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Variable Immunodominance Hierarchies for H2-M3-Restricted N-Formyl Peptides Following Bacterial Infection¹

Sections of Infectious Diseases and Immunobiology, Yale University School of Medicine, New Haven, CT 06520

	Abstract

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H2-M3-restricted presentation of N-formyl methionine (f-Met) peptides to CD8⁺ T cells provides a mechanism for selective recognition of bacterial infection. In this report we demonstrate that Listeria monocytogenes infection induces distinct CD8⁺ T cell populations specific for each of the known Listeria-derived formyl methionine peptides presented by M3. The sum H2-M3-restricted, Listeria-specific T cell response constitutes a major fraction of the total CD8⁺ T cell response to primary infection. H2-M3-restricted T cell populations expand synchronously in vivo and achieve peak frequencies 2 days earlier than MHC class Ia-restricted T cell populations. Although cross-recognition of different f-Met peptides by M3-restricted T cells was previously described, costaining of CD8⁺ T cells ex vivo with H2-M3 tetramers complexed with different f-Met peptides shows that the majority of Listeria-specific, M3-restricted CD8⁺ T cells are peptide specific. In contrast to the highly predictable size and immunodominance hierarchies of MHC class Ia-restricted T cell responses, the magnitudes of T cell responses specific for H2-M3-restricted peptides are remarkably variable between genetically identical mice. Our findings demonstrate that H2-M3-restricted T cell responses are distinct from classically restricted T cell responses to bacterial infection.

	Introduction

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Initiation with N-formyl methionine (f-Met)⁴ distinguishes bacterial from nearly all eukaryotic protein synthesis. The vertebrate immune system has exploited this distinction by evolving specialized mechanisms to recognize f-Met peptides. The N-formyl peptide receptor (FPR) (1), for example, is a chemotactic receptor found on neutrophils and monocytes that leads these cells along gradients of f-Met peptides toward sites of bacterial infection. Genetic disruption of the murine FPR demonstrates the importance of this receptor in immune responses to bacterial infection; mice deficient in FPR exhibit diminished neutrophil chemotaxis in response to the chemotactic f-MLF peptide and are more susceptible to infection with the Gram-positive bacterium Listeria monocytogenes (2).

Another immune mechanism that targets N-formyl peptides involves CD8⁺ T cell recognition of peptide bound to the murine MHC class Ib molecule H2-M3 (3). Binding by H2-M3 is highly dependent on the presence of formyl methionine at the peptide N terminus (4, 5); while M3 can present nonformylated peptides, it does so 10²- to 10⁴-fold less efficiently (6, 7). Only 13 mitochondrial proteins can supply endogenous f-Met peptides, and most of these are bound by H2-M3 with low affinity (8). Despite the paucity of self f-Met peptide and the resulting low cell surface expression of H2-M3 (9, 10), selection of H2-M3-restricted CD8⁺ T cells results at least in part from thymic selection on H2-M3 molecules presenting a mitochondrial peptide (11, 12). Exogenous addition of N-formyl peptide results in rapid trafficking of H2-M3 to the cell surface from a peptide-receptive intracellular pool (10), suggesting that presentation of bacterial f-Met peptide is likely to occur rapidly upon infection, with little competition from endogenous peptides.

Early work addressing T cell responses to L. monocytogenes infection demonstrated that some CD8⁺ T cells were not restricted by classical MHC class I molecules (13, 14) and that adoptive transfer of immune T cells provided protection to naive, MHC-disparate mice (15, 16). An explanation for this MHC-unrestricted recognition was provided when H2-M3 was shown to present L. monocytogenes-derived peptides to CD8⁺ T cells (17, 18); three Listeria-derived N-formyl epitopes have since been identified (7, 19, 20). We recently demonstrated that H2-M3-restricted T cells specific for the Listeria-derived peptide fMIGWIIA expand earlier than MHC class Ia-restricted T cells in response to primary infection, but fail to expand substantially upon reinfection with L. monocytogenes (21).

In this study we have generated H2-M3/peptide tetramers with each of the known L. monocytogenes-derived f-Met peptides. These tetramers stain CTL lines with specificity that is influenced by peptide length. Previous reports indicated that cross-recognition of N-formyl peptides is a feature of H2-M3-restricted T cell responses (22, 23); we detected a minor degree of cross-reactivity among in vitro restimulated CTL lines and found only minimal cross-recognition of listerial f-Met epitopes by directly isolated H2-M3-restricted T cells. Over the course of our studies we detected remarkable variability in the magnitude of H2-M3-restricted, L. monocytogenes-specific T cell responses, suggesting that environmental factors influence the M3-restricted T cell repertoire. Our findings indicate that three different L. monocytogenes-derived f-Met peptides induce distinct CD8⁺ effector T cell populations following primary Listeria infection and, in addition, highlight the substantial diversity of CD8⁺ T cell populations responding to bacterial infection.

	Materials and Methods

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Mice, bacteria, and bacterial infection

Female CB6F₁/J, C57BL/6, BALB/c, and C3H/HeJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). L. monocytogenes were grown in brain-heart infusion (strain 10403s from Daniel Portnoy, University of California, Berkeley, CA). Salmonella typhimurium (strain SL3261, AroA mutant from B. A. Stocker, (24)) were grown in Luria Bertoni medium. Mice were infected with 2 x 10³ Listeria or 1 x 10⁵ Salmonella for primary infection. Listeria-immune mice were reinfected with 1 x 10⁵ L. monocytogenes to investigate recall responses. On the indicated day, splenocytes were dissociated through wire mesh followed by lysis of RBC with ammonium chloride-Tris.

Generation of tetrameric MHC/₂-microglobulin (₂m)/peptide complexes

Tetramers were produced as described previously (21, 25). Briefly, MHC heavy chains and ₂m purified from inclusion bodies were refolded with 60 µg/ml listeriolysin O (LLO)_91–99 peptide (for H2-K^d) or fMIGWII, fMIGWIIA, fMIVIL, fMIVTLF, or fMFINRW (cytochrome c oxidase subunit I; COI) peptide (for H2-M3). Peptides were synthesized by Research Genetics (Huntsville, AL). Refolding was performed in the presence of protease inhibitors (pepstatin (1 µg/ml), leupeptin (1 µg/ml), and PMSF (0.4 mM)). Soluble monomeric H2-M3/₂m/peptide complexes were purified by gel filtration over a Superdex 200HR column (Pharmacia Biotech, Piscataway, NJ), followed by in vitro biotinylation at 20°C for 12 h in the presence of 15 µg of BirA enzyme (AVIDITY, Boulder, CO), 80 µM biotin, 10 mM ATP, 10 mM MgOAc, 20 mM bicine, and 10 mM Tris-HCl (pH 8.3). Complexes were purified again by gel filtration to remove excess biotin and tetramerized with PE- or allophycocyanin (APC)-conjugated streptavidin (Molecular Probes, Eugene, OR) at a 4/1 molar ratio. Following purification by gel filtration, tetramers were stored at a concentration of 5 mg/ml at 4°C in PBS (pH 8.0) with 0.02% sodium azide, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 0.5 mM EDTA.

In vitro restimulation of splenocytes

Approximately 3–4 x 10⁷ splenocytes from L. monocytogenes-infected mice (days 6–7 postinfection) were resuspended in 5 ml of RP10⁺ (RPMI 1640 supplemented with 10% FCS, L-glutamine (0.2 µg/ml), HEPES (1.2 µg/ml, pH 7.5), 2-ME (50 µm), penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (50 µg/ml; Life Technologies, Gaithersburg, MD). Syngeneic stimulator cells from naive mice were irradiated (3000 rad), pulsed with 10^-6 M (fMIGWII, fMIVIL, fMIVTLF), or 10^-9 M (LLO_91–99) (26) peptide for 1 h at 37°C, and washed to remove unbound peptide. Approximately 3 x 10⁷ of the prepared stimulator cells were resuspended in 5 ml of RPMI 1640 supplemented with 10% FCS, L-glutamine (0.2 µg/ml), HEPES (1.2 µg/ml, pH 7.5), 2-ME (50 µm), penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (50 µg/ml) and added to 5 ml of immune splenocytes in a T25 cell culture flask. T cell cultures were supplemented with 5% T-STIM Culture Supplement (rat, with Con A; Collaborative Biomedical Products, Bedford, MA). To prevent mitogenic stimulation, Con A was inactivated by the addition of 0.05 M methyl--D-mannopyranoside. Cultures were restimulated weekly with 3 x 10⁷ peptide-coated syngeneic splenocytes in the presence of 5% T-STIM. Testing of tetramer specificity and CTL assays were performed after 2–3 wk of in vitro restimulation.

Enrichment and tetramer staining of CD8⁺ T cells

For some experiments magnetically activated cell sorting (MACS; Miltenyi, Bergisch Gladbach, Germany) was used to enrich splenocytes for CD8⁺ T cells by negative selection of CD4⁺, MHC class II⁺, and MAC1⁺ cells as previously described (21). Before staining, about 3 x 10⁵ enriched cells (per well in a 96-well plate) were blocked with unconjugated streptavidin (0.5 mg/ml; Molecular Probes, Eugene, OR) and Fc-block (PharMingen) in staining buffer (SB; PBS, 0.5% BSA, and 0.02% sodium azide, pH 7.45) for 20 min on ice. Splenocytes were then stained for 1 h on ice with anti-CD62L-FITC (clone MEL-14; PharMingen, San Diego, CA), anti-CD8-CyChrome (clone 53-6.7; PharMingen), and PE- or APC-conjugated H2-M3/N-formyl peptide or PE-conjugated H2-K^d/LLO_91–99 tetrameric complexes (0.25–0.5 mg/ml). For double-tetramer staining (Fig. 5), H2-M3/fMIGWII-APC staining in the presence of anti-CD62L and anti-CD8 was followed by incubation with PE-conjugated tetramers (in SB) for another hour. Cells were washed three times in SB between tetramer stainings (when applicable) and before fixation in 1% paraformaldehyde/PBS (pH 7.45). For cell sorting, tetramer staining of enriched CD8⁺ T cells from each spleen was performed in a single tube using a buffer of PBS supplemented with 5% FCS. For some ex vivo experiments, unenriched splenocytes (1 x 10⁶/well) were stained as described above with anti-CD62L-FITC, anti-CD8-APC (clone 53-6.7; PharMingen), PE-conjugated tetramers, and propidium iodide (PI; Fig. 8) or ethidium monoazide bromide (EMA; Fig. 2; Molecular Probes) to exclude dead cells. This setup was also used when CTL lines were stained. When PI was used, cells were not fixed. All data were acquired using a FACSCalibur flow cytometer and analyzed using CellQuest software (Becton Dickinson, Mountain View, CA).

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Ex vivo purified, H2-M3-restricted, peptide-specific T cell populations sorted from Listeria-infected mice exhibit minimal cross-recognition of other Listeria-derived N-formyl epitopes. Splenocytes were harvested and combined from six CB6F1/J mice 6 days after infection with 2000 L. monocytogenes. Cells enriched for CD8+ T cells by MACS were stained with Abs to CD62L, CD8, and H2-M3/fMIGWII (A) or H2-M3/fMIVTLF (B) PE-conjugated tetramer. Activated (CD62Llow), tetramer-positive CD8+ T cells were FACS sorted and placed in CTL assays with 51Cr-labeled, peptide-coated p815 target cells. The x- and y-axes show target peptide and percent specific lysis, respectively. E:T cell ratios were 3:1 for fMIGWII (A) and 1.4:1 for fMIVTLF (B) sorted cells.

View larger version (44K): [in this window] [in a new window]   FIGURE 8. The magnitudes of H2-M3-restricted T cell responses to L. monocytogenes can differ dramatically among genetically identical mice. CB6F1/J and C3H/HeJ mice were infected with 2000 L. monocytogenes. A, On day 6 postinfection, unenriched splenocytes were stained with anti-CD62L, anti-CD8, H2-M3/fMIGWII tetramers, and PI. Dot plots are gated on live (PI-negative) CD8+ T cells, with anti-CD62L and tetramer staining on the x- and y-axes, respectively. In the upper quadrants, the percentages of tetramer-positive (CD62Llow (activated) or CD62Lhigh) T cells among CD8+ splenocytes are indicated. B, Whole splenocytes (day 6 postinfection) were incubated with fMIGWII peptide in vitro for 5 h as described in Materials and Methods. After stimulation, cells were stained with anti-CD8 and, following fixation and permeabilization, anti-IL4 (C3H mouse only) and anti-IFN- or anti-TNF-. Dot plots are gated on CD8+ T cells. Cytokine staining is shown on the y-axis for IL-4 and on the x-axis for IFN- (left column) and TNF- (right column). The percentages of CD8+ T cells that secrete IFN- and TNF- in response to peptide stimulation are indicated. CB6 and C3H mice were 13 and 22 wk of age, respectively, at the time of harvest. C, Scattergram of splenocytes harvested on days 5 (n = 5), 6 (n = 11), and 7 (n = 12) postinfection from CB6F1/J mice and on day 6 postinfection from C3H/HeJ mice (n = 9). Whole splenocytes harvested 5 or 6 days after infection were stained as described in A above, with Kd/LLO91–99 (CB6 only) or M3/fMIGWII tetramers and PI or EMA. Cells harvested on day 7 postinfection (CB6F1) were MACS-enriched (see Materials and Methods) for CD8+ cells and stained with Kd/LLO91–99 or M3/fMIGWIIA tetramers (no PI/EMA). Each dot represents a single mouse. CB6 mice were 8–13 wk of age, and fMIGWII(A) and LLO91–99 stainings were performed on the same mice. C3H/HeJ mice were 10–28 wk of age. The percentage of epitope-specific, CD62Llow T cells (among CD8+ cells) is shown on the y-axis. Days postinfection are shown on the x-axis.

View larger version (57K): [in this window] [in a new window]   FIGURE 2. T cell populations specific for each of the known Listeria-derived peptides can be detected ex vivo from mice infected with L. monocytogenes using tetrameric MHC/peptide reagents. Female CB6F1/J mice were infected with 2 x 103 L. monocytogenes (left column) or 1 x 105 S. typhimurium (AroA-; right column), and splenocytes were harvested 7 days later. Unenriched splenocytes were stained with anti-CD62L, anti-CD8, EMA, and H2-Kd/LLO91–99 or one of the three H2-M3/N-formyl peptide tetramers noted to the left. Dot plots are gated on live (EMA-negative) CD8+ T cells. The activation marker CD62L and tetramer staining are found on the x- and y-axes, respectively. Plots are representative of two mice per group.

Unenriched splenocytes (5 x 10⁶ cells/well in a 24-well plate) were incubated with 10^-6 M LLO_91–99 or fMIGWII peptide (or without peptide) in the presence of 1 µl/ml GolgiPlug (brefeldin A; PharMingen) for 5 h at 37°C. Stimulated cells (1–2 x 10⁶/well in a 96-well plate) were blocked as described above and stained with anti-CD8-CyChrome (clone 53-6.7; PharMingen) for 30 min on ice. Following washes in SB, cells were fixed and permeabilized in Cytofix/Cytoperm solution (PharMingen) and stained following the manufacturer’s instructions with anti-IFN--FITC (clone XMG1.2; PharMingen), anti-TNF--FITC (clone MP6-XT22; PharMingen), or the suggested FITC-conjugated isotype control (clone R3-34; PharMingen). In some experiments (Fig. 8, C3H), cells were simultaneously stained with anti-IL4-APC (clone 11B11; PharMingen). Splenocytes were resuspended in SB for acquisition as described above.

Chromium release assays

CTL assays were performed as previously described (27). Briefly, P815 target cells (American Type Culture Collection, Manassas, VA) were labeled with ⁵¹Cr and washed. CTL lines were incubated with 1 x 10⁴ labeled targets and a titration of the indicated peptide (or without peptide) at a constant E:T cell ratio for 4–5 h at 37°C. FACS-sorted cells were incubated with 5 x 10³ labeled target cells alone or in the presence of 10^-6 M of the target peptide for 7–7.5 h at 37°C. The percent specific lysis was calculated as previously described (27) based on the amount of ⁵¹Cr released into the supernatant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Staining of CTL lines and splenocytes from L. monocytogenes-infected mice with H2-M3 tetramers refolded with Listeria-derived f-Met peptides

The H2-M3 class Ib molecule presents three known L. monocytogenes-derived peptides, fMIGWII(A), fMIVIL, and fMIVTLF, to CD8⁺ T cells (see Table I). In previous studies we demonstrated that H2-M3/fMIGWIIA tetramers specifically stain both fMIGWIIA-specific CTL lines and Ag-specific CD8⁺ T cells from Listeria-infected mice (21). To further investigate M3-restricted T cell responses to bacteria infection, we generated H2-M3 tetramers with additional L. monocytogenes f-Met peptides. Because it is not known whether the H2-M3-restricted epitope derived from LemA is six or seven amino acids in length (7), we made tetramers with both the hexamer (fMIGWII) and the heptamer (fMIGWIIA). These tetramers stain peptide-restimulated CTL lines with specificity (Fig. 1). A fMIGWII peptide-specific line (Fig. 1A) stained with both M3/fMIGWII tetramers (92%) and M3/fMIGWIIA tetramers (77%). Cell lines restimulated with fMIGWIIA also stained with M3/fMIGWII tetramers, often to a higher degree than with M3/f-MIGWIIA tetramers (data not shown). CTL lines specific for fMIVIL stained only with M3/fMIVIL tetramers (Fig. 1B), while fMIVTLF-specific CTL lines (Fig. 1C) stained with both M3/fMIVTLF tetramers (80%) and, to a lesser extent, M3/fMIVIL tetramers (36%). These data indicate that some H2-M3-restricted CD8⁺ T cells recognize peptides promiscuously, detecting multiple peptides that are similar (e.g., fMIVIL and fMIVTLF) or differ in length (fMIGWII and fMIGWIIA).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. H2-M3 tetramers refolded with the listerial peptides fMIGWII, fMIGWIIA, fMIVIL, and fMIVTLF stain in vitro restimulated CTL lines with specificity. CTL lines specific for fMIGWII (A), fMIVIL (B), fMIVTLF (C), and LLO91–99 (D) were generated from splenocytes of L. monocytogenes-infected CB6F1/J mice by in vitro peptide restimulation. Cells were stained with Abs to CD62L (L-selectin) and CD8, PI, and H2-M3/N-formyl peptide or H2-Kd/LLO91–99 PE-conjugated tetramers (key to the right of histograms). Histograms are gated on live CD8+ T cells with tetramer staining on the x-axis and cell counts on the y-axis. Results are representative of several lines tested.

Cross-recognition of N-formylated listerial epitopes by H2-M3-restricted T cells in vitro

CTL clones specific for fMIGWII have been reported to cross-react with other N-formyl peptides (23). We found that staining with H2-M3 tetramers complexed with different f-Met peptides lacks the fidelity of staining with H2-K^d tetramers. Fig. 1A provides an example of cross-reactive staining; both M3/fMIVIL and M3/fMIVTLF tetramers stained this fMIGWII-specific CTL line with low intensity (each <20%). Cross-reactive staining with H2-M3 or H2-K^d tetramers was never detected during extensive analysis of CTL lines restimulated in vitro with LLO_91–99 or other H2-K^d-restricted peptides (Fig. 1D) (21, 25). These data suggest that peptide cross-reactivity may be a feature of H2-M3-restricted T cell populations that distinguishes them from classically restricted T cells.

To further investigate cross-reactivity of H2-M3-restricted T cells, we generated T cell lines by stimulating immune splenocytes in vitro with each of the L. monocytogenes f-Met peptides and assayed them in cytolytic assays for peptide recognition. Fig. 3A demonstrates that a CTL line restimulated in vitro with fMIGWII recognized target cells coated with fMIGWII and also recognized the fMIGWIIA peptide at roughly 10-fold higher concentrations. Interestingly, this fMIGWII-stimulated CTL line also recognized fMIVIL at roughly 1000-fold higher peptide concentrations. Similarly, CTL lines specific for fMIVIL and fMIVTLF recognized other Listeria-derived, N-formyl peptides at high concentrations (Fig. 3 and data not shown), generally requiring peptide concentrations 1–3 logs greater than the stimulating peptide. Despite consistent staining of a subset of fMIVTLF-restimulated CTL with H2-M3/fMIVIL tetramers (Fig. 1C and data not shown), fMIVTLF-specific CTL lines did not exhibit significant recognition of the fMIVIL peptide in CTL assays (Fig. 3C and data not shown). The reason for this disparity in tetramer staining and in vitro cytolytic activity is unclear. CTL lines restimulated with the L. monocytogenes f-Met peptides did not lyse targets in the presence of high concentrations of the COI self peptide (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 3. CTL lines specific for H2-M3-restricted, L. monocytogenes-derived peptides exhibit cross-recognition of other N-formyl listerial epitopes at high peptide concentrations. In vitro peptide-restimulated CTL lines specific for fMIGWII (A), fMIVIL (B), and fMIVTLF (C) were established from Listeria-infected CB6F1/J mice. CTL lines were tested against 51Cr-labeled P815 targets coated with titrations of fMIGWII, fMIGWIIA, fMIVIL, or fMIVTLF peptide as indicated. The target peptide concentration and percent specific lysis are shown on the x- and y-axes, respectively.

Although our analysis of CTL lines supports the idea that peptide cross-reactivity is a feature of H2-M3-restricted T cells, we were concerned that in vitro cultivation of CTL might bias the T cell population and either diminish or amplify cross-reactive populations. To further investigate cross-recognition, we performed direct ex vivo analyses of H2-M3-restricted T cell populations. Immune splenocytes were double stained with PE- and APC-conjugated H2-M3 tetramers complexed with different L. monocytogenes f-Met peptides. Essentially all fMIGWII-specific cells costained with M3/fMIGWII-PE and M3/fMIGWII-APC tetramers (Fig. 4), demonstrating the feasibility of double staining. As might be expected from the in vitro data presented above, M3/fMIGWII and M3/fMIGWIIA tetramers largely stained the same T cell population; nearly all cells that stained with M3/fMIGWIIA tetramers costained with M3/fMIGWII tetramers, but some fMIGWII-specific T cells did not bind the M3/fMIGWIIA tetramer. This result suggests that fMIGWII, and not fMIGWIIA, is the epitope that primes T cells in vivo. The percentage of double-positive cells (upper right region, Fig. 4) was greater for H2-M3/fMIGWII costaining with M3/fMIVIL and M3/fMIVTLF tetramers (0.46 and 0.42%, respectively) than for costaining with H2-K^d/LLO_91–99 (0.13%), suggesting that a small portion of fMIGWII-specific T cells may be cross-reactive in vivo.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. T cell populations detected by H2-M3 tetramers containing fMIGWII and fMIGWIIA are largely overlapping, but only minor cross-recognition among other Listeria-derived N-formylated epitopes is detected by double staining of splenocytes from infected mice. Splenocytes from three CB6F1/J mice infected with 2000 L. monocytogenes were harvested 6 days after infection, combined, and enriched for CD8+ T cells by MACS. Cells were stained with anti-CD62L, anti-CD8, and H2-M3/N-formyl peptide or H2-Kd/LLO91–99 tetramers conjugated with PE or APC. All dot plots are gated on CD8+ T cells. Tetramer staining with PE- and APC-conjugated reagents is shown on the x- and y-axes, respectively. The top row of dot plots represents single-tetramer staining with PE tetramers (specific tetramers indicated above plots). Single-tetramer staining with H2-M3/fMIGWII-APC is shown in the left-most dot plot of the bottom row. The remaining plots of the bottom row are double stained with H2-M3/fMIGWII-APC and the PE tetramer indicated above. Percentages of tetramer-positive cells among CD8+ splenocytes are indicated in the defined regions.

Kinetics of Listeria-specific H2-M3-restricted T cell responses specific for different N-formyl peptides following L. monocytogenes infection

H2-M3-restricted T cells specific for the Listeria-derived N-formyl peptide fMIGWIIA expand earlier than H2-K^d-restricted T cell populations following primary L. monocytogenes infection, while little expansion of fMIGWIIA-specific cells is detected after reinfection (21) (Fig. 6A). H2-M3-restricted T cell populations specific for the Listeria-derived fMIVIL (Fig. 6B) and fMIVTLF (Fig. 6C) peptides share the same characteristics of early expansion during primary infection, peaking between days 5 and 7 postinfection. In contrast, H2-K^d-restricted T cells specific for LLO_91–99 peak 2 days later, (Fig. 6D) (21, 25). Like fMIGWIIA-specific T cells, M3-restricted populations specific for fMIVIL and fMIVTLF fail to expand dramatically in response to re-exposure to L. monocytogenes. Although recall populations peak 5 days after reinfection, the magnitude of H2-M3-restricted memory responses is approximately the same as that of the primary responses (Fig. 6, A–C). As shown previously (21, 25), H2-K^d-restricted cells exhibit responses to reinfection that are characteristic of immunological memory, expanding more rapidly and to a much greater magnitude than the primary response (Fig. 6D). Thus, H2-M3-restricted T cell populations expand and contract synchronously in response to L. monocytogenes infection, with kinetics distinct from those of class Ia-restricted T cell responses.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. The distinct H2-M3-restricted T cell populations specific for known Listeria-derived epitopes expand synchronously following primary infection, but do not undergo enhanced expansion upon reinfection. Splenocytes from CB6F1/J mice were harvested on the indicated day after primary infection with 2 x 103 L. monocytogenes or reinfection with 1 x 105 bacteria (7 wk later). Cells were enriched for CD8+ T cells and stained with anti-CD8, anti-CD62L, and H2-M3/fMIGWIIA (A), H2-M3/fMIVIL (B), H2-M3/fMIVTLF (C), or H2-Kd/LLO91–99 (D) tetramers. Days postinfection are shown on the x-axes, and the absolute number of tetramer-positive cells per spleen (CD8+ and activated) is shown on the y-axes. Graphs show an average of three mice per day (with SD). Kinetics during primary infection were confirmed by an independent experiment under identical conditions. Error bars for primary infection (filled symbols) have a shorter cap length than bars for recall infection (open symbols).

Despite sharing the same H2-M3 allele, C57BL/6 (BL/6) and C3H/HeJ (C3H) mice mount substantially larger M3-restricted, fMIGWIIA-specific T cell responses following primary L. monocytogenes infection than are detected in BALB/c mice (21). To determine whether this strain difference is a general feature of M3 responses, splenocytes from Listeria-infected, age-matched BALB/c, C3H, and BL/6 mice were stained directly ex vivo with H2-M3/fMIGWIIA (Fig. 7A), fMIVIL (Fig. 7B), or fMIVTLF (Fig. 7C) tetramers on day 7 after infection. BALB/c mice exhibited small M3 restricted populations for all the known epitopes (Fig. 7, A–C), suggesting that H2-M3-restricted T cell responses may not play as great a role in these mice as in other strains. C3H and BL/6 mice (Fig. 7, A–C, center and right columns, respectively) generated substantial responses specific for all the Listeria-derived N-formyl peptides, with consistently larger fMIGWII- and fMIVTLF-specific T cell populations than fMIVIL-specific populations. Similar differences between mouse strains were found on days 5 and 9 postinfection (data not shown). Cell populations staining with M3/fMIGWII tetramers follow the same trends among mouse strains as shown for the fMIGWIIA variant of the epitope (data not shown). Of note, while C3H mice generated fMIVTLF-specific T cell populations larger than those found in BL/6 mice in the experiment shown, this is not a consistent feature of M3-restricted responses in these strains. We have detected remarkable variability in the relative magnitudes of T cell responses specific for fMIGWII and fMIVTLF (see below). Splenocytes from uninfected mice did not stain with any of the tetramers tested (data not shown) (21), and no staining was detected with M3 tetramers complexed with the COI self peptide (Fig. 7D) (21).

View larger version (20K): [in this window] [in a new window]   FIGURE 7. H2-M3-restricted T cell responses to L. monocytogenes-derived N-formyl peptides differ in magnitude in BALB/c, C3H/HeJ, and C57BL/6 mice. Splenocytes were harvested from female, age-matched BALB/c, C3H/HeJ, and C57BL/6 mice (left, center, and right columns, respectively) 7 days after infection with 2 x 103 L. monocytogenes. Cells enriched for CD8+ T cells were stained with anti-CD62L and anti-CD8 Abs and H2-M3/fMIGWIIA (A), H2-M3/fMIVIL (B), H2-M3/fMIVTLF (C), or H2-M3/fMFINRW (D; COI self) tetramers. For each mouse strain (x-axes), the absolute number of CD8+, activated (CD62Llow), tetramer-positive cells in the spleen was calculated (y-axes). Graphs show the average of two mice, and SDs are indicated.

During the course of our studies, we have noticed that the magnitude of H2-M3-restricted T cell responses can vary dramatically between genetically identical mice. Relatively typical frequencies of H2-M3-restricted T cell populations are shown in Figs. 2 and 5. However, compared with the highly reproducible MHC class Ia (H2-K^d)-restricted T cell responses to L. monocytogenes, the magnitudes of H2-M3-restricted T cell populations are far less predictable. As shown in Fig. 8A, comparison of two CB6 mice (from the same cohort) revealed disparate fMIGWII responses; while only 1.82% of CD8⁺ T cells in the first mouse were fMIGWII specific, in the second CB6 mouse >7% of CD8⁺ T cells were fMIGWII specific. Importantly, T cell responses specific for the H2-K^d-restricted LLO_91–99 epitope were nearly identical in these two mice (results not shown), indicating that there were no disparities in L. monocytogenes infection. Mice occasionally generate extraordinarily large fMIGWII-specific T cell responses during primary Listeria infection, as demonstrated in a C3H mouse in Fig. 8A. In this mouse nearly 20% of CD8⁺ T cells were activated (CD62L^low) and stained with fMIGWII tetramers, while another 11% were CD62L^high and fMIGWII tetramer positive. We also performed intracellular cytokine staining assays for T cell production of TNF- and IFN-. As demonstrated in Fig. 8B, the frequencies of fMIGWII-specific T cells detected by intracellular cytokine staining closely reflect those measured by H2-M3 tetramer staining. It is unclear whether the distinct CD62L^high, tetramer-positive population in the C3H mouse contributes to cytokine production; while the frequency of cytokine-producing cells is nearly identical with that of the CD62L^low population, it is not uncommon for us to detect <100% of the CD62L^low tetramer-positive cells by the intracellular cytokine secretion assay (Fig. 8, A and B, CB6 mice). In any case, this result demonstrates that the variability detected by tetramer staining truly represents differences in the number of Ag-specific CD8⁺ T cells that have expanded in response to L. monocytogenes infection in these different animals.

Although a degree of variability is inherent in all in vivo experiments, the magnitudes of H2-M3-restricted T cell responses are less predictable than those of H2-K^d (MHC class Ia)-restricted responses following L. monocytogenes infection. Fig. 8C (left panels) demonstrates the range of T cell response magnitudes to K^d/LLO_91–99 and M3/fMIGWII(A) on days 5, 6, and 7 postinfection. Although the percentage of CD62L^low, LLO_91–99 epitope-specific T cells (among CD8⁺ cells) varied somewhat from mouse to mouse, M3/fMIGWII(A)-specific responses were clearly more variable. C3H/HeJ mice demonstrated even greater variability in the M3-restricted response to Listeria infection, mounting responses ranging from 1.3% to nearly 20% (CD62L^low only) of CD8⁺ T cells (Fig. 8C, right panel).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
H2-M3-restricted presentation of N-formyl peptides to CD8⁺ T cells is a specialized mechanism for T cell recognition of bacterial infection. In this report we demonstrate that distinct H2-M3-restricted T cell populations, specific for several N-formyl peptides, are synchronous in their response to infection with L. monocytogenes. These MHC class Ib-restricted T cell populations expand earlier than class Ia-restricted populations and, in aggregate, account for a large fraction of the activated CD8⁺ T cell pool following primary bacterial infection. Our studies have also revealed a remarkable variability in the magnitude of M3-restricted responses to Listeria among genetically identical mice, suggesting that environment has a much larger impact on M3-restricted CD8⁺ T cell populations than on conventional CD8⁺ T cell responses. These findings add to our understanding of H2-M3-restricted T cell responses to bacterial infection and further distinguish this MHC class Ib-restricted T cell subset from other T cell responses to infection.

The groove of H2-M3, unlike that of MHC class Ia molecules, is open at the C terminus of the bound peptide. This property, in combination with predominant peptide anchoring at the f-Met, allows H2-M3 to bind peptides with a wide range of lengths. When the LemA epitope was identified (7), it was concluded that the formylated hexamer, fMIGWII, or the heptamer, fMIGWIIA, is the natural epitope presented by M3 during L. monocytogenes infection; the CTL clone used in the study recognized both forms of the peptide equivalently. Our studies, both in vitro and ex vivo, demonstrate that some LemA-specific, H2-M3-restricted T cells can distinguish between the hexamer and heptamer peptides. This is demonstrated most convincingly by double tetramer staining of splenocytes from L. monocytogenes-infected mice (Fig. 4). Only 60% of the LemA-specific cells that recognize fMIGWII also recognize fMIGWIIA. In contrast, all cells that stain with H2-M3/fMIGWIIA tetramers also stain with H2-M3/fMIGWII tetramers. These findings indicate that the length of bound peptide influences T cell recognition, a finding that has been previously described only for T cell recognition of MHC class II/peptide complexes (28). In addition, these studies strongly suggest that the peptide presented in vivo is the hexamer peptide, fMIGWII.

Previous investigations of H2-M3-restricted T cell clones demonstrated a remarkable degree of peptide cross-recognition (23). Several features of H2-M3 may account for this promiscuity. First, cell surface expression of H2-M3 is normally low, increasing dramatically in the presence of f-Met peptides. It is possible that low levels of H2-M3 expression could result in diminished induction of peripheral tolerance, forming a population of T cells with specificity for the MHC class Ib molecule but lower peptide selectivity than most T lymphocytes. A second possible explanation for the cross-reactivity demonstrated by H2-M3-restricted T cells relates to the unusual structure of H2-M3. Specifically, the crystallization of H2-M3 revealed that M3 binds peptides quite deeply the pocket (29), resulting in a relatively small amount of exposed area. Thus, it is possible that the surface topology of H2-M3 complexed with different hydrophobic L. monocytogenes peptides is sufficiently similar to account for the cross-recognition of N-formyl peptides by M3-restricted T cells.

Our CTL lines lysed target cells in the presence of relatively high concentrations of heterologous N-formyl peptides (Fig. 3) and stained, to a small degree, with heterologous M3 tetramers (Fig. 1). However, the CTL lines recognized homologous peptide at over 1000-fold lower concentrations, suggesting that a significant role for cross-recognition of heterologous peptides during in vivo T cell responses to L. monocytogenes infection is unlikely. This conclusion is further supported by our findings that only minimal cross-recognition is detected ex vivo by CTL assay (at high peptide concentrations) with FACS-sorted cells (Fig. 5) or by double staining with different H2-M3/peptide tetramers (Fig. 4). Although the degree of cross-recognition reported here and by others distinguishes H2-M3-restricted T cell responses from conventional MHC class Ia responses, our studies demonstrate that the majority of H2-M3-restricted CD8⁺ T cells responding to Listeria infection are peptide specific.

During the early adaptive response to L. monocytogenes infection (days 5–7 postinfection), H2-M3-restricted populations specific for the Listeria-derived epitopes fMIGWII(A), fMIVIL, and fMIVTLF generally reach magnitudes greater than the immunodominant H2-K^d-restricted response toward LLO_91–99. Thus, the sum M3-restricted T cell response constitutes a relatively large portion of the activated CD8⁺ T cell pool early during primary Listeria infection. H2-M3-restricted T cells specific for the three known f-Met epitopes account for nearly 20% of the activated (CD62L^low) CD8⁺ T cells in the L. monocytogenes-infected mouse shown in Fig. 2. The percentage of H2-M3-restricted cells among activated CD8⁺ T cells would be even greater in some mice (see Fig. 8). These findings, together with the synchronous early kinetics of H2-M3-restricted T cells during primary infection, support the idea that M3-restricted T cells may play an important role in early defense against bacterial infection.

H2-K^d-restricted CD8⁺ T cell responses to L. monocytogenes infection are highly consistent in both magnitude and immunodominance hierarchy (25). Infection of the H2^d mouse strains BALB/c, B10.D2, and DBA/2 results in the expansion of H2-K^d-restricted CD8⁺ T cell populations similar in frequency (data not shown). In this report we demonstrate that, in contrast, H2-M3-restricted T cell responses to Listeria infection differ in magnitude between genetically disparate mice even though they share the same H2-M3 allele (Fig. 7). However, even more remarkable is the variability of H2-M3-restricted T cell responses among genetically identical mice. T cell populations specific for the fMIGWII epitope typically constitute 2–5% of the CD8⁺ T cell pool at the peak of the response to L. monocytogenes infection, but frequencies as high as 30% (of CD8⁺ T cells) have been detected in some mice (Fig. 8). There is also variability in immunodominance among the Listeria-specific, H2-M3-restricted T cell populations; for example, in different experiments fMIGWII-specific (see Fig. 4) and fMIVTLF-specific (Fig. 2) T cell populations have been dominant. The striking variability of H2-M3-restricted T cell responses in genetically identical mice supports the idea that environmental influences have a large impact on M3-restricted T cell responses to bacterial infection.

Several additional lines of evidence suggest that cross-recognition of N-formyl epitopes from environmental bacteria may modulate the repertoire of H2-M3-restricted T cells. Lenz and Bevan (22) reported that M3-restricted T cell responses specific for the fMIGWII and fMIVIL epitopes from Listeria could be detected in naive mice housed in non-specific-pathogen-free conditions. We found that H2-M3-restricted T cell populations generated in response to L. monocytogenes infection tend to be smaller in young (6-wk-old) mice than in older mice (K. Kerksiek and E. G. Pamer, unpublished observations). Our results here (Fig. 6) and in a previous report (21) demonstrate that M3-restricted responses to primary Listeria infection occur more rapidly than class Ia (H2-K^d)-restricted responses. These findings are consistent with the hypothesis that environmental bacteria influence the repertoire of H2-M3-restricted T cells in the naive mouse.

We searched the protein databases SWISS-PROT and TrEMBL for sequences identical with the three known Listeria-derived H2-M3-restricted epitopes. No identity was found to the peptides MIGWII or MIVTLF among bacterial sequences. The search for identity to MIVIL identified only one amino-terminal match, to a protein from a thermophilic archebacterial species, an unlikely commensal inhabitant of mice. Despite the negative results of our searches, there is still reason to believe that peptides from environmental bacteria may have an impact on M3-restricted T cells. For one, the list of sequenced bacterial genomes is quite limited, especially for nonpathogenic commensal bacteria. Additionally, while we searched the sequence databases for peptide identity, it is clear that promiscuity is factor in M3-restricted T cell recognition of N-formyl epitopes. Thus, nonidentical but cross-reactive peptides may influence the repertoire of H2-M3-restricted T cells in L. monocytogenes-naive mice.

H2-M3-restricted T cell populations form a unique subset of the CD8⁺ T cell pool, expanding more rapidly than other Listeria-specific T cells in response to L. monocytogenes infection and, at the peak of their expansion, constituting a large portion of the activated CD8⁺ T cells. Studies of immune responses to Listeria infection have identified cytolysis (30), IFN- (31, 32), and TNF- (33, 34) as critical determinants for effective clearance of the bacterium. Listeria-specific, H2-M3-restricted T cells are able to perform all these functions, and their presence in large numbers at such an early point in the adaptive immune response indicates that they may form an important arm of the immune response to bacterial infection.

	Footnotes

 
¹ This work was supported by Public Health Services Grant 1RO1AI-42135. K.M.K. is supported by National Institutes of Health Training Grant 5T32AI07019. D.H.B is a recipient of a Howard Hughes Fellowship for Physicians.

² Current address: Institut fir Mikrobiologie, Immunologie und Hygiene, Technische Universitat Munchen, Klinikum rechts der Isar, Trogerstrasse 9, Munich, Germany.

³ Address correspondence and reprint requests to Dr. Eric G. Pamer, Infectious Diseases Service and Laboratory of Antimicrobial Immunity, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.

⁴ Abbreviations used in this paper: f-Met, N-formyl methionine; FPR, N-formyl peptide receptor; APC, allophycocyanin; ₂m, ₂-microglobulin; COI, cytochrome c oxidase subunit I; LLO, listeriolysin O; MACS, magnetically activated cell sorting; PI, propidium iodide; EMA, ethidium monoazide bromide; SB, staining buffer.

Received for publication July 27, 2000. Accepted for publication October 18, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Murphy, P. M.. 1994. The molecular biology of leukocyte chemoattractant receptors. Annu. Rev. Immunol. 12:593.[Medline]
2. Gao, J.-L., E. J. Lee, P. M. Murphy. 1999. Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor. J. Exp. Med. 189:657.[Abstract/Free Full Text]
3. Lindahl, K. F., D. E. Byers, V. Dabhi, R. Hovik, E. P. Jones, G. P. Smith, C.-R. Wang, H. Xiao, M. Yoshino. 1997. H2-M3, a full service class Ib histocompatibility antigen. Annu. Rev. Immunol. 15:851.[Medline]
4. Shawar, S. M., R. G. Cook, J. R. Rodgers, R. R. Rich. 1990. Specialized functions of MHC class I molecules. I. An N-formyl peptide receptor is required for construction of the class I antigen Mta. J. Exp. Med. 171:897.[Abstract/Free Full Text]
5. Shawar, S. M., J. M. Vyas, J. R. Rodgers, R. G. Cook, R. R. Rich. 1991. Specialized functions of major histocompatibility complex class I molecules. II. Hmt binds N-formylated peptides of mitochondrial and prokaryotic origin. J. Exp. Med. 174:941.[Abstract/Free Full Text]
6. Smith, G. P., V. M. Dabhi, E. G. Pamer, K. F. Lindahl. 1994. Peptide presentation by the MHC class Ib molecule, H2-M3. Int. Immunol. 6:1917.[Abstract/Free Full Text]
7. Lenz, L. L., B. Dere, M. J. Bevan. 1996. Identification of an H2-M3-restricted Listeria epitope: implications for antigen presentation by M3. Immunity 5:63.[Medline]
8. Dabhi, V. M., R. Hovik, L. V. Kaer, K. F. Lindahl. 1998. The alloreactive T cell response against the class Ib molecule H2-M3 is specific for high affinity peptides. J. Immunol. 161:5171.[Abstract/Free Full Text]
9. Vyas, J. M., R. R. Rich, D. D. Howell, S. M. Shawar, J. R. Rodgers. 1994. Availability of endogenous peptides limits expression of an M3a-Ld major histocompatibility complex class I chimera. J. Exp. Med. 179:155.[Abstract/Free Full Text]
10. Chiu, N. M., T. Chun, M. Fay, M. Mandal, C.-R. Wang. 1999. The majority of H2-M3 is retained intracellularly in a peptide-receptive state and traffics to the cell surface in the presence of N-formylated peptides. J. Exp. Med. 190:423.[Abstract/Free Full Text]
11. Berg, R. E., M. F. Princiotta, S. Irion, J. A. Moticka, K. R. Dahl, U. W. Staerz. 1999. Positive selection of an H2–M3 restricted T cell receptor. Immunity 11:33.[Medline]
12. Chiu, N. M., B. Wang, K. M. Kerksiek, R. Kurlander, E. G. Pamer, C.-R. Wang. 1999. The selection of M3-restricted T cells is dependent on M3 expression and presentation of N-formylated peptides in the thymus. J. Exp. Med. 190:1869.[Abstract/Free Full Text]
13. Libero, G. D., S. H. Kaufmann. 1986. Antigen-specific Lyt-2⁺ cytolytic T lymphocytes from mice infected with the intracellular bacterium Listeria monocytogenes. J. Immunol. 137:2688.[Abstract]
14. Brown, M. L., P. E. Fields, R. J. Kurlander. 1992. Metabolic requirements for macrophage presentation of Listeria monocytogenes to immune CD8 cells. J. Immunol. 148:555.[Abstract]
15. Kaufmann, S. H. E., H.-R. Rodewald, E. Hug, G. D. Libero. 1988. Cloned Listeria monocytogenes specific non-MHC-restricted Lyt-2⁺ T cells with cytolytic and protective activity. J. Immunol. 140:3173.[Abstract]
16. Lukacs, K., R. J. Kurlander. 1989. MHC-unrestricted transfer of antilisterial immunity by freshly isolated immune CD8 spleen cells. J. Immunol. 143:3731.[Abstract]
17. Kurlander, R. J., S. M. Shawar, M. L. Brown, R. R. Rich. 1992. Specialized role for a murine class I-b MHC molecule in prokaryotic host defenses. Science 257:678.[Abstract/Free Full Text]
18. Pamer, E. G., C. R. Wang, L. Flaherty, K. F. Lindahl, M. J. Bevan. 1992. H2–M3 presents a Listeria monocytogenes peptide to cytotoxic T lymphocytes. Cell 70:215.[Medline]
19. Gulden, P. H., P. Fischer, N. E. Sherman, W. Wang, V. H. Engelhard, J. Shabanowitz, D. H. Hunt, E. G. Pamer. 1996. A Listeria monocytogenes pentapeptide is presented to cytolytic T lymphocytes by the H2-M3 MHC class Ib molecule. Immunity 5:73.[Medline]
20. Princiotta, M. F., L. L. Lenz, M. J. Bevan, U. D. Staerz. 1998. H2–M3 restricted presentation of a Listeria-derived leader peptide. J. Exp. Med. 187:1711.[Abstract/Free Full Text]
21. Kerksiek, K. M., D. H. Busch, I. M. Pilip, S. E. Allen, E. G. Pamer. 1999. H2-M3 restricted T cells: rapid effector function during primary bacterial infection and diminished memory responses. J. Exp. Med. 190:195.[Abstract/Free Full Text]
22. Lenz, L. L., M. J. Bevan. 1997. CTL responses to H2–M3-restricted Listeria epitopes. Immunol. Rev. 158:115.[Medline]
23. Nataraj, C., G. R. Huffman, R. J. Kurlander. 1998. H2M3wt-restricted, Listeria monocytogenes-immune CD8 T cells respond to multiple formylated peptides and to a variety of Gram-positive and Gram-negative bacteria. Int. Immunol. 10:7.[Abstract/Free Full Text]
24. Hoiseth, S., B. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291:238.[Medline]
25. Busch, D. H., I. M. Pilip, S. Vijh, E. G. Pamer. 1998. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8:353.[Medline]
26. Busch, D. H., E. G. Pamer. 1998. MHC class I/peptide stability: implications for immunodominance, in vitro proliferation and diversity of responding CTL. J. Immunol. 160:4441.[Abstract/Free Full Text]
27. Busch, D. H., E. G. Pamer. 1998. Killer cell assays. S. Kaufmann, ed. In Methods in Microbiology Vol. 25:237.-256. Academic Press, New York.
28. Moudgil, K. D., I. S. Grewal, P. E. Jensen, E. E. Sercarz. 1996. Unresponsiveness to a self-peptide of mouse lysozyme owing to hindrance of T cell receptor-major histocompatibility complex/peptide interaction caused by flanking epitopic residues. J. Exp. Med. 183:535.[Abstract/Free Full Text]
29. Wang, C. R., A. R. Castano, P. A. Peterson, C. Slaughter, K. F. Lindahl, J. Deisenhofer. 1995. Nonclassical binding of formylated peptide in crystal structure of the MHC class Ib molecule H2-M3. Cell 82:655.[Medline]
30. Kagi, D., B. Ledermann, K. Burki, H. Hengartner, R. F. Zinkernagel. 1994. CD8 T cell-mediated protection against an intracellular bacterium by perforin-dependent cytotoxicity. Eur. J. Immunol. 24:3068.[Medline]
31. Buchmeier, N. A., R. D. Schreiber. 1985. Requirement of endogenous interferon- production for resolution of Listeria monocytogenes infection. Proc. Natl. Acad. Sci. USA 82:7404.[Abstract/Free Full Text]
32. Harty, J. T., M. J. Bevan. 1995. Specific immunity to Listeria monocytogenes in the absence of IFN-. Immunity 3:109.[Medline]
33. Havell, E.. 1987. Production of tumor necrosis factor during murine listeriosis. J. Immunol. 139:4225.[Abstract]
34. Pfeffer, K., T. Matsuyama, T. Kundig, A. Wakeham, K. Kishihara, A. Shahinian, K. Wiegmann, P. Ohashi, M. Kronke, T. Mak. 1993. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell 73:457.[Medline]

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