Identification of the mechanisms underlying the survival of effector T cells and their differentiation into memory T lymphocytes are critically important to understanding memory development. Because cytokines regulate proliferation, differentiation, and survival of T lymphocytes, we hypothesized that cytokine signaling dictates the fate of effector T cells. To follow cytokine receptor expression during T cell responses, we transferred murine TCR transgenic T cells into naive recipients followed by immunization with peptide emulsified in adjuvant or pulsed on dendritic cells. Our findings did not correlate IL-7R α-chain and IL-2R β-chain expression on effector CD8+ cells with the generation of memory T lymphocytes. However, we could correlate the extent of IL-7Rα expression down-regulation on effector T cells with the level of inflammation generated by the immunization. Furthermore, our findings showed that the maintenance of a high level of IL-7R expression by effector T cells at the peak of the response does not preclude their death. This suggests that maintenance of IL-7R expression is not sufficient to prevent T cell contraction. Thus, our results indicate that expression of the IL-7R is not always a good marker for identifying precursors of memory T cells among effectors and that selective expression of the IL-7R by effector T cells should not be used to predict the success of vaccination.

Following Ag encounter, T lymphocytes undergo massive clonal proliferation and differentiation into effector cells. After elimination of the pathogen, most effector T cells die by apoptosis. A few differentiate into memory T cells that are responsible for long-term protection of the organism against reinfection. According to the dominant paradigm, memory T lymphocytes directly derive from effector T cells that survived the contraction phase (1, 2, 3, 4, 5). However, we still do not understand how effector T cell fate is determined during the contraction phase of the immune response.

Identification of the mechanism determining which effector T cells survive and become memory T lymphocytes is critically important to understanding memory T cell development. Because cytokines regulate the proliferation, differentiation, and survival of T lymphocytes, we hypothesized that cytokine signaling dictates the fate of effector T cells. We postulated that memory T cells emerge from effector T cells that have modified the expression of cytokine receptors involved in the apoptosis of CD8+ effector T cells (IL-2R) or in the generation (IL-7R) and survival (IL-15R) of CD8+ memory T cells (6, 7, 8, 9, 10, 11, 12, 13, 14, 15). To follow cytokine receptor expression during T cell response to Ag, we adoptively transferred TCR transgenic T cells into naive hosts and immunized recipients with the antigenic peptide emulsified in adjuvant or pulsed on dendritic cells (DCs).4 In these models, we were unable to correlate the expression of IL-7R with the generation of long-lived functional memory T cells. Moreover, the levels of expression of IL-7Rα were similar on live and apoptotic effector T cells after immunization with Ag emulsified in adjuvant. Furthermore, IL-7Rα expression was maintained at a high level on effector T cells at the peak of the response following immunization with peptide-pulsed DCs and without influencing the extent of T cell contraction. Recently, Kaech et al. (16) suggested that increased expression of the IL-7R identifies the effector CD8+ T cells that differentiate into memory T cells. In line with this, Badovinac et al. (17) have observed that the absence of T cell contraction correlated with the maintenance of IL-7Rα expression by effector T cells at the peak of the immune response. In contrast, our results indicate that IL-7R expression cannot reliably identify precursors of memory T cells among effectors. Therefore, the selective expression of IL-7Rα by effector T lymphocytes cannot be used to predict the extent of T cell contraction and the success of vaccination.

C57BL/6 mice, 2C mice, and B6.SJL mice were bred at the Guy-Bernier Research Center. Mice expressing a tetracycline-inducible TCR specific for the OVA peptide SIINFEKL in the context of Kb (18) were bred to Cα-deficient mice (Vβ5LTAOCα−/−).

Supernatants of hybridomas were used: H129 (anti-CD4), M5/114 (anti-MHC class II), RA3-6B2 (anti-B220), and 1B2 (anti-2C TCR). The following Abs were used: anti-CD8 (Caltag Laboratories), anti-CD44 (Caltag Laboratories), anti-Ly6C (BD Biosciences), anti-CD122 (anti-IL-2Rβ; BD Biosciences), anti-CD127 (anti-IL-7Rα, A7R34; eBioscience), anti-IFN-γ (Caltag Laboratories), rat anti-mouse IgG1 (BD Biosciences), goat F(ab′)2 anti-rat IgG (H+L) (Caltag Laboratories), and anti-CD45.2 (BD Biosciences). Streptavidin-PE or -PerCP (BD Biosciences) was used to detect biotinylated Abs. The OVA257–264 (SIINFEKL) and the SYRGL (SIYRYYGL) peptides were synthetized at the peptide core facility, Laval University (Quebec, Canada).

Lymph node cells from 2C mice were stained with anti-CD4, anti-class II, and anti-B220 Abs. Stained cells were depleted by cell sorting to obtain purified 2C CD8+ T cells. A total of 5 × 105 2C T cells were injected i.v. in C57BL/6 mice. Recipients were immunized 3 days later either by s.c. injection of 75 μg of SYRGL peptide (SIYRYYGL) emulsified in CFA (Sigma-Aldrich) at the base of the tail or by i.v. injection of 5 × 105 mature DCs pulsed with 2 μg/ml SYRGL peptide. We injected 106 lymph node CD8+ T cells from female Vβ5LTAOCα−/− (CD45.2+) mice i.v. in female B6.SJL (CD45.1+) hosts. Two days later, mice were immunized i.v. with 5 × 105 male B6.SJL mature DCs loaded with 2 μg/ml OVA peptide (OVA257–264).

Bone marrow cells (2 × 105 cells/ml) were plated on 6-well plates in complete RPMI 1640 (supplemented with 10% FCS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10 mM HEPES, 10 μM 2-ME, and penicillin-streptomycin). GM-CSF (500 U/ml) and IL-4 (250 U/ml) were added on days 0, 2, 3, and 6 of culture. To induce maturation of DCs, 1 μg/ml LPS was added on day 6. On day 7, cells were pulsed for 4 h with 2 μg/ml OVA257–264 or SYRGL peptide. Cultures were underlaid on 14.7% nycodenz (Sigma-Aldrich) and centrifuged at 1200 × g for 20 min. DCs were collected from the interface and washed three times in PBS before injection (19).

Mice were anesthetized with ketamine (150 mg/kg) and xylazine (30 mg/kg). A ventral midline incision was made and abdominal skin was retracted to expose the peritoneal wall. PBS (5 ml) was then injected in the peritoneal cavity using a 21-gauge needle. The abdomen was massaged gently, and the peritoneal fluid was withdrawn. The procedure was repeated once.

Mice were anesthetized with ketamine (150 mg/kg) and xylazine (30 mg/kg). A ventral midline incision was made, skin was retracted, and ribs were cut to see the heart and lungs. Portal vein was cut just under the right lung, and 10 ml of PBS were injected into the right ventricle using a 26-gauge needle. Infused lungs were collected in 3 ml of PBS, dissociated in small pieces using 21-gauge needles, and incubated for 30 min at 37°C with 2 mg/ml collagenase (Sigma-Aldrich). After incubation, lung pieces were crushed and cells were filtered through a nylon mesh.

Cells were incubated with 5 μl of annexin V (AnV; BioSource International) and 5 μg/ml propidium iodide (PI) in 50 μl of AnV binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) for 15 min at room temperature.

Splenocytes were stimulated with 1 μg/ml peptide in complete RPMI 1640 for 6 h at 37°C. For the last 2 h, 10 μg of brefeldin A (Sigma-Aldrich) per milliliter of cells were added. Cells were fixed in 2% formaldehyde in PBS 1.2× for 20 min at room temperature. Cells (106) were stained with anti-IFN-γ Abs diluted in 0.5% saponin (Sigma-Aldrich) for 30 min at room temperature. Cells were washed twice without saponin before cell surface staining.

To study memory T cell development, we transferred 2C TCR transgenic CD8+ T cells into C57BL/6 mice. Three days later, we immunized with the specific antigenic peptide SYRGL (20, 21) emulsified in CFA. Five days after immunization, 2C T cells (CD8+1B2+) represented 1–2% of lymph node cells in immunized recipients, but no 2C cells were detected in control mice (Fig. 1,A). The kinetics of the immune response were established to determine the onset of the contraction phase in our model. The peak of the response occurs at day 5 postimmunization followed by the contraction phase of the response up to day 10 (Fig. 1,B). Finally, 1 mo after immunization, 2C cells were CD44highLy6Chigh, as expected for genuine CD8+ memory T cells (Fig. 1,C). Moreover, these cells produced IFN-γ ex vivo when stimulated for 6 h with Ag (Fig. 1 C). These 2C cells were long-lived functional memory T cells because they were still detectable 3 mo after immunization and were able to rapidly produce IFN-γ (not shown). Altogether, our model leads to the generation of a population of functional CD8+ memory T cells that allows us to monitor the expression of cytokine receptors on effector cells during an immune response.

FIGURE 1.

Model to study memory CD8+ T cell development. Sorted 2C cells (5 × 105) were transferred into C57BL/6 mice, followed 3 days later by immunization with 75 μg of the SYRGL peptide emulsified in CFA. A, Five days after immunization, expansion of 2C T cells isolated from inguinal lymph nodes was assessed by staining with anti-CD8 and anti-2C TCR (1B2) Abs. B, Time course of 2C CD8+ T cell expansion. The number of CD8+1B2+ T cells in inguinal lymph nodes is shown as a function of the day postimmunization. Each dot represents a mouse. C, Presence of functional 2C memory T cells 1 mo after immunization. Lymph node cells were stained with anti-CD8, anti-CD44, anti-Ly6C, and 1B2 Abs. Ly6C and CD44 surface expressions on the CD8+1B2+ T cells are shown (right panels). Splenocytes were restimulated in vitro in the presence or absence of 1 μg/ml SYRGL for 6 h; IFN-γ production (left panels) was assessed by intracellular staining. The percentage of 2C T cells is shown when applicable.

FIGURE 1.

Model to study memory CD8+ T cell development. Sorted 2C cells (5 × 105) were transferred into C57BL/6 mice, followed 3 days later by immunization with 75 μg of the SYRGL peptide emulsified in CFA. A, Five days after immunization, expansion of 2C T cells isolated from inguinal lymph nodes was assessed by staining with anti-CD8 and anti-2C TCR (1B2) Abs. B, Time course of 2C CD8+ T cell expansion. The number of CD8+1B2+ T cells in inguinal lymph nodes is shown as a function of the day postimmunization. Each dot represents a mouse. C, Presence of functional 2C memory T cells 1 mo after immunization. Lymph node cells were stained with anti-CD8, anti-CD44, anti-Ly6C, and 1B2 Abs. Ly6C and CD44 surface expressions on the CD8+1B2+ T cells are shown (right panels). Splenocytes were restimulated in vitro in the presence or absence of 1 μg/ml SYRGL for 6 h; IFN-γ production (left panels) was assessed by intracellular staining. The percentage of 2C T cells is shown when applicable.

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It is well established that IL-7 is required for the generation of CD8+ memory T cells (8), that IL-15 promotes the survival of CD8+ memory T lymphocytes (9, 10, 11, 12, 13, 15), and that IL-2 signals can induce cell death (6, 7). Thus, we postulated that those effector T cells that survive the contraction phase and become memory T cells are the ones that have altered the expression profile of IL-2R, IL-7R, and IL-15R to receive optimal differentiation and survival signals. To verify this hypothesis, the surface expression of IL-7Rα and IL-2Rβ (components of the IL-2R and IL-15R) on 2C cells were assessed during T cell response to Ag. Fig. 2,A shows the expression of these receptors on 2C cells (CD8+1B2+) from inguinal lymph nodes relative to recipient CD8+ T cells (CD8+1B2) that do not respond to Ag. The expression of IL-2Rβ increased at day 5 postimmunization on 2C cells relative to recipient CD8+ T cells (Fig. 2,A). This up-regulation persisted during the contraction phase of the immune response, albeit to lower levels than on day 5. In contrast, IL-7Rα expression decreased on 2C cells following immunization. Between days 7 and 20, the expression of the latter was down-regulated on most of the 2C cells, whereas a small proportion of TCR transgenic T cells maintained levels of expression comparable to naive CD8+ T cells (Fig. 2,A). Importantly, all 2C T cells present at the peak of the response and during the contraction phase have seen their Ag because they all have up-regulated the expression level of IL-2Rβ (Fig. 2,A) and CD44 (data not shown). Thus, this rules out the possibility that some of the cells expressing high levels of IL-7Rα represent cells that were not activated following immunization. Moreover, the lower levels of IL-7Rα expression on most of the activated 2C cells cannot be attributed to reduced levels of expression of this receptor on naive 2C T cells because the latter expressed similar levels of IL-7Rα as polyclonal CD8+ T cells from C57BL/6 mice (not shown). To take into account the variations in staining intensities on recipient cells from one day to another, we normalized the results obtained in Fig. 2,A by dividing the mean fluorescence intensity (MFI) of receptor expression on 2C cells by the MFI on recipient CD8+ T cells. A ratio superior to one demonstrates increased expression of the receptor on 2C cells, whereas a ratio lower than one represents down-regulation of the receptor. As shown in Fig. 2 B, the IL-7Rα ratios are always less than one. This means that the expression of this receptor is reduced after immunization. The down-regulation becomes more important over time and stabilizes around day 15. These results show a modulation of IL-7Rα expression during the immune response; however, unlike previous reports (16, 22), we did not observe increased IL-7Rα expression on effector cells during the contraction phase of the T cell response.

FIGURE 2.

Cytokine receptor expression on effector 2C CD8+ T cells after immunization with peptide emulsified in CFA. 2C cells (5 × 105) were transferred into C57BL/6 mice, followed 3 days later by immunization with 75 μg of SYRGL peptide in CFA. At various days after immunization, lymph node cells were stained with anti-IL-2Rβ or anti-IL-7Rα, anti-CD8, anti-CD44, and 1B2 (anti-2C TCR) Abs. A, IL-2Rβ and IL-7Rα expressions on CD8+1B2+ and CD8+1B2 cells are shown relative to isotype control staining. Results are representative of three independent experiments. B, Quantification of IL-7Rα expression by 2C cells during an antigenic response. MFI of IL-7Rα expression on CD8+1B2+ T cells was normalized to MFI of IL-7Rα expression on the CD8+1B2 T cells. The MFI ratio for each mouse is shown over time.

FIGURE 2.

Cytokine receptor expression on effector 2C CD8+ T cells after immunization with peptide emulsified in CFA. 2C cells (5 × 105) were transferred into C57BL/6 mice, followed 3 days later by immunization with 75 μg of SYRGL peptide in CFA. At various days after immunization, lymph node cells were stained with anti-IL-2Rβ or anti-IL-7Rα, anti-CD8, anti-CD44, and 1B2 (anti-2C TCR) Abs. A, IL-2Rβ and IL-7Rα expressions on CD8+1B2+ and CD8+1B2 cells are shown relative to isotype control staining. Results are representative of three independent experiments. B, Quantification of IL-7Rα expression by 2C cells during an antigenic response. MFI of IL-7Rα expression on CD8+1B2+ T cells was normalized to MFI of IL-7Rα expression on the CD8+1B2 T cells. The MFI ratio for each mouse is shown over time.

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Although these data show no correlation between IL-7Rα expression on effector T cells during the contraction phase and memory T cell generation, they do not rule out the possibility that IL-7Rα down-regulation through the contraction phase results from sustained antigenic stimulation of effector cells due to persistence of Ag when mice are immunized with CFA emulsion. Thus, we have evaluated IL-7Rα expression on effector 2C T cells during the contraction phase in a model where Ag does not persist. To do so, we adoptively transferred purified 2C CD8+ T cells into female C57BL/6 mice as previously described with the exception that instead of immunizing with peptide emulsified in CFA, we used peptide-pulsed mature bone marrow-derived DCs. Two days after adoptive transfer, mice were immunized by i.v. injection of 5 × 105 male DCs pulsed with 2 μg/ml SYRGL peptide. We chose to immunize female mice with male DCs to provide CD4 help, which is necessary for proper development of functional and long-lived memory CD8+ T cells (23, 24, 25, 26). Importantly, 2C T cells are not cross-reactive to the male Ag as shown by their lack of expansion after immunization with male DCs that were not pulsed with SYRGL peptide (Fig. 3,A, right panels). However, immunization with peptide-pulsed mature DCs leads to the expansion of 2C T cells (CD8+1B2+) and to the development of long-lived memory T cells (Ly6Chigh), as shown in Fig. 3,A. Moreover, these memory T cells are functional because they rapidly, within 6 h, produced IFN-γ ex vivo following peptide stimulation (Fig. 3,A). Because this model leads to the generation of a population of functional CD8+ memory T cells, we further monitored the expression of the IL-7Rα on effector cells during the contraction phase of the immune response. As shown in Fig. 3, B and C, IL-7Rα expression was maintained at high levels on effector 2C T cells at the peak (day 5) of the antigenic response and during all of the contraction phase (from days 5 to 14). To correct for variations in IL-7Rα staining intensity that occur from one day to another, we always compared IL-7Rα expression on 2C effector T cells (CD8+1B2+) with the recipient naive CD8+ T cells (CD8+1B2) from the same mouse (Fig. 3,B). Importantly, the expression of IL-7Rα on recipient cells was unaffected by the immunization (data not shown). Moreover, Fig. 3,C illustrates that a much higher number of CD8+1B2+ effector T cells maintained IL-7R expression than the number of effector CD8+ T cells that are predicted to survive to the contraction phase of the T cell response. Although IL-7Rα expression was maintained at a high level on effector 2C T cells, it was interesting that we still observed a normal contraction of the T cell response (Fig. 3 D), suggesting that IL-7 signals alone are not sufficient for the survival of effector T cells. Thus, the maintenance of high levels of IL-7Rα at the surface of effector T cells does not in our models allow for the identification of memory T cell precursor within the CD8+ effector pool and does not prevent contraction of the T cell response.

FIGURE 3.

IL-7Rα expression is maintained on effector 2C T cells during the contraction phase that occurs following immunization with peptide-pulsed mature DCs. A, Generation of 2C CD8+ effector and memory T cells after immunization with peptide-pulsed DCs. First, 106 purified 2C CD8+ T cells (1B2+) were transferred into C57BL/6 hosts (1B2). Three days posttransfer, mice were immunized with 5 × 105 mature DCs loaded with 2 μg/ml SYRGL peptide or unpulsed DCs. Days 5 and 54 after immunization, responsive CD8+ T cells were detected by staining lymph node cells with anti-CD8 and 1B2 Abs. Percentages and numbers of 2C CD8+ T cells (1B2+) from one lymph node are indicated on each dot plot. Histogram (bottom) shows the phenotype of effector and memory T cells. Ctrl, control. B, IL-7Rα expression on 2C effector T cells during the contraction phase. Effector T cells were generated as described in A. At various days after immunization, lymph node cells were stained with anti-2C TCR (1B2), anti-CD8, and anti-IL-7Rα Abs. IL-7Rα expressions on effector T cells (CD8+1B2+) and naive C57BL/6 recipient T cells (CD8+1B2) are shown relative to isotype control staining for two independent mice of three. C, A high level of IL-7Rα expression on 2C effectors does not exclusively identify CD8+ memory T cell precursors. The percentage of 2C T cells expressing IL-7Rα at the same level as naive CD8+ T cells (IL-7Rαhigh cells) and the percentage of 2C memory precursor effector T cells are shown over time. D, Kinetics of T cell contraction. The number of Ag-specific T cells (CD8+1B2+) present in lymph nodes (mesenteric, inguinal, and brachial) at different days after immunization is shown.

FIGURE 3.

IL-7Rα expression is maintained on effector 2C T cells during the contraction phase that occurs following immunization with peptide-pulsed mature DCs. A, Generation of 2C CD8+ effector and memory T cells after immunization with peptide-pulsed DCs. First, 106 purified 2C CD8+ T cells (1B2+) were transferred into C57BL/6 hosts (1B2). Three days posttransfer, mice were immunized with 5 × 105 mature DCs loaded with 2 μg/ml SYRGL peptide or unpulsed DCs. Days 5 and 54 after immunization, responsive CD8+ T cells were detected by staining lymph node cells with anti-CD8 and 1B2 Abs. Percentages and numbers of 2C CD8+ T cells (1B2+) from one lymph node are indicated on each dot plot. Histogram (bottom) shows the phenotype of effector and memory T cells. Ctrl, control. B, IL-7Rα expression on 2C effector T cells during the contraction phase. Effector T cells were generated as described in A. At various days after immunization, lymph node cells were stained with anti-2C TCR (1B2), anti-CD8, and anti-IL-7Rα Abs. IL-7Rα expressions on effector T cells (CD8+1B2+) and naive C57BL/6 recipient T cells (CD8+1B2) are shown relative to isotype control staining for two independent mice of three. C, A high level of IL-7Rα expression on 2C effectors does not exclusively identify CD8+ memory T cell precursors. The percentage of 2C T cells expressing IL-7Rα at the same level as naive CD8+ T cells (IL-7Rαhigh cells) and the percentage of 2C memory precursor effector T cells are shown over time. D, Kinetics of T cell contraction. The number of Ag-specific T cells (CD8+1B2+) present in lymph nodes (mesenteric, inguinal, and brachial) at different days after immunization is shown.

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Because we did not observe any enrichment for cells selectively expressing the IL-7R during the contraction phase, we further investigated cytokine receptor expression levels in another model. We transferred Vβ5LTAOCα−/− T cells (18), specific for the OVA peptide 257–264 in the context of Kb, into B6.SJL female recipients followed by immunization with peptide-pulsed male DCs. Again, we immunized female mice with male DCs to provide CD4 help. As shown in Fig. 4,A, immunization with peptide-pulsed mature DCs leads to the expansion of Vβ5LTAOCα−/− T cells (CD45.2+) and to the development of long-lived memory T cells (Ly6Chigh) with the peak of the T cell response occurring at day 4 after immunization (Fig. 4,B). Moreover, our protocol leads to the generation of functional memory T cells because they make efficient recall response (data not shown). The expression of IL-7Rα by Vβ5LTAOCα−/− effector T cells was subsequently determined before (day 3) and at the peak of the response (day 4) and during the contraction phase (days 5, 7, 10, and 14) in lymph nodes and spleen. We did not observe a selective expression of the IL-7R by a subpopulation of effector cells (Fig. 5 A), thus confirming our observations in the 2C model.

FIGURE 4.

Generation of Vβ5LTAOCα−/−CD8+ effector and memory T cells after immunization with peptide-pulsed DCs. A, Immunization with peptide-pulsed DCs leads to the expansion of Vβ5LTAOCα−/− T cells and to the development of memory T cells. First, 106 CD8+ (CD45.2+) T cells from Vβ5LTAOCα−/− mice were transferred into B6.SJL hosts (CD45.1+). On day 2 posttransfer, mice were immunized with 5 × 105 mature DCs loaded with OVA257–264 (DCs OVA257–264) or unpulsed DCs. On days 4 and 60 after immunization, responsive CD8+ T cells were detected by staining lymph node cells with anti-CD45.2 and anti-CD8 Abs. Percentages and numbers of OVA-specific CD8+ T cells (CD45.2+) from one lymph node are indicated on each dot plot. Histogram (bottom) shows the phenotype of effector and memory T cells. B, Kinetics of the T cell response. The percentages of Ag-specific Vβ5LTAOCα−/− T cells (CD8+CD45.2+) recovered from lymph nodes at different days after immunization are shown.

FIGURE 4.

Generation of Vβ5LTAOCα−/−CD8+ effector and memory T cells after immunization with peptide-pulsed DCs. A, Immunization with peptide-pulsed DCs leads to the expansion of Vβ5LTAOCα−/− T cells and to the development of memory T cells. First, 106 CD8+ (CD45.2+) T cells from Vβ5LTAOCα−/− mice were transferred into B6.SJL hosts (CD45.1+). On day 2 posttransfer, mice were immunized with 5 × 105 mature DCs loaded with OVA257–264 (DCs OVA257–264) or unpulsed DCs. On days 4 and 60 after immunization, responsive CD8+ T cells were detected by staining lymph node cells with anti-CD45.2 and anti-CD8 Abs. Percentages and numbers of OVA-specific CD8+ T cells (CD45.2+) from one lymph node are indicated on each dot plot. Histogram (bottom) shows the phenotype of effector and memory T cells. B, Kinetics of the T cell response. The percentages of Ag-specific Vβ5LTAOCα−/− T cells (CD8+CD45.2+) recovered from lymph nodes at different days after immunization are shown.

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FIGURE 5.

No selective expression of IL-7Rα on Vβ5LTAOCα−/− T cells during the contraction phase. A, IL-7Rα expression on Vβ5LTAOCα−/− effector T cells during the contraction phase. Effector T cells were generated as described in Fig. 4 A. At various days after immunization, lymph node (LN) and spleen cells were stained with anti-CD8, anti-CD45.2, anti-CD45.1, and anti-IL-7Rα Abs. IL-7Rα expressions on effector T cells (CD8+CD45.2+) and naive B6.SJL recipient T cells (CD8+CD45.1+) are shown relative to isotype control staining for one representative mouse. B, Quantification of IL-7Rα expression by Vβ5LTAOCα−/− lymph node T cells during an antigenic response. MFI of IL-7Rα expression on Vβ5LTAOCα−/− T cells (CD45.2+) was normalized to MFI of IL-7Rα expression on the recipient CD8+ T cells (CD45.2). The MFI ratio for each mouse is shown over time. C, A high percentage of early effector Vβ5LTAOCα−/− T cells maintained IL-7Rα expression after immunization with peptide-pulsed DCs. The percentage of Vβ5LTAOCα−/− effector T cells expressing IL-7Rα at the level of the one observed on naive CD8+ T cells is shown over time. D, A high level of IL-7Rα expression on Vβ5LTAOCα−/− effectors does not exclusively identify CD8+ memory T cell precursors. The percentage of Vβ5LTAOCα−/− T cells expressing IL-7Rα at the same level as naive CD8+ T cells (IL-7Rαhigh cells) and the percentage of Vβ5LTAOCα−/− memory precursor effector T cells are shown over time.

FIGURE 5.

No selective expression of IL-7Rα on Vβ5LTAOCα−/− T cells during the contraction phase. A, IL-7Rα expression on Vβ5LTAOCα−/− effector T cells during the contraction phase. Effector T cells were generated as described in Fig. 4 A. At various days after immunization, lymph node (LN) and spleen cells were stained with anti-CD8, anti-CD45.2, anti-CD45.1, and anti-IL-7Rα Abs. IL-7Rα expressions on effector T cells (CD8+CD45.2+) and naive B6.SJL recipient T cells (CD8+CD45.1+) are shown relative to isotype control staining for one representative mouse. B, Quantification of IL-7Rα expression by Vβ5LTAOCα−/− lymph node T cells during an antigenic response. MFI of IL-7Rα expression on Vβ5LTAOCα−/− T cells (CD45.2+) was normalized to MFI of IL-7Rα expression on the recipient CD8+ T cells (CD45.2). The MFI ratio for each mouse is shown over time. C, A high percentage of early effector Vβ5LTAOCα−/− T cells maintained IL-7Rα expression after immunization with peptide-pulsed DCs. The percentage of Vβ5LTAOCα−/− effector T cells expressing IL-7Rα at the level of the one observed on naive CD8+ T cells is shown over time. D, A high level of IL-7Rα expression on Vβ5LTAOCα−/− effectors does not exclusively identify CD8+ memory T cell precursors. The percentage of Vβ5LTAOCα−/− T cells expressing IL-7Rα at the same level as naive CD8+ T cells (IL-7Rαhigh cells) and the percentage of Vβ5LTAOCα−/− memory precursor effector T cells are shown over time.

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To take into account the variations in staining intensities on recipient cells from one day to another, we normalized the results obtained in Fig. 5,A by dividing the MFI of receptor expression on Vβ5LTAOCα−/− T cells by the MFI on recipient CD8+ T cells. As shown in Fig. 5,B, IL-7Rα expression is more down-regulated early in the response (days 3 and 4) but rapidly returned to the level of naive T cells (day 5, ratio of 1). Unlike the report of Kaech et al. (16), more effector Vβ5LTAOCα−/− CD8+ T cells maintained a high level of IL-7Rα expression than expected to survive to the contraction phase of the T cell response (Fig. 5, C and D). For example, at the peak of the T cell response (day 4), 70% of the effector T cells expressed IL-7Rα at a level similar to naive T cells (Fig. 5,C), although only 7–9% of these effectors will survive to become memory T cells (Fig. 5,D). Thus, this clearly illustrates a lack of correlation between the level of IL-7Rα expression and the survival of effector CD8+ T cells during the contraction of the T cell response (Fig. 5 D).

Interestingly, like in the 2C model, IL-7Rα expression was maintained at a high level in Vβ5LTAOCα−/− effectors without affecting the contraction of the T cell response (Fig. 4 B). Moreover, Vβ5LTAOCα−/− effector T cells also behave similarly to 2C cells in relation to IL-2Rβ expression levels during the contraction phase (not shown).

It was also possible that we were missing the effector T cells expressing a low level of the IL-7Rα due to their migration to peripheral nonlymphoid sites. To rule out this possibility, we followed IL-7Rα expression by effector Vβ5LTAOCα−/− CD8+ T cells that migrated to the lungs and peritoneal cavity. As shown if Fig. 6,A, the patterns of expression of IL-7Rα by effector T cells during the expansion and contraction phases was similar in the lungs and peritoneal cavity to patterns observed in lymph nodes and spleen (Fig. 5,A). We also observed a similar kinetic of T cell expansion and death as the one observed in lymph nodes and spleen (Fig. 6, B and C). Therefore, our data demonstrate a lack of correlation between IL-7Rα expression and effector CD8+ T cell survival.

FIGURE 6.

No selective expression of IL-7Rα on Vβ5LTAOCα−/− effector T cells from peripheral nonlymphoid organs. A, IL-7Rα expression on Vβ5LTAOCα−/− effector T cells from peripheral nonlymphoid organs during the expansion and contraction phase of the antigenic response. Effector T cells were generated as described in Fig. 4 A. At various days after immunization, lymphoid cells obtained from the lungs and the peritoneal cavity were stained with anti-CD8, anti-CD45.2, anti-CD45.1, and anti-IL-7Rα Abs. IL-7Rα expression by effector T cells (CD8+CD45.2+) and naive B6.SJL recipient T cells (CD8+CD45.1+) is shown relative to isotype control staining for one representative mouse. B and C, Kinetics of the T cell response in nonlymphoid organs. The percentages (B) and the numbers (C) of Ag-specific Vβ5LTAOCα−/− T lymphocytes (CD8+CD45.2+) recovered from lungs and peritoneal cavity are shown over time. Each dot represents the percentage or the number of Ag-specific T cells per mouse from a pool of three mice.

FIGURE 6.

No selective expression of IL-7Rα on Vβ5LTAOCα−/− effector T cells from peripheral nonlymphoid organs. A, IL-7Rα expression on Vβ5LTAOCα−/− effector T cells from peripheral nonlymphoid organs during the expansion and contraction phase of the antigenic response. Effector T cells were generated as described in Fig. 4 A. At various days after immunization, lymphoid cells obtained from the lungs and the peritoneal cavity were stained with anti-CD8, anti-CD45.2, anti-CD45.1, and anti-IL-7Rα Abs. IL-7Rα expression by effector T cells (CD8+CD45.2+) and naive B6.SJL recipient T cells (CD8+CD45.1+) is shown relative to isotype control staining for one representative mouse. B and C, Kinetics of the T cell response in nonlymphoid organs. The percentages (B) and the numbers (C) of Ag-specific Vβ5LTAOCα−/− T lymphocytes (CD8+CD45.2+) recovered from lungs and peritoneal cavity are shown over time. Each dot represents the percentage or the number of Ag-specific T cells per mouse from a pool of three mice.

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To determine whether the expression of IL-7Rα dictates the fate of effector cells, we evaluated receptor expression levels between dying (apoptotic) effector T cells and those surviving to become memory T cells. Fig. 7 shows normalized levels of expression of IL-7Rα on apoptotic (AnV+PI) and nonapoptotic (AnVPI) effectors. Apoptotic 2C cells express higher levels of the receptor compared with nonapoptotic cells (Fig. 7). This is not due to an increase in background staining on apoptotic cells because there was no difference in staining for other cell surface markers (CD8, TCR) on apoptotic and nonapoptotic cells (data not shown). Similarly, the IL-2Rβ expression level was also higher on apoptotic effector T cells (not shown). These results show that 2C effector T cells that survive the contraction phase and become memory cells express lower or similar levels of the IL-2Rβ and IL-7Rα than effector cells that die at the termination of the response. Unexpectedly, these findings demonstrate a lack of correlation between high IL-7Rα expression and effector T cell survival and thus memory T cell development.

FIGURE 7.

Live effector 2C T cells do not express IL-7Rα at higher levels. IL-7Rα expression on apoptotic (AnV+) vs live (AnV) effector 2C CD8+ T cells during the immune response. At various days after immunization with peptide pulsed in CFA, lymph node cells were stained with anti-IL-7Rα, anti-CD8, and 1B2 Abs and with AnV and PI to detect apoptotic cells. MFI of IL-7Rα expression on CD8+1B2+PI T cells was normalized to MFI of the CD8+1B2PI T cells to obtain a ratio for the AnV+ or the AnV T cells. Each dot represents a mouse.

FIGURE 7.

Live effector 2C T cells do not express IL-7Rα at higher levels. IL-7Rα expression on apoptotic (AnV+) vs live (AnV) effector 2C CD8+ T cells during the immune response. At various days after immunization with peptide pulsed in CFA, lymph node cells were stained with anti-IL-7Rα, anti-CD8, and 1B2 Abs and with AnV and PI to detect apoptotic cells. MFI of IL-7Rα expression on CD8+1B2+PI T cells was normalized to MFI of the CD8+1B2PI T cells to obtain a ratio for the AnV+ or the AnV T cells. Each dot represents a mouse.

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In this report, we demonstrate that 5 days after immunization IL-2Rβ is strongly up-regulated, preceding the contraction phase following an antigenic response. This correlates with the role of IL-2 in apoptosis of effector T cells (6, 7). Effector T cells that are destined to die during the contraction phase may require high levels of IL-2Rβ at the peak of the response to provide optimal death signals, mediated by IL-2. However, IL-2Rβ expression remains high during the course of the contraction phase relative to naive cells. This can be explained by the fact that the IL-2Rβ constitutes part of the IL-15R because IL-15 is necessary for memory CD8+ T cell survival (9, 10, 11, 12, 13, 14, 15). Therefore, IL-2Rβ expression levels may be elevated on effector T cells to allow them to survive the contraction phase and differentiate into memory cells. Thus, the generated memory T cells will be able to receive the IL-15 survival signal. Unfortunately, this marker cannot be used to identify effector CD8+ T cells differentiating into memory cells because IL-2Rβ is already expressed on all effector cells at the peak of the response and maintained uniformly during the contraction phase.

IL-7Rα is constitutively expressed on naive T cells but is down-regulated following T cell stimulation (8, 16, 22, 27). It is believed that this occurs due to the production of IL-2 by activated T cells that then leads to the down-regulation of IL-7Rα expression (27). Effector T cells have decreased levels of IL-7R at their surface, as demonstrated in vivo (8). IL-7 signaling is known to be required for proper development of memory T cells from effectors (8). We hypothesized that effector T cells expressing higher level of IL-7R, either by maintaining or by regaining its expression, would constitute a small population endowed with the ability to survive the contraction phase to become memory T lymphocytes. To support this, we would expect to see an enrichment of cells expressing high levels of IL-7Rα during the contraction phase. However, using two different TCR transgenic models, we saw no enrichment of IL-7Rα expression on effector CD8+ cells during the contraction phase. This is in striking contrast to the report of Kaech et al. (16) who observed a selective enrichment of CD8+ T cells expressing IL-7R during the contraction phase of the T cell response to lymphocytic choriomeningitis virus (LCMV). One major difference between our models and that used by Kaech et al. (16) is the level of down-regulation of IL-7R. Our findings demonstrate that, at the peak of the antigenic response, few of the effector T cells have down-regulated IL-7Rα without completely losing its expression (Figs. 2 A, 3B, 5A, and 6), whereas IL-7Rα expression was completely abrogated on LCMV-specific CD8+ effector cells (16). The degree to which the IL-7Rα-chain differs in its down-regulation in the different models is probably due to the extent of T cell expansion and thus IL-2 production. In the LCMV model used by Kaech et al., CD8+ T cell expansion is massive, reaching over 20 × 106 Ag-specific CD8+ T cells in the spleen (16). However, in our models of transfer of TCR transgenic CD8+ T cells followed by immunization with peptide emulsified in CFA or pulsed on DCs, the levels of T cell expansion are much lower (<106 cells per spleen). We believe that the massive T cell expansion that occurs during LCMV infection leads to the production of very high levels of IL-2 that is then capable of efficiently abrogating IL-7Rα expression on LCMV-specific effector CD8+ T cells at the peak of the response. To further survive and differentiate into memory T cells, these effectors must reexpress IL-7Rα. Only a small percentage that does so will survive the contraction phase. This explains the selective enrichment of IL-7Rα+ CD8+ T cells observed by Kaech et al. Because IL-7Rα is only partially down-regulated by effector CD8+ T cells in our models, this precludes the use of IL-7Rα expression in identifying memory precursors among CD8+ effector cells during the contraction phase. Indeed, our findings demonstrate that all effector T cells should be able to receive IL-7 signals to further survive and differentiate into memory cells. This indicates that other factors besides IL-7 participate actively in the fate of effector T lymphocytes or that stochastic mechanisms regulate the differentiation of effector CD8+ T cells into memory cells. Notably, our results also show that the maintenance of high levels of IL-7Rα expression by effector T cells at the peak of the antigenic response does not prevent the contraction of the T cell response in our models. This is in contrast with the recent observation of Badovinac and collaborators (17) that correlates a lack of T cell contraction with the maintenance of high level IL-7Rα expression by effector CD8+ T cells. Moreover, they (17) have shown that early inflammation mediated by IFN-γ production regulates the contraction phase, maybe by influencing IL-7Rα expression by effectors. Because we observed efficient T cell contraction even if effector T cells express IL-7Rα at high levels, it seems unlikely that inflammation is the sole factor controlling IL-7Rα expression at the peak of the T cell response. Moreover, our data suggest that other events independent of IL-7Rα expression need to occur to allow effector T cell survival and their further differentiation into memory T cells. The identification of these other signals that either protect effector T cells from death or promote their survival and differentiation will bring valuable information about the mechanism by which effector T cells avoid T cell death and become memory T cells.

Intriguingly, we have observed that, during the contraction phase, live effector T cells express lower levels of IL-7Rα than apoptotic effector T cells (Fig. 7). This is in sharp contrast to the notion that cells expressing higher levels of IL-7Rα will receive more survival signals from IL-7. In light of the recent report of Park and collaborators (28) showing that IL-7 signals down-regulate IL-7Rα transcription and cell surface expression, it is tempting to speculate that the reduced expression of IL-7Rα by live effector T cells compared with that undergoing apoptosis reflect the fact that the former have received IL-7 survival signals.

Interestingly, our results clearly show that abrogation of IL-7Rα expression is not a prerequisite for the induction of cell death of effector CD8+ T lymphocytes. The induction of proapoptotic molecules in effector T cells, such as the selective increases in caspase-3 expression (29), FasL, and TNFRs (30, 31), is probably sufficient to induce T cell death even in the presence of a survival signal provided by IL-7.

We thank the members of the laboratory for discussions, S. Ouellet for cell sorting, and L. Sabbagh for critical reading of the manuscript. We thank C. Perreault for reviewing the manuscript and for kindly providing 2C mice and the 1B2 hybridoma. We acknowledge the editorial assistance of Judith Kashul.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Canadian Institutes of Health Research (CIHR). N.L. is a New Investigator of the CIHR.

4

Abbreviations used in this paper: DC, dendritic cell; AnV, annexin V; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; PI, propidium iodide.

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