CD8 T cell populations restricted by H2-M3 MHC class Ib molecules expand rapidly during primary Listeria monocytogenes infection but only minimally upon reinfection. In contrast, CD8 T cells restricted by MHC class Ia molecules undergo more delayed expansion during primary infection but rapid and robust expansion following reinfection. In this study we demonstrate that primary H2-M3-restricted CD8 T cell responses are unaffected by the frequency of naive MHC class Ia-restricted T cells during L. monocytogenes infection. The magnitude of H2-M3-restricted memory responses, in contrast, is down-modulated by increasing frequencies of MHC class Ia-restricted effector T cells following secondary systemic infection. Suppression by MHC class Ia-restricted T cells, however, is not a universal feature of MHC class Ib-restricted memory responses. Primary systemic L. monocytogenes infection followed by secondary tissue infection, for example, results in robust expansion of H2-M3-restricted memory T cells in draining lymph nodes, despite the activation of MHC class Ia-restricted memory T cell responses. Thus, whereas MHC class Ia-restricted memory T cell populations predominate in spleens following systemic reinfection, H2-M3-restricted memory T cell responses remain prominent in lymph nodes draining localized infections. Our studies demonstrate that interactions between CD8 T cell populations can differ, depending on the status of the responding T cells (naive vs memory) and the route of reinfection. These results may have important implications for prime-boost vaccination strategies.

Pathogen -specific CD8 T lymphocyte populations are often complex. One level of complexity reflects T cell specificity for multiple pathogen-derived Ags and restriction by an array of MHC class Ia molecules. Following bacterial infections, responding CD8 T cell populations can also be subdivided on the basis of restriction by conventional MHC class Ia molecules or atypical MHC class Ib molecules (1). Even greater complexity is introduced by the broad repertoire of T cell receptors, each with distinct affinities for cognate MHC class I/peptide complexes, expressed on CD8 T cell populations specific for a particular epitope (2). How different pathogen-specific T cells relate to one another during infection is an important topic of investigation. Many studies support the concept that pathogen-specific T cells compete for Ag, APCs, lymphoid space, and cytokines (2, 3, 4). However, general rules for competition between T lymphocytes during immune responses to pathogens remain elusive.

In some circumstances, high-affinity T cells specific for dominant epitopes suppress the expansion of T cells specific for subdominant epitopes. Such competition is believed to be at the level Ag presentation by dendritic cells (DCs),3 and infusion of additional Ag-pulsed DCs can overcome suppression (4). In other circumstances, such as during the CD8 T cell response to lymphocytic choriomeningitis virus infection, increasing the number of naive, epitope-specific T cells suppressed the endogenous response to the same, but not heterologous viral epitopes (3). Why some T cells predominate and others fall behind is complex and influenced by multiple factors, including TCR affinity, TCR frequency, Ag prevalence, Ag persistence, and Ag-processing efficiency (2).

Although evidence for competition among MHC class Ia-restricted CD8 T cells has been demonstrated in lymphocytic choriomeningitis virus and following immunization with OVA (3, 4), in other systems competition has been less apparent. For example, deletion of immunodominant MHC class Ia-restricted epitopes from Listeria monocytogenes, an intracellular bacterium that is cleared from mice by CD8 T cells, did not increase the magnitude of T cell responses specific for subdominant epitopes (5). Along similar lines, the dominance hierarchy of four MHC class Ia-restricted CD8 T cell populations established during primary infection was maintained following reinfection, suggesting that immunodominant populations do not prevent the expansion of subdominant memory T cell populations (6). In contrast, within Ag-specific T cell populations, repeat infection results in a population of T cells with a narrower TCR repertoire and net higher affinity, suggesting that higher affinity T cells have a selective advantage (7, 8). In the setting of T cell affinity maturation, however, it remains unclear whether higher affinity T cells suppress lower affinity populations, or simply proliferate more rapidly or extensively than low-affinity T cells. Nevertheless, at least among MHC class Ia-restricted T cells, suppression of subdominant by dominant responses is not a substantial factor following L. monocytogenes infection.

Infection of mice with L. monocytogenes also primes H2-M3-restricted CD8 T cells (9, 10). H2-M3-restricted CD8 T cells expand more rapidly than MHC class Ia-restricted CD8 T cells during primary infection and give rise to memory T cells, which are activated but do not expand following secondary infection (11, 12). Diminished expansion of H2-M3-restricted memory T cells has been attributed to suppression by robust MHC class Ia-restricted memory T cell response (13). Thus, whereas dominant MHC class Ia-restricted memory T cells do not suppress subdominant MHC class Ia responses, they very effectively down-regulate expansion of H2-M3-restricted memory T cell populations. The reason for this disparity remains unclear.

To characterize the interactions between MHC class Ia- and H2-M3-restricted T cells during L. monocytogenes infection, we investigated the expansion of adoptively transferred, H2-M3-restricted TCR transgenic (tg) T cells during primary and recall infection. We find that naive MHC class Ia- and H2-M3-restricted do not suppress each other during primary infection with L. monocytogenes. In contrast, and consistent with a previous report (13), H2-M3-restricted memory T cells are suppressed by MHC class Ia-restricted memory responses. Although suppression by MHC class Ia-restricted T cells is a prominent feature of H2-M3-restricted T cells in the spleen, in the lymph node H2-M3-restricted T cells appear to be on equal footing with MHC class Ia-restricted T cells. Thus, competition between H2-M3- and MHC class Ia-restricted T cell subsets differs, depending on their anatomic location and their activation status.

L. monocytogenes strain 10403S was originally provided by Daniel Portnoy (University of California, Berkeley, CA), and the recombinant L. monocytogenes strain-expressing OVA (LmOVA) was provided by Hao Shen (University of Pennsylvania, Philadelphia, PA). Bacteria were grown in brain-heart infusion broth (BD Biosciences). Mice were immunized by i.v. injection with the indicated doses into the lateral tail vein or by s.c. injection into the hind footpad. Spleens and popliteal lymph nodes were harvested at the indicated time points after immunization or cell transfer. Bacterial numbers in spleen and lymph nodes were determined by homogenizing tissues in PBS containing 0.1% Triton X-100 and plating on brain-heart infusion agar plates. Splenocytes were dissociated through a wire mesh, and lymph nodes were macerated with frosted glass slides (Fisher Scientific). Erythrocytes were lysed with ammonium chloride, and splenocytes and lymph node cells were resuspended in RPMI 1640/10% FCS (Invitrogen Life Technologies).

C57BL/6, B6.PL, and C57BL/6-OT-1 tg mice were obtained from The Jackson Laboratory. C57BL/6-C10.4 TCR tg mice were kindly provided by Uwe Staerz (14). The OT-1 and C10.4 TCR tg lines were crossed onto the B6.PL background. Sex-matched animals were used in all of the experiments and maintained under specific pathogen-free conditions at the animal facilities of Memorial Sloan-Kettering Cancer Center.

Splenocyte suspensions from OT-1 or C10.4 TCR tg B6.PL mice were resuspended at 2 × 106 cells/ml in PBS and adoptively transferred into congenic C57BL/6 recipients by injection of 500 μl (1 × 106 cells/mouse, respectively) into the lateral tail vein. Before transfer, the naive surface phenotype of the T cells (CD25low, CD44int, CD62Lhigh, CD69low) was confirmed by flow cytometry.

DCs were generated by culturing C57BL/6 bone marrow cells in complete RPMI 1640 supplemented with 2% GM-CSF-containing culture supernatant from the P815 cell line (kindly provided by Lisa Denzin, Memorial Sloan-Kettering Cancer Center, New York, NY). Nonadherent cells were washed off after 2 days, and media were replaced every 2 days. After 5 days, DCs were matured with 100 ng/ml LPS (Sigma-Aldrich) and washed multiple times. Cells were then pulsed with 10−6 M peptide for 1 h at 37°C and washed. Before i.v. injection of 106 cells per mouse, the activated surface phenotype (CD40high, CD80high, CD86high, MHC class IIhigh) of DCs was confirmed by flow cytometry.

Peptides were synthesized by the Memorial Sloan-Kettering Cancer Center microchemistry core facility. The following mAbs directed to mouse cell surface Ags were purchased from BD Pharmingen: anti-CD8α-FITC, -PE, -PerCP, -APC (clone 53-6.7), anti-CD62L-FITC, -PE (MEL-14), anti-CD25-PE (PC61), anti-CD44-PE (IM7), anti-CD69-PE (H1.2F3), anti-CD127-PE (SB)/199), anti-Thy1.1-PerCP (OX-7), anti-Thy1.2-APC (53-2.1), anti-CD4-FITC (H129.19), anti-CD40-FITC (HM40-3), anti-CD80-FITC (16-10A1), anti-CD86-FITC (53-5.8), anti-H2-Kb-FITC (AF6-88.5), anti-I-Ab-FITC (AF6-120.1), anti-H2-M3 (130), anti-Armenian and Syrian hamster-IgG-PE (mixture) (G70-204, G94-90.5), anti-CD11c-APC (HL3), anti-CD11b-PerCP (M1/70), anti-Vβ5 TCR-FITC (MR9-4), anti-Vβ8 TCR-FITC (MR5-2). PE-conjugated streptavidin tetramers of H2-M3 complexed with fMIGWII, H2-Kd complexed with LLO91–99, and H2-Kb complexed with SIINFEKL peptide for detecting epitope-specific T cell populations were generated as described previously (15). For flow cytometric analysis, ∼5 × 106 cells were aliquoted per staining well of a 96-well plate. After incubation at 4°C for 20 min with unconjugated streptavidin (0.5 mg/ml; Molecular Probes) and Fc-block (BD Pharmingen) in FACS SB (PBS (pH 7.4), 0.5% BSA, and 0.02% sodium azide), cells were incubated for 30 min (1 h for stainings including tetramers) with saturating concentrations of the various mAbs and tetramers. Labeled cells were washed with SB and analyzed on a BD-LSR flow cytometer (BD Biosciences) using CellQuest (BD Biosciences) and FlowJo software (Tree Star).

Cells were washed three times with PBS and resuspended at 2 × 107 cells/ml in PBS containing 4 μM CFSE (Molecular Probes). After incubation at 37°C for 10 min in the dark, cells were immediately washed with cold RPMI 1640/10% FCS and counted before resuspension in PBS and i.v. injection into mice.

Analysis of in vivo cytolytic activity was conducted as described previously (16). C57BL/6 splenocytes were divided into two populations and labeled with either a high concentration (4 μM) or a low concentration (0.25 μM) of CFSE. Next, CFSEhigh cells were pulsed as indicated with 10−6 M fMIVTLF or SIINFEKL peptide for 1 h at 37°C in the dark, whereas CFSElow cells remained nonpulsed. After washing, CFSElow cells were mixed with equal numbers of CFSEhigh cells before injecting 2 × 107 of this cell suspension per mouse. Recipient spleens were taken 15 h later for flow cytometric analysis to measure in vivo killing as indicated by loss of the peptide-pulsed population(s) relative to the control population. The percentage-specific lysis was calculated according to the formula: (1 − (ratio unprimed/ratio primed) × 100), where the ratio unprimed = the percentage of peptide-nonpulsed/the percentage of peptide-pulsed cells remaining in noninfected recipients, and the ratio unprimed = the percentage of peptide-nonpulsed/the percentage of peptide-pulsed cells remaining in infected recipients.

Although the magnitude of MHC class Ia-restricted T cell responses following L. monocytogenes infection is very predictable, the magnitude of H2-M3-restricted T cell responses is more variable. To improve the reproducibility of H2-M3-restricted T cell responses we adoptively transferred naive C10.4 TCR tg T cells, which are specific for L. monocytogenes-derived fMIVTLF presented by H2-M3, into Thy 1.2 congenic recipient mice before infection (14). C10.4 T cells were tracked with anti-Thy1.1 Abs. Mice were infected with a strain of OVA-expressing L. monocytogenes, LmOVA, and CD8 T cells specific for the immunodominant SIINFEKL epitope were stained with H2-Kb Ova257–264 tetramers (17). To determine whether adoptively transferred C10.4 T cells expand and contract with kinetics similar to endogenous H2-M3-restricted T cells, we infected recipient mice with a sublethal dose of LmOVA and measured C10.4 and SIINFEKL-specific T cell expansion on sequential days following bacterial inoculation. Similar to endogenous H2-M3-restricted T cells (11), adoptively transferred C10.4 TCR tg T cells reached peak frequencies 5 days following bacterial inoculation. In contrast, H2-Kb/SIINFEKL-specific T cell populations reached maximum size 7 days after primary LmOVA infection (Fig. 1). H2-M3- and H2-Kb-restricted T cells both give rise to long-term memory populations. Although MHC class Ia-restricted T cells undergo marked expansion 5 days after re-infection with LmOVA, H2-M3-restricted CD8+ T cells do not (Fig. 1). These results demonstrate that adoptively transferred C10.4 CD8 T cells recapitulate the responses of endogenous H2-M3-restricted T cells.

FIGURE 1.

Diminished secondary expansion of H2-M3-restricted CD8+ T cells following L. monocytogenes reinfection. Splenocytes from C10.4 B6.PL TCR tg mice were adoptively transferred into congenic C57BL/6 recipients. Recipients were infected 24 h later with 5000 LmOVA and C10.4 and SIINFEKL-specific CD8+ T cells were quantified in the spleen, either prior to infection, or 5, 7, and 21 days later, as indicated. Three weeks after primary infection, mice were rechallenged with 1 × 105 LmOVA, and memory expansion was analyzed 5 days later (21 + 5). C10.4 T cells were detected by staining for Thy1.1, and OVA-specific T cells were detected by H2-Kb/SIINFEKL tetramer staining. Plots are gated on live CD8 T cells and are representative of three mice per time point. The percentage of total CD8 T cells is indicated in the CD62L-negative upper quadrant.

FIGURE 1.

Diminished secondary expansion of H2-M3-restricted CD8+ T cells following L. monocytogenes reinfection. Splenocytes from C10.4 B6.PL TCR tg mice were adoptively transferred into congenic C57BL/6 recipients. Recipients were infected 24 h later with 5000 LmOVA and C10.4 and SIINFEKL-specific CD8+ T cells were quantified in the spleen, either prior to infection, or 5, 7, and 21 days later, as indicated. Three weeks after primary infection, mice were rechallenged with 1 × 105 LmOVA, and memory expansion was analyzed 5 days later (21 + 5). C10.4 T cells were detected by staining for Thy1.1, and OVA-specific T cells were detected by H2-Kb/SIINFEKL tetramer staining. Plots are gated on live CD8 T cells and are representative of three mice per time point. The percentage of total CD8 T cells is indicated in the CD62L-negative upper quadrant.

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The distinct expansion kinetics of H2-M3- and MHC class Ia-restricted T cells during primary L. monocytogenes infection may result from differences intrinsic to the T cell populations, or may result from differences in the presentation of these Ags. In vivo priming of MHC class Ia- and H2-M3-restricted CD8 T cells following L. monocytogenes infection requires CD11c-expressing DCs (18). We decided to determine whether the in vivo expansion kinetics of H2-M3- and H2-Kb-restricted T cells differed when immunization was performed with peptide-coated DCs (Fig. 2). We found that peptide-coated DCs efficiently prime H2-Kb- and H2-M3-restricted T cells and result in peak populations of effector T cells 5 days following immunization. In contrast to immunization with DCs, infection with LmOVA resulted in delayed H2-Kb-restricted T cell responses. The distinct expansion kinetics of CD8 T cells following primary immunization with LmOVA vs peptide-pulsed DCs might reflect a lag in the presentation of Ag following bacterial infection, which, at early time points, involves low numbers of bacteria that may have to replicate in vivo to exceed threshold quantities of Ag required for T cell priming. These results suggest, however, that presentation of MHC class Ia-restricted epitopes requires greater in vivo bacterial growth than presentation of H2-M3-restricted epitopes. Furthermore, this experiment suggests that the respective primary expansion kinetics of H2-Kb and H2-M3-restricted CD8 T cells do not result from differences intrinsic to the T cells, but rather reflect differences in the presentation of SIINFEKL and fMIVTLF following infection with LmOVA.

FIGURE 2.

Distinct expansion of H2-M3- and H2-Kb-restricted CD8+ T cells following primary immunization with LmOVA vs peptide-pulsed DCs. A total of 1 × 105 splenocytes from C10.4 B6.PL TCR tg mice was transferred into congenic C57BL/6 recipients. Recipients were immunized with 5000 LmOVA or 1 × 106 LPS-activated bone marrow-derived DCs pulsed with 1 × 10−6 M fMIVTLF or SIINFEKL peptide. Primary expansion of H2-M3- and H2-Kb-restricted CD8+ T cells was assessed at the indicated time points. Representative plots from groups of three mice are gated on live CD8 T cells, and C10.4 T cells are detected by Thy1.1 staining and OVA-specific T cells by tetramer staining. The percentages of total CD8 T cells in the upper quadrants are indicated.

FIGURE 2.

Distinct expansion of H2-M3- and H2-Kb-restricted CD8+ T cells following primary immunization with LmOVA vs peptide-pulsed DCs. A total of 1 × 105 splenocytes from C10.4 B6.PL TCR tg mice was transferred into congenic C57BL/6 recipients. Recipients were immunized with 5000 LmOVA or 1 × 106 LPS-activated bone marrow-derived DCs pulsed with 1 × 10−6 M fMIVTLF or SIINFEKL peptide. Primary expansion of H2-M3- and H2-Kb-restricted CD8+ T cells was assessed at the indicated time points. Representative plots from groups of three mice are gated on live CD8 T cells, and C10.4 T cells are detected by Thy1.1 staining and OVA-specific T cells by tetramer staining. The percentages of total CD8 T cells in the upper quadrants are indicated.

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CD8 T cells are postulated to compete for priming following infection with an antigenically complex pathogen (2). It is unclear, however, whether H2-M3- and MHC class Ia-restricted T cells compete during the primary immune response to L. monocytogenes infection. To examine this issue, we altered the precursor frequency of naive H2-M3 and SIINFEKL-specific CD8 T cells in mice before infection by adoptively transferring TCR tg T cells into recipient mice. Recipient mice were then either immunized with peptide-coated DCs or LmOVA, and the responding T cell populations were measured. Transfer of 105 naive, SIINFEKL-specific OT-1 cells into recipient mice did not diminish the proliferation of C10.4 T cells following immunization with DC coated with fMIVTLF, fMIVTLF plus SIINFEKL, or LmOVA (Fig. 3,A). The presence of OT-1 cells did not diminish the number of responding H2-M3-restricted CD8 T cells (Fig. 3,B), despite the massive expansion of SIINFEKL-specific T cells (Fig. 3,C). Similarly, expansion of H2-Kb:SIINFEKL-specific T cells (Fig. 3,D) is not affected by increased frequencies of naive fMIVTLF-specific T cells (Fig. 3 E). These experiments demonstrate that naive H2-M3- and H2-Kb-restricted CD8+ T cells do not inhibit each other during the primary response to L. monocytogenes infection. Thus, competition does not appear to be a significant factor during priming of naive T cells or their subsequent expansion.

FIGURE 3.

Naive H2-M3-restricted CD8+ T cells successfully compete with naive MHC class Ia-restricted CD8+ T cells for priming. A, CFSE-labeled C10.4 TCR tg splenocytes (1 × 105) were transferred into recipient mice that had either not received (top row) or received (bottom row) 1 × 105 naive OT-1 T cells. Mice were then immunized with DCs without peptide or peptide-pulsed DCs (fMIVTLF, or fMIVTLF and SIINFEKL) or infected with 5000 LmOVA, as indicated above the histograms, and the proliferation of C10.4 T cells in the spleen, as measured by CFSE dilution, was analyzed by flow cytometry. Histograms are gated on Thy1.1-expressing C10.4 T cells. Representative plots from groups of three mice per condition are shown. The total number of C10.4 T cells (B) and OT-1 T cells (C) following DC or LmOVA immunization are shown. The results represent the mean of three mice per group. The impact of 1 × 105 adoptively transferred, naive C10.4 T cells on primary SIINFEKL-specific T cell responses following immunization with 5000 LmOVA is shown in D. Panel E shows the number of responding C10.4 cells.

FIGURE 3.

Naive H2-M3-restricted CD8+ T cells successfully compete with naive MHC class Ia-restricted CD8+ T cells for priming. A, CFSE-labeled C10.4 TCR tg splenocytes (1 × 105) were transferred into recipient mice that had either not received (top row) or received (bottom row) 1 × 105 naive OT-1 T cells. Mice were then immunized with DCs without peptide or peptide-pulsed DCs (fMIVTLF, or fMIVTLF and SIINFEKL) or infected with 5000 LmOVA, as indicated above the histograms, and the proliferation of C10.4 T cells in the spleen, as measured by CFSE dilution, was analyzed by flow cytometry. Histograms are gated on Thy1.1-expressing C10.4 T cells. Representative plots from groups of three mice per condition are shown. The total number of C10.4 T cells (B) and OT-1 T cells (C) following DC or LmOVA immunization are shown. The results represent the mean of three mice per group. The impact of 1 × 105 adoptively transferred, naive C10.4 T cells on primary SIINFEKL-specific T cell responses following immunization with 5000 LmOVA is shown in D. Panel E shows the number of responding C10.4 cells.

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Previous studies have suggested that memory H2-M3-restricted T cell responses are suppressed by memory MHC class Ia-restricted T cells (13). To confirm this finding with H2-M3-restricted C10.4 TCR tg T cells, we performed adoptive transfer experiments and immunized mice sequentially with LmOVA and peptide-coated DCs, as outlined in Fig. 4,A. To test whether C10.4 T cells are capable of re-expanding in vivo after secondary Ag encounter, we first primed mice with LmOVA or fMIVTLF or SIINFEKL-coated DCs. Three weeks after priming, immunized mice were infected with LmOVA or immunized with peptide-coated DCs and C10.4, and SIINFEKL-specific T cell responses were measured 5 days later (Fig. 4). SIINFEKL-specific memory T cells expanded irrespective of whether mice were initially immunized with LmOVA or SIINFEKL-pulsed DCs. In contrast, C10.4 memory T cells only expanded in the absence of a MHC class Ia-restricted memory response, i.e., after fMIVTLF/DC immunization followed by LmOVA infection. To determine whether fMIVTLF/DC immunization primes H2-M3-restricted memory T cell populations with enhanced proliferative potential, we primed mice with LmOVA and then immunized mice with fMIVTLF-coated DCs. fMIVTLF-specific memory T cells expanded following DC immunization, demonstrating that H2-M3-restricted C10.4 memory T cells induced by L. monocytogenes infection can undergo memory expansion. H2-M3-restricted memory T cell expansion, however, appears to be suppressed by MHC class Ia-restricted CD8 T cell responses.

FIGURE 4.

H2-M3-restricted memory expansion is restored in the absence of a MHC class Ia-restricted CD8+ T cell response. A total of 1 × 106 splenocytes from C10.4 B6.PL TCR tg mice was transferred into congenic recipients. Mice were immunized i.v. with peptide-pulsed DCs or 5000 LmOVA and rechallenged with peptide-pulsed DCs or 100,000 LmOVA as specified in A. The total numbers of H2-M3- and H2-Kb-restricted primary effector T cells (E1) on day 7, memory T cells (M) on day 21, and secondary effector T cells (E2) on day 5 post-rechallenge were determined in the spleen, and the ratios of E2:E1 and E2:M were calculated (A). H2-M3-restricted C10.4 T cell responses (B) and SIINFEKL-specific responses (C) are shown following the immunization strategies described in A. Representative plots from groups of three mice are shown and are stained and gated as described in the legend to Figure 1.

FIGURE 4.

H2-M3-restricted memory expansion is restored in the absence of a MHC class Ia-restricted CD8+ T cell response. A total of 1 × 106 splenocytes from C10.4 B6.PL TCR tg mice was transferred into congenic recipients. Mice were immunized i.v. with peptide-pulsed DCs or 5000 LmOVA and rechallenged with peptide-pulsed DCs or 100,000 LmOVA as specified in A. The total numbers of H2-M3- and H2-Kb-restricted primary effector T cells (E1) on day 7, memory T cells (M) on day 21, and secondary effector T cells (E2) on day 5 post-rechallenge were determined in the spleen, and the ratios of E2:E1 and E2:M were calculated (A). H2-M3-restricted C10.4 T cell responses (B) and SIINFEKL-specific responses (C) are shown following the immunization strategies described in A. Representative plots from groups of three mice are shown and are stained and gated as described in the legend to Figure 1.

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To further characterize suppression of H2-M3-restricted memory T cell responses by memory MHC class Ia-restricted CD8 T cell responses, mice were immunized with LmOVA and then reimmunized with DCs pulsed with fMIVTLF alone or both fMIVTLF and SIINFEKL (Fig. 5). fMIVTLF-specific memory CD8+ T cells expanded following immunization with fMIVTLF-coated DCs. The activation of H2-Kb-restricted memory T cells by immunization with SIINFEKL and fMIVTLF-coated DCs significantly decreased the expansion of fMIVTLF-specific T cells. These data confirm the findings of Harty and colleagues (13) and further support the conclusion that activated MHC class Ia-restricted effector T cells inhibit secondary expansion of H2-M3-restricted memory T cells.

FIGURE 5.

Competition between H2-M3- and MHC class Ia-restricted memory T cells. A total of 1 × 106 C10.4 splenocytes was adoptively transferred into congenic C57BL/6 recipients. Recipient mice were infected with 5000 LmOVA, and 3 wk later mice were immunized i.v. with DCs in the absence of Ag, or DCs pulsed with fMIVTLF or fMIVTLF plus SIINFEKL. Memory expansion of C10.4 T cells and SIINFEKL-specific T cells was analyzed 5 days later. The percentage of total CD8 T cells specific for SIINFEKL or fMIVTLF is indicated. Each bar represents the mean and SD of three mice per group.

FIGURE 5.

Competition between H2-M3- and MHC class Ia-restricted memory T cells. A total of 1 × 106 C10.4 splenocytes was adoptively transferred into congenic C57BL/6 recipients. Recipient mice were infected with 5000 LmOVA, and 3 wk later mice were immunized i.v. with DCs in the absence of Ag, or DCs pulsed with fMIVTLF or fMIVTLF plus SIINFEKL. Memory expansion of C10.4 T cells and SIINFEKL-specific T cells was analyzed 5 days later. The percentage of total CD8 T cells specific for SIINFEKL or fMIVTLF is indicated. Each bar represents the mean and SD of three mice per group.

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Previous studies have suggested that H2-M3-restricted memory T cells have diminished effector functions (13). In contrast, H2-M3-restricted T cells contribute to protection against secondary L. monocytogenes infection (12, 19). To investigate the in vivo cytolytic activity of H2-M3-restricted memory T cells, we immunized mice with LmOVA or fMIVTLF-pulsed DCs, rechallenged with LmOVA, and then measured in vivo epitope-specific cytolytic activity 3 and 5 days later. Although very little if any fMIVTLF-specific in vivo lysis was detected 30 days after priming with DC or LmOVA, 3 days after rechallenge with LmOVA in vivo cytolytic activity increased markedly in primed but not unprimed mice (Fig. 6). This result demonstrates that cytolytic effector function of H2-M3-restricted memory cells can be induced by reinfection. Because rechallenge with LmOVA induces expansion of fMIVTLF-specific T cells in DC, but not LmOVA primed mice, in vivo cytolytic activity is greater in DC-primed mice (Fig. 6, middle panels).

FIGURE 6.

H2-M3-restricted memory T cells are cytolytic and contribute to the elimination of Ag-bearing cells. C57BL/6 mice were immunized with 1 × 106 DCs pulsed with fMIVTLF peptide, infected with 5000 LmOVA, or left untreated. Thirty days later, 107 CFSEhigh, fMIVTLF peptide-pulsed splenocytes, and 107 CFSElow, nonpulsed C57BL/6 splenocytes were coinjected into recipients that were uninfected (left plots), or rechallenged with 1 × 106 LmOVA either three days (middle plots) or 5 days (right plots) prior to target cell infusion. In vivo cytolysis of target cells was assessed 18 h after their infusion. Histograms are gated on CFSE+ splenocytes in recipient mice. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of total CFSE+ donor cells recovered.

FIGURE 6.

H2-M3-restricted memory T cells are cytolytic and contribute to the elimination of Ag-bearing cells. C57BL/6 mice were immunized with 1 × 106 DCs pulsed with fMIVTLF peptide, infected with 5000 LmOVA, or left untreated. Thirty days later, 107 CFSEhigh, fMIVTLF peptide-pulsed splenocytes, and 107 CFSElow, nonpulsed C57BL/6 splenocytes were coinjected into recipients that were uninfected (left plots), or rechallenged with 1 × 106 LmOVA either three days (middle plots) or 5 days (right plots) prior to target cell infusion. In vivo cytolysis of target cells was assessed 18 h after their infusion. Histograms are gated on CFSE+ splenocytes in recipient mice. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of total CFSE+ donor cells recovered.

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Most studies of primary and memory CD8 T cell responses to L. monocytogenes infection have focused on the spleen. Because DC populations in spleen and lymph nodes differ (20), we decided to investigate memory CD8 T cell responses in lymph nodes following s.c. infection with L. monocytogenes. As a first step, we characterized the localization of naive, CFSE-labeled C10.4 and OT-1 T cells 24 h following adoptive transfer in lymph nodes (Fig. 7,A, upper panels). Both T cell populations trafficked similarly to lymph nodes and spleen. Five days following i.v. infection, C10.4 and OT-1 T cells had proliferated, as determined by CFSE dilution, and, in the spleen, down-regulated CD62L expression. Although i.v. infection primed T cells in the lymph node, a larger proportion than in the spleen remained unactivated. s.c. infection of the footpad also primed naive C10.4 and OT-1 T cells in the draining lymph node. Quantitative cultures revealed that footpad infection with L. monocytogenes does not result in detectable dissemination to spleen (Fig. 7 B), suggesting that activated T cells in the spleen following s.c. infection trafficked there from the draining lymph node. The majority of T cells proliferating in lymph nodes expressed intermediate to high levels of CD62L, whereas most C10.4 and OT-1 cells in the spleen had down-regulated CD62L, irrespective of the route of infection.

FIGURE 7.

Primary expansion of H2-M3- and H2-Kb-restricted CD8+ T cells 5 days after i.v. or s.c. LmOVA infection. A, Adoptive transfer of CFSE-labeled splenocytes (1 × 106) from C10.4 B6.PL or OT-1 B6.PL TCR tg mice into congenic mice. The recipients were injected with PBS (noninfected), or infected either i.v. with 5000 LmOVA or s.c. in the footpad with 1 × 106 LmOVA. Spleens and popliteal lymph nodes were harvested 5 days later, and the expansion of adoptively transferred CD8+ T cells was assessed by CFSE dilution plotted on the horizontal axis. The dot plots are gated on Thy1.1+ CD8+ T cells and were normalized to 1000 events per plot. The numbers in the quadrants indicated the percentage of CD8+ T cells. Representative plots from three mice per group are shown. B, The number of bacteria cultured from draining lymph nodes and spleen at the indicated times following footpad inoculation with L. monocytogenes.

FIGURE 7.

Primary expansion of H2-M3- and H2-Kb-restricted CD8+ T cells 5 days after i.v. or s.c. LmOVA infection. A, Adoptive transfer of CFSE-labeled splenocytes (1 × 106) from C10.4 B6.PL or OT-1 B6.PL TCR tg mice into congenic mice. The recipients were injected with PBS (noninfected), or infected either i.v. with 5000 LmOVA or s.c. in the footpad with 1 × 106 LmOVA. Spleens and popliteal lymph nodes were harvested 5 days later, and the expansion of adoptively transferred CD8+ T cells was assessed by CFSE dilution plotted on the horizontal axis. The dot plots are gated on Thy1.1+ CD8+ T cells and were normalized to 1000 events per plot. The numbers in the quadrants indicated the percentage of CD8+ T cells. Representative plots from three mice per group are shown. B, The number of bacteria cultured from draining lymph nodes and spleen at the indicated times following footpad inoculation with L. monocytogenes.

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To determine whether H2-M3-restricted memory CD8 T cells undergo expansion in lymph nodes, we primed mice by i.v. infection with LmOVA and then re-challenged them s.c. The s.c. rechallenge of mice resulted in a dramatic expansion of C10.4 T cells in draining lymph nodes, whereas rechallenge with i.v. bacteria did not (Fig. 8 A). Of note, primary s.c. infection of naive mice resulted is substantially smaller C10.4 T cell responses in the draining node, indicating that s.c. challenge of i.v.-primed mice resulted in a memory T cell response in the draining lymph node. The i.v. infection with LmOVA also did not result in the expansion of SIINFEKL-specific memory T cells in the lymph node, whereas s.c. reinfection did result in the expansion of SIINFEKL-specific T cells in the draining lymph node. These results demonstrate that suppression of H2-M3-restricted memory T cells by MHC class Ia-restricted T cells differs between spleen and lymph nodes.

FIGURE 8.

Expansion of H2-M3-restricted memory CD8+ T cells in draining lymph nodes following footpad immunization with L. monocytogenes. Splenocytes (1 × 106) from C10.4 B6.PL TCR tg mice were transferred into C57BL/6 mice. Twenty-four hours post-transfer, recipient mice were immunized i.v. with 5000 LmOVA, and 27 days later were reinfected either s.c. in the footpad with 1 × 106 LmOVA or i.v. with 1 × 105 LmOVA. Spleens and popliteal lymph nodes were harvested 5 days later, and the expansion of H2-M3:fMIVTLF- and H2-Kb:SIINFEKL-specific CD8+ T cells was assessed by FACS analysis. Total number of H2-M3:fMIVTLF- and H2-Kb:SIINFEKL-specific CD8+ T cells in popliteal lymph nodes (A) and spleens (B) of memory mice 5 days following secondary LmOVA infection are plotted. The values represent the mean and SD of three mice per group.

FIGURE 8.

Expansion of H2-M3-restricted memory CD8+ T cells in draining lymph nodes following footpad immunization with L. monocytogenes. Splenocytes (1 × 106) from C10.4 B6.PL TCR tg mice were transferred into C57BL/6 mice. Twenty-four hours post-transfer, recipient mice were immunized i.v. with 5000 LmOVA, and 27 days later were reinfected either s.c. in the footpad with 1 × 106 LmOVA or i.v. with 1 × 105 LmOVA. Spleens and popliteal lymph nodes were harvested 5 days later, and the expansion of H2-M3:fMIVTLF- and H2-Kb:SIINFEKL-specific CD8+ T cells was assessed by FACS analysis. Total number of H2-M3:fMIVTLF- and H2-Kb:SIINFEKL-specific CD8+ T cells in popliteal lymph nodes (A) and spleens (B) of memory mice 5 days following secondary LmOVA infection are plotted. The values represent the mean and SD of three mice per group.

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The distinct kinetics of H2-M3- and MHC class Ia-restricted CD8 T cell expansion during primary and secondary L. monocytogenes infection remain puzzling. In this report, we confirm previous studies showing suppression of H2-M3-restricted memory responses by MHC class Ia-restricted effector T cells (13), and further demonstrate that this internecine suppression is not a feature of primary immune responses to L. monocytogenes infection. Analysis of T cell responses in different anatomic sites revealed that suppression of H2-M3-restricted memory T cell expansion, while a prominent feature in spleen, does not occur in draining lymph nodes following tissue infection with L. monocytogenes. The disparities in H2-M3-restricted memory T cell responses in lymph node vs spleen, and the restriction of H2-M3-restricted T cell suppression to memory but not naive cells may provide some insights into the mechanisms that account for the kinetic differences between these two CD8 T cell responses.

Previous studies demonstrated that priming of naive CD8 T cells following L. monocytogenes infection occurs during the first 1–2 days of infection, and that primed T cells undergo proliferation and differentiation independent of ongoing infection (21, 22, 23). Priming of MHC class Ia-restricted T cells is accelerated by immunization with peptide-coated DCs, with peak expansion occurring 5 days following DC inoculation, as opposed to 7 days following L. monocytogenes infection (Fig. 2). The kinetics of H2-M3-restricted T cell expansion, in contrast, are similar following L. monocytogenes infection or immunization with peptide-coated DCs. Although this result suggests that bacterial formyl-methionine peptides are presented in immunologically relevant quantities more rapidly than peptides bound by MHC class Ia molecules, it also indicates that H2-M3-restricted T cells have a head start during primary infection, potentially making them, at least for a few days, less vulnerable to inhibition by MHC class Ia-restricted T cells. The relatively delayed activation of MHC class Ia-restricted T cells might explain the absence of suppression of H2-M3-restricted T cells by increased numbers of naive OT-1 cells during primary L. monocytogenes infection. Interestingly, H2-M3-restricted T cells cease expansion 5 days following primary infection, at the time when frequencies of activated MHC class Ia-restricted T cells increase to levels detectable by MHC tetramer staining or ELISPOT assays (6, 24). It is possible, therefore, that expanding MHC class Ia-restricted T cell populations down-regulate further expansion of H2-M3-restricted T cell populations.

Careful studies have demonstrated that MHC class Ia-restricted T cells suppress H2-M3-restricted memory T cell responses in vivo (13). Using adoptively transferred C10.4 T cells, we demonstrate similar inhibition by MHC class Ia-restricted T cells and also show that H2-M3-restricted memory T cells are capable of expanding in the absence of activated MHC class Ia-restricted T cells. Although these dynamics are a consistent feature of CD8 T cell responses in spleens of systemically reinfected mice, we find that the interactions between memory MHC class Ia- and H2-M3-restricted T cells are distinct in draining lymph nodes following s.c. L. monocytogenes infection (Fig. 8). Although it remains unclear why H2-M3-restricted memory T cell responses are robust in lymph nodes and not in spleen, we can propose several possible scenarios. The first possibility is that differences in splenic and lymph node T cell populations account for this disparity. Recent studies indicate that central memory T cells, which are more prevalent in lymph nodes than spleen, are less cytotoxic than effector memory T cells, which are more prevalent in spleen (25). Thus, it is possible that APCs are destroyed more rapidly in spleen than in lymph nodes, limiting the activation of memory H2-M3-restricted T cells. This model, would require the activation of MHC class Ia-restricted memory T cells to be less dependent on the duration of Ag presentation than H2-M3-restricted memory T cells. Although we are unaware of enhanced survival of lymph node APCs relative to splenic APCs, studies of the duration of in vivo Ag presentation in lymph nodes (26) suggest that it is more prolonged than in the spleen (16, 27). A second explanation for enhanced H2-M3-restricted memory T cells in lymph nodes is that Ag may be delivered from infected s.c. tissues for a longer period of time, thus enabling more prolonged or repeated stimulation of H2-M3-restricted T cells. Analysis of Ag delivery from s.c. tissues to draining lymph nodes, indeed, has demonstrated that successive waves of APCs provide distinct stimuli to responding T cells (28). Thus, it is possible that repeated presentation of H2-M3-restricted peptides by different APCs following s.c. infection provides a superior stimulus to H2-M3-restricted memory T cells and drives them to proliferate.

Regardless of the mechanism, our results provide a new picture of complex memory T cell responses to reinfection via distinct routes. The finding that systemic immunization followed by localized immunization provides superior expansion of an otherwise largely unresponsive CD8 T cell population suggests that routes of reimmunization may be of greater importance than commonly appreciated. Our findings suggest that prime-boost immunization strategies might be enhanced by sequential systemic and local administration of vaccines.

We thank Ewa Menet, An Tran, and Maggie Zhong for excellent technical support, and members of the laboratory for insightful discussions.

The authors have no financial conflict of interest.

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

This work was supported by National Institutes of Health Grant AI49602 and by a Cancer Research Institute predoctoral fellowship (to A.P.).

3

Abbreviations used in this paper: DC, dendritic cell; tg, transgenic; int, intermediate; SB, staining buffer.

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