No abstract

Infection of mice with Salmonella typhimurium gives rise to a disease similar to human typhoid fever caused by S. typhi. Since S. typhimurium is a facultative intracellular bacterium, the requirement of B cells in the immune response against S. typhimurium is a longstanding matter of debate. By infecting mice on a susceptible background and deficient in B cells (Igμ−/− mice) with different strains of S. typhimurium, we could for the first time formally clarify the role of B cells in the response against S. typhimurium. Compared with Igμ+/+ mice, LD50 values in Igμ−/− mice were reduced during primary, and particularly secondary, oral infection with virulent S. typhimurium. After systemic infection, Igμ−/− mice cleared attenuated aroA− S. typhimurium, but vaccine-induced protection against systemic infection with virulent S. typhimurium involved both B cell-dependent and -independent effector mechanisms. Thus, B cell-mediated immunity plays a distinct role in control of S. typhimurium in susceptible mice.

CD4+ T cell help is important for the generation of CD8+ T cell responses. We used depleting anti-CD4 mAb to analyze the role of CD4+ T cells for memory CD8+ T cell responses after secondary infection of mice with the intracellular bacterium Listeria monocytogenes, or after boost immunization by specific peptide or DNA vaccination. Surprisingly, anti-CD4 mAb treatment during secondary CD8+ T cell responses markedly enlarged the population size of antigen-specific CD8+ T cells. After boost immunization with peptide or DNA, this effect was particularly profound, and antigen-specific CD8+ T cell populations were enlarged at least 10-fold. In terms of cytokine production and cytotoxicity, the enlarged CD8+ T cell population consisted of functional effector T cells. In depletion and transfer experiments, the suppressive function could be ascribed to CD4+CD25+ T cells. Our results demonstrate that CD4+ T cells control the CD8+ T cell response in two directions. Initially, they promote the generation of a CD8+ T cell responses and later they restrain the strength of the CD8+ T cell memory response. Down-modulation of CD8+ T cell responses during infection could prevent harmful consequences after eradication of the pathogen.

Objective Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits DOX therapy. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) appear to be involved in DOX cardiotoxicity. Ca/calmodulin dependent protein kinase II (CaMKII) can be activated by ROS through oxidation and is known to contribute to myocardial dysfunction through Ca leakage from the sarcoplasmic reticulum (SR). Rationale We hypothesized that CaMKII contributes to DOX-induced defects in intracellular Ca ([Ca]i) handling. Methods Cardiac myocytes were isolated from wild-type (WT) adult rat hearts and from mouse hearts lacking the predominant myocardial CaMKII isoform (CaMKIIδ−/−, KO) vs. WT. Isolated cardiomyocytes were investigated 30 min after DOX (10 µmol/L) superfusion, using epifluorescence and confocal microscopy. Intracellular ROS-generation ([ROS]i) and [Ca]i handling properties were assessed. In a subset of experiments, KN-93 or AIP (each 1 µmol/L) were used to inhibit CaMKII. Melatonin (Mel, 100 µmol/L) served as ROS-scavenger. Western blots were performed to determine the amount of CaMKII phosphorylation and oxidation. Results DOX increased [ROS]i and led to significant diastolic [Ca]i overload in rat myocytes. This was associated with reduced [Ca]i transients, a 5.8-fold increased diastolic SR Ca leak and diminished SR Ca content. ROS-scavenging partially rescued Ca handling. Western blots revealed increased CaMKII phosphorylation, but not CaMKII oxidation after DOX. Pharmacological CaMKII inhibition attenuated diastolic [Ca]i overload after DOX superfusion and led to partially restored [Ca]i transients and SR Ca content, presumably due to reduced Ca spark frequency. In line with this concept, isoform-specific CaMKIIδ-KO attenuated diastolic [Ca]i overload and Ca spark frequency. Conclusions DOX exposure induces CaMKII-dependent SR Ca leakage, which partially contributes to impaired cellular [Ca]i homeostasis. Pharmacological and genetic CaMKII inhibition attenuated but did not completely abolish the effects of DOX on [Ca]i. In light of the clinical relevance of DOX, further investigations seem appropriate to determine if CaMKII inhibition could reduce DOX-induced cardiotoxicity.

Infection of mice with Salmonella enterica serotype Typhimurium induces a strong Th1 cell response that is central for the control of infection. We infected mice of a resistant background with a virulent strain of S. enterica serovar Typhimurium and analyzed the kinetics and magnitude of the T-cell response. After infection, the majority of CD4 ؉ and CD8 ؉ splenocytes acquired an activated phenotype, as indicated by expression levels of CD44 and CD62L. In addition, after 3 to 4 weeks of infection, more than 20% of the CD4 ؉ and more than 30% of the CD8 ؉ T cells produced gamma interferon (IFN-␥) in response to short-term polyclonal stimulation. In contrast, we detected only a moderate (two-to threefold) expansion of both T-cell populations, and BrdU incorporation revealed that there was either no or only a limited increase in the in vivo proliferation of CD4؉ and CD8 ؉ T cells, respectively. Our results indicate that although an unexpectedly large population of both CD4؉ and CD8 ؉ T cells is activated and acquires the potential to secrete IFN-␥, this activation is not paralleled by substantial expansion of these T-cell populations.

Infection of mice with the intracellular bacterium Listeria monocytogenes results in a strong CD8+ T cell response that is critical for efficient control of infection. We used CD28-deficient mice to characterize the function of CD28 during Listeria infection, with a main emphasis on Listeria-specific CD8+ T cells. Frequencies and effector functions of these T cells were determined using MHC class I tetramers, single cell IFN-γ production and Listeria-specific cytotoxicity. During primary Listeria infection of CD28−/− mice we observed significantly reduced numbers of Listeria-specific CD8+ T cells and only marginal levels of specific IFN-γ production and cytotoxicity. Although frequencies were also reduced in CD28−/− mice during secondary response, we detected a considerable population of Listeria-specific CD8+ T cells in these mice. In parallel, IFN-γ production and cytotoxicity were observed, revealing that Listeria-specific CD8+ T cells in CD28−/− mice expressed normal effector functions. Consistent with their impaired CD8+ T cell activation, CD28−/− mice suffered from exacerbated listeriosis both after primary and secondary infection. These results demonstrate participation of CD28 signaling in the generation and expansion of Ag-specific CD8+ T cells in listeriosis. However, Ag-specific CD8+ T cells generated in the absence of CD28 differentiated into normal effector and memory T cells.

The immune response against the intracellular bacterium Listeria monocytogenes involves both CD4+ and CD8+ T cells. We used the MHC class II-presented peptide listeriolysin189–201 to characterize the organ-specific CD4+ T cell response during infection. Systemic listeriosis resulted in a strong peptide-specific CD4+ T cell response with frequencies of 1/100 and 1/30 CD4+ splenocytes at the peak of primary and secondary response, respectively. This response was not restricted to lymphoid organs, because we detected specific CD4+ T cells in all tissues analyzed. However, the tissue distribution of the T cell response was dependent on the route of infection. After i.v. infection, the strongest CD4+ T cell response and the highest levels of memory cells were observed in spleen and liver, the major sites of L. monocytogenes replication. After oral infection, we detected a strong response in the liver, the lamina propria, and the intestinal epithelium. These tissues also harbored the highest frequencies of listeriolysin189–201-specific CD4+ memory T cells 5–8 wk post oral infection. Our results show that kinetics and magnitude of the CD4+ T cell response and the accumulation of CD4+ memory T cells depend on the route of infection and are regulated in a tissue-specific way.

Effective priming of T cell responses depends on cognate interactions between naive T cells and professional antigen-presenting cells (APCs). This contact is the result of highly coordinated migration processes, in which the chemokine receptor CCR7 and its ligands, CCL19 and CCL21, play a central role. We used the murine Listeria monocytogenes infection model to characterize the role of the CCR7/CCR7 ligand system in the generation of T cell responses during bacterial infection. We demonstrate that efficient priming of naive major histocompatibility complex (MHC) class Ia–restricted CD8+ T cells requires CCR7. In contrast, MHC class Ib–restricted CD8+ T cells and MHC class II–restricted CD4+ T cells seem to be less dependent on CCR7; memory T cell responses are independent of CCR7. Infection experiments with bone marrow chimeras or mice reconstituted with purified T cell populations indicate that CCR7 has to be expressed on CD8+ T cells and professional APCs to promote efficient MHC class Ia–restricted T cell priming. Thus, different T cell subtypes and maturation stages have discrete requirements for CCR7.