Organ-Specific CD4  T Cell Response During Listeria monocytogenes Infection 1 Mischo Kursar, 2 * Kerstin Bonhagen, 2† Anne Ko ¨hler,* Thomas Kamradt, † Stefan H. E. Kaufmann,* and Hans-Willi Mittru ¨ cker 3 * The immune response against the intracellular bacterium Listeria monocytogenes involves both CD4  and CD8  T cells. We used the MHC class II-presented peptide listeriolysin 189 –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 listeriolysin 189 –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. The Journal of Immunology, 2002, 168: 6382– 6387. T he course of Listeria monocytogenes infection has been extensively characterized in the mouse (1). After oral up- take, bacteria reach the small intestine, from where they translocate across the intestinal mucosa into the Peyer’s patches (PP). 4 From there, bacteria spread via the mesenteric lymph nodes (MLN) into spleen and liver. In these organs, L. monocytogenes infects and replicates in macrophages and hepatocytes, and for several days high numbers of bacteria can be isolated from in- fected organs. Eventually, the acquired immune response controls infection, and bacteria are eradicated from the organism by day 10 of infection (1). The cytosolic habitat of L. monocytogenes promotes processing and presentation of listerial Ags through the MHC class I pathway. As a consequence, a potent CD8 + T cell response is induced which is critical for antilisterial defense (1, 2). Different MHC class I presented T cell epitopes expressed by wild type, and recombinant L. monocytogenes strains have been used to analyze the Listeria- specific CD8 + T cell response (3–7). These studies show strong CD8 + T cell responses in spleen, liver, lamina propria, and intes- tinal epithelium. However, there were differences in the magnitude and kinetics of responses between different organs, indicating an organ-specific regulation of CD8 + T cells (4 –7). L. monocytogenes infection also induces a CD4 + T cell re- sponse, and there is evidence that CD4 + T cells participate in protection (1, 8, 9). Transfer of CD4 + T cells from infected into naive mice confers partial protection in recipients and MHC class II-deficient mice, or mice in which CD4 + T cells were depleted by Ab treatment suffer from reduced protection against listeriosis (2, 9, 10). During L. monocytogenes infection, CD4 + T cells differ- entiate into Th1 cells and production of Th1 cell-derived cyto- kines, such as IFN-, is regarded central to CD4 + T cell-mediated protection (1, 11). There is very limited information on the kinet- ics, magnitude, and the tissue distribution of the CD4 + T cell response against L. monocytogenes (8), and only recently immu- nodominant CD4 + T cell epitopes have been identified which al- low tracking of Listeria-specific CD4 + T cells during infection (12–15). In this study, we use an immunodominant CD4 + T cell epitope derived from listeriolysin (LLO) to characterize and quantify a Listeria-specific CD4 + T cell response in different lymphoid and nonlymphoid organs after both oral and systemic infection. Materials and Methods Antibodies Rat IgG Abs, anti-CD16/CD32 mAb (clone: 2.4G2), anti-IFN- mAb (clone: R4-6A2, rat IgG1), and anti-CD4 mAb (clone: YTS191.1) were purified from rat serum or hybridoma supernatants with protein G-Sepha- rose. Abs were Cy5- or FITC-conjugated according to standard protocols. FITC-conjugated anti-TNF- mAb (clone: MP6-XT22, rat IgG1), PE-con- jugated anti-IL-10 mAb (clone: JES5-16E3, rat IgG2b), PE-conjugated anti- IL-2 mAb (clone: JES6-5h4, rat IgG2b), FITC- and PE-conjugated rat IgG1 isotype control mAb (clone: R3-34), and rat IgG2b isotype control mAb (clone: A95-1) were purchased from BD PharMingen (San Diego, CA). Infection of mice C57BL/6 mice were bred in our facility, and experiments were conducted according to the German animal protection laws. Mice were infected with L. monocytogenes strain EGD. Bacteria were grown overnight in tryptic soy broth (TSB), washed twice in PBS, aliquoted in PBS/10% glycerol, and stored at -80°C. Aliquots were thawed and bacterial titers were deter- mined by plating serial dilutions on TSB agar plates. For i.v. infection, bacteria were diluted and injected in a volume of 200 l PBS into the *Max-Planck-Institute for Infection Biology and † Deutsches Rheumaforschungszen- trum, Berlin, Germany Received for publication November 6, 2001. Accepted for publication April 11, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 M.K. was supported by the Graduiertenkolleg 276/2, and this work will be part of his PhD thesis. 2 M.K. and K.B. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Hans-Willi Mittru ¨cker, Max- Planck-Institute for Infection Biology, Schumannstr. 21/22, 10117 Berlin, Germany. E-mail address: mittruecker@mpiib-berlin.mpg.de 4 Abbreviations used in this paper: PP, Peyer’s patch; IEL, intraepithelial lymphocyte; LLO, listeriolysin; MLN, mesenteric lymph node; p.o., per os; TSB, tryptic soy broth. The Journal of Immunology Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00