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The Journal of Immunology, 2003, 171: 2832-2839.
Copyright © 2003 by The American Association of Immunologists

Sustained Response Initiation Is Required for T Cell Clonal Expansion But Not for Effector or Memory Development In Vivo 1

Leo Lefrançois2, Amanda Marzo and Kristina Williams

Department of Medicine, Division of Immunology, University of Connecticut Health Center, Farmington, CT 06030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The factors determining whether an immune response is productive are poorly understood. To understand the circumstances affecting the early stage of the immune response which determine whether memory is generated, the CD8 T cell response was mapped in detail following immunization with live or heat-killed bacteria. Our results demonstrate that even in response to a weak immunogen, functional memory cell development is linked to effector cell induction in lymphoid and nonlymphoid tissues. The main defect in the response to killed microorganisms is inefficient induction of clonal expansion. This failure is due to a contracted, but costimulation-dependent activation phase in the lymphoid tissues, resulting in rapid but abortive growth. Conversely, the response to live bacteria is characterized by protracted early T cell sequestration in lymphoid tissues. Thus, memory development requires effector induction, while optimal clonal expansion is regulated by the duration of response initiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The goal of vaccination is to generate sufficient long-term immunological memory to provide protection upon encounter with an infectious organism. Thus, effective vaccination results in development of memory T and B cells that are capable of rapidly responding to infection and, given sufficient numbers, can provide sterilizing immunity (1, 2, 3). There are two types of memory T cells now recognized based on their anatomic location and the level of effector function detectable (4, 5, 6, 7). Central memory cells reside in lymphoid tissues while effector memory cells are found in nonlymphoid tissues such as the lung and intestinal lamina propria (5, 6). Central memory cells can rapidly acquire effector function upon Ag encounter while effector memory cells exert immediate effector activity in response to Ag.

The identification of memory T cell subsets in vivo fostered reexamination of the origin and lineage relationships of effector and memory T cells. A longstanding question has been whether memory T cells are invariably derived from effector cells or are derived from a second lineage that bypasses the effector stage (8, 9, 10, 11). Since nonlymphoid CD8 memory T cells exhibit lytic activity identical to that of primary effectors (6, 12), it follows that such memory cells are derived from effector cells. Conversely, central memory cells may be derived from activated cells that do not traverse an effector phase (3, 12). However, the inability to precisely identify activated T cells destined to become memory cells in vivo has not allowed resolution of this question. In the case of CD8 T cells, there is reasonable evidence derived from in vitro and in vivo studies that memory cells are derived from effector cells. Brief optimal activation of CD8 T cells results in programmed, Ag-independent expansion and induction of effector function (13, 14, 15, 16). In addition, transfer of effector cells in vivo results in development of CD4 and CD8 memory T cells, further supporting the concept that memory cells are derived from effector cells (17, 18, 19). Recent analysis of gene expression in effector and memory cells further suggests that memory cells undergo gradual development from the effector population (19). It should also be noted that the generation of memory cells does not always follow effector cell induction. Thus, in vivo activation of CD8 T cells in the absence of Th activity results in substantial effector cell development without apparent memory cell production (20) or may result in functionally defective " lethargic" cells (21).

Evidence also exists showing that in some cases the development of memory T cells does not require prior effector cell generation. In vitro activation of CD8 T cells in the presence of IL-15 does not induce effector function but transfer of such cells in vivo results in memory T cell development (22). Although IL-15 is required for CD8 memory cell homeostasis in vivo (23, 24), whether it participates in memory cell lineage decisions remains to be seen. In the case of CD4 T cells, isolation and transfer of in vitro-derived effector cells does not generate memory while transfer of noneffectors resulted in memory development (25). Generation of effector and memory CD4 T cells also appears to be much more heterogeneous than for CD8 T cells (26, 27, 28), underscoring the possibility that distinct rules may apply for memory cell production in each subset.

The context in which the immunogen is recognized also regulates the efficiency of effector cell generation and memory cell production (29). Infection with viruses and bacteria, which induce substantial inflammation, generates large populations of effector and memory cells (30, 31, 32, 33), while immunization with protein Ags or killed organisms, particularly in the absence of adjuvants, generally results in poor protection and deletion or anergy of T cells (3, 34, 35, 36, 37, 38). Thus, understanding why attenuated vaccines are generally ineffective will provide rationales for augmentation of particular stages of the immune response. Recently, it has been proposed that vaccination with heat-killed Listeria monocytogenes (HKLM) 3 does not induce CD8 effector cells while apparently generating a significant memory cell population, suggesting that memory cells are not derived from effector cells in this system (39). Such a model would allow determination of the factors required in vivo for driving effector vs memory T cell induction. We have now used this system to compare the requirements for generation of Ag-specific effector and memory CD4 and CD8 T cells in lymphoid and nonlymphoid tissues in response to heat killed (HK) or live Listeria monocytogenes (LM). The results indicated that optimal clonal expansion observed with infection was characterized by sustained T cell residence in the lymphoid tissue as compared with a contracted initiation phase in the response to HKLM. Nevertheless, the limited clonal expansion observed in response to HKLM resulted in memory T cell development that was linked to effector cell induction in the primary response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6J and CB6F1 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The OT-I mouse line (40) was generously provided by Dr. W. R. Heath (WEHI, Parkville, Australia) and Dr. F. Carbone (Monash Medical School, Prahan, Victoria, Australia) and was maintained as a C57BL/6-Ly5.2 line on a RAG-/- background.

Infection with rLM-OVA

A strain of recombinant LM-producing OVA (rLM-OVA) was produced as previously described using a truncated OVA cDNA fused to a signal sequence to allow secretion of OVA (28). Mice were infected i.v. with 1 x 103 CFU rLM-OVA or 1 x 109 HKLM-OVA for primary infections or with 1 x 104 CFU for recall responses. HKLM was produced by incubating bacteria from log phase cultures at 70°C for 3 h. Efficiency of killing was assessed by plating undiluted preparations on brain-heart infusion agar for 48 h at 37°C. Bacterial titers within tissues were determined by homogenizing the tissue in PBS containing 1% saponin, plating serial dilutions of homogenate on brain-heart infusion agar containing 5 µg/ml erythromycin and incubating for 2 days at 37°C.

Isolation of lymphocyte populations

Single-cell suspensions were prepared from lymph nodes and spleens using a cell strainer (BD Labware, Franklin Lakes, NJ). Lymphocytes were isolated from small intestine lamina propria and liver as previously described (6, 41). To obtain lymphocytes from lungs, anesthetized mice were perfused with PBS containing 75 U/ml heparin until the lungs were cleared of blood and white in color. The lungs were removed and cut into small pieces and stirred at 37°C for 30 min in HBSS containing 1.3 mM EDTA. Lymphocytes were released from the tissue by digestion with 150 U/ml collagenase (Life Technologies, Grand Island, NY) in RPMI 1640 containing 1 mM MgCl2, 1 mM CaCl2, and 5% FCS at 37°C for 30 min. Released cells were pooled and then mashed through a cell strainer (BD Labware).

Immunofluorescence analysis

At the indicated times after infection, lymphocytes were isolated and OVA-specific CD8 T cells were detected using an H-2Kb tetramer containing the OVA-derived peptide SIINFEKL produced as previously described (42, 43). For staining, lymphocytes were suspended in PBS/0.2% BSA/0.1% NaN3 (PBS/BSA/NaN3) at a concentration of 1 x 106–1 x 107 cells/ml, followed by incubation at room temperature for 1 h with OVA-tetramer-APC plus the appropriate dilution of anti-CD8-PE (clone 53.6.7; BD PharMingen, San Diego, CA). Cells were washed with PBS/BSA/NaN3 and stained with FITC-conjugated anti-CD11a and PerCP-conjugated anti-CD4 (clone RM4-5; BD PharMingen) and incubated at 4°C for 20 min, washed, and fixed in 3% paraformaldehyde in PBS. Relative fluorescence intensities were measured with a FACSCalibur (BD Biosciences, San Jose, CA). Data were analyzed using WinMDI software (J. Trotter, The Scripps Clinic, La Jolla, CA).

Intracellular detection of IFN-{gamma}

Lymphocytes were isolated from the indicated tissues and cultured for 5 h with 1 µg/ml Golgistop (BD PharMingen), with or without 10 µg/ml listeriolysin O (LLO)190–201 peptide (44) or 1 µg/ml of the OVA-derived peptide SIINFEKL. Peptides were purchased from Research Genetics (Huntsville, AL). After culture, cells were stained for surface molecules, then fixed, and cell membranes were permeabilized in cytofix/cytoperm solution (BD PharMingen) and stained with anti-IFN-{gamma}-FITC (XMG1.2, 5 µg/ml; BD PharMingen) or control rat IgG1 FITC (R3-34, 5 µg/ml; BD PharMingen). Cells were then washed and the fluorescence intensity was measured on a FACSCalibur.

CFSE labeling of cells and adoptive transfer

C57BL/6-Ly5.2 OT-I-RAG-/- CD8 cells were resuspended in HBSS at a concentration of 10 x 106 cells/ml and then warmed to 37°C. Cells were incubated for 10 min with CFSE (0.01 mM; Molecular Probes, Eugene, OR) followed by two washes with HBSS (45). CFSE-labeled cells (1–5 x 106 cells) were resuspended in PBS and adoptively transferred into C57BL/6J-Ly5.1 mice by i.v. injection. At the indicated times after immunization/infection, cells were isolated and analyzed for the presence of donor cells by flow cytometric analysis of Ly5.2 expression and CFSE intensity.

In vivo cytotoxicity assay

This assay was performed essentially as previously described (13, 46). Normal spleen cells were labeled to low (0.25 µm) or high (2.5 µm) CFSE levels and CFSEhigh cells were incubated with 1 µg/ml SIINFEKL peptide for 45 min at 37°C. Equal numbers (5 x 106) of each population were mixed and injected i.v. into OT-I-transferred mice that were immunized 3 days previously or into OT-I-transferred unprimed mice. Four hours later, spleen cells were analyzed for the presence of CFSEhigh and CFSElow populations: percent lysis = [1 - (ratio unprimed/ratio primed)] x 100. Ratio = percent CFSElow/percent CFSEhigh.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The primary and memory response to HKLM immunization is kinetically and quantitatively distinct from the response to LM infection

We examined the kinetics of the CD8 T cell response in the spleen and the lung following HKLM or live LM immunization, since a substantial portion of the response to LM infection is focused in nonlymphoid tissues (6, 47). Ag-specific CD8 T cells were identified using an H-2Kb tetramer containing the OVA-derived SIINFEKL peptide and activation status was initially measured by CD11a expression levels. Five days after immunization similar percentages of OVA-specific CD8 T cells were present in the spleens and lungs of mice receiving either HKLM or live LM (Fig. 1) and all tetramer+ cells expressed high levels of CD11a. Interestingly, the CD8 response to HKLM immunization declined from this point, dropping to 0.4 and 0.16% of splenic and 2.7 and 2.0% of lung CD8 T cells on days 7 and 9, respectively. In contrast, the spleen and lung response to live infection increased dramatically from days 5 to 7. On day 9, the splenic response had begun to decline but the percentage of tetramer+ cells continued to increase in the lung, in agreement with our previous results showing that CD8 responses in nonlymphoid tissues peak later and are more protracted than those in the spleen (6, 43). When the total number of tetramer+ cells in the spleen and lung were quantitated (Fig. 2) on day 5, there was no statistical difference between the number of cells in either organ regardless of whether live or HKLM was administered. In contrast, the number of Ag-specific cells declined thereafter with HKLM immunization, but substantially increased with live LM infection. These results demonstrated that the initial response to live or HKLM was quantitatively similar, but that subsequent clonal expansion was impaired following HKLM vaccination.



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  		FIGURE 1. Distinct kinetics of the endogenous CD8 response to HK or live LM immunization. C57BL/6J mice were immunized i.v. with 1 x 109 HK or 1 x 103 live LM-OVA. On the indicated days later, spleen and lung CD8 T cells were stained with OVA-tetramer and anti-CD11a and analyzed by flow cytometry. Plots shown are of gated CD8+ cells and the values indicate the percentage of tetramer+ CD8+ T cells. Control staining using unimmunized mice or an irrelevant tetramer was <0.05%. Plots shown are representative of the three mice used for each time point.

 


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  		FIGURE 2. Priming with live or HKLM initially induces similar numbers of endogenous Ag-specific CD8 T cells. C57BL/6J mice were immunized i.v. with 1 x 109 HK or 1 x 103 live LM-OVA. On the indicated days later, spleen and lung CD8 T cells were stained with OVA-tetramer and anti-CD11a and analyzed by flow cytometry. Total cell numbers were then calculated based on cell counts from the spleen or lung and the percentage of tetramer+ cells. Values shown (±SE) are derived from three mice per time point. *, Not statistically significant based on Student’s t test.

 
A previous study suggested that comparable populations of memory CD8 T cells were generated following live vs HKLM immunization but this was not directly measured (39). To do so, we quantitated tetramer+ cells 3 wk after immunization (Fig. 3). LM infection resulted in the appearance of memory cells in the spleen and lung. HKLM immunization also induced a population of memory cells which on average was 10- to 20-fold smaller than that observed after LM infection. Thus, the size of the memory population correlated with the size of the peak effector population, comprising ~5–10% of that value. Similar results were obtained using CB6F1 mice and quantitation of the LLO-specific CD8 response, indicating that our results were not unique to OVA (data not shown).



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  		FIGURE 3. HKLM immunization generates small populations of lymphoid and nonlymphoid memory cells. Mice were immunized as in Fig. 1 and lymphocytes were analyzed 3 wk after infection. Plots shown are of gated CD8+ cells and the values indicate the percentage of tetramer+ CD8+ T cells.

 
The CD8 T cell recall response to primary HKLM immunization is robust but delayed

Since memory cells could be generated after HKLM immunization, it was of interest to test whether protection could be afforded. Live LM- or HKLM-primed mice were secondarily infected and bacterial counts from spleen and liver were performed 2 and 4 days after infection (Fig. 4). As compared with unprimed control LM-infected mice, HKLM- or live LM-primed mice were able to provide substantial and statistically significant protection against infection at both time points, although the bacterial counts had greatly declined by day 4 postinfection (Fig. 4). The differences in bacterial counts between HK- and live LM-primed mice were not significantly different in the spleen on day 2 or in the liver or spleen on day 4 but were significantly different in the liver on day 2. Thus, both forms of immunization afforded protection, although HKLM priming was slightly less effective than live infection. These results may differ from those of a previous report examining protection using this system (39) due to the different mouse strains used (B6 in our case and CB6F1 or BALB/c used in Ref.39).



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  		FIGURE 4. HKLM priming protects against secondary infection. Three weeks after priming with HK or live LM-OVA, mice were challenged with 1 x 104 live LM-OVA and 2 and 4 days later bacterial counts were performed on splenic and liver tissue. Values shown are the mean and SE of values derived from analysis of four mice per group.

 
The fact that some degree of bacterial clearance occurred after HKLM immunization suggested that challenged HKLM-primed mice had mounted a secondary response. To test this possibility, HKLM-immunized mice were infected with LM and the response was measured in the blood over time. Mice initially infected with LM mounted a rapid recall response to a secondary infection which peaked on day 4 with ~50% of peripheral blood CD8 T cells tetramer+ (Fig. 5). In contrast, the recall response of mice primed with HKLM was delayed ~2–3 days but then rapidly increased and by day 7, >30% of CD8 T cells were tetramer+. At all time points beyond day 5, the recall response of HKLM-primed mice was substantially greater than that of a primary response, indicating that a true secondary response had occurred. These findings indicated that although the magnitude of the recall response to HKLM vs live LM may be similar at later time points, mounting a response to live LM in HKLM-primed mice required significantly more time in keeping with the smaller number of memory cells available at the time of secondary infection.



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  		FIGURE 5. HKLM priming results in a robust but delayed recall response to secondary infection. Mice primed with the indicated immunogen were infected 3 wk later with 1 x 104 LM-OVA. The primary response was generated via infection with 1 x 104 live LM-OVA. PBL were analyzed on the indicated days after secondary infection for the presence of tetramer+ cells. Values are the percentage of CD8 T cells that are tetramer+ ± SE from groups of four mice per time point.

 
HKLM immunization induces small numbers of memory CD4 T cells

One possible reason for the poor expansion of CD8 T cells responding to HKLM was that the CD4 response was defective. We quantitated the CD4 response to LLO by intracellular IFN-{gamma} analysis following short in vitro incubation with a previously described LLO peptide (44). After primary immunization with HKLM, Ag-specific CD4 T cells could not be detected at any time point (day 9 shown, Fig. 6). Following LM infection however, 1% of splenic and 4% of lung CD4 T cells responded to the LLO peptide (Fig. 6, right panels). Given the poor CD8 T cell response to HKLM, generation of only small numbers of Ag-specific CD4 T cells was perhaps the reason for our inability to detect IFN-{gamma}-producing cells. To test this possibility, recall responses were measured 5 days after secondary infection. At this time point after primary infection, LLO-specific CD4 T cells were detectable but comprised <0.1 and <0.5% of splenic and lung CD4 T cells, respectively (data not shown). Therefore, the appearance of large numbers of LLO-specific CD4 T cells in the spleens and lungs of HKLM- or live LM-immunized mice following secondary infection indicated that a substantial recall response had occurred (Fig. 6). Indeed, a larger population of Ag-specific CD4 T cells was present in HKLM-primed mice compared with infected mice. This result also demonstrated that HKLM immunization generated functional memory cells able to produce cytokines upon challenge. Ergo, as with the CD8 response, normal CD4 memory cells were generated following HKLM immunization but in small numbers.



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  		FIGURE 6. HKLM immunization primes small numbers of LLO-specific CD4 T cells. Mice were immunized with 1 x 109 HKLM or 1 x 103 live LM (primary) or 1 x 104 live LM (recall, 2 mo after priming). Cells from spleen (Spln) and lung were stimulated with LLO peptide in vitro, and intracellular IFN-{gamma} along with CD4 and CD44 expression was measured by flow cytometry 9 days after primary immunization or 5 days after secondary infection. Ag-specific CD4 T cells 5 days after primary infection were <0.5% of CD4 T cells (data not shown). Analysis of gated CD4 T cells is shown and values indicate percentage of CD4 T cells producing IFN-{gamma}.

 
Sustained T cell activation in the spleen drives optimal clonal expansion

Analysis using tetramers or intracellular cytokine staining does not allow inspection of early events in the response. To do so, we used the adoptive transfer of CFSE-labeled OVA-specific TCR-transgenic CD8 T cells (OT-I) into normal mice followed by immunization with live or HKLM (Fig. 7). Forty-eight hours after LM infection, none of the cells in the blood had divided. In contrast, HKLM immunization had induced considerable clonal expansion, with 12% of the CD8 cells being OT-I cells and most of these had divided five to eight times. These cells had up-regulated CD44 expression and had down-regulated CD62 ligand expression (data not shown). Twenty-four hours later, the number of OT-I cells in HKLM-immunized mice had declined by ~50% whereas in LM-infected mice a massive expansion of OT-I cells had occurred, with nearly all cells CFSE negative (note y-axis scales). This trend continued to day 4 with declining OT-I numbers in HKLM-immunized mice and increasing OT-I numbers in infected mice. Similar to the studies using tetramers, small numbers of OT-I memory cells were detected following HKLM immunization (data not shown). Thus, just as with the endogenous response to HKLM, the OT-I cell proliferative response was rapid, but abortive.



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  		FIGURE 7. HKLM immunization induces rapid but abortive proliferation of CD8 T cells. C57BL/6-Ly5.1 mice received CFSE-labeled B6-Ly5.2 OT-I T cells and 1 day (d) later were immunized with 1 x 109 HK or 1 x 103 live LM. At the indicated times, PBL were analyzed for the presence of OT-I cells and CFSE intensity by flow cytometry. Values indicate the percentage of OT-I cells among total CD8 T cells.

 
One possible explanation for the above finding was that OT-I cells were encountering Ag at different times with the different immunogens. However, OT-I cells in the spleen 24 h after live or HKLM immunization had increased in size to the same extent, although no division had yet occurred in either case (Fig. 8, top, and data not shown). These results indicated that regardless of immunogen, most OT-I cells had encountered Ag during the 24 h after immunization. The finding that no OT-I division could be detected in the blood at 48 h after infection but that in the succeeding 24 h a large scale expansion had occurred suggested that dividing cells were sequestered in the lymphoid tissue. Therefore, we analyzed OT-I cells in the spleen at 48 and 72 h after immunization with live or HKLM (Fig. 8). Interestingly, only limited numbers of OT-I cells could be isolated from the spleen 2 days after LM infection and approximately one-half of these had divided two to four times and were blasting (below the horizontal line indicates the size of naive cells). Although some of the undivided cells were blasting, a substantial proportion remained small (Fig. 8) and had not modulated activation markers. In contrast, as seen in the blood, HKLM immunization resulted in substantially more clonal expansion at 48 h with most cells having divided three to four times. (It should be noted that no activated T cells were present in the lymph nodes or any other tissues at this time point with live or HKLM immunization.) As with live LM infection, a small population of undivided, naive phenotype cells was evident (see below). Twenty-four hours later (3 days postimmunization), a large population of CFSElow OT-I cells was present in the spleens of LM-infected mice, indicating that at 48 h, cells undergoing blastogenesis were present in the spleen but were not released by standard isolation procedures, since it is highly unlikely that most cells underwent more than eight divisions in a 24-h time period. The latter is likely the result of sequestration of activated T cells that we and others (48, 49) have observed early after immunization and was perhaps an indication of heightened T cell-APC interactions as recently described (50). Interestingly, at 72 h after HKLM immunization, most OT-I cells had ceased dividing and were small in size. By day 4, the CFSE intermediate cells in HKLM-immunized mice had disappeared, with the majority of the remaining cells either undivided (naive) or CFSElow (Fig. 9A). Thus, HKLM immunization did not result in sustained T cell activation in the spleen, but rather induced transient blastogenesis.



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  		FIGURE 8. Initiation and outcome of early response to live vs HK LM immunization. C57BL/6-Ly5.1 mice received CFSE-labeled B6-Ly5.2 OT-I T cells and 1 day (D) later were immunized with 1 x 109 HK or 1 x 103 live LM. At the indicated times, splenocytes were analyzed for the presence of OT-I cells and CFSE intensity by flow cytometry. Upper panels, Black histograms represent gated OT-I cells from immunized mice and red histograms represent OT-I cells from unimmunized mice. Values indicate the percentage of OT-I cells among total CD8 T cells. Cells below the horizontal line in the dot plots exhibit forward scatter characteristic of naive cells. The percentage of OT-I cells among CD8 T cells in unimmunized mice was ~2% (data not shown).

 


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  		FIGURE 9. Recruitment of Ag-specific T cells and costimulation dependence of the anti-HKLM response. A, OT-I-transferred mice were immunized with HK or live LM and 4 days later the number of undivided cells was measured in the spleen by flow cytometry. Values indicate the total number of CFSEhigh cells. B, One day before and each day thereafter HKLM-immunized mice that had received CFSE-labeled OT-I cells, were treated with CTLA4-Ig (200 µg/day) or control (mutant CTLA4-Ig unable to bind B7). Four days after immunization, the CFSE intensity of OT-I cells in the spleen was analyzed by flow cytometry.

 
One possible reason for the lack of a sustained OT-I response to HKLM was that only a small number of cells was recruited into the response. To test this, we measured the number of undivided CFSEhigh cells in the spleens of mice 4 days after immunization (Fig. 9A). Although the size of the populations of CFSElow cells was substantially different between the two groups, the number of undivided cells was equivalent in each. This result indicated that a similar number of OT-I cells had been recruited into the primary response regardless of whether HK or live LM was administered.

The lack of proper costimulation, perhaps as a result of Ag presentation by nonprofessional APCs, could also potentially explain the poor response to HKLM. To determine whether the OT-I cell response to HKLM was costimulation dependent, we administered CTLA4-Ig to block CD28-B7 interactions. In control mice, although only small numbers of cells were remaining, a population of CFSElow OT-I cells was evident in the spleen 4 days after HKLM immunization (Fig. 9B). In the presence of CTLA4-Ig, this population was substantially decreased and most cells had not divided. Perhaps due to increased precursor frequency obtained with adoptive transfer, in our hands the OT-I response to live LM infection was only inhibited ~30–40% with CTLA4-Ig (data not shown), although the endogenous response is blocked much more effectively (A.M. and L.L., unpublished observations). Nevertheless, CD28-mediated costimulation was essential to mount the CD8 response to HKLM.

HKLM immunization induces CD8 effector cells

To this point, our data indicated that the response to HKLM was characterized by the generation of small numbers of primary and memory T cells. The possibility existed that the primary Ag-specific cells generated were not effectors, as was suggested from previous results using adoptive transfer of TCR-transgenic T cells (39). The poor expansion of CD8 T cells responding to HKLM makes performance of cytotoxicity assays in vitro problematic. To circumvent this issue, we used a sensitive in vivo assay to measure CTL activity. Two populations of splenocytes were labeled with either high or low CFSE concentrations. CFSEhigh cells were coated with the SIINFEKL peptide, mixed with an equal number of uncoated CFSElow cells, and transferred to mice 3 days after immunization of OT-I-transferred mice. Four hours later, the ratio of CFSEhigh:CFSElow cells is an indication of CTL activity, since peptide-coated targets are destroyed. In LM-OVA-infected mice, 87% lysis occurred while in HKLM-immunized mice, 35% lysis was detected (Fig. 10). Therefore, HKLM immunization induced effector CTL.



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  		FIGURE 10. HKLM immunization generates lytic effectors. Mice received OT-I cells followed by immunization with HK or live LM-OVA or nothing. Three days later, a mixture of CFSEhigh (peptide-coated) and CFSElow (uncoated) spleen cells were injected and 4 h later the CFSE-labeled populations in the spleen were quantitated. The lysis values were calculated as described in Materials and Methods.

 
To further substantiate that the response to HKLM generated effector cells, we measured Ag-specific IFN-{gamma} production by endogenous CD8 T cells. Six days after HK or live LM immunization, populations of tetramer+ cells were evident in the spleen and lung (Fig. 11). When stimulated with SIINFEKL peptide, a similar number of CD8 T cells from each organ produced IFN-{gamma}, whether the mice were immunized with HKLM or were infected with LM (Fig. 11). Primary OT-I cells responding to HKLM also produced IFN-{gamma} ex vivo (data not shown). Therefore, the two hallmarks of CD8 T cell effector function, lytic activity and cytokine production, were induced in response to HKLM immunization.



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  		FIGURE 11. HKLM immunization induces endogenous cytokine-producing effector CD8 T cells. Mice were immunized with HK or live LM and 6 days later spleen and lung CD8 T cells were analyzed for tetramer reactivity directly ex vivo and intracellular IFN-{gamma} production after in vitro peptide stimulation. Analysis of gated CD8+ cells is shown and the values indicate the percentage of either tetramer+ or IFN-{gamma}+ cells among CD8 T cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results indicated that a killed bacterial vaccine induced small numbers of effector and memory cells, and the latter were able to provide significant protection against a challenge infection. Our kinetic analysis showed that HKLM-primed mice mounted a robust CD8 T cell recall response but that this response was delayed as compared with that of live LM-primed mice. In addition, LLO-specific CD4 T cells were not detectable after HKLM priming but mounted a vigorous secondary response after challenge infection. These results were explained by the generation of small numbers of memory cells in response to HKLM, since expansion of a smaller initial Ag-specific population will appear to lag behind the clonal expansion of a larger starting population. Nevertheless, at later time points when the response of live LM-primed mice is declining, the response to HKLM was numerically similar and this could explain the previously reported results of Lauvau et al. (39) derived from a single time point after secondary infection.

Although immunization with HKLM resulted in a poor primary response, the Ag-specific CD8 T cells generated either from endogenous or adoptively transferred precursors exhibited effector functions. Thus, the hallmark CD8 T cell functions of lytic activity and IFN-{gamma} production were mediated by the small population of HKLM-primed CD8 T cells. Similarly, the CD8 memory cells generated via HKLM priming were also able to produce IFN-{gamma} upon stimulation (data not shown). In addition, CD4 memory cells generated by HKLM priming also produced IFN-{gamma} during the recall response (Fig. 6). These data indicated that HKLM priming was capable of inducing effector cells and, subsequently, CD4 and CD8 memory T cells were formed, including effector-memory cells in the nonlymphoid tissues such as the lung. Although available technology does not allow the precise identification of memory cell precursors during an in vivo immune response, these results strongly suggested that the memory cells generated were derived from the effector population, especially since nearly all splenic HKLM-primed CD8 T cells produced IFN-{gamma} (Fig. 11). The fact that protection against secondary infection was similar in HK or live LM-primed mice suggested that the number of memory cells induced by HKLM priming was sufficient to provide protection before our ability to detect the responding cells. This finding agrees well with previous results using DNA vaccination where small numbers of memory cells are not protective but mount a robust recall response (51). As suggested in that report, memory cell proliferation may not actually be needed for protection if sufficient numbers are generated by optimal priming. Thus, as recently discussed, the quantity of memory cells is as critical as their functional qualities (52).

The underlying reason that HKLM vaccination resulted in poor T cell expansion appears to be linked to events early in the primary response. CD8 T cell encounter with HKLM-derived Ag resulted in a rapid expansion of T cells within 48 h in the OT-I transfer system. In contrast, at the same time point, little OT-I cell division was noted after LM infection (Figs. 7 and 8). However, 24 h later, a large population of OT-I cells that had divided extensively were evident in the blood and spleen. Since it is unlikely that this extent of proliferation occurred in only 24 h and activated cells were not found elsewhere in the body, then dividing cells must have been present at 48 h after infection but were sequestered in the spleen. Even at 48 h only a small number of dividing cells could be isolated from the spleen presumably due to strong T cell-DC interactions that resisted simple mechanical disruption. Thus, the length of time that Ag-specific T cells were present in the secondary lymphoid organs appeared to be much longer after live vs HKLM immunization. This result is in keeping with the demonstration that sustained TCR engagement in vitro is required for optimal T cell activation (53, 54, 55) but this has not been directly demonstrated in vivo. Our results indicated that HKLM immunization resulted in blastogenesis that was rapidly curtailed, in contrast to the sustained programmed expansion of CD8 T cells noted after infection with LM or viruses or after strong in vitro stimulation (14, 56, 57). This hypothesis is also supported by the possibility that HKLM may not activate APCs as effectively as live LM. Recent elegant studies using macrophages derived from TLR2-/- mice and Myd88-/- mice, the latter of which lack the ability to signal through several Toll-like receptors (TLRs), showed that macrophage activation by HKLM was primarily TLR2 mediated while live LM triggered activation via multiple TLRs (58, 59). Thus, a lack of sufficient TLR signaling after HKLM immunization could explain the poor T cell response since provoking an optimal innate response can be closely linked to the efficiency of the adaptive immune response (38).

Overall, our findings indicated that, at least in some cases, the poor response engendered by attenuated vaccines is due to ineffectual initial priming resulting in limited T cell expansion. Nevertheless, effector T cells were spawned and their numbers correlated closely with the memory population produced, providing support for the theory that CD8 memory cells develop from effector cell precursors in vivo. These memory cells were also capable of robust expansion and effector cell generation and the secondary response generated a substantial memory cell pool whether HKLM or live LM was the initial immunogen (data not shown). Thus, a prime-boost regimen could perhaps employ less attenuated microbes in the secondary challenge to drive generation of protective immune memory. Alternatively, adjuvants such as CpG DNA could be coupled with killed vaccines (60) to promote efficient effector cell expansion to produce sufficient numbers of memory cells.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI41576 and DK45260. Back

2 Address correspondence and reprint requests to Dr. Leo Lefrançois, Department of Medicine, Division of Immunology, University of Connecticut Health Center, Farmington, CT 06030-1319. E-mail address: llefranc{at}neuron.uchc.edu Back

3 Abbreviations used in this paper: HKLM, heat-killed Listeria monocytogenes; LLO, listeriolysin O; TLR, Toll-like receptor. Back

Received for publication April 17, 2003. Accepted for publication July 15, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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