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The Journal of Immunology, 2008, 180, 2855 -2862
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Listeria Infections

Constitutive Expression of IL-7 Receptor {alpha} Does Not Support Increased Expansion or Prevent Contraction of Antigen-Specific CD4 or CD8 T Cells following Listeria monocytogenes Infection1

Jodie S. Haring*, Xuefang Jing*, Julie Bollenbacher-Reilley{ddagger}, Hai-Hui Xue*, Warren J. Leonard{ddagger} and John T. Harty2,*,{dagger}

* Department of Microbiology, Carver School of Medicine and {dagger} Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA 52242; and {ddagger} Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20824


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Expression of IL-7R{alpha} (CD127) has been suggested as a major determinant in the survival of memory T cell precursors. We investigated whether constitutive expression of IL-7R{alpha} on T cells increased expansion and/or decreased contraction of endogenous Ag-specific CD4 and CD8 T cells following infection with Listeria monocytogenes. The results indicate that constitutive expression of IL-7R{alpha} alone was not enough to impart an expansion or survival advantage to CD8 T cells responding to infection, and did not increase memory CD8 T cell numbers over those observed in wild-type controls. Constitutive expression of IL-7R{alpha} did allow for slightly prolonged expansion of Ag-specific CD4 T cells; however, it did not alter the contraction phase or protect against the waning of memory T cell numbers at later times after infection. Memory CD4 and CD8 T cells generated in IL-7R{alpha} transgenic mice expanded similarly to wild-type T cells after secondary infection, and immunized IL-7R{alpha} transgenic mice were fully protected against lethal bacterial challenge demonstrating that constitutive expression of IL-7R{alpha} does not impair, or markedly improve memory/secondary effector T cell function. These results indicate that expression of IL-7R{alpha} alone does not support increased survival of effector Ag-specific CD4 or CD8 T cells into the memory phase following bacterial infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Generation of memory CD4 and CD8 T cells that are capable of providing protection to the host against secondary pathogen challenge remains the ultimate goal of vaccination. After encountering Ag and receiving requisite costimulatory signals, naive T cell precursors undergo rapid expansion culminating in a peak in numbers after 5–7 days. Following expansion, Ag-specific T cells enter the contraction phase during which 90–95% of the cells die via apoptosis. The small subset of T cells that survive contraction are the initial memory cells, which will evolve into the long-term, self-sustaining memory population capable of providing protection against future infection (1). To incorporate optimal memory T cell development into vaccine design, the factors responsible for the selection of effector cells that survive the contraction phase following Ag-induced expansion to seed the initial memory pool must be understood.

IL-7 has long been known to be required for the survival and homeostatic proliferation of naive T cells (2, 3, 4, 5). More recently, IL-7 has been implicated as a prosurvival cytokine important for early memory CD8 T cell selection and maintenance (6, 7, 8). IL-7R{alpha} is dynamically regulated and has been shown to be potently down-regulated by human peripheral blood lymphocytes in response to treatment with IL-2 (9). Up-regulation of IL-7R{alpha} in B and T lymphocytes is dependent, at least in part, on the Ets family transcription factors PU.1 (10) and GABP (11). It has been demonstrated (6, 7) in some model systems that expression of IL-7R{alpha} by Ag-specific CD8 T cells at the peak of expansion identifies a population enriched in memory cell precursors. However, it is still not completely clear how absolute a role IL-7R{alpha} expression plays in selecting memory CD8 T cell precursors, because it has also been demonstrated that under some circumstances IL-7R{alpha}+ cells die during the contraction phase (12, 13) and IL-7R{alpha}low cells can survive contraction (14). A recent report addressed this question directly and demonstrated that constitutive expression of IL-7R{alpha} in P14 transgenic CD8 T cells, specific for the glycoprotein33–41 epitope from lymphocytic choriomeningitis virus did not lead to increased survival of cells during the contraction phase following viral infection. This report also found no major differences in the expression of surface markers that would indicate a more rapid generation of a memory phenotype in cells with constitutive expression of IL-7R{alpha} suggesting that the unwavering ability to signal through the IL-7R does not significantly alter the developmental fate of Ag-specific CD8 T cells (15).

The importance of IL-7/IL-7R signaling in the regulation of CD4 T cell responses has also been established. IL-7R signaling is required for the generation of an Ag-specific CD4 T cell response following L. monocytogenes infection. Mice with a knock-in mutation in IL-7R{alpha} (IL-7R{alpha}449F) that has been shown to prevent STAT5 activation (16) have normal thymocyte and peripheral T cell development, but do not make a detectable CD4 T cell response after bacterial infection. Ag-specific CD8 T cells are generated in these mice, but memory is poorly maintained despite Bcl-2 expression that is similar to that detected in wild-type (WT)3 Ag-specific CD8 T cells (17). IL-7 is required for the long-term maintenance of memory CD4 T cells (18, 19) and it has recently been shown that signals delivered by IL-2 early during activation institute a program that results in re-expression of IL-7R{alpha} at later times during the response. When primed with peptide-coated dendritic cells in the absence of IL-2 or IL-2R{alpha}, CD4 T cells do not re-express IL-7R{alpha}, which results in poor survival of memory CD4 T cells (20). It has not previously been investigated how constitutive expression of IL-7R{alpha} by Ag-specific CD4 T cells would influence contraction/memory cell selection of this population.

In the current study, we used mice with constitutive expression of IL-7R{alpha} in T cells to study what effect this had on the response kinetics of endogenous Ag-specific CD4 and CD8 T cell responses following infection of mice with L. monocytogenes. Specifically, we tested the hypothesis that IL-7R{alpha} was the sole selective factor for memory cell precursors by investigating whether constitutive expression of IL-7R{alpha} resulted in increased survival of endogenous Ag-specific CD4 and CD8 T cells during the contraction phase following bacterial infection. Importantly, we also examined the ability of memory Ag-specific CD4 and CD8 T cells generated in IL-7R{alpha} transgenic (Tg) mice to protect and undergo secondary expansion after lethal bacterial challenge. Our results support and extend the conclusion of the previous study using P14 Tg CD8 T cells (15) and show that constitutive expression of IL-7R{alpha} does not impart an expansion, or survival advantage to endogenous Ag-specific CD8 T cells, nor do IL-7R{alpha} Tg Ag-specific CD8 T cells exhibit any major changes in phenotype indicative of altered memory development after bacterial infection. In addition, our results demonstrate that constitutive expression of IL-7R{alpha} results in slightly prolonged expansion of Ag-specific CD4 T cells compared with WT T cells; however, there was no overall survival benefit and no protection from waning memory levels at later times after infection. Memory IL-7R{alpha} Tg Ag-specific CD4 and CD8 T cells expanded appropriately after secondary bacterial challenge and immunized IL-7R{alpha} Tg mice were fully protected. Overall, our data support the conclusion that expression of IL-7R{alpha} by itself is not sufficient to promote survival of effector T cells during the contraction and memory phases following infection.


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

To create IL-7R{alpha} transgenic mice, murine IL-7R{alpha} cDNA was amplified from mouse splenic T cells by RT-PCR, and the sequence was verified after cloning into pCR2.1 vector by TA cloning (Invitrogen). The cDNA was then subcloned into a CD4 enhancer/promoter transgenic vector (21), which lacks the CD4 silencer located in intron 1 and thus directs transgene expression in T lineage cells (Fig. 1A). The plasmid harboring the IL-7R{alpha} cDNA was then linearized and injected into C57BL/6 oocytes. The progenies were screened for the presence of the transgene by Southern blotting (data not shown). Two positive mice were used as founders and bred on the C57BL/6 background. All the infection experiments were done on mice after four to five generations of backcrossing. WT mice in these experiments were littermate controls. L. monocytogenes-infected mice were housed in accordance with biosafety regulations. All animal experiments followed approved Institutional Animal Care and Use Committee protocols.


Figure 1
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  		FIGURE 1. IL-7R{alpha} Tg mice have increased expression of IL-7R{alpha} in developing and mature T cells. A, Schematic showing the structure of transgenic constructs. B, Increased expression of IL-7R{alpha} in the Tg mice. Thymocytes and splenocytes were isolated from IL-7R{alpha} Tg mice and littermate controls, and stained with anti-CD4, CD8, and IL-7R{alpha} Abs. The expression levels of IL-7R{alpha} on thymocyte subsets and splenic CD4 and CD8 T cells are shown, presented by mean fluorescence intensities and percentages of IL-7R{alpha}-expressing cells. Representatives from at least three independent experiments are shown. Shaded histograms are IL-7R{alpha} staining and open histograms are isotype control staining.

 
Bacteria and infection of mice

L. monocytogenes that expresses OVA was a gift from Dr. Hao Shen (University of Pennsylvania, Philadelphia, PA) and Dr. Leo Lefrancois (University of Connecticut, Farmington, CT) (22). An attenuated version of this strain was created by introducing an in-frame deletion in the actA gene as previously described (23) (referred to as actA– LM-OVA). IL-7R{alpha} Tg and control littermates were given a primary infection of 5 x 106 actA- LM-OVA. Immunized mice (day 42 after primary infection) were challenged with 7 x 105 virulent LM-OVA. Bacteria were grown and quantified as previously described (24, 25). All infections were performed via i.v. injection. The number of LM-OVA present in the spleens and livers of challenged mice was determined on day 3 following infection as previously described (24).

Antibodies

The following Abs were used to stain for surface markers in combination with intracellular cytokines to identify Ag-specific T cells: PerCP anti-mouse CD4 (clone RM4–5), PerCP anti-mouse CD8 (clone Ly-2), FITC anti-mouse Thy 1.2 (clone 53–2.1), PE anti-mouse CD27 (clone LG.3A10), PE anti-mouse CD43 (clone 1B11), PE anti-mouse CD62L (clone MEL-14), PE anti-mouse CD127 (clone 12–1271-83'; eBioscience), PE anti-mouse IL-2 (clone JES6–5H4; BioLegend), allophycocyanin anti-mouse IFN-{gamma} (clone XMG1.2; BioLegend). All Abs were from BD Pharmingen unless indicated otherwise. To prevent cleavage of surface CD62L during peptide stimulation, cells were preincubated with 0.1 mM TAPI-2 (Peptides International) for 30 min before the addition of peptide as previously described (26).

Detection and calculation of the number of Ag-specific CD4 and CD8 T cells

Ag-specific CD4 and CD8 T cells were detected by intracellular staining for IFN-{gamma} and, in some cases IL-2, as previously described (27) after a 5.5-h stimulation with either 5 µM listeriolysin O (LLO)190–201 (NEKYAQAYPNVS) or 200 nM OVA257–264 (SIINFEKL) peptide in the presence of brefeldin A (BioLegend). The total number of Ag-specific T cells per spleen was calculated by multiplying the frequency of CD4 or CD8+Thy1.2+IFN-{gamma}+ cells after stimulation with specific peptide by the total number of splenocytes. The number of cells producing cytokine in the absence of peptide was subtracted.

Tetramer analysis and detection of intracellular phospho-STAT 5

MHC class I tetramers specific for OVA257–264 were prepared according to published protocols (28). The method used to detect intracellular phospho-STATs has been previously described (29), but was modified slightly to incorporate tetramer staining to detect Ag-specific CD8 T cells. In short, splenocytes from Tg and WT control mice were rested at 37°C in medium containing 2% serum for 2 h before stimulation with 5 ng/ml rIL-7 (R&D Systems) or IL-15 (PeproTech) for 30 min. Following stimulation, cells were surface stained with APC-OVA257–264 tetramer and PerCP anti-mouse CD8 (clone Ly-2) for 30 min on ice, washed once, then processed for intracellular staining as previously described (29). Abs used were rabbit anti-mouse phospho STAT 5 (Y694) (Cell Signaling Technology) and Alexa Fluor 488 F(ab)2 fragment of goat anti-rabbit IgG (Invitrogen). Samples were analyzed by flow cytometry on a FACS Calibur (BD Biosciences) immediately following the completion of staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Description of IL-7R{alpha} Tg mice

To achieve ectopic expression of IL-7R{alpha} specifically in T cells, we used a CD4 enhancer/promoter transgenic vector (21). This vector fused the CD4 enhancer and CD4 promoter, and also contained a deletion of a silencer in intron 1 that allowed the transgene to be expressed in both CD4 and CD8 T cells (Fig. 1A). The construct was injected into oocytes, and two founder lines were established. IL-7R{alpha} expression is dynamically regulated during thymus ontogeny. IL-7R{alpha} in nontransgenic controls was highly expressed in most immature thymocytes that were double negative for CD4 and CD8 expression, completely turned off in CD4 and CD8 double positive (DP) thymocytes, and up-regulated again in CD4 or CD8 single positive cells (30). The expression of IL-7R{alpha} persisted in mature CD4 and CD8 T cells where it maintains naive T cell survival and homeostasis (5, 31). In IL-7R{alpha} Tg mice, IL-7R{alpha} expression was increased throughout thymocyte development, especially in DP thymocytes (Fig. 1B). The expression of IL-7R{alpha} in splenic T cells was also increased based on mean fluorescence intensity. Consistent with previous findings (32), constitutive expression of IL-7R{alpha} resulted in a slight decrease of thymic cellularity at 6–8 wk of age (97.4 ± 15.4 x 106 in Tg and 146.7 ± 13.2 x 106 in WT; n = 8), probably due to the increased consumption of IL-7 by IL-7R{alpha}-expressing DP cells. However, this did not alter the output of T cells in the periphery. Total numbers of splenocytes were not statistically different between Tg and WT mice 6–10 wk of age (3.9 ± 1.2 x 107 and 5.0 ± 1.1 x 107, respectively; p = 0.13). Total numbers of CD4 (7.5 ± 1.7 x 106 in Tg and 7.1 ± 1.3 x 106 in WT mice; p = 0.67) and CD8 T cells (4.2 ± 0.7 x 106 in Tg and 4.1 ± 1.0 x 106 in WT mice; p = 0.82) were also not different. Both founder lines of Tg mice showed indistinguishable IL-7R{alpha} expression levels and patterns, and thus the following infection experiments were done on one line only.

Constitutive expression of IL-7R{alpha} does not alter the expansion or death of Ag-specific CD8 T cells following infection with L. monocytogenes

Expression of IL-7R{alpha} by a small frequency of effector CD8 T cells at the peak of Ag-induced expansion has been suggested to permit these cells to survive the contraction phase and become memory Ag-specific CD8 T cells (6, 7). Also, signals transmitted through the intracellular domains of the IL-7R ({alpha}- and {gamma}c-chains) appear to enhance expansion of Ag-specific T cells after viral infection (33). IL-7R{alpha} Tg mice were infected with L. monocytogenes to determine whether constitutive expression of IL-7R{alpha} on endogenous CD8 T cells would result in increased memory development due to greater expansion, decreased contraction, or both. The IL-7R{alpha} Tg and WT littermate control mice were on a C57BL/6 background; therefore, actA– LM-OVA was used to infect the mice so the OVA257–264-specific CD8 T cell could be tracked using intracellular cytokine staining for IFN-{gamma}. As shown in Fig. 2A, there were nearly equivalent frequencies of OVA257–264-specific CD8 T cells in representative WT and IL-7R{alpha} Tg mice on day 7 post infection (p.i.). This translated into similar total numbers of OVA257–264-specific CD8 T cells in the spleens of control and Tg mice at all time points evaluated (from days 7 to 42) after primary infection with actA– LM-OVA (Fig. 2B). These results confirm the findings presented in an earlier report (15), which demonstrated no survival advantage in IL-7R{alpha} Tg P14 T cells during contraction after viral infection, and extend the same conclusions to the endogenous CD8 T cell response in a bacterial infection model.


Figure 2
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  		FIGURE 2. Constitutive expression of IL-7R{alpha} does not prevent contraction of Ag-specific CD8 T cells. IL-7R{alpha} Tg and control mice were infected with actA– LM-OVA and the response to OVA257–264 was monitored in the spleen at several times p.i. using intracellular cytokine staining for IFN-{gamma}. A, Example of ICS for IFN-{gamma} at day 7 p.i. Dot plots were first gated on CD8+ cells. Frequencies are Thy 1.2+IFN-{gamma}+ cells within the CD8+ population. B, Total number of OVA257–264-specific CD8 T cells in the spleens of IL-7R{alpha} Tg (squares) and control (triangles) mice at the days p.i. indicated on the x-axis calculated as described in the Materials and Methods section. Each time point is an average of three to six mice. C, D, F–H, Expression of surface markers on OVA257–264-specific CD8 T cells. Histograms are all examples of staining on day 7 p.i. and are first gated on CD8+Thy 1.2+IFN-{gamma}+ cells. Open histograms are isotype Ab staining. Shaded histograms are staining for the marker indicated on the x-axes of the histograms. Frequencies represent the frequency of CD8+Thy 1.2+IFN-{gamma}+ cells within the gate shown. Graphs are frequencies of gated cells at the times p.i. indicated on the x- axes. Each time point is an average of 3–6 mice. The * symbol indicates that the values for IL-7R{alpha} Tg and WT mice on that time point were significantly different (p < 0.05) as determined by unpaired t test. E, Dot plots are examples of staining on d7 p.i. and are first gated on CD8+Thy 1.2+ cells. Frequencies are IFN-{gamma}+ or IFN-{gamma}+IL-2+ cells within the CD8+Thy 1.2+ population. Graph is frequency of IL-2+ cells within the CD8+Thy 1.2+IFN-{gamma}+ (Ag-specific) populations at the times p.i. indicated on the x-axis. Each time point is an average of three to six mice.

 
Given that IL-7R{alpha} expression has been implicated in identifying memory CD8 T cell precursors we next wanted to determine whether constitutive expression of IL-7R{alpha} would alter the progression of endogenous OVA257–264-specific CD8 T cells from an effector phenotype to a memory phenotype. We evaluated OVA257–264-specific CD8 T cells in WT and IL-7R{alpha} Tg mice for expression of several surface markers that are normally associated with a memory-like phenotype at several times p.i. Representative profiles for Ag-specific (CD8+Thy1.2+IFN-{gamma}+) WT and IL-7R{alpha} Tg mice day 7 p.i. are shown in Fig. 2, C–H (top), whereas time course data from multiple mice are shown in bottom graphs. As expected there was significant down-regulation of IL-7R{alpha} by WT Ag-specific CD8 T cells during their expansion followed by gradual reacquisition of IL-7R{alpha} expression as the cells progressed to memory. IL-7R{alpha} expression in the Tg mice was relatively stable and consistently higher than in WT mice (Fig. 2C). There was slight down-regulation of IL-7R{alpha} by Ag-specific CD8 T cells in Tg mice, which was especially notable on day 10 p.i. This could be the result of down-regulation of nontransgene derived IL-7R{alpha}, or a limitation of the Ab used to detect surface expression. We do not feel that this is significant to the interpretation of the results because the Tg T cells still expressed much higher levels of IL-7R{alpha} compared with WT T cells at times p.i. that corresponded to the contraction phase. In addition, as will be discussed later, Tg T cells were capable of receiving IL-7 signals during the contraction phase whereas WT T cells were not (Fig. 3). Despite the disparity in IL-7R{alpha} expression, there were no significant differences in the frequency of KLRG1+ (Fig. 2D), IL-2+ (Fig. 2E), CD27high (Fig. 2F), CD43high (Fig. 2G), or CD62Lhigh (Fig. 2H) OVA257–264-specific CD8 T cells between WT and IL-7R{alpha} Tg mice on day 7 p.i. or any other day that was analyzed, suggesting that constitutive expression of IL-7R{alpha} does not dramatically alter the rate at which Ag-specific CD8 T cells acquire memory characteristics.


Figure 3
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  		FIGURE 3. The Tg IL-7R{alpha} transduces IL-7 signals to Ag-specific CD8 T cells during contraction and does not alter the ability of these cells to make IFN-{gamma} after peptide stimulation. IL-7R{alpha} Tg and control mice were infected with actA– LM-OVA and the response to OVA257–264 was monitored in the spleens at day 7, day 11, and day 14 p.i. using MHC class I tetramer staining and ICS for IFN-{gamma} on splenocytes from the same mice in parallel. A, Representative example of MHC class I tetramer staining on splenocytes from mice day 7 p.i. Dot plots were first gated on CD8+ cells. Frequencies are Thy 1.2+/Tetramer+ cells within the CD8+ population. B, Total numbers of OVA257–264-specific CD8 T cells calculated using MHC class I tetramer staining (triangles) or ICS for IFN-{gamma} (circles) in the samples indicated on the x-axis. There were three mice per group per time point. C–E, Intracellular staining for phospho-STAT5 on day 7 (C), day 11 (D), and day 14 (E) p.i. All histograms are gated on OVA257–264-tetramer positive cells. Shaded histograms are phospho-STAT5 staining of unstimulated T cells. Open histograms are phospho-STAT5 staining of cells stimulated with the cytokine indicated in the upper right hand corner of the histogram overlay. The histograms presented are representative of three mice per group per time point.

 
The Tg IL-7R{alpha} does not alter the ability of Ag-specific CD8 T cells to produce IFN-{gamma} in an Ag-specific manner and is fully functional to transduce IL-7 signals

Enumerating Ag-specific CD8 T cells using intracellular cytokine staining (ICS) for IFN-{gamma} after peptide stimulation assumes that there would not be a difference in the ability of Tg and WT T cells to respond to Ag by making this important effector cytokine. To physically, rather than functionally, show that there were no differences in expansion or contraction of Ag-specific CD8 T cells between Tg and WT control mice we tracked the OVA257–264 response after actA– LM-OVA infection using MHC class I tetramers and ICS for IFN-{gamma} in parallel. As presented in Fig. 3A, the tetramer clearly identified OVA257–264-specific CD8 T cells and this staining showed nearly identical frequencies of Ag-specific CD8 T cells in Tg and WT control mice day 7 p.i. indicating that there was similar expansion in both sets of mice. When the total numbers of Ag-specific CD8 T cells were determined in the same splenocyte preparations generated from Tg and WT mice using either MHC class I tetramer staining or ICS for IFN-{gamma} on days 7, 11, and 14 p.i. there was no statistical difference between the numbers generated using either methodology (Fig. 3B). These data indicate that that the Tg IL-7R{alpha}-chain does not alter the ability of the Tg T cells to make IFN-{gamma} after peptide stimulation and confirm the previous conclusion that there are no differences in expansion or contraction between the Tg and WT Ag-specific CD8 T cells.

We next wanted to demonstrate that the Tg IL-7R{alpha}-chain was functional and that its expression resulted in the ability of Tg Ag-specific CD8 T cells to receive IL-7 signals during the contraction phase when the majority of WT T cells could not. To accomplish this, we combined tetramer staining with intracellular staining for phospho-STAT5 after IL-7 stimulation of splenocytes from mice days 7, 11, and 14 p.i. The Tg IL-7R{alpha}-chain expressed by Ag-specific CD8 T cells was fully functional as demonstrated by the shift in phospho-STAT5 staining after stimulation of splenocytes from mice day 7 p.i. with IL-7 (Fig. 3C). The level of IL-7 responsiveness in the Tg T cells did not change appreciably from day 7 through day 14 p.i., (Fig. 3, C–E), which was predicted because the level of IL-7R{alpha} did not change dramatically on the cells at these times p.i. (Fig. 2C). In contrast, WT Ag-specific CD8 T cells were unresponsive to IL-7 on day 7 p.i. (Fig. 3C), most likely due to significant down-regulation of IL-7R{alpha} at this time point. As the cells progressed through the contraction phase, and progressively up-regulated IL-7R{alpha}, their level of IL-7 responsiveness increased as demonstrated by the shift in phospho-STAT5 detected after stimulation with IL-7 (Fig. 3, D and E). Both Tg and WT Ag-specific CD8 T cells did not change their level of responsiveness to IL-15 during the contraction phase, indicating that phospho-STAT5 could be detected in WT cells if the cytokine receptor was present (Fig. 3, C–E). The ability to respond to IL-15 also demonstrated that Tg T cells were receptive to another cytokine whose receptor included the {gamma}c and the expression of the Tg IL-7R{alpha}-chain was not sequestering all available {gamma}c expressed by the cells. Tg T cells also responded to IL-2 and IL-4 similarly to WT Ag-specific CD8 T cells (data not shown).

In summary, these data demonstrate that, despite the consistent expression of a fully functional IL-7R{alpha}-chain, Tg Ag-specific CD8 T cells exhibit a pattern of expansion and contraction after L. monocytogenes infection that is nearly indistinguishable from WT T cells.

IL-7R{alpha} Tg Ag-specific CD4 T cells exhibit a slightly prolonged expansion phase, but contract normally

IL-7 has also been shown to be important for the development of long-lived memory CD4 T cells (18, 19, 34). Thus, we also evaluated the CD4 T cell response to LLO190–201 in IL-7R{alpha} Tg and WT mice. Similar to what was observed with CD8 T cell responses, there were nearly identical frequencies and total numbers of LLO190–201-specific CD4 T cells in WT and IL-7R{alpha} Tg mice d7 p.i. (Fig. 4A) despite vastly different expression patterns of IL-7R{alpha} at this time point (Fig. 4C). In contrast to Ag-specific CD8 T cells, IL-7R{alpha} Tg LLO190–201-specific CD4 T cells continued to expand until day 8 p.i., whereas the WT T cells had already started to contract at this time point indicating that constitutive expression of IL-7R{alpha} prolonged the ability of CD4 T cells to expand following activation with Ag. However, this increased ability to proliferate was short-lived because by day 10 p.i. both WT and IL-7R{alpha} Tg LLO190–201-specific CD4 T cells had dramatically contracted (Fig. 4B). Of note, constitutive expression of IL-7R{alpha} throughout their development and transition to memory was also unable to protect memory Ag-specific CD4 T cells from slowly decaying over time at the same rate as WT T cells. These results indicate that, although IL-7R{alpha} Tg Ag-specific CD4 T cells did exhibit a slightly prolonged expansion phase, overall, constitutive expression of IL-7R{alpha} did not permit increased survival of T cells during the contraction phase or generation of a more stable long-term memory Ag-specific CD4 T cell population.


Figure 4
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  		FIGURE 4. IL-7R{alpha} Tg CD4 T cells expand slightly longer than control T cells, but contract normally. IL-7R{alpha} Tg and control mice were infected with actA– LM-OVA and the response to LLO190–201 was monitored in the spleen at several times p.i. using intracellular cytokine staining for IFN-{gamma}. A, Example of ICS for IFN-{gamma} at day 7 p.i. Dot plots were first gated on CD4+ cells. Frequencies are Thy 1.2+IFN-{gamma}+ cells within the CD4+ population. B, Total number of LLO190–201-specific CD4 T cells in the spleens of IL-7R{alpha} Tg (squares) and control (triangles) mice at the days p.i. indicated on the x-axis calculated as described in the Materials and Methods section. Each time point is an average of three to six mice. C, E–F, Expression of surface markers on LLO190–201-specific CD4 T cells. Histograms are all examples of staining on day 7 p.i. and are first gated on CD4+Thy 1.2+IFN-{gamma}+ cells. Open histograms are isotype Ab staining. Shaded histograms are staining for the marker indicated on the x-axes of the histograms. Frequencies represent the frequency of CD4+Thy 1.2+IFN-{gamma}+ cells within the gate shown. Graphs are frequencies of gated cells at the times p.i. indicated on the x-axes. Each time point is an average of three to six mice. The asterisk (*) indicates that the values for IL-7R{alpha} Tg and WT mice on that time point were significantly different (p < 0.05) as determined by unpaired t test. D, Dot plots are examples of staining on day 7 p.i. and are first gated on CD4+Thy 1.2+ cells. Frequencies are IFN-{gamma}+ or IFN-{gamma}+IL-2+ cells within the CD4+Thy 1.2+ population. Graph is frequency of IL-2+ cells within the CD4+Thy 1.2+IFN-{gamma}+ (Ag-specific) populations at the times p.i. indicated on the x-axis. Each time point is an average of three to six mice.

 
We also evaluated LLO190–201-specific CD4 T cells for expression of a panel of markers to determine whether constitutive expression of IL-7R{alpha} altered the phenotype of responding cells. Despite a consistently high expression of IL-7R{alpha}, we did not detect a significant difference in frequency of IL-2+ (Fig. 3D), CD27high (Fig. 4E), or CD62Lhigh (Fig. 4F) IL-7R{alpha} Tg LLO190–201-specific CD4 T cells compared with WT T cells. Thus, IL-7R{alpha} expression by itself cannot dictate memory Ag-specific CD4 T cell development.

IL-7R{alpha} Tg memory CD4 and CD8 T cells are protective against secondary infection, but they do not expand greater than WT cells

We wanted to determine whether memory T cells generated in the IL-7R{alpha} Tg mice were of sufficient quality to expand normally after secondary infection and provide protection equally as well as the same number of WT memory T cells. Immunized WT and IL-7R{alpha} Tg mice were challenged with 7 x 105 virulent LM-OVA (~7 LD50) day 42 after primary infection. Secondary T cell expansion was determined by intracellular cytokine staining on day 5 p.i. (Fig. 5, A and B) and protection was determined by evaluating bacterial numbers (CFUs) in spleens (Fig. 5C) and livers (Fig. 5D) of naive and immunized mice on day 3 postchallenge. Both IL-7R{alpha} Tg LLO190–201-specific CD4 (Fig. 5A) and IL-7R{alpha} Tg OVA257–264-specific CD8 (Fig. 5B) T cells expanded after secondary infection. The degree of expansion was similar to what was observed in WT mice. Immunized IL-7R{alpha} Tg mice were as equally protected as WT mice, which is indicated by the CFU counts in the spleens (Fig. 5C) and livers (Fig. 5D) of challenged mice day 3 p.i. Naive mice challenged with the same dose of LM-OVA had succumbed to infection by this time point, which is generally associated with a high number (>108) of bacteria in each organ. These results suggest that constitutive expression of IL-7R{alpha} on memory T cells does not impede, nor improve, secondary expansion and protection from lethal virulent bacterial challenge.


Figure 5
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  		FIGURE 5. IL-7R{alpha} Tg Ag-specific T cells expand after secondary infection and convey protection. Immunized IL-7R{alpha} Tg and control mice day 42 after primary infection were challenged with a high dose of virulent LM-OVA. A, Memory levels and secondary expansion of LLO190–201-specific CD4 T cells in the spleens of control (filled bars) and IL-7R{alpha} Tg (striped bars) mice 5 days after challenge. Numbers of Ag-specific T cells were determined using ICS for IFN-{gamma}. Three mice were used in each group. B, Memory levels and secondary expansion of OVA257–264-specific CD8 T cells in the spleens of control (filled bars) and IL-7R{alpha} Tg (striped bars) mice 5 days after challenge. Numbers of Ag-specific T cells were determined using ICS for IFN-{gamma}. Three mice were used in each group. C, CFU counts in the spleens of control (triangles) and IL-7R{alpha} Tg (circles) mice day 3 post challenge. Each symbol represents one mouse. Data are reported as number of CFU/spleen of each animal. LOD, Limit of detection. D, CFU counts in the livers of control (triangles) and IL-7R{alpha} Tg (circles) mice day 3 post challenge. Each symbol represents one mouse. Data are reported as number of CFU/g liver.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
T cells rely on survival signals to navigate through thymic developmental stages, to endure in the periphery until called upon to respond to Ags, to persist during the contraction phase to become long-lived memory cells, and to maintain memory cell numbers for periods of time that can approach the lifespan of the host. Before methods can be developed to improve the survival of beneficial T cells, such as memory T cells, and to eliminate excess T cells, such as after pathogen clearance, we must first understand what, how, and when survival signals are delivered to T cells.

IL-7 is an important survival cytokine for T cells during multiple phases of their existence. Early reports suggested that IL-7R{alpha} expression marked early memory precursors at the peak of expansion (6, 7). However, until recently it had not been directly tested whether expression of IL-7R{alpha} alone was the sole factor that determined which cells survive contraction after Ag-induced expansion to become memory cells, and which cells die. Our results substantiate recent findings, that IL-7R{alpha} alone does not permit survival of Ag-specific CD8 T cells during contraction (Ref. 15 and Fig. 2). In addition, our study provides evidence that constitutive IL-7R{alpha} expression by Ag-specific CD4 T cells also does not increase survival of this population during contraction (Fig. 4). Effector CD4 and CD8 T cells with constitutive expression of IL-7R{alpha} acquired a memory phenotype, as determined by CD27, CD43, CD62L, and IL-2 staining, with the same kinetics as WT T cells indicating that persistent IL-7R{alpha} expression does not change the developmental pattern of Ag-specific memory cells. Thus, IL-7R{alpha} expression itself does not determine memory cell development.

It has been demonstrated in a recent study that surface expression and mRNA levels of IL-7R{alpha} are specifically down-regulated in lymph node-derived CD4 and CD8 T cells after in vitro culture with the prosurvival cytokines IL-2, IL-4, IL-6, IL-7, and IL-15 (35). It was suggested that this suppression of IL-7R{alpha} was a mechanism to preserve the availability of IL-7 for T cells that had not received recent survival signals and was important for the maintenance of naive T cells because lower numbers of splenocytes were found in mice with over-expression of a IL-7R{alpha} transgene (35). In our experiments, age-matched IL-7R{alpha} Tg and WT littermate control mice did not have statistically different numbers of CD4 or CD8 T cells in their spleens suggesting that the inability to down-regulate IL-7R{alpha} did not create an imbalance of available IL-7 for the maintenance of naive T cells.

It seems unlikely that limited availability of IL-7 is what ultimately determines the number of T cells that survive. In preliminary experiments using real-time PCR, we detected stable amounts of IL-7 mRNA in the spleen days 1–10 after L. monocytogenes infection that were ~2–5-fold decreased when compared with a naive spleen sample (data not shown). In another study, treatment with exogenous IL-7 after vaccinia virus infection did prolong the survival of Ag-specific CD4 T cells; however, neutralization of IL-7 did not increase the frequency of cells that underwent contraction (36). These data would suggest that limiting availability of IL-7 does not fully explain the selection of memory CD4 T cells. The dynamic pattern of IL-7R{alpha} expression demonstrated by Ag-specific CD8 T cells has been shown to be independent of IL-7 following vesicular stomatitis virus infection. Therefore, in this model, IL-7R{alpha} expressing cells are not selected by IL-7 binding to the receptor (37). It remains to be determined whether cognate Ag recognition directly influences IL-7R{alpha} expression, or whether there is also down-regulation of IL-7R{alpha} by bystander T cells after an infection. It has been shown that the inflammatory environment that exists during Ag-specific CD8 T cell priming influences the expression of IL-7R{alpha}. When T cells are primed with peptide-coated DCs, or in antibiotic pretreated mice infected with L. monocytogenes, both of which represent a very low inflammatory environment, Ag-specific CD8 T cells do not down-regulate IL-7R{alpha} expression as dramatically as when they are primed in a more highly inflammatory setting (13, 38). Taking these data together, it appears that IL-7R{alpha} expression may be incorporated into the programmed response of T cells, which is dictated by the priming event.

Our data, in combination with others, implies that we should reconsider the idea that IL-7/IL-7R{alpha} interactions are a selective mechanism for memory T cell precursors. Expression of IL-7R{alpha} by effector cells is not the only key factor to promote transition from the effector to memory phase, but remains critical to support the long-term survival of memory cells.


   Acknowledgments
 
We thank Dr. Chengyu Liu (Transgenic Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD) for performing the oocyte injection and establishing IL-7R{alpha} transgenic line, and Dr. Dan Littman (New York University School of Medicine, New York, NY) for providing the CD4 enhancer/promoter transgenic vector. We also thank Rebecca Podyminogin (University of Iowa, Iowa City, IA) for excellent laboratory assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 Grants AI42767, AI46653, and AI50073 (to J.T.H.), and American Cancer Society Grant PF-05-142-01-LIB (to J.S.H.). Back

2 Address correspondence and reprint requests to Dr. John T. Harty, Department of Microbiology, 3-512 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242. E-mail address: john-harty{at}uiowa.edu Back

3 Abbreviations used in this paper: WT, wild type; Tg, transgenic; DP, double positive; p.i., post infection; ICS, intracellular cytokine staining; LLO, listeriolysin O. Back

Received for publication August 28, 2007. Accepted for publication November 26, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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