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The Journal of Immunology, 2003, 171: 5454-5460.
Copyright © 2003 by The American Association of Immunologists

Increased CD8+ T Cell Memory to Concurrent Infection at the Expense of Increased Erosion of Pre-existing Memory: The Paradoxical Role of IL-151

Yvan Chapdelaine2, Dean K. Smith3, Joao A. Pedras-Vasconcelos4, Lakshmi Krishnan and Subash Sad5

Laboratory of Cellular Immunology, Institute for Biological Sciences, National Research Council of Canada, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The use of cytokines during vaccination, particularly IL-15, is being considered due to the unique ability of IL-15 to enhance the proliferation of memory CD8+ T cells. However, as homeostatic mechanisms limit excessive lymphocyte expansion, we addressed the consequences of this enhancement of T cell memory by IL-15. Infection of mice with either recombinant Mycobacterium bovis (BCG) expressing IL-15 (BCG-IL-15) or BCG and purified IL-15 resulted in an increased CD44, IL-2R{beta} expression and increased frequency of IFN-{gamma}-secreting CD8+ T cells. Surprisingly, the enhancement of memory to concurrent infection by IL-15 exacerbated the attrition of pre-existing memory. Infection of mice with Listeria monocytogenes expressing OVA resulted in potent OVA257–264-specific CD8+ T cell memory, and a challenge of these mice with either BCG-IL-15 or BCG and purified IL-15 resulted in an increased erosion of OVA257–264-specific CD8+ T cell memory, relative to BCG. Enhancement in the erosion of OVA-specific CD8+ T cell memory by BCG-IL-15 resulted in a consequently greater impairment in protection against a challenge with OVA-expressing tumor cells. We thus raise important questions regarding vaccinations that are aimed at maximizing T cell memory without considering the impact on pre-existing T cell memory.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The importance of T cell memory for the control of intracellular pathogens and tumors is well documented (1, 2, 3); however, the mechanisms that govern the induction and maintenance of memory T cells are not clear. Although memory T cells can be maintained in the absence of Ag, in the face of immune challenge with distinct pathogens, attrition of an otherwise stable memory has been documented (4, 5). Following challenge with lymphocytic choriomeningitis virus, 40–60% of CD8+ T cells appear to be virus specific, yet <5% of these cells survive following pathogen clearance (6, 7). With the resolution of the virus, a new homeostasis of the memory pool is established, but virus-specific CD8+ T cells remain as high as 10% of the memory pool (8). However, this stable memory pool is subject to attrition, and a single heterologous viral infection can reduce Ag-specific CD8+ T cell memory by 2- to 5-fold, depending on the virus involved (4, 5). We have previously reported this erosion of pre-existing T cell memory also occurs during heterologous bacterial infections, as a challenge of mice with the intracellular pathogen, Mycobacterium bovis (BCG),6 causes an erosion of CD8+ T cell memory generated previously against another intracellular pathogen, Listeria monocytogenes (LM), resulting in a compromised protective efficacy against a tumor challenge (9). In this heterologous bacterial model of attrition, we now evaluate the influence of the cytokine IL-15, which promotes memory CD8+ T cell responses (10, 11), and ask whether the presence of IL-15 during the secondary heterologous infection maintains or improves pre-existing T cell memory.


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

Female C57BL/6 mice were obtained from Charles River Laboratory (St-Constant, Canada). B6.PL mice expressing Thy-1.1, and OVA257–264-specific TCR transgenic mice (OT-1) expressing Thy-1.2 were obtained from the The Jackson Laboratory (Bar Harbor, ME). Frozen stocks of BCG, BCG-IL-15, and BCG-OVA (described previously (12)) were thawed, resuspended in PBS-T (PBS containing 0.025% Tween 80), and injected into mice via the lateral tail vain. Control mice were injected with PBS-T. For induction of OVA257–264-specific CD8+ T cell response, LM-OVA (described previously (12)) was diluted in saline (0.9%) and injected via the lateral tail vein.

Plasmids and bacterial strains

Cloning was performed in Escherichia coli HB101 strain using 25 µg/ml Kanamycin with pMV261 vector (13). BCG (Pasteur strain) was grown, as described previously (12). BCG selection medium contained 15 µg/ml Kanamycin (Sigma-Aldrich, St. Louis, MO). Plasmid DNA (5 µg) was introduced in BCG by electroporation (12), and plated on solid medium containing Kanamycin (15 µg/ml). Single colonies were used to inoculate liquid cultures. Generation of LM-expressing OVA has been described previously (12).

Cloning mouse IL-15 cDNA

Total RNA was isolated from mouse peritoneal exudate cells using the SNAP RNA isolation kit (Invitrogen, Carlsbad, CA). IL-15 coding sequence (gb:U14332) was obtained by cDNA synthesis and PCR (RT-PCR, Titan kit; Boehringer Mannheim, Indianapolis, IN) on mouse peritoneal exudate cells. The gene coding for Ag 85B (Rv1886c) was amplified from Mycobacterium tuberculosis DNA. IL-15-derived PCR products were fused to the Ag 85B secretion signal by overlap PCR using different sets of primers for each linker sequence to be inserted. The fusion PCR products were cut with the MscI and EcoRI restriction enzymes, and cloned in the pMV261 vector, using the same restriction sites. All inserts were verified by sequencing. IL-15 activity was measured by HT2 cell bioassay (14).

Assessment of T cell responses and flow cytometry

Cell culture medium (R8) consisted of RPMI 1640 (Life Technologies, Grand Island, NY), containing 8% heat-inactivated FBS (HyClone, Logan, UT). CD8+ T cells were purified using a CELLection biotin binder kit (Dynal, Great Neck, NY) (9). Spleen cells were stimulated (5 x 105/well) with LM-Ag or BCG-Ag (described previously (9)), or with plate-bound anti-CD3 Ab. Production of IFN-{gamma}, in the 24- and 72-h culture supernatant, was measured by ELISA (14). Frequency of IFN-{gamma}-secreting T cells was evaluated by ELISPOT assay (12). Intracellular IFN-{gamma} staining was performed using the Cytofix/Cytoperm kit (BD PharMingen, San Diego, CA), according to manufacturer’s instructions. For evaluation of CD44 and IL-2R expression, 106 cells were stained using FITC-labeled anti-CD4 and anti-CD8, and PE-labeled anti-CD44 (IM7.8.1), anti-IL-2R{alpha} (7D4), and anti-IL-2R{beta} (TM-{beta}1) Abs (BD PharMingen) for 30 min on ice. Cells were washed, fixed, and analyzed on EPICS XL flow cytometer.

Adoptive transfer experiments

Spleens were removed from the OT-1 transgenic mice, and CD8+ T cells were purified, as mentioned above. Purified CD8+ T cells (5 x 106/mouse) along with the various pathogens were resuspended in 200 µl of HBSS and injected into mice via the lateral tail vein. The numbers of donor OT-1 CD8+ T cells in recipient B6.PL mice were evaluated by removing the spleens from recipient mice, and staining spleen cells (107/aliquot) with anti-Thy-1.2 PE (BD PharMingen) and anti-CD8 FITC. After 30 min, cells were washed and fixed, and 100,000 gated CD8+ T cell events were acquired on the EPICS XL flow cytometer.

Cytotoxicity assay

A total of 3 x 106 spleen cells or 0.5 x 106 purified CD8+ T cells from immunized C57BL/6 mice was incubated with 5 x 105 irradiated (10,000 rad) OVA-expressing target cells (EG7 cells) in 10 ml of R8 and IL-2 (0.1 ng/ml). The total number of spleen cells in each flask was normalized to 30 x 106 cells by adding the required numbers of syngeneic spleen cells from unimmunized mice. Cultures were placed in 25-cm2 tissue culture flasks kept upright. After 5 days (37°C, 8% CO2), cells were harvested, washed, counted, and used as effectors in a standard 51Cr release CTL assay against 51Cr-labeled EL-4 cells in the presence or absence of OVA257–264. One lytic unit is defined as the number of effector cells per 106 spleen cells that yield 25% lysis of a population of 2.5 x 104 target cells.

Cell lines

P815, EL-4, and Yac1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and maintained in R8.EG7 cells (15), and B16OVA cells (16) expressing the gene for OVA were obtained from ATCC and E. Lord (University of Rochester, New York), respectively, and maintained in R8 containing 400 µg/ml G418. HT-2 cells were obtained from T. Mosmann (University of Rochester) and maintained in R8 containing 1 ng/ml IL-2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IL-15 by BCG

The sequence coding for murine IL-15 was fused to the Ag 85B secretion signal from M. tuberculosis, and inserted in the E. coli-BCG shuttle vector pMV261 (13) to generate the plasmid pMIL15B. Western blot analysis of BCG-IL-15 revealed the presence of multiple bands (Fig. 1A, lane 1), which are absent from control BCG extracts (Fig. 1A, lane 2). The 10-kDa band (b) comigrates with its purified homologue (lane 3), and would therefore represent the active cytokine. The other products 5, 13 (putative precursor), 17, and 24 kDa, are probably inactive. The secretion of biologically active IL-15 in the supernatant over time by BCG harboring the plasmid pMIL15B was determined (Fig. 1B), and IL-15 expression closely followed the growth curve of BCG-IL-15. These results indicate that the processing of the fusion protein in BCG is inefficient. To obtain a rBCG strain that would express IL-15 more efficiently, a synthetic IL-15 construct was derived from the human IL-15 sequence secreting 10 times more IL-15 activity. However, this high IL-15-expressing BCG was cleared much more rapidly in mice, and failed to improve the resulting T cell response (see Discussion).



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  		FIGURE 1. Expression of IL-15 by BCG. BCG-IL-15 (lane 1) and BCG (lane 2) extracts were subject to Western blotting using a rabbit anti-IL-15 polyclonal Ab (A). Mouse rIL-15 (10 ng, lane 3) is shown as a positive control (arrow). The estimated size of each IL-15-specific band is 5.2 (a), 9.1 (b), 17.5 (c), and 24.5 kDa (d). The growth and activity of BCG-IL-15 in vitro were measured by monitoring OD600 and IL-15 bioactivity in the culture supernatant (B). C57BL/6 mice were injected i.v. with PBS, 105 BCG, or BCG-IL-15, and the bacterial burden (C) and NK-dependent cytolytic activity (D) measured at various time intervals.

 
We determined the relative growth of BCG and BCG-IL-15 in C57BL/6 mice infected with BCG or BCG-IL-15 (Fig. 1C). During the first 2 wk of infection, both BCG- and BCG-IL-15-infected mice exhibited similar bacterial burdens. At 4 wk, BCG-IL-15-infected mice had lower bacterial burden relative to BCG-infected mice. We also measured NK-dependent cytolytic activity during these time intervals against the NK-sensitive (Yac1) vs NK-resistant (P815) target cells. All the experimental groups exhibited <5% cytotoxicity toward P815 target cells. Overall, the level of NK-dependent cytotoxicity observed against the Yac1 target cells was low. At early time intervals, BCG-IL-15-infected mice exhibited an elevated cytotoxic activity in comparison with BCG-infected controls (Fig. 1D). At later time intervals, cytotoxic activity mediated by the spleen cells from both the BCG- and BCG-IL-15-infected mice decreased.

Enhancement of cellular immune response by BCG-IL-15

We measured the bacterial burden and cellular immune responses at 4 wk (Fig. 2, A–D) and 9 wk (Fig. 2, E–I) after infection. At 4 wk, spleen cells from BCG-IL-15-infected mice in comparison with BCG-infected controls exhibited a reduced bacterial burden (Fig. 2A) and produced higher levels of IFN-{gamma} in response to anti-CD3 (Fig. 2B) and BCG-Ag (Fig. 2C). Furthermore, the numbers of CD8+ T cells expressing CD44, which is considered to be expressed on all memory T cells irrespective of their activation status (1, 17, 18, 19), also increased in BCG-IL-15-infected mice (Fig. 2D). Similar results were obtained at 9 wk after infection (Fig. 2, E–H). Additionally, CD8+ T cells purified from BCG-IL-15-infected mice at 9 wk produced higher IFN-{gamma} levels after anti-CD3 stimulation (Fig. 2I).



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  		FIGURE 2. Enhanced acquired immune response induced by BCG-IL-15. C57BL/6 mice were infected with 106 bacteria (i.v.). Splenocytes were harvested at 4 wk (A–D) and 9 wk (E–I) to determine bacterial burden (A, E) and cellular immune responses (B–D, F–I). Spleen cells (0.5 x 106 cells/well, B, C, F, G) or purified CD8+ T cells (1 x 105/well, I) were stimulated with plate-bound anti-CD3 Ab (B, F, and I) or BCG-Ag (C, G) and IFN-{gamma} produced in the culture supernatant measured by ELISA. Flow cytometry analysis of CD4+ and CD8+ T cells was performed, and the percentage of CD44high cells was determined (D and H).

 
Effect of BCG-IL-15 on pre-existing CD8+ T cell responses

We have previously reported the development of potent OVA257–264-specific CD8+ T cell response by infection of C57BL/6 mice with LM-OVA (12), and a subsequent erosion of this response after a challenge with BCG (9). In the current study, we used a 10-fold reduced dose of BCG (105 i.v.) that is expected to cause less erosion of pre-existing memory so that the effects of IL-15 could be discerned. Challenge of LM-OVA-injected mice with BCG resulted in a reduction in OVA-specific CD8+ (Fig. 3A) T cell response, whereas a challenge with BCG-IL-15 resulted in an even greater erosion of this response. We also tested OVA257–264-specific cytotoxicity after bulk spleen cells (Fig. 3, B and D) or purified CD8+ T cells (Fig. 3, C and E) were restimulated with OVA-expressing target cells for 5 days in the presence of IL-2. Percentage of cytotoxicity data (Fig. 3, B and C) was converted into lytic units (Fig. 3, D and E), which takes into account the numbers of effectors required to kill a defined number of targets. By this analysis, the drop in the CTL response can be appreciated more quantitatively. A challenge of LM-OVA-injected mice with BCG-IL-15 resulted in an even greater erosion of OVA257–264-specific CD8+ T cell response relative to BCG. These results suggest that BCG-IL-15 induces attrition of pre-existing CD8+ T cell memory rather than promoting their maintenance and/or expansion.



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  		FIGURE 3. Effect of BCG-IL-15 challenge on pre-existing CD8+ T cell memory. C57BL/6 mice were infected with LM-OVA (5 x 103) and challenged on day 15 with PBS, BCG (105), or BCG-IL-15 (105). At day 45, spleens were harvested, and spleen cells (5 x 105/well) were stimulated with OVA257–264 (A) for IFN-{gamma} production. Cytolytic activity of spleen cells (B, D) or purified CD8+ T cells (C, E) was tested, as described in Materials and Methods. The percentage of cytotoxicity data (B, C) was also converted into lytic units (D, E). The percentage of cytotoxicity against control EL-4 cells was <15% in all the experimental groups.

 
We next evaluated the consequences of this increased attrition of pre-existing T cell memory by IL-15 in a tumor model in which the presence of OVA257–264-specific CD8+ T cells is critical for protection (12, 20). As mentioned above, a reduced dose of BCG and BCG-IL-15 was used so that BCG itself induces less erosion and the effects of IL-15 could be discerned. Naive control mice (PBS injected) showed the most rapid tumor growth (Fig. 4). Mice preimmunized with LM-OVA exhibited a profound delay in tumor growth, suggesting that OVA257–264-specific CD8+ T cells provide substantial protection against B16OVA tumor challenge. A challenge of LM-OVA-immunized mice with BCG reduced this protection considerably, advancing tumor onset by ~8–10 days. Remarkably, a challenge with BCG-IL-15 had an even stronger impact: onset of tumor in BCG-IL-15 group was similar to that of naive mice. On day 75, all BCG-IL-15-challenged mice had developed measurable tumors, while the tumors in all the BCG-challenged animals remained too small to be measured. Compared with LM-OVA-preimmunized mice, statistical analysis indicates p values of 0.0002, 0.008, and 0.09 for naive and BCG-IL-15- and BCG-infected animals, respectively. Only naive and BCG-IL-15 groups are significantly different (p < 0.05) from LM-OVA-infected group.



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  		FIGURE 4. Effect of BCG-IL-15 challenge on pre-existing antitumor immunity. C57BL/6 mice were infected with LM-OVA (5 x 103). On day 30, mice were injected (i.v.) with PBS, BCG (105), or BCG-IL-15 (105). On day 60, B16-OVA tumor cells (1 x 106 cells/mouse) were grafted by s.c. injection in the rear dorsal region. Tumor size (mm2) was obtained by multiplying longitudinal and lateral diameters, which were determined commencing at day 65. Values of p of various experimental groups are indicated relative to LM-OVA-injected group.

 
We then re-evaluated the role of IL-15 in this heterologous bacterial model of attrition to determine whether increased memory erosion is due to IL-15 or due to a unique property of the BCG-IL-15 construct. Commencing with BCG challenge, a group of mice also received four injections (2/wk) of purified rIL-15. At day 45, we measured the frequency of T cells specific for LM-Ag, BCG-Ag, and OVA257–264 by ELISPOT assay (Fig. 5A). When spleen cells were stimulated with the BCG-Ag, increased numbers of IFN-{gamma}-secreting cells were detectable in BCG-injected mice that also received IL-15. Furthermore, increased numbers of CD44high (Fig. 5B), IL-2R{beta}high (Fig. 5C), and IFN-{gamma}+ (Fig. 5D) CD8+ T cells were present in BCG + IL-15-injected mice. These results suggest that IL-15 enhances T cell response against BCG. In contrast, when the frequency of IFN-{gamma}-secreting T cells was measured against the native (LM-Ag) and recombinant (OVA257–264) Ags of LM, IL-15 administration during the BCG challenge enhanced the reduction in the frequency of IFN-{gamma}-secreting T cells (Fig. 5A). Furthermore, OVA257–264-specific cytolytic activity (Fig. 5, E and F) was reduced when IL-15 was administered during BCG challenge.



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  		FIGURE 5. Effect of rIL-15 administration on pre-existing and ongoing CD8+ T cell responses. C57BL/6 mice were preimmunized with LM-OVA (5 x 103) and challenged with BCG (105) on day 15. A group of BCG-challenged mice was also injected with four doses of purified IL-15 (5 µg each) on days 15, 19, 22, and 26. Spleens were harvested on day 45, and the frequency of IFN-{gamma}-secreting cells (per 1 x 106 spleen cells) was evaluated by ELISPOT assay (A). The numbers of CD8+ T cells expressing high levels of CD44 (B) and IL-2R{alpha}/{beta} (C) were evaluated. Spleen cells were stimulated with PMA (5 ng/ml) and calcium ionophore (A23187, 0.5 µg/ml), and the numbers of IFN-{gamma}-expressing CD8+ T cells were enumerated (D). Cytotoxicity toward OVA257–264-pulsed targets was evaluated, as described in Materials and Methods, and expressed as percentage of cytotoxicity (E) and lytic units (F). The percentage of cytotoxicity against control EL-4 cells was <15%.

 
We evaluated the influence of IL-15 on concurrent and pre-existing CD8+ T cell memory using an adoptive transfer model, wherein the specificity of the transferred CD8+ T cells is known. Because donor OT-1 CD8+ T cells (expressing a TCR of OVA257–264) are Thy-1.2+ and recipient mice B6.PL express Thy-1.1, the fate of the transferred donor CD8+ T cells could be evaluated using an anti-Thy-1.2 Ab. IL-15 administration in the context of a single infection, LM-OVA (Fig. 6A) or BCG-OVA (Fig. 6B), resulted in an enhancement in the numbers of donor OVA257–264-specific CD8+ T cells, suggesting that IL-15 enhances CD8+ T cell memory against a concurrent (acute or chronic) infection. In contrast, administration of IL-15 during a challenge of LM-OVA-infected mice with BCG resulted in a greater erosion of the donor OVA257–264-specific CD8+ T cells (Fig. 6C). These results clearly demonstrate that while IL-15 promotes CD8+ T cell memory to concurrent infection, this comes at an even greater erosion of pre-existing CD8+ T cell memory.



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  		FIGURE 6. Enhancement of CD8+ T cell memory by IL-15 increases the erosion of pre-existing CD8+ T cell memory. B6.PL recipient mice (Thy-1.1) were injected (i.v.) with A, LM-OVA (5 x 103) or B, BCG-OVA (105) and 5 x 106 purified CD8+ T cells from OVA257–264 TCR transgenic mice (OT-1) expressing Thy-1.2. Mice were also injected with PBS or four doses of purified mouse IL-15 (5 µg/injection) on days 7, 11, 15, and 18. Spleens were harvested on day 20, and the numbers of donor CD8+ T cells (Thy-1.2) were evaluated. In another experiment (C), B6.PL recipient mice (Thy-1.1) were injected with LM-OVA and purified CD8+ T cells from OT-1 mice (Thy-1.2), as described above, and mice were challenged on day 10 with PBS or BCG. A group of BCG-challenged mice also received four doses of IL-15 (days 10, 13, 17, and 20). On day 25, spleens were harvested, and the numbers of donor Thy-1.2+ cells were evaluated in the gated CD8+ T cell population.

 
In the preceding experiments, heterologous infectious challenges were done soon after the primary infection (~second week after the first infection). We therefore determined whether the erosion of pre-existing memory would also occur if the timing of the heterologous infectious challenge was delayed. We infected C57BL/6 mice with LM-OVA and challenged them at early interval (Fig. 7A), or late interval (Fig. 7B) with BCG, or BCG + IL-15. Thirty days after the challenge, we determined the frequency and the numbers of OVA257–264-specific CD8+ T cells in various experimental groups. It is clear from Fig. 7 that the timing of the heterologous infection does not seem to influence the extent of erosion of pre-existing T cell memory.



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  		FIGURE 7. The timing of heterologous infectious challenge does not influence the degree of erosion of pre-existing T cell memory. C57BL/6 mice were preimmunized with LM-OVA (5 x 103) and challenged with BCG (105) on day 7 (A) or 45 (B). A group of BCG-challenged mice was also injected with four doses (2/wk) of purified IL-15 (5 µg each). Thirty days after the BCG challenge, spleens were harvested from the various experimental groups. The frequency of IFN-{gamma}-secreting cells (per 1 x 106 spleen cells) was evaluated by ELISPOT assay, and the numbers of OVA257–264-specific CD8+ T cells per spleen were determined.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
From the host’s perspective, there is a clear selective advantage for the maintenance of memory against life-threatening pathogens, while also having the capacity to respond to new challenges. Although the current efforts in vaccinology are geared toward enhancing vaccine efficacy by maximizing T cell responses to vaccines, particularly against cancer, it becomes important to discern the consequences of generating potent T cell responses. IL-15 is one of the key cytokines that promotes CD8+ T cell memory (3, 10, 11) and consequently enhances vaccine efficacy (21, 22, 23); however, it is unclear what the consequences of excessive memory expansion might be! Our results reveal that although IL-15 enhances CD8+ T cell memory (10, 21, 23, 24), such enhancement of T cell memory comes at an increased expense of a greater erosion of pre-existing T cell memory.

The elegant work on the attrition of pre-existing T cell memory during subsequent heterologous infections was first reported in viral infection models (4, 5), in which a single infection was found to reduce the previously established CD8+ T cell memory by 2- to 5-fold. We have previously reported that attrition of established T cell memory also occurs during bacterial infections, as a challenge of LM-infected mice with BCG results in an erosion of pre-existing memory CD8+ T cells against LM, and that this erosion of memory is not limited only to the spleen (9). Which cells within the memory T cell pool are eliminated in the absence of their cognate Ag may depend on a variety of factors, including the phase of cell cycle and/or the cell’s activation status, in addition to the balance between pro- and antiapoptotic regulatory factors in the T cells, before their encounter with the secondary BCG infection. In the viral attrition model, apoptosis of pre-existing memory CD8+ T cells was reported to be due to the expression of IFN-{alpha}/{beta}, and not IFN-{gamma} or Fas ligand (FasL) (25). Indeed, the bystander activation of memory phenotype CD8+ T cells was previously reported to occur mainly due to the expression of IFN-{alpha}/{beta} during viral and bacterial infections (26, 27), and it was reported later that the activation of memory CD8+ T cells by IFN-{alpha}/{beta} was actually due to the stimulation of IL-15 production by APCs in response to IFN-{alpha}/{beta} (10). Because IL-15 may be produced endogenously by APCs that are activated chronically by BCG (28), it is quite likely that even with unmodified BCG, IL-15 produced in a chronic and sustained manner facilitates the attrition of memory. Our results report that the same cytokine, IL-15, that induces the proliferation of memory CD8+ T cells also facilitates the erosion of pre-existing memory CD8+ T cells. Thus, despite the memory-promoting function of IL-15, enhancement of the erosion of pre-existing memory by IL-15 suggests that the broader network of homeostatic control mechanisms supersedes. Support for this comes from our observations that the erosion of pre-existing memory by BCG occurs even in mice deficient in inducible NO synthase, TNFR, IFN-{gamma}, FasL, and perforin (our unpublished observations). Thus, the various conventional mediators of cell death, which may be expressed chronically during BCG infection (29, 30, 31), do not appear to influence the erosion of pre-existing memory. Even with a single infection, the mechanisms that drive T cells to commit to apoptosis are not clear, as apoptosis remains unaltered in FasL- and TNFR-deficient mice (32). In other studies, IFN-{gamma} (33, 34, 35) and perforin (33, 36) were shown to be the important mediators of effector T cell death. Even during infection with BCG, IFN-{gamma} was shown to be the main mediator of effector CD4+ T cell death, as infection of IFN-{gamma}-deficient mice with BCG resulted in a profound accumulation of CD44highCD4+ T cells (37). This IFN-{gamma}-mediated T cell death in BCG-infected mice appeared to be partly due to the production of high levels of nitrite ions (37). Because the erosion of pre-existing T cell memory occurred even in the absence of these mediators, it is likely that the elimination of effector T cells during a single infection vs the erosion of pre-existing memory T cells during a subsequent heterologous infection is mediated by distinct mechanisms that are not clear yet.

Compared with the TCR-induced T cell activation, little is known about the mechanisms of action of IL-15. In this context, it was recently reported (38) that both anti-CD3 and IL-15 induce highly similar responses in memory CD8+ T cells as measured by cellular proliferation, gene expression changes, synthesis of effector molecules (IFN-{gamma}, TNF, granzyme B, and perforin), and induction of cytotoxicity. Of the 189 genes whose expression changed in CD8+ memory T cells after IL-15 and anti-CD3 stimulation, 77% of the genes exhibited a highly similar pattern of expression after IL-15 and anti-CD3 treatment. Thus, IL-15 not only acts as a crucial growth factor, but it also is an Ag-independent activator of effector functions of memory CD8+ T cells. Besides memory CD8+ T cells, IL-15 also enhances the activation of NK cells (39, 40). We also observed an enhancement of NK cytotoxicity in mice infected with BCG-IL-15. Thus, the presence of high levels of IL-15 during infection of mice with BCG results in potent activation of CD8+ T cells and NK cells, which may facilitate the erosion of memory, unless the Ag is present to sustain the proliferation of memory T cells, as we have previously reported that only the presence of a cross-reactive Ag can prevent the erosion of pre-existing memory T cells in this heterologous model of attrition (12). Although we have shown that IL-15 is partially responsible for causing the erosion of pre-existing CD8+ T cell memory, however, this occurred during a challenge with the intracellular bacterium (BCG) that induces a chronic infection. It is unclear whether IL-15 will have similar effects during other bacterial or viral challenges that induce acute infections. In this context, IFN-{alpha}/{beta}, which causes IL-15 activation, was reported to induce erosion of pre-existing memory CD8+ T cells during acute viral infection (25).

Although, in the absence of direct tetramer staining, we cannot definitively conclude that pre-existing memory T cells are deleted, however, several lines of evidence suggest that pre-existing memory T cells are likely to be deleted. If significant numbers of LM-specific T cells were simply anergized in BCG-IL-15-infected mice, then the IL-2 and Ag used during the CTL restimulations would most likely have released them from that state (41, 42). Our previous results (43) and those of others (44) have indicated that anergy in CD8+ T cells impairs cytokine production, but not cytolytic activity. Furthermore, IL-15-injected mice exhibit an increase in the number of CD44-expressing CD8+ T cells and an increase in the number of BCG-Ag-specific IFN-{gamma}-secreting T cells. Additionally, the production of enhanced IFN-{gamma} levels from purified T cells (in the BCG-IL-15-challenged mice) after stimulation with anti-CD3, but reduced LM-OVA-specific IFN-{gamma} after stimulation with OVA peptide, argues against anergy and strongly supports deletion of LM-specific T cells. The reduction of tumor immunity in vivo, which was associated with the reduction of OVA-specific T cell responses, is also strongly indicative of Ag-dependent attrition. Finally, an enhancement in the erosion of the transferred OT-1 CD8+ T cells in BCG + IL-15 group of mice in comparison with BCG-infected controls indicates that IL-15 exacerbates the attrition of pre-existing CD8+ T cell memory.

Higher viral burden in vivo correlates directly with stronger acquired immune responses (45, 46). Similarly, we have also noted that a higher BCG burden in vivo results in the development of a stronger T cell response (12, 47). With BCG-IL-15 infection, enhancement of cellular immune response occurred even when the bacterial burden was lower (at day 30), suggesting that the expression of appropriate cytokines can overcome the requirement of high pathogen burden for generation of optimal cellular immune responses. Because the expression of IL-15 by BCG-IL-15 was low in vitro, we generated rBCG expressing ~10-fold higher levels of IL-15 than the one reported in this study. However, the expression of high levels of IL-15 resulted in a rapid clearance and poor growth of the modified BCG, which impaired the generation of acquired immune response (data not shown). Thus, a reduction in bacterial burden due to expression of high levels of cytokines defeats the purpose of using live bacteria for chronic cytokine delivery. Under physiologic circumstances, appropriate cytokines are produced in high amounts local to the site of Ag, often acting in concert with Ag-driven signals to generate effector responses (48). Although cytokines can be administered systemically, such systemic administration of cytokines produces high concentrations of cytokines in the vasculature at sites distant from the Ag, and often suboptimal levels in tissues at the site of Ag (49). Thus, when purified IL-15 was injected in vivo, potent effects on CD8+ T cells occurred only when microgram quantities were injected systemically (10). In contrast, chronic bacteria that persist in lymphoid tissues may deliver cytokines on a continuous basis at the site of Ag presentation, and induce potent effects even at lower cytokine concentrations.

BCG has been used extensively for cancer treatment, with repeated doses as high as 1010, although the mechanisms involved are not well established (50, 51, 52). The use of cytokine-expressing BCG has been suggested for treatment of cancer. For example, combined IL-2 and BCG can improve BCG therapy (53). Similarly, IL-15-expressing BCG may enhance antitumor immune responses by enhancing NK and CD8+ T cell responses. However, our study raises important questions regarding immunization protocols that are aimed at maximizing T cell memory to ongoing vaccines without considering the impact that these regimens may have on pre-existing T cell memory.


   Acknowledgments
 
We thank Henk van Faassen and Renu Dudani for technical services, and Dr. Hao Shen and Dr. W. R. Jacobs for providing pJJD-OVA and pMV261 plasmids, respectively. We also thank Dr. R. J. North and Dr. Edward J. Pearce for helpful discussions.


   Footnotes
 
1 This work was supported by funds from the National Research Council of Canada. This is National Research Council Publication 42481. Back

2 Current address: PHYTOBIOTECH, St-Martin, Laval, Quebec, Canada. Back

3 Current address: Biologic and Genetic Therapies Directorate, Health Canada, Ottawa, Canada. Back

4 Current address: Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892. Back

5 Address correspondence and reprint requests to Dr. Subash Sad, Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Room 4105, Ottawa, Ontario, Canada K1A 0R6. E-mail address: subash.sad{at}nrc-cnrc.gc.ca Back

6 Abbreviations used in this paper: BCG, Mycobacterium bovis; FasL, Fas ligand; LM, Listeria monocytogenes. Back

Received for publication March 13, 2003. Accepted for publication September 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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