Persistent Ehrlichia chaffeensis infection occurs in the absence of functional major histocompatibility complex class II genes.

Human monocytic ehrlichiosis is an emerging tick-borne disease caused by the rickettsia Ehrlichia chaffeensis. We investigated the impact of two genes that control macrophage and T-cell function on murine resistance to E. chaffeensis. Congenic pairs of wild-type and toll-like receptor 4 (tlr4)- or major histocompatibility complex class II (MHC-II)-deficient mice were used for these studies. Wild-type mice cleared the infection within 2 weeks, and the response included macrophage activation and the synthesis of E. chaffeensis-specific Th1-type immunoglobulin G response. The absence of a functional tlr4 gene depressed nitric oxide and interleukin 6 secretion by macrophages and resulted in short-term persistent infections for > or =30 days. In the absence of MHC-II alleles, E. chaffeensis infections persisted throughout the entire 3-month evaluation period. Together, these data suggest that macrophage activation and cell-mediated immunity, orchestrated by CD4(+) T cells, are critical for conferring resistance to E. chaffeensis.     

Increased host resistance against Pneumocystis carinii pneumonia in gammadelta T-cell-deficient mice: protective role of gamma interferon and CD8(+) T cells

Although a clear relationship between alphabeta T-cell receptor-positive (alphabeta-TCR(+)) CD4(+) T cells and susceptibility to Pneumocystis carinii infection exists, the role of other T-cell subsets is less clearly defined. Previous studies have shown that gammadelta-TCR(+) T cells infiltrate into the lung during P. carinii pneumonia. Therefore, the present study examined the role of gammadelta-TCR(+) T cells in host defense against P. carinii pneumonia. C57BL/6 (control) and B6.129P2-Tcrd(tm1Mom) (gammadelta-TCR(+) T-cell-deficient) mice were inoculated intratracheally with P. carinii. At specific time points, mice were sacrificed and analyzed for P. carinii burden, T-cell subsets, and cytokine levels in lung tissue. Analysis of P. carinii burden showed a more rapid and complete resolution of infection in gammadelta-TCR(+) T-cell-deficient mice than in C57BL/6 controls. This augmented resolution was associated with elevated gamma interferon (IFN-gamma) levels in bronchoalveolar lavage fluid predominantly produced by CD8(+) T cells, as well as an increased recruitment of CD8(+) T cells in general. In separate experiments, neutralization of IFN-gamma or depletion of CD8(+) T cells early during infection abolished the augmented resolution previously observed in gammadelta-TCR(+) T-cell-deficient mice. These results show that the presence of gammadelta-TCR(+) T cells modulates host susceptibility to P. carinii pneumonia through interactions with pulmonary CD8(+) T cells and tissue production of IFN-gamma.     

Role for T cell-independent B cell activity in the resolution of primary rotavirus infection in mice.

We examined the importance of T cell-independent B cell activity in the resolution of primary murine (EDIM) rotavirus infection in adult mice. We showed that Rag 1 (C57BL / 6 background) and Rag 2 (BALB / c background) knockout mice, which lack both T and B cells, chronically shed high levels of rotavirus Ag in stool samples following oral inoculation. However, nude mice (BALB / c and C57BL / 6 backgrounds) and alpha beta TCR knockout mice (C57BL / 6 background) chronically shed 100-fold lower levels of virus in stool samples. Thus, B cells appeared to sharply reduce the level of chronic rotavirus shedding by a T cell-independent mechanism. C57BL / 6 mice depleted of CD4(+) cells or both CD4(+) and CD8(+) cells were also unable to resolve primary rotavirus infection but chronically shed equally low levels of rotavirus Ag in stool samples, whereas mice depleted of only CD8(+) cells resolved infection. Similar results were obtained with a second rotavirus strain (EC(w)) in which virus was shed chronically in stool samples at low levels in alpha beta TCR knockout mice and at high levels in Rag 1 knockout mice. Virus-specific intestinal IgA was readily detected in mice lacking thymic T cells and alpha beta T cells and in mice depleted of CD4(+) cells but levels were 95 % reduced in comparison to immunocompetent control mice. Together, these results show that B cells lacking CD4(+) T cell help have the capacity to substantially reduce rotavirus shedding, possibly through the production of T cell-independent IgA to rotavirus, but full resolution requires alpha beta T cells.     

Minor genomic differences between related B6 and B10 mice affect severity of schistosome infection by governing the mode of dendritic cell activation

Infection with the helminth Schistosoma mansoni results in hepatointestinal granulomatous inflammation mediated by CD4 T cells directed against parasite eggs. The severity of disease varies greatly in humans and mice; however, the genetic basis of such a heterogenous immune response remains poorly understood.   Here we show that, despite their close genetic relationship, C57BL/10SnJ (B10) mice developed significantly more pronounced immunopathology and higher T helper 17 cell responses than C57BL/6J (B6) mice. Similarly, live egg‐stimulated B10‐derived dendritic cells (DCs) produced significantly more IL‐1β and IL‐23, resulting in higher IL‐17 production by CD4 T cells.  Gene expression analysis disclosed a heightened proinflammatory cytokine profile together with a strikingly lower expression of Ym1 in B10 versus B6 mice, consistent with failure of B10 DCs to attain alternative activation. To genetically dissect the differential response, we developed and analyzed congenic mouse strains that capture major regions of allelic variation, and found that the level of inflammation was controlled by a relatively small number of genes in a locus mapping to chromosome 4 117–143 MB. Our study has thus identified novel genomic regions that regulate the severity of the schistosome infection by way of controlling the mode of DC activation and consequent CD4 T‐cell subset development.     

Host defense against Mycobacterium avium does not have an absolute requirement for major histocompatibility complex class I-restricted T cells.

The role of CD8(+) T cells was evaluated in a mouse model of disseminated Mycobacterium avium infection. C57BL/6J and C57BL/6Jbeta2-/- (beta2-/-) mice were infected intravenously, and the number of viable bacteria in each liver and spleen was determined. No significant difference between the number of bacteria in the two strains of mice was observed at 2, 4, 6, and 8 weeks after infection. Histopathological examination of granulomas from C57BL/6J and beta2-/- mice did not show any difference either in the number of organisms per granuloma or in the size of the granulomas. Investigation of the cytokine profile in the spleen demonstrated that the beta2-/- strain of mice produced a significantly lower amount of gamma interferon at 8 weeks after infection and significantly increased concentrations of tumor necrosis factor alpha compared with that from the wild-type mouse. Interleukin-12 and transforming growth factor beta1 levels did not differ between the two strains of mice at 2, 4, 6, and 8 weeks. Although previous work had found that host response against Mycobacterium tuberculosis involves major histocompatibility complex class I-restricted T cells, our results indicate that chronic deficiency of CD8(+) T cells does not lead to a different expression of the disease and that if CD8(+) T cells are involved in the host response, their function can be assumed by other immune cells.     

Angiostrongylus costaricensis infection in C57BL/6 mice: MHC-II deficiency results in increased larval elimination but unaltered mortality

During experimental Angiostrongylus costaricensis infections in several inbred mouse strains, genetic factors as well as different cytokine secretion patterns have recently been shown to play a role in the outcome of infection in terms of morbidity and mortality, e.g. BALB/c mice show a high and C57BL/6 mice a low mortality during the acute phase of infection. In this study, C57BL/6 MHC-II knockout mice infected with A. costaricensis did not show increased mortality during the acute phase of infection when compared with wild-type mice. Furthermore, MHC-II knockout mice showed a strongly diminished parasite-specific humoral and cellular immune response, which can be explained by the nearly complete lack of CD4+ T cells in the periphery. This defect in MHC-II genes, the lack of CD4+ T cells, and the resulting cellular and humoral unresponsiveness resulted in a three times higher output of first-stage larvae in feces compared with wild-type animals. The results indicate that during experimental A. costaricensis infection a parasite-specific immune response, directed via MHC-II molecules and CD4+ T cells, is not essential for the survival of C57BL/6 mice during the acute phase of infection, whereas the elimination of first-stage larvae seems to be regulated by a MHC-II- and CD4+ T-cell-dependent mechanism.     

Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance.

Previous studies have shown that the capacity to clear an attenuated strain of Salmonella typhimurium after the second week of infection varies widely among mouse strains. Bacterial clearance is mediated by CD4+ T cells and is regulated in part by the H-2 complex. The aim of the present study was to compare the patterns of cytokine mRNA expression in the spleens of C57BL/6 (H-2b) and CBA (H-2k) mice, which exhibit a low and a high rate of bacterial clearance, respectively. A transient increase in interleukin-12 (IL-12) mRNA levels was found in both mouse strains. Gamma interferon (IFN-gamma) gene expression was higher and more sustained in C57BL/6 than in CBA mice. No increase in IL-4 mRNA was detected. A transient increase in IL-10 mRNA was found in C57BL/6 mice. Separation of spleen cells into CD4+ and CD4- fractions showed that CD4+ T cells produced the bulk of IFN-gamma in both mouse strains and of IL-10 in C57BL/6 mice. Infection of H-2 congenic mice induced a higher level of IFN-gamma mRNA expression by CD4+ T cells in mice with a low rate of clearance (H-2b) than in mice with a high rate of clearance (H-2q). Treatment of infected C57BL/6 mice with anti-IFN-gamma or anti-CD4 monoclonal antibodies indicated that IFN-gamma participates in resistance in the early phase of infection, but not in bacterial clearance, and that CD4+ T cells mediate bacterial clearance during the 3rd week of infection. Taken together, these results suggest that defective bacterial clearance in H-2b mice is not linked to defective IFN-gamma production and that CD4+ T cells mediate bacterial clearance by an IFN-gamma-independent mechanism.     

Completely allogeneic spleen cells induced cytolytic neonatal tolerance to alloantigens,but failed to establish allo-helper interactions with host T cells.

The injection of spleen cells from F1 mice into-newborns from a parental strain results in the establishment of cytolytic tolerance to donor alloantigens and the development of a lupus-like disease. This syndrome is the consequence of the recognition by alloreactive host CD4+ T cells of discordant major histocompatibility complex (MHC) class II antigens on semi-allogeneic donor B cells. We have analysed whether completely allogeneic spleen cells are as able as semi-allogeneic spleen cells to induce cytolytic tolerance to donor alloantigens and to co-operate with alloreactive T cells for autoantibody production. BALB/c mice were injected at birth with Thy-1-depleted spleen cells from (C57BL/6 x BALB/c)F1 or C57BL/6 mice, either alone or in combination. Cytolytic tolerance was always induced, as manifested by persistence of chimerism and acceptance of skin allografts. However, only F1 semi-allogeneic B cells were activated by alloreactive host T cells to produce anti-DNA IgG antibody. The deficient co-operation between BALB/c CD4+ T cells and completely allogeneic C57BL/6 B cells was confirmed after neonatal injection of (C57BL/ 6 x BALB/c)F1(Igha) spleen cells together with C57BL/6(Ighb) spleen cells. These mice developed anti-DNA antibodies bearing only the Igha allotype. Similar results were observed in experiments of allogeneic interaction in vitro, in which BALB/c CD4+ T cells were cocultured with either (C57BL/6 x BALB/c)F1 or C57BL/6 B cells. The present results demonstrate that completely allogeneic spleen cells efficiently induced cytolytic unresponsiveness to donor alloantigens, but B cells contained in this spleen cell population were unable to establish allo-helper interactions with alloreactive CD4+ T cells, suggesting that cytolytic and helper T-cell interactions involved in alloreactivity may be different.     

Distinct and synergistic roles of FcγRIIB deficiency and 129 strain-derived SLAM family proteins in the development of spontaneous germinal centers and autoimmunity

The inhibitory IgG Fc receptor (FcγRIIB) deficiency and 129 strain-derived signaling lymphocyte activation molecules (129-SLAMs) are proposed to contribute to the lupus phenotype in FcγRIIB-deficient mice generated using 129 ES cells and backcrossed to C57BL/6 mice (B6.129.RIIBKO). In this study, we examine the individual contributions and the cellular mechanisms by which FcγRIIB deficiency and 129-derived SLAM family genes promote dysregulated spontaneous germinal center (Spt-GC) B cell and follicular helper T cell (Tfh) responses in B6.129.RIIBKO mice. We find that B6 mice congenic for the 129-derived SLAM locus (B6.129-SLAM) and B6 mice deficient in FcγRIIB (B6.RIIBKO) have increased Spt-GC B cell responses compared to B6 controls but significantly lower than B6.129.RIIBKO mice. These data indicate that both FcγRIIB deficiency and 129-SLAMs contribute to elevated Spt-GC B cell responses in B6.129.RIIBKO mice. However, only 129-SLAMs contribute significantly to augmented Tfh responses in B6.129.RIIBKO mice, and do so by a combination of T cell-dependent effects and enhanced B cell and DC-dependent antigen presentation to T cells. Elevated Spt-GC B cell responses in mice with FcγRIIB deficiency and polymorphic 129-SLAMs were associated with elevated metabolic activity, improved GC B cell survival and increased differentiation of naïve B cells into GC B cell phenotype. Our data suggest that the interplay between 129-SLAM expression on B cells, T cells and DCs is central to the alteration of the GC tolerance checkpoint, and that deficiency of FcγRIIB on B cells is necessary to augment Spt-GC responses, pathogenic autoantibodies, and lupus disease.     

Effects of in vivo T lymphocyte subset depletion on mycobacterial infections in mice.

The relative importance of CD4+ and CD8+ T cell subsets in the expression of acquired resistance to systemic infection by Mycobacterium kansasii was determined. T cell subsets were depleted in thymectomized C57BL/6 mice by the intravenous administration of monoclonal antibodies directed against the relevant T cell determinants. Depletion of the CD4+ subset exacerbated the severity of the infection in intravenously challenged mice. This effect was apparent in the first 2 weeks of the infection and persisted throughout the 12 weeks of the study. On the other hand, depletion of the CD8+ cells had no apparent effect on the growth curves. Infections by Mycobacterium tuberculosis Erdman or bacille Calmette-Guérin (BCG) Pasteur were also substantially enhanced by CD4 depletion, but not by the depletion of CD8+ cells. The effect of subset depletion on infections by M. tuberculosis and BCG was examined in both innately susceptible C57BL/6 mice and innately resistant B6D2 mice.     

Strain differences in mouse cellular responses to Mycobacterium lepraemurium and BCG subcutaneous infections. I. Analysis of cell surface phenotype in local granulomas.

C57BL/6, BALB/c and CBA mice were subcutaneously infected with either Mycobacterium lepraemurium (MLM) or BCG, and studied for bacillary growth, granuloma size of infected footpads and draining lymph nodes (DLN), and DLN cell surface phenotype. Whereas, BCG-infected mice controlled the infection and developed early and large granulomas, MLM-infected mice exhibited major strain variations in their resistance to the infection, as well as in the granuloma size and kinetics. C57BL/6 mice, highly resistant, displayed early and regressive granulomas; BALB/c mice showed lower resistance and early granulomas that grew continuously; CBA mice, highly susceptible, developed late, soft, phagocyte-rich granulomas. Important strain differences in lymph node lymphocyte subset distribution could be observed prior to any infection: C57BL/6 mice displayed higher B cell percentages than both of the other strains and BALB/c mice showed the highest CD4/CD8 ratios, followed by CBA and C57BL/6 mice. BCG and MLM infections both induced similar changes of these parameters in all three strains: that is a decrease of the B cell percentage and a decrease of the CD4/CD8 ratio, and the strain differences observed in uninfected mice persisted. On the other hand, DLN cells stimulated by the infecting bacillus and interleukin 2 also displayed an increase of the CD8 T cell percentage as compared with normal lymph node cells, but this phenomenon was much less pronounced in BALB/c mice, whether infected by MLM or BCG, and in MLM-infected CBA mice, than in BCG- or MLM-infected C57BL/6 (B6) mice. Thus the ability of C57BL/6 mice to generate an early and persistent CD8 T cell response to mycobacteria may contribute to their resistance to MLM.     

Psoriasiform dermatitis susceptibility in Itgb2(tm1Bay) PL/J mice requires low-level CD18 expression and at least two additional loci for progression to severe disease

Itgb2(tm1Bay) PL/J mice express low levels of the beta(2) integrins and, unlike Itgb2(tm1Bay) C57BL/6J mice, spontaneously develop psoriasiform dermatitis with several similarities to human psoriasis. To define the genetic requirements for skin disease susceptibility we analyzed more than 500 F2 progeny from an Itgb2(tm1Bay) (PL/J x C57BL/6J) intercross. We found that 23.5% developed chronic inflammatory skin disease, although significant differences in severity were observed. Another CD18 mutation, Itgb2(tm2Bay), has now been generated that completely eliminates CD18 expression. Surprisingly, of 10 Itgb2(tm2Bay) homozygote PL/J N4 mice generated, none showed clinical or histopathological evidence of disease. However, Itgb2(tm1Bay)/Itgb2(tm2Bay) PL/J mice developed dermatitis indistinguishable from Itgb2(tm1Bay) PL/J mice. In addition, approximately half of Itgb2(tm1Bay)/Itgb2(tm2Bay) (C57BL/6J x PL/J)F1 mice were found to develop mild psoriasiform dermatitis identical to the early stages of disease seen in Itgb2(tm1Bay) PL/J mice. Collectively, these results suggest a complex inheritance pattern of psoriasiform dermatitis in this model that involves lowered, but not absent, CD18 expression and at least two additional PL/J loci for the development of severe disease. The susceptibility allele can act in either a heterozygous or homozygous state, dependent on the level of CD18 expression.     

Heme oxygenase-1 attenuates ovalbumin-induced airway inflammation by up-regulation of foxp3 T-regulatory cells, interleukin-10, and membrane-bound transforming growth factor- 1

Cumulative evidence suggests the up-regulation of interleukin (IL)-10 and T-regulatory (Treg) cells is implicated in anti-inflammatory effect of heme oxygenase-1 (HO-1). Thus, we postulated that induction of HO-1 could augment IL-10 and transforming growth factor (TGF)-beta production and foxp3+CD4+CD25+ Treg cell function, thereby leading to attenuation of airway inflammation. In this study, CD4+CD25+ Treg cells isolated from mouse spleen were either transfected with a HO-1 expression vector (pcDNA3HO-1) or treated with a HO-1 inducer (hemin). Up-regulation of HO-1 enhanced foxp3 expression and IL-10 secretion in the Treg cells in vitro. Next, BALB/c, C57/B6.129, and IL-10-deficient B6.129P2-Il10tm1Cgn/J mice were challenged by ovalbumin to induce airway inflammation. Consistent with in vitro findings, hemin treatment resulted in induction of HO-1 and foxp3 and production of IL-10 and membrane-bound TGF-beta1 in vivo. This was further correlated with decrease of ovalbumin-specific immunoglobulin E level and eosinophil infiltration in bronchial alveolar lavage fluid from the asthmatic mice. Furthermore, hemin significantly enhanced the biological activity of CD4+CD25+ Treg cells. This protective effect was specifically blocked by Sn-protoporphyrin, a HO-1 enzymatic inhibitor. Finally, hemin failed to up-regulate the function of CD4+CD25+ Treg cells from IL-10-deficient mice. Our study indicates that HO-1 exerts its protective effect on asthma through a mechanism mediated by foxp3+CD4+CD25+ Treg cells, IL-10, and membrane-bound TGF-beta1.     

CD4+ helper T cells are required for resistance to a highly metastatic murine tumor

A role of CD4+ T helper cells in induction of tumor transplant rejection leading to complete regression of a highly metastatic DBA/2 mouse lymphoma was analyzed. Using an anti-CD4 monoclonal antibody (GK1.5) which eliminates T helper cells in vivo and in vitro, we found that CD4+ cells are required for tumor resistance in syngeneic DBA/2 mice or allogeneic but major histocompatibility complex-identical B10.D2 mice. In contrast, in allogeneic C57BL/6 mice tumor rejection was independent of CD4+ cells. An analogous requirement for immune CD4+ cells for in vitro induction of CD8+ tumor-specific cytotoxic T cells was found in these respective strains. The requirement for immune CD4+ cells in vitro could be replaced by recombinant interleukin 2. These results demonstrate a role of CD4+ regulatory T cells and T-T cell cooperation in the induction of anti-tumor immunity and tumor rejection, and point to possible therapeutic interventions in the afferent phase of anti-tumor immune responses.     

Characterization of the CD8+ T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice.     

Mechanisms of immunity to Ehrlichia muris: a model of monocytotropic ehrlichiosis

Ehrlichia species can cause life-threatening infections or chronic persistent infections. Mechanisms of protective immunity were examined in an Ehrlichia muris mouse model of monocytotropic ehrlichiosis. C57BL/6 mice possessed strong genetic resistance to E. muris of an undetermined mechanism. CD8 T lymphocytes were particularly important, as revealed by 81% fatalities for E. muris-infected, major histocompatibility complex class I gene knockout mice compared with no deaths for wild-type C3H mice. Moreover, 80% of C3H mice depleted of CD8 and CD4 cells died of E. muris infection compared with only 44% of CD4 cell-depleted mice. CD8 T lymphocytes were demonstrated for the first time in an Ehrlichia infection to exhibit cytotoxic T-lymphocyte activity against Ehrlichia-infected target cells. Both gamma interferon and tumor necrosis factor were shown to play synergistic roles in protective immunity in vivo for the first time, as demonstrated by 75% fatalities when both cytokines were neutralized compared with minimal mortality when they were depleted separately. Passive transfer of antibodies, but not Fab fragments, to E. muris protected C3H/SCID mice against lethal infection. The mechanism of increased susceptibility (22% lethality) of C57BL/6 major histocompatibility complex class II gene knockout mice and CD4 cell-depleted C3H mice (i.e., through a gamma interferon or antibody mechanism), as well as the more important role of CD8 T lymphocytes (in the form of cytotoxic T-lymphocyte activity and/or gamma interferon production), remains to be elucidated. Protective immunity against monocytotropic E. muris is mediated by a combination of CD8 and CD4 T lymphocytes, gamma interferon, tumor necrosis factor alpha, and antibodies.     

Immunological factors that affect the in vivo fate of T7 phage in the mouse

Phage display is a powerful method to study organ and tissue specific addresses. As part of our studies on the in vivo panning of tissue-homing peptide libraries, we examined the survival of T7 phage in the blood of C57BL/6J mice to estimate the half-life of T7 phage and the factors responsible for its inactivation. Amplified and purified T7 phage particles with or without random peptide library inserts were injected intravenously into the tail vein of wild-type (C57BL/6J) and immunocompromized (C57BL/6J) female mice. In wild-type mice, both the parent phage as well as phage carrying a peptide library were eliminated quickly from the blood, with only approximately 1% survival of detectable infectious phage after 60 min of injection. In SCID (C57BL/6J-Prkdc(scid)) mice, phage titers were stable over the same period of time with or without peptide library, suggesting a role for either B- or T cells or both in phage inactivation. The presumed role of B cell was indicated by demonstration of stable phage in the B-cell deficient mouse (C57BL/10-Igh-6(tm1Cgn)). In other immunocompromized mice, the phage titers were unstable, similar to that found in wild-type mice. In no case, was there a difference between phage with or without random peptide library. These data indicate that the presence of random C-X7-C peptides on the T7 phage coat protein does not affect the clearance of the phage in murine blood. Most likely, host immune factors play a role in the neutralization of T7 phage in blood by reacting with B-cell dependent immunoglobin.