th1 immune responses in C57BL/6 mice

Tuberculosis (TB) remains to be an enormous global health problem. The inconsistent protection efficacy of Bacille Calmette-Guérin (BCG) calls for new vaccines for TB. One choice to improve the efficacy of BCG vaccine is recombinant BCG (rBCG). Experimental evidences have revealed that Ag85B, ESAT-6 and Rv3620c are important immunodominant antigens of Mycobacterium tuberculosis. In this study, we have constructed a novel rBCG expressing fusion protein Ag85B-ESAT6-Rv3620c and evaluated the immunogenicity of this rBCG in C57BL/6 mice. Results show that there is a strong TB-specific CD4⁺ and CD8⁺ T lymphocytes proliferation in mice immunized with this rBCG vaccine. A single dose immunization of rBCG could induce a significantly strong Th1 immune response characterized by an increasing ratio of antigen-specific IgG2b/IgG1 as well as a high expression level of Th1 cytokines such as IFN-γ, TNF-α and IL-2. This conclusion was confirmed by a decreased secretion of Th2 cytokine IL-10. Moreover, this rBCG induced a strong humoral response in mice with an increasing antigen-specific IgG titer. Therefore, we concluded that this rBCG could significantly increase both Th1 type cellular immune response and antigen-specific humoral response compared with BCG. The above observations demonstrated that rBCG::Ag85B-ESAT6-Rv3620c is a potential candidate vaccine against M. tuberculosis for further study.     

Bartonella henselae-Specific Cell-Mediated Immune Responses Display a Predominantly Th1 Phenotype in Experimentally Infected C57BL/6 Mice

Immune responses of the immunocompetent host to Bartonella henselae infection were investigated in the murine infection model using C57BL/6 mice. Following intraperitoneal infection with human-derived B. henselae strain Berlin-1, viable bacteria could be recovered from livers and spleens during the first week postinfection, while Bartonella DNA remained detectable by PCR in the liver for up to 12 weeks after infection. Granulomatous lesions developed in livers of infected mice, reached maximal density at 12 weeks after infection, and persisted for up to 20 weeks, indicating that B. henselae induced a chronic granulomatous hepatitis in the immunocompetent murine host. T-cell-mediated immune responses were analyzed in vitro by means of spleen cell proliferation and cytokine release assays as well as analysis of immunoglobulin G (IgG) isotypes. Spleen cells from infected mice proliferated specifically upon stimulation with heat-killed Bartonella antigen. Proliferative responses were mainly mediated by CD4+ T cells, increased during the course of infection, peaked at 8 weeks postinfection, and decreased thereafter. Gamma interferon, but not interleukin-4, was produced in vitro by spleen cells from infected animals upon stimulation with Bartonella antigens. Bartonella-specific IgG was detectable in serum of infected mice by 2 weeks, and the antibody concentration peaked at 12 weeks postinfection. IgG2b was the prominent isotype among the Bartonella-specific serum IgG antibodies. These data indicate that B. henselae induces cell-mediated immune responses with a Th1 phenotype in immunocompetent C57BL/6 mice.     

Acute Immune Response to Mycobacterium massiliense in C57BL/6 and BALB/c Mice

Mycobacterium massiliense is an environmental opportunistic pathogen that has been associated with soft tissue infection after minor surgery. We studied the acute immune response of C57BL/6 and BALB/c mice infected intravenously with 10⁶ CFU of an M. massiliense strain isolated from a nosocomial infection in Brazil. The results presented here show that M. massiliense is virulent and pathogenic to both C57BL/6 and BALB/c mice, inducing a granulomatous inflammatory reaction that involves the activation of macrophages, dendritic cells, and natural killer cells induced by gamma interferon and interleukin-17 (IL-17) in C57BL/6 mice and by IL-12 in BALB/c mice.Mycobacteria that do not belong to the complex Mycobacterium tuberculosis are known as nontuberculous mycobacteria (NTM) or atypical mycobacteria. NTM are ubiquitous microorganisms found worldwide in soil and water (3, 23, 38). These environmental mycobacteria are considered emerging and environmental opportunistic pathogens (6, 23).Mycobacterium massiliense is an environmental nonphotochromogenic, rapidly growing Mycobacterium strain that has been associated with soft-tissue infection after minor surgery or intramuscular injection (3, 5, 17, 22, 26, 46) and with pulmonary infection due to diseases, such as cystic fibrosis (29, 41). This species differs only slightly from Mycobacterium abscessus, sharing a 99.6% sequence identity of their 16S rRNA genes; genetic differences can be observed by comparative sequence analysis of the rpoB and hsp65 genes (1, 25, 42). Infections with these agents tend to respond poorly to macrolide-based chemotherapy (3), even though the organisms are susceptible to clarithromycin (15, 44, 47).M. massiliense infection mainly affects immunocompetent individuals and occasionally is associated with disseminated disease (8). An outbreak of M. massiliense occurred in Goiania, Brazil, where 30 individuals were infected after undergoing knee joint and laparoscopic surgery (5). Despite the fact that the infected individuals were from different hospitals, a unique M. massiliense strain was identified and characterized by pulsed-field gel electrophoresis.Disease pathogenesis involves host-pathogen interactions that directly affect parasite clearance. Typically, when environmental bacteria are passively introduced into the host, rapid bacterial clearance occurs due to an efficient innate immune response (30). Nonetheless, accidental infections with M. massiliense have been described as having a chronic evolution and, in some cases, the disease is disseminated irrespective of the host''s immune status. Such findings raise the possibility that this species is more virulent and/or pathogenic than other environmental mycobacteria, such as M. chelonae and M. abscessus.Recently, a murine model of M. abscessus infection was described, and isogenic mice were shown to be good models to address the immune response of the host (34, 39). In the present study, we analyzed the immune response of C57BL/6 and BALB/c mice infected with a clinical isolate of M. massiliense obtained from the recent outbreak in Goiania, Brazil. We show here that M. massiliense is virulent and pathogenic to both C57BL/6 and BALB/c mice, inducing a granulomatous inflammatory reaction that involves the activation of macrophages, dendritic cells (DCs), and natural killer (NK) cells induced mainly by gamma interferon (IFN-γ) and interleukin-17 (IL-17) in C57BL/6 mice and by IL-12 in BALB/c mice.     

Modulation of Cell-Mediated Immune Response in B16F-10 Melanoma-Induced Metastatic Tumor-Bearing C57BL/6 Mice by Sulforaphane

Effect of sulforaphane on cell-mediated immune response (CMI) was studied in B16F-10 melanoma-induced metastasis-bearing C57BL/6 mice. Administration of sulforaphane significantly enhanced natural killer (NK) cell activity in metastatic tumor-bearing animals (43.17% cell lysis, on day 5) and the activity was observed earlier than in tumor-bearing control animals (maximum of 9.76% cell lysis, on day 9). Antibody-dependent cellular cytotoxicity also was enhanced significantly in metastatic tumor-bearing animals (41.20% cell lysis on day 9) after sulforaphane administration compared with untreated control tumor-bearing animals (maximum of 12.62% cell lysis on day 15). An early antibody-dependent complement-mediated cytotoxicity also was observed in sulforaphane-treated tumor-bearing animals (26% cell lysis, on day 15). Administration of sulforaphane significantly enhanced the production of IL-2 and IFN-γ in metastatic tumor-bearing animals. In addition, sulforaphane significantly downregulated the serum levels of proinflammatory cytokines such as IL-1β, IL-6, TNF-α, and GM-CSF during metastasis. These data clearly suggest that sulforaphane effectively inhibited the spread of metastatic tumor cells through the stimulation of CMI, upregulation of IL-2 and IFN-γ, and downregulation of proinflammatory cytokines IL-1β, IL-6, TNF-α, and GM-CSF.     

Immune Function in Adult C57BL/6J Mice Following Exposuke to Ukethan Pre-OK Postnatally

Administration of urethan (LTRE or ethyl carbamate) to mice results in the development of a variety of tumors, and, in certain strains of mice, marked suppression of the immune response. Perinatal exposure of mice to URE has been found to result in increased tumor induction compared to exposure of adult animals. In the present study, the effects of perinatal exposure to UKE on the development of immunorompetence was investigated. Pregnant mice were injected with total doses of either 0.5 or 1.0 mg URE/g of body weight over days 7-16 of gestation or pups of nontreated dams were administered a total dose of 2.0 mg URE/g of body weight over postpartum days 5-14. Postnatal exposure to URE suppressed NK (natural killer) cell activity but left intact other measured parameters of the host defense system. Prenatal exposure, on the other hand, resulted in elevated leukocyte counts and a trend toward increased spleen and thymus size in offspring of treated mothers. Humoral immune function, as measured by the IgM response to sheep erythrocytes, was suppressed in pups from dams injected with a total of 1.0 mg/g URE. These results indicate that marked differences in immunopharmacologic effects may be observed if chemical exposure occurs at different times during the ontogeny of the immune system.     

Antibody class switching differs among SJL, C57BL/6 and 129 mice

Inbred mouse strains used for gene manipulation studies vary in many respects, including immune system function. These differences can interfere with data interpretation unless the mice are well backcrossed. Here, we show that antibody class switching to IgG3 in cultured splenic B cells from Swiss James Lambert (SJL) and 129/Sv mice is 2- to 6-fold less efficient compared with C57BL/6 (B6). Under optimal stimulation conditions, IgA switching is also 2- to 19-fold lower in SJL and 129/Sv B cells. Splenic B cells from SJL mice express higher levels of CD19 and CD21 compared with B6, and their CD21(high)CD23(low) B cells have little CD9 expression, suggesting atypical marginal zone (MZ) B cells. However, sort purification of splenic B cell subsets did not equalize in vitro class switching to IgG3 or IgA between SJL and B6. 129/Sv spleens have a 3-fold greater number of MZ B cells compared with B6, with similar CD9 expression. Poor IgG3 switching by 129/Sv B cells is specific to CD23(high) follicular B cells, whereas similar changes in IgA switching are seen in both CD21(high) and CD23(high) B cell subsets from 129/Sv. Therefore, the functions and phenotypes of mature B cells differ among three common strains of mice.     

Recombinant Mycobacterium bovis BCG Expressing the Chimeric Protein of Antigen 85B and ESAT-6 Enhances the Th1 Cell-Mediated Response

The chimeric protein that relies on the T-cell epitopes of antigen 85B (Ag85B) and the 6-kDa early secreted antigen target (ESAT-6) has been demonstrated to augment the Th1 immune response. In this study, we developed a recombinant Mycobacterium bovis BCG (rBCG) strain that secretes the chimeric protein of Ag85B and ESAT-6 (rBCG-A_N-E-A_C). Immunization with this rBCG strain induced stronger antigen-specific gamma interferon (IFN-γ) activities, as determined by an enzyme-linked immunospot assay, and higher levels of antigen-specific CD4⁺ and CD8⁺ T-cell responses than those in the control groups immunized with either rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) or BCG. Likewise, rBCG-A_N-E-A_C significantly increased the level of production of the major Th1 cytokines IFN-γ and tumor necrosis factor alpha in splenocyte cultures to levels comparable to those elicited by control BCG. Moreover, the antigen-specific immunoglobulin 2c (IgG2c)/IgG1 ratio for mice immunized with rBCG-A_N-E-A_C was also much higher than the ratios for the other immunized groups. Together, these results indicate that this rBCG-A_N-E-A_C strain enhances the Th1 cell-mediated response and may serve as a potential vaccine against M. tuberculosis.Mycobacterium bovis bacillus Calmette Guérin (BCG) is the only vaccine against tuberculosis (TB) currently available and exhibits various levels of efficacy for the prevention of pulmonary TB (range, 0 to 80%) in different trials (9). BCG has a protective effect in children, particularly against tuberculous meningitis; however, it does not satisfactorily prevent the development of pulmonary TB in adults and fails to protect individuals against reinfection (1). Given the rate of mortality from TB worldwide, with more 8 million new cases and 2 million deaths occurring annually (2), newer strategies need to be implemented to improve BCG or vaccines more effective than BCG urgently need to be developed.One approach that might be used to increase the efficacy of BCG could be to construct a recombinant BCG (rBCG) which either overexpresses immunogenic antigens or modulates the ensuing immune response (8). rBCG vaccines are attractive because of the widespread experience with their use, the known immunogenicity associated with protection against the worst forms of the disease in children, and the safety profiles of standard BCG strains (13). Two rBCG vaccines have been entered into clinical trials. This includes rBCG30, which expresses the antigen 85B (Ag85B) protein, and ΔureC hly-positive rBCG, which expresses listeriolysin and which is urease deficient (12, 15). It is hoped that these vaccines will provide a strong and perhaps longer-lasting immune response than that achieved with the conventional BCG vaccine.The most effective defined-antigen TB vaccines will likely require the induction of both cell-mediated and humoral immune responses. Ag85B and the 6-kDa early secreted antigen target (ESAT-6) have been identified as two of the most promising vaccine candidates which are strongly recognized by T lymphocytes (3, 19). In a previous study, we relied on the T-cell epitopes of Ag85B and ESAT-6 to design a chimeric protein by inserting ESAT-6 into Ag85B from amino acids 167 to 182 and demonstrated that this recombination of Ag85B and ESAT-6 could improve the immunogenicity and enhance the T-helper type 1 (Th1) cell-mediated immune response (27). This finding prompted us to explore further the efficacy of rBCG overexpressing this chimeric protein. In this study, we constructed rBCG expressing chimeric protein Ag85B_N-ESAT-6-Ag85B_C (rBCG-A_N-E-A_C) and further compared the immune response to that protein with that to rBCG expressing the Ag85B-ESAT-6 fusion protein (rBCG-A-E) and BCG.     

Evaluation of a recombinant BCG expressing antigen Ag85B and PPE protein Rv3425 from DNA segment RD11 of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in C57BL/6 mice

Antigen 85B (Ag85B) is an important immunodominant antigen of Mycobacterium tuberculosis, and is a very promising vaccine candidate molecule. Rv3425 is a member of the subgroup 3 of the PPE family, which does not exist in all BCG strains. In this study we constructed a new rBCG which included this united gene (Ag85B-Rv3425). The level of antigen-stimulated T cells expressing IFN-γ was significantly higher in the C57BL/6 mice vaccinated with rBCG::Ag85B-Rv3425 than with BCG. In addition, the sera from mice immunized with rBCG::Ag85B-Rv3425 revealed an increase in the specific immunoglobulin G titers than that from mice immunized with BCG. Antigen specific IgG subclass analysis showed that rBCG::Ag85B-Rv3425 tended to facilitate IgG2a production, suggesting enhancement of predominant Th1 response which in turn may facilitate increased production of protective IFN-γ. These results suggested that this rBCG::Ag85B-Rv3425 could be a strong vaccine candidate for further study. Jiu ling Wang and Ya qing Qie are contributed equally to this study.     

Age-Dependent Changes in Isotype Expression and Down-Regulation of C57BL/6 Mice

Age-related changes in antibody response and tolerance inducibility are polymorphic; in this paper isotype changes in ageing C57BL/6 mice are examined. Female C57BL/6 mice of various ages were immunized with either heat-aggregated RGG (a-RGG) or phosphorylcholine conjugate of RGG (PC-RGG); other animals of the same ages were given aggregate free RGG, followed by injections with either aggregated RGG or haptenated RGG. Sera from these four groups were analysed for antibody isotype. The data presented here indicate that age-related changes in isotype predominance and magnitude are different for different determinants. The capacity to be down-regulated appeared to undergo different age-related changes with different isotypes: there is split tolerance in isotypes. Age-dependent changes in T- and B-cell tolerance could be deduced by comparing responses of animals to hapten and to carrier determinants. In 5-week-old animals tolerance to hapten was more profound than tolerance to carrier. It was concluded that T-cell regulation dominated the response at this age. With advancing age, i.e. by 95 weeks, tolerance is observed in response to hapten but not in response to carrier determinants. We concluded that suppressor cells were induced by aggregate free RGG and affected the response of 'naive' but not of 'experienced' B cells.     

Effect of Short Term Cocaine Administration On the Immune System of Yg and Old C57BL/6 Female Mice

Abstract

It has been shown that either cocaine or aging alone can alter the immune system. Our objective was to study if the immune system of aging mice was more susceptible to the effect of cocaine than the immune system of young mice. We used a short term (20 days) cocaine daily administration protocol. Cocaine only decreased the absolute number of Thy 1^±, CD4^±, CD8^±, IL-2R^±, Mac 1^± and B cells, in the spleen of old mice. Old untreated mice had a lower number of Thy 1^± cells in the thymus, and a higher number of cells expressing IL-2R. Cocaine decreased the number of Thy 1^± cells in the thymus of both age groups. Old mice showed a lower number of IgA^± plasma cells in the intestinal lamina propria (ILP) than young mice. Short term cocaine administration provoked a decrease in the number of CD4^± cells in young mice ILP and of CD8^± cells in old mice ILP. Our data suggest that cocaine can potentiate the effect of aging on the thymus and on the mucosal immune system. Taken together, our findings indicate that aging and cocaine can potentiate each other to impairing the host immune system.     

Mechanisms Involved in Age-dependent Decline of Immune Responsiveness and Apparent Resistance against Tolerance Induction in C57BL/6 Mice

The plaque-forming antibody response of C57BL/6 mice to rabbit gamma globulin (RGG) decreases as a function of ae. RGG in tolerogenic form induces tolerance of young mice but sensitizes older animals. If antigen is administered together with lipopolysaccharide, the age-dependent decline in immune responsiveness is not observed, nor are older animals sensitized by tolerogen. The age-dependent decline in immune responsiveness is due to a loss of T helper capacity; sensitization by tolerogen is attributable to a subpopulation of B cells which becomes sensitized by the tolerogen. Older animals, treated with tolerogen, show a degree of central tolerance of T cells and a relatively slight, age-dependent diminution in colchicine- or cyclophosphamide-sensitive precursors of suppressor cells.     

Differences in Measures of Exploration and Fear in MHC-Congenic C57BL/6J and B6-H-2K Mice

MHC-congenic mice prefer to mate with mice of different MHC types and are able to discriminate between MHC-congenic mice by their urine odors, but nothing is known about behavioral differences among MHC-congenic mice. The present experiments examine the strain and sex differences in MHC-congenic C57BL/6J and B6-H-2K mice with respect to exploration and fear motivated behaviors in the elevated plus maze, the elevated zero maze, and the open field. In the elevated plus maze and elevated zero maze, C57BL/6J mice spent more time in the open areas than B6-H-2K mice, suggesting that they were less fearful and more exploratory. No sex differences for exploration were found but male mice defecated more than females in the elevated plus maze. In the open field there were no significant strain or sex differences in measures of exploration or fear. There were no strain differences in the investigation of a novel object placed into the open field, but males investigated the novel object more than females. These results indicate that there are differences in exploratory and fear behavior in MHC-congenic mice that may contribute to mate preferences and that behavioral differences in these mice might be further examined advantageously.     

Prime-Boost Immunization of Codon Optimized HIV-1 CRF01_AE Gag in BCG with Recombinant Vaccinia Virus Elicits MHC Class I and II Immune Responses in Mice

The HIV-1 CRF01_AE gag gene was modified by codon restriction for Mycobacterium spp. and transformed into BCG; and it was designated as rBCG/codon optimized gagE. This produced 11 fold higher HIV-1 gag protein expression than the recombinant native gene rBCG/HIV-1gagE. In mice, CTL activity could be induced either by a single immunization of the codon optimized construct or by using it as a priming antigen in the prime-boost modality with recombinant Vaccinia virus expressing native HIV-1 gag. Specific secreted cytokine responses were also investigated. Only when rBCG gag was codon optimized did the prime-boost immunization produce significantly enhanced IFN-γ and IL-2 secretion indicating recognition via CD4+ and CD8+ T cells, and these responses seemed to be codon optimized immunogen dose-responsive. On contrary, the prime-boost vaccination using an equal amount of native rBCG/HIV-1gagE instead, or a single rBCG/codon optimized gagE immunization, had no similar effect on the cytokine secretion. These findings suggest that the use of recombinant codon BCG construct with recombinant Vaccinia virus encoding CRF01_AE gag as the prime-boost HIV vaccine candidate, will induce CD4+ Th1 and CD8+ T cell cytokine secretions in addition to enhancing CD8+ CTL response.     

Immune response induced by three Mycobacterium bovis BCG substrains with diverse regions of deletion in a C57BL/6 mouse model

This study was performed to examine the adaptive immune response generated by three Mycobacterium bovis bacillus Calmette-Guérin (BCG) substrains to determine if the number of genomic regions of deletion played a significant role in determining the magnitude of the immune response or affected their ability to reduce the bacterial burden following low-dose aerosol challenge with a virulent M. tuberculosis strain. BCG Connaught, Pasteur, and Sweden were chosen as representative substrains, as they possessed many, intermediate, and few regions of deletion, respectively, as a result of changes in the genome in various regions. Mice were vaccinated subcutaneously and were then examined at 14, 21, and 42 days postvaccination. BCG was observed in the spleen, lung, and lymph nodes. BCG Connaught induced a greater pulmonary T-cell response than the other two substrains at day 14 postvaccination, although by 42 days postvaccination activated T-cell levels dropped to the levels observed in control mice for all three substrains. Among the three substrains, BCG Connaught induced significantly greater levels of interleukin-12 in bone marrow-derived macrophage cultures. Mice challenged at days 14, 21, and 42 postvaccination displayed an equal capacity to reduce the bacterial burden in the lungs and spleen. The data provide evidence that although the BCG substrains generated qualitatively and quantitatively different immune responses, they induced similar reductions in the bacterial burden against challenge with a virulent M. tuberculosis strain in the mouse model of tuberculosis. The data raise questions about the assessment of vaccine immune responses and the relationship to a vaccine's ability to reduce the bacterial burden.     

Evaluation of Acute Immunotoxicity of Aerosolized Aflatoxin B(1) in Female C57BL/6N Mice

There is evidence for immunotoxicity of aflatoxin B1 (AFB(1)) in chronic animal feeding studies; however, little information is available as to the effects of inhalation exposure. This study evaluated the acute affects of aerosolized AFB(1) on systemic immune function of female C57BL/6N mice following a single aerosol exposure. Mice were exposed in nose-only inhalation tubes to 0, 2.86, 6.59 and 10 mug AFB(1) aerosol/L air for 90 minutes. A negative control group of untreated mice and a positive control group of cyclophosphamide-treated mice were included to account for day to day variation. Three days following exposure, mice were sacrificed and body, liver, lung, thymus and spleen weights, and complete blood counts and white blood cell differentials were measured. Splenocytes were isolated for flow cytometric analysis of CD4(+) and CD8(+) lymphocytes, CD19(+) B-cells and natural killer cells (NK 1.1(+)). The effect of AFB(1) on humoral immunity was assessed by measuring serum anti-keyhole limpet hemocyanin (KLH) IgM levels. Of the tissues examined, only the thymus weight of AFB(1) exposed mice decreased significantly compared to naive mice; however, the decrease was not dose related and was also observed in the 0 AFB(1) aerosol control group. A decrease in the mean white blood cell count of treated vs. naive mice was observed at all dose levels but was clearly not dose related and was statistically significant only in the 0 and 2.86 mug/L groups. Red blood cell and platelet counts and white blood cell differentials were not significantly affected by AFB(1). The number of CD4(+) (helper T-cells), CD8(+) (cytotoxic T-cells) and CD19(+) (B-cells) decreased in spleens of AFB(1) aerosol exposed mice compared to naive mice; however, the decrease was not dose-related and was also observed in the 0 AFB(1) exposure group. Dose-related changes in the CD4(+)/CD8(+) T-lymphocyte ratios were not observed. The IgM response to KLH was not significantly different in AFB(1) compared to naive mice, suggesting that AFB(1) did not effect antigen-specific antibody production. Based on the results of this study, a single AFB(1) inhalation exposure up to 10 mug/L for 90 minutes (CxT = 900 mug .min/L) did not significantly alter the immune parameters measured in this study. The aerosol vehicle (ethanol) and/or stress could have masked subtle AFB(1)-dependent changes in thymus and spleen weights, and in splenic lymphocyte subpopulations. However, for other immunological parameters, such as the IgM response to KLH, there was clearly no significant effect of AFB(1) aerosol exposure.     

The Influence of T Cells on the Immunoglobulin Repertoire and the Affinity Maturation of the Immune Response Against Dextran B512 in C57BL/6 Mice

A collection of hybridomas from C57BL/6 mice producing antibodies to dextran B512 was analysed and found to reflect the immune response in vivo with regard to immunoglobulin class expression, T cell dependency and antibody affinity. IgM-, IgG-3, and IgG-2b, and IgA-producing hybridomas were found. IgG3-producing hybridomas were obtained from nude mice, indicating T cell independent IgG3 synthesis. All monoclonal antibodies were of kappa light chain. A major anti-dextran idiotype was expressed in many monoclonals. Secondary immune responses to dextran were also suppressed at the hybridoma cell level. However, hybridomas from secondary responses produced antibodies expressing the major idiotype, suggesting that anti-idiotype mediated suppression was not responsible for the reduced secondary response. Most monoclonals belonged to the VHJ558 family, but the IgG3-producing hybridomas showed a preferential use of genes from the VHX24 family. All monoclonals were directed against internal structures of the dextran molecule. The affinity for dextran of the IgG antibodies produced in secondary immune responses was drastically increased, even when the mice were immunized with thymus-independent forms of dextran, indicating that T helper cells need not be involved in affinity maturation of the immune response.     

Vaccinated C57BL/6 Mice Develop Protective and Memory T Cell Responses to Coccidioides posadasii Infection in the Absence of Interleukin-10

High concentrations of lung tissue-associated interleukin-10 (IL-10), an anti-inflammatory and immunosuppressive cytokine, correlate with susceptibility of mice to Coccidioides spp. infection. In this study, we found that macrophages, dendritic cells, neutrophils, and both CD8⁺ and CD4⁺ T cells recruited to Coccidioides posadasii-infected lungs of nonvaccinated and vaccinated mice contributed to the production of IL-10. The major IL-10-producing leukocytes were CD8⁺ T cells, neutrophils, and macrophages in lungs of nonvaccinated mice, while both Foxp3⁺ and Foxp3⁻ subsets of IL-10⁺ CD4⁺ T cells were significantly elevated in vaccinated mice. Profiles of the recruited leukocytes in lungs revealed that only CD4⁺ T cells were significantly increased in IL-10^−/− knockout mice compared to their wild-type counterparts. Furthermore, ex vivo recall assays showed that CD4⁺ T cells isolated from vaccinated IL-10^−/− mice compared to vaccinated wild-type mice produced significantly higher amounts of IL-2, gamma interferon (IFN-γ), IL-4, IL-6, and IL-17A in the presence of a coccidioidal antigen, indicating that IL-10 suppresses Th1, Th2, and Th17 immunity to Coccidioides infection. Analysis of absolute numbers of CD44⁺ CD62L⁻ CD4⁺ T effector memory T cells (T_EM) and IFN-γ- and IL-17A-producing CD4⁺ T cells in the lungs of Coccidioides-infected mice correlated with better fungal clearance in nonvaccinated IL-10^−/− mice than in nonvaccinated wild-type mice. Our results suggest that IL-10 suppresses CD4⁺ T-cell immunity in nonvaccinated mice during Coccidioides infection but does not impede the development of a memory response nor exacerbate immunopathology of vaccinated mice over at least a 4-month period after the last immunization.