Anne Köhler

Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis

Mycobacterium bovis bacille Calmette–Guérin (BCG) is the most widely used live bacterial vaccine. However, limited information is available correlating route and dose of vaccination and induction of specific T cell responses with protection against tuberculosis. We compared efficacy of oral and systemic vaccination and correlated vaccine-induced T cell responses with protection in experimental tuberculosis of mice. After oral and systemic vaccination, we observed profound differences in persistence and dissemination of BCG and frequencies and location of specific IFN-γ-secreting CD4+ and CD8+ T cells. Yet, both vaccination routes caused comparable levels of protection against aerosol challenge with Mycobacterium tuberculosis. Protection correlated best with rapid accumulation of specific CD8+ T cells in infected tissues of challenged mice. In contrast, specific IFN-γ production by CD4+ T cells reflected the load of M. tuberculosis rather than the strength of protection. Our data question the measurement of IFN-γ secretion by CD4+ T cells and emphasize the need for new biomarkers for evaluation of tuberculosis vaccine efficacies.

Regulatory CD4+CD25+ T Cells Restrict Memory CD8+ T Cell Responses

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.

Cutting Edge: Role of B Lymphocytes in Protective Immunity Against Salmonella typhimurium Infection

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.

Characterization of the Murine T-Lymphocyte Response to Salmonella enterica Serovar Typhimurium Infection

ABSTRACT 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-γ) 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-γ, this activation is not paralleled by substantial expansion of these T-cell populations.

Role of CD28 for the Generation and Expansion of Antigen-Specific CD8+ T Lymphocytes During Infection with Listeria monocytogenes

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.

Organ-Specific CD4+ T Cell Response During Listeria monocytogenes Infection

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.

Inducible Costimulator Protein Controls the Protective T Cell Response Against Listeria monocytogenes

The inducible costimulator protein (ICOS) was recently identified as a costimulatory molecule for T cells. Here we analyze the role of ICOS for the acquired immune response of mice against the intracellular bacterium Listeria monocytogenes. During oral L. monocytogenes infection, low levels of ICOS expression were detected by extracellular and intracellular Ab staining of Listeria-specific CD4+ and CD8+ T cells. Blocking of ICOS signaling with a soluble ICOS-Ig fusion protein markedly impaired the Listeria-specific T cell responses. Compared with control mice, the ICOS-Ig treated mice generated significantly reduced numbers of Listeria-specific CD8+ T cells in spleen and liver, as determined by tetramer and intracellular cytokine staining. In contrast, the specific CD8+ T cell response in the intestinal mucosa did not appear to be impaired by the ICOS-Ig treatment. Analysis of the CD4+ T cell response revealed that ICOS-Ig treatment also affected the specific CD4+ T cell response. When restimulated with listerial Ag in vitro, reduced numbers of CD4+ T cells from infected and ICOS-Ig-treated mice responded with IFN-γ production. The impaired acquired immune response in ICOS-Ig treated mice was accompanied by their increased susceptibility to L. monocytogenes infection. ICOS-Ig treatment drastically enhanced bacterial titers, and a large fraction of mice succumbed to the otherwise sublethal dose of infection. Thus, ICOS costimulation is crucial for protective immunity against the intracellular bacterium L. monocytogenes.

Substantial in vivo proliferation of CD4(+) and CD8(+) T lymphocytes during secondary Listeria monocytogenes infection.

In mice Listeria monocytogenes infection induces a strong T cell response. In an attempt to quantitatively analyze the magnitude and kinetics of the CD4+ and CD8+ T cell response during L. monocytogenes infection in vivo we used a T cell transfer system that is independent of in vitro cell culture techniques and information about the identity of immunogenic T cell epitopes. Our results demonstrate substantial expansion of the in vivo primed and transferred T cell populations in response to L. monocytogenes. At the peak of response, transferred T cells represented more than one third of the total CD4+ and CD8+ T cell populations in blood and spleen of recipient mice. After stimulation in vitro, 40 % of these CD4+ T cells responded to heat‐killed listeriae with the production of IFN‐γ. Thus, our results reveal that in addition to the large CD8+ T cell population an almost equally large population of Listeria‐reactive CD4+ T cells is generated in response to L. monocytogenes infection.

Depletion of CD4+ T Cells during Immunization with Nonviable Listeria monocytogenes Causes Enhanced CD8+ T Cell-Mediated Protection against Listeriosis

Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8+ T cell response and consequently only limited protection against subsequent L. monocytogenes infection. We have recently demonstrated that depletion of regulatory CD4+ T cells during immunization significantly enhances CD8+ T cell responses. In the present study, we determined the impact of CD4+ T cell depletion on the CD8+ T cell response against heat-killed Listeria. Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8+ T cells and improved protection against subsequent infection with L. monocytogenes. During challenge infection, numbers of Listeria-specific CD8+ T cells were enhanced, and these cells expressed effector functions in terms of IFN-γ production. In summary, we demonstrate that combining nonviable L. monocytogenes vaccination and CD4+ T cell depletion improves generation of long-lasting and functional Listeria-specific CD8+ memory T cells.

TLR2-, TLR4- and Myd88-independent acquired humoral and cellular immunity against Salmonella enterica serovar Typhimurium

Toll-like receptors (TLR) are central to pathogen recognition by the innate immune system. However, their role in the generation of acquired immunity is less clear. Using experimental Salmonella enterica serovar Typhimurium (ST) infection of mice, we determined the role of TLR2, TLR4 and of the TLR adaptor protein Myeloid differentiation factor 88 (MyD88) in the generation of specific T- and B-cell responses against this pathogen. When infected with an attenuated ST strain, mice deficient in TLR4, TLR2 plus TLR4 or MyD88 suffered from exacerbated bacterial burden and delayed clearance. However, all mutant mice not only survived infection but also generated normal antibody responses. Compared to wild-type mice, TLR2+4 deficient mice displayed even enhanced CD4(+) T(H)1 and CD8(+) T cell responses. In contrast, T-cell responses in TLR2 deficient and MyD88 deficient mice were similar to those observed in wild-type controls. Overall, T- and B-cell responses were functional and provided protection against a challenge infection with virulent ST. Our results demonstrate that in the ST infection model, MyD88 as well as TLR2 and TLR4 were largely dispensable for the induction of protective acquired immunity.