Memory CD44int CD8 T cells show increased proliferative responses and IFN-{gamma} production following antigenic challenge in vitro

Maria Pihlgren, Christophe Arpin, Thierry Walzer, Martine Tomkowiak, Annie Thomas, Jacqueline Marvel and Patrice M. Dubois

Immunologie Cellulaire, Laboratoire de Biologie Moléculaire et Cellulaire, Ecole Normale Supérieure de Lyon, CNRS UMR 49, 46 allée d'Italie, 69364 Lyon Cedex 07, France

Correspondence to: J. Marvel


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
F5 TCR transgenic mice challenged in vivo with peptide generate long-lived primed CD8 T cells that hyper-proliferate in response to peptide in vitro. These primed CD8 T cells can be subdivided into three distinct populations on the basis of CD44 cell surface expression. In this report, we show that among primed CD8 T cells, those expressing intermediate levels of CD44 appear to be true memory T cells by the measurement of a variety of characteristics. Indeed, these cells hyper-proliferate in response to peptide re-stimulation in vitro, and produce IFN-{gamma} with faster kinetics and at higher levels than naive populations in vitro. We also show that CD8 T cells expressing high levels of CD44 express several activation markers and cycle in vivo in the absence of antigen. However, this population is unable to respond to peptide stimulation in vitro as measured by both proliferation and IFN-{gamma} secretion. The origin and specificity of these cells is unknown. These results provide evidence that memory CD8 T cells are functionally different from naive CD8 T cells both in terms of proliferation and cytokine secretion. They identify the CD8/CD44int T cells as the population responsible for hyper-reactivity in vitro.

Keywords: cell surface molecules, cytokines, memory, T lymphocytes


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Secondary immune responses are faster and more efficient than primary responses, a phenomenon attributed to immunological memory. For T cell memory, two major hypotheses, not mutually exclusive, have emerged to explain the increased speed and efficiency of secondary responses. One hypothesis states that an increase in the number of antigen-specific precursors accounts for the more efficient memory response. Indeed, an increase in the number of reactive lymphocytes following the primary response has been demonstrated for both CD4 and CD8 T cells (13). The second hypothesis claims that memory T cells are intrinsically different from naive T cells. For example, memory T cells may have a lower threshold for activation than naive T cells. This was suggested by early studies showing increased responsiveness and IFN-{gamma} secretion by T cells expressing activation/memory T cell markers such as LFA-3, CD44 or CD45RO (47). However, interpretation of these results was controversial as in those experiments the activation stage of the cells was not studied and more importantly one could never exclude the possibility that a subset of T cells with the highest reactivity towards antigen was expanded following priming. Over the last few years, studies in TCR transgenic mice have provided some experimental evidence suggesting that T cells acquire a lower activation threshold following priming. For example, CD4 memory T cells specific for a chicken ovalbumin peptide have been shown to proliferate in response to lower doses of peptide antigen than naive CD4 T cells in vitro (8). Moreover, when compared to primed CD4 T cells, proliferation of CD4 naive T cells is more dependent on co-stimulation (9,10). For CD8 T cells it has been shown that, in vivo, TCR transgenic CD8 memory T cells give a more sustained but slower proliferative response than naive CD8 T cells and have different survival requirements (11,12). In vitro CD8 memory cells have a lower activation threshold than naive CD8 T cells (13,14).

We have used F5 TCR transgenic mice to study the characteristics of CD8 memory T cells. CD8 T cells of these mice are specific for an influenza nucleoprotein-derived peptide (15). Intraperitoneal injection of influenza virus nucleoprotein peptide (residues 366–374) in F5 mice leads to proliferation of the majority of CD8 T cells, as measured by BrdU incorporation (13). Activated F5 CD8 T cells also acquire the ability to lyse peptide-loaded targets (15). Primed CD8 T cells from thymectomized F5 mice immunized 6 weeks earlier are resting. However, in vitro they respond to lower doses of peptide than naive CD8 T cells in the presence of exogenous IL-2, indicating that these cells have acquired a new activation threshold (13).

In primed mice, three populations of CD8 T cells can be identified on the basis of CD44 expression, i.e. CD8 T cells expressing low, intermediate or high levels of CD44. Six weeks after priming 30–60% of CD8 T cells express intermediate levels of CD44. CD8 T cells expressing low and high levels of CD44 are also present in naive mice, indicating that these populations do not represent antigen-primed CD8 T cells. However, as CD8 memory T cells identified in other systems express high levels of CD44 it was possible that some CD8 CD44high T cells were in fact memory T cells. We have therefore characterized CD8 T cells expressing different levels of CD44 with respect to proliferation and cytokine secretion in vitro, surface phenotype, and turnover in vivo.

In this study we show that CD8 T cells expressing intermediate levels of CD44 are responsible for the hyper-reactive proliferative responses and cytokine production seen in bulk cultures of primed CD8 T cells. In contrast, CD8 T cells expressing high levels of CD44 do not proliferate in response to antigenic peptide challenge in vitro despite the fact that these cells show an activated phenotype in vivo.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and immunizations
C57BL/10 F5 TCR transgenic mice and RAG–/– F5 mice were a gift from D. Kioussis (16). All mice were bred in the institute's animal facility. Thymectomies were performed on 5- to 7-week-old mice which were then allowed to recover for at least 4 weeks before immunization. Mice were injected with 50 nmol of the A/NT/60/68 influenza virus nucleoprotein peptide: Ala-Ser-Asn-Glu-Asn-Met-Asp-Ala-Met NP-(366–374) (Neosystems Laboratoire, Strasbourg, France) in PBS in the peritoneal cavity. Mice were primed 5–12 weeks prior to experiments. Control mice were either not treated or injected with PBS alone. All primed and naive F5 mice were thymectomized.

Fluorescence staining and flow cytometry sorting
For sorting, cells from mesenteric, inguinal and salivary lymph nodes of F5 transgenic mice were pooled and washed once with DMEM (Gibco/BRL, Gaithersburg, MD) supplemented with 2% FCS (TechGen International, Paris, France), 50 µg/ml gentamycin (Gibco/BRL), 10 mM HEPES, 2 mM L-glutamine and 50 µM ß-mercaptoethanol. Cells were incubated with YTS169.4–FITC (anti-CD8; Caltag, South San Francisco CA) and IM7.8.1–biotin (anti-CD44, prepared in house) diluted in supplemented DMEM for 45 min at 4°C. After one wash, cells were incubated with avidin–phycoerythrin (PE; Caltag) for 30 min at 4°C. Cells were analyzed using the FACStar (Becton Dickinson, Mountain view, CA) calibrated with Fluoresbrite plain YG 2.0 micron microspheres (Polysciences, Warrington, PA) and CellQuest software (Becton Dickinson Immunocytometry systems, San Jose, CA). Viable lymphocytes are shown after gating on forward and side scatter, and cells were sorted into round-bottomed 96-well plates (1000 cells/well) using Clone-Cyt Software (Becton Dickinson).

For analysis of surface markers, spleen cells were stained as described (13). The following antibodies were used: YTS169.4–PE (anti-CD8) and IM7.8.1–FITC (anti-CD44) from Caltag; 5H4–FITC (anti-IL-2Rß) and Jo2–biotin (anti-Fas) from PharMingen (San Diego, CA); IM7.8.1–biotin (anti-CD44), 143.4.2–biotin (anti-Ly6.2C), YN1.1.7–biotin (anti-ICAM-1), FD441.8–biotin (anti-LFA-1), RR3.15–biotin (anti-Vß11) and 145.2C11–FITC (anti-CD3) were prepared in-house. Avidin–TriColor (Caltag) was used to reveal biotin-coupled antibodies. BrdU labeling and staining were performed as previously reported (13). Cells were analyzed using the FACScan (Becton Dickinson) and CellQuest software (Becton Dickinson Immunocytometry Systems). Viable lymphocytes are shown after gating on forward and side scatter.

Cell culture
Cells were cultured in DMEM supplemented with 6% FCS, 50 µg/ml gentamycin, 10 mM HEPES, 2 mM L-glutamine and 50 µM ß-mercaptoethanol. For the proliferation assays 1x103 sorted F5 transgenic T cells were activated with various concentrations of nucleoprotein peptide in the presence of 5x105 irradiated (30 Gy) C57BL/10 spleen cells in the presence of 10% supernatant containing IL-2 (17).

For RT-PCR analysis, 1x106 F5 transgenic spleen cells were activated in 24-well plates in the presence of 2x106 irradiated (30 Gy) C57BL/10 spleen cells with 30 nM nucleoprotein peptide or with 1 nM nucleoprotein in the presence of 2.5% supernatant containing IL-2.

For ELISA assays, 5x104 F5 transgenic spleen cells were activated in 96-well plates with various concentrations of nucleoprotein peptide in the presence of 2x105 irradiated (30 Gy) C57BL/10 spleen cells and in the presence or absence of 5% supernatant containing IL-2.

In vitro proliferation assays
Proliferation was measured by two different techniques giving similar results. Cells were either pulsed for 16 h with 0.5 µCi [3H]thymidine/well (2.0 Ci/mmol; Amersham, Les Ulis, France) on day 6 and harvested on day 7. Alternatively, the proliferation was evaluated on day 7 using a MTT colorimetric assay. Briefly, 150 µg of MTT was added to the cultures and the plates were incubated for 4 h at 37°C. The supernatants were discarded and the crystals dissolved by adding 100 µl of DMSO containing 0.04 N HCl. The optical density was measured at 490 nm with 650 nm as reference wavelength.

Semi-quantitative RT-PCR analysis
Cells were applied to Ficoll-Hypaque gradient centrifugation and total cellular RNA was isolated by the RNA Now method according to the manufacturer's instruction (Biogentex, Seabrook, TX). Annealing of 250 ng of oligo(dT) (Promega) to various concentrations of cellular RNA and 200 fg of standard RNA [pMus3 (18)] was carried out at 65°C for 15 min. cDNA was synthesized in 20 µl of reaction volume containing 20 U RNasin (Promega), 10 mM dithiothreitol, 2 mM dNTPs and 200 U MMLV reverse transcriptase (Gibco/BRL) in 1xFirst Strand Buffer (Gibco/BRL) for 1 h at 37°C. The reaction was terminated by a 10 min incubation at 70°C. DNA amplification was carried out in 1xPCR buffer supplemented with 400 µM dNTPs, 1.5 mM MgCl2, 100 ng each of 5'- and 3'-specific primers, 0.25 or 0.5 U Taq polymerase (Goldstar Eurogentec, Seraing, Belgium), and 2 µl of the cDNA reaction. The reaction was amplified in 33 sequential cycles with a Perkin Elmer 2400 Thermal Cycler. The cycles were at 94°C for 30 s, 55°C for 30 s and 72°C for 1 min for IL-2, IL-4, IL-5 and tumor necrosis factor (TNF)-{alpha}. For IFN-{gamma} the cycles were carried out at 94°C for 1 min, 60°C for 1 min and 72°C for 1.5 min. Using these conditions, the standard plasmid RNA amplification obtained with the IL-2, IL-4, IL-5, IFN-{gamma} and TNF-{alpha} primers was equivalent. Hence, comparison of the expression level of these different cytokines was possible. For quantitative experiments the numbers of cycles were 30 or 33 for IFN-{gamma}, 33 for IL-2 and TNF-{alpha}, and 26 for ß2-microglobulin. In these experiments, the standard plasmid RNA was included in all samples to verify equivalent amplification in different tubes (data not shown). The primer sequences and expected amplicons have been described elsewhere (18,19). PCR products were analyzed by agarose gel (2%) electrophoresis in the presence of ethidium bromide.

ELISA assay
Cell supernatants were collected after 4 days of bulk culture or after 6 days of culture of sorted cells and IFN-{gamma} content was measured by capture ELISA. Ninety-six-well ELISA plates (Corning, New York, NY) were coated with 0.2 µg of R4-6A2 antibody (Rat-IgG1, made in-house) per well diluted in 0.1M NaHCO3 (pH 8.2) by overnight incubation at 4°C. After two washes with PBS/0.05% Tween 20, plates were blocked with PBS/10% newborn calf serum for 2 h at room temperature. Plates were washed twice with PBS/Tween 20 and incubated overnight at 4°C with supernatants. Dilutions of recombinant murine IFN-{gamma} (Boehringer Mannheim) were used to establish a standard curve. After four washes with PBS/Tween 20, 0.1 µg of biotinylated AN18 antibody (rat IgG1, made in-house) diluted in PBS/10% newborn calf serum was added per well and plates were incubated at room temperature for 45 min. Plates were washed 6 times with PBS/Tween 20 and incubated with avidin–peroxidase (Sigma, St Louis, MO) diluted 1/1000 in PBS/10% newborn calf serum for 30 min at room temperature. After 8 washes with PBS/Tween 20, 30 µg of the substrate ABTS (Sigma) diluted in 0.1 M citric acid, pH 4.35, containing 0.03% H2O2 was added per well which were read after 10–80 min at 410 nM.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD8 T cells showing hyper-proliferation express intermediate levels of CD44
To investigate the proliferation of CD8 T cells expressing low, intermediate or high levels of CD44 from naive and primed mice, subsets of CD8 cells defined by the level of CD44 expression were sorted using a FACStar (Fig. 1A and BGo). Primed cells were derived from thymectomized F5 TCR transgenic mice injected with peptide in the peritoneal cavity 5–12 weeks before the experiments. Cells were activated in vitro with different concentrations of peptide in the presence of IL-2 and proliferation was measured after 7 days. As expected, the total population of primed CD8 T cells responded to 10-fold lower concentrations of peptide when compared to their naive counterparts (Fig. 1CGo). Primed CD8 T cells expressing intermediate levels of CD44 showed a strong hyper-reactivity compared to the equivalent naive CD8 CD44int population (Fig. 1EGo). The small difference between naive and primed CD8 cells expressing low levels of CD44 was not significant (Fig. 1DGo). Neither naive nor primed CD8 T cells expressing high levels of CD44 responded to peptide, indicating that these cells were not triggered by peptide in vitro (Fig 1FGo). These results show that CD8 T cells expressing intermediate levels of CD44 are the major subset responsible for the hyper-reactivity of primed CD8 T cells. We cannot exclude that the lack of proliferation observed with CD8 CD44high cells was due to negative signaling mediated by the anti-CD8 or anti-CD44 antibodies used for sorting. However, the hyper-reactivity of primed cells in vitro was not affected by staining with anti-CD8 and anti-CD44 followed by cell sorting (Fig. 1CGo and data not shown), indicating that the CD8 CD44 high cells do not contribute significantly to that functional phenotype.



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Fig. 1. Primed CD8 T cells expressing intermediate levels of CD44 are hyper-reactive to antigen. Lymph nodes cells from naive (A) or primed (B) F5 mice were sorted directly into 96-well plates. Cells were gated for only CD8 (R5) or for CD8 and low (R2), intermediate (R3) or high (R4) CD44 levels. One thousand sorted cells were stimulated with different concentrations of peptide in the presence of IL-2 and irradiated B10 spleen cells as described in Methods. Proliferation was measured using a MTT colorimetric assay after 7 days in culture. The proliferation of CD8 (C), CD8 CD44low (D) CD8 CD44int (E) and CD8 CD44high (F) cells from naive (white circles) and primed (black squares) mice is shown. One representative experiment out of five is shown. In all experiments, cells were pooled from at least two naive or two primed mice respectively.

 
CD8 T cells expressing high levels of CD44 show an activated phenotype
Spleen cells from naive and primed mice were stained for CD8, CD44 and a number of other surface molecules (Fig. 2Go). As previously described (13), the CD8 CD44int T cell subset found in primed mice expressed increased levels of Ly6C compared to naive CD8 CD44low T cells. In contrast, CD8 CD44high T cells differed markedly from naive CD8 CD44low and primed CD8 CD44int cells as they expressed higher levels of IL-2Rß, ICAM-1, LFA-1 and Ly6C, and lower levels of CD3 and Vß11. All subsets expressed similar levels of Fas. The CD8 CD44high T cell subset from naive or primed mice showed similar staining patterns. Thus, the CD8 CD44high subset found in both naive and primed animals expresses a panel of activation markers usually associated with an activated state.



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Fig. 2. CD8 T cells expressing high levels of CD44 show an activated phenotype. Spleen cells from naive or primed thymectomized F5 mice were triple stained for CD8 and CD44 in combination with Ly6C, CD3, Vß11, Fas, LFA-1, ICAM-1 or IL-2Rß. (A) Cells were gated for CD8 at the acquisition level and 22% log contours are shown. The difference in CD44 intensity is due to the use of either CD44–FITC (in combination with Ly6C, Vß11, Fas, LFA-1 and ICAM-1) or CD44–biotin and avidin–TriColor (in combination with CD3 and IL-2Rß). (B and C) CD44 expression as revealed with CD44–FITC (B) or CD44–biotin and avidin–TriColor (C). Using the markers shown, the sizes of the three CD8+ subsets defined by CD44 were 67 ± 2 and 35.5 ± 1.5% of CD44low; 17.5 ± 1.5 and 51 ± 1% of CD44int; and 16 ± 2 and 14 ± 1% of CD44high in naive and primed mice respectively. Results for one representative experiment out of three are shown.

 
Previous results suggested that CD44int cells were resting in vivo. Indeed, most of the cells which were labeled using a BrdU pulse during the primary response maintained their BrdU content for up to 6 weeks. However, the cycling status of the different CD8 subsets defined by expression levels of CD44 was not measured in these experiments (13). We therefore investigated the turnover rate of these different subsets in vivo using long-term BrdU labeling. Thymectomized F5 mice primed with peptide 4 weeks earlier were fed continuously with BrdU for 1 or 3 weeks. As shown in Fig. 3, Goa significant fraction of the CD8 CD44high T cells subset incorporated BrdU. Up to 11% of the CD44high CD8 T cell subset had incorporated BrdU after 1 week of labeling. This number reached 33% after 3 weeks of labeling in vivo. In contrast, <1% of CD8 CD44low and CD8 CD44int incorporated BrdU over a 3 week labeling period. These results confirm our previous findings which showed that CD44int CD8 T cells were not cycling in vivo in the absence of antigen. In addition, we show that the CD8 CD44high T cells which are found in both naive and primed animals cycle in vivo.



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Fig. 3. CD8 T cells expressing high levels of CD44 proliferate in vivo. Thymectomized F5 mice were primed with peptide and 4 weeks later were given BrdU in the drinking water for 1 or 3 weeks. Cells were triple stained for CD8, CD44 and gated for CD8 at the acquisition level. The contour plots for CD44 versus BrdU (upper row) as well as the histograms for the CD44 staining (lower row) are shown. One out of four mice are shown for each time point. The percentage of CD44low/int and CD44high CD8 T cells stained by the anti BrdU antibody, in control mice not treated with BrdU, was 0.3 and 0.2% respectively.

 
In F5 mice, CD8 T cells could express an endogenous {alpha} chain, generating a TCR with a new antigenic specificity which could then trigger their activation. If so, this population would not be present in F5 mice back-crossed into the RAG–/– background. Surprisingly, a population of CD8 T cells expressing high levels of CD44 was clearly detectable in RAG–/– F5 mice. In addition, this population also expressed higher levels of IL-2Rß, ICAM-1, LFA-1 and Ly6C, and lower levels of CD3 and Vß11 compared to CD8 CD44low T cells (Fig. 4Go).



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Fig. 4. CD8 T cells expressing high levels of CD44 are present in RAG–/– F5 mice. Spleen cells from RAG–/– F5 mice were triple stained for CD8, CD44 and Ly6C, LFA-1, ICAM-1, IL-2Rß, CD3 or Vß11. (A) Cells were gated for CD8 at the acquisition level and 20% log contours are shown. CD44–FITC was used in combination with Ly6C, LFA-1 and ICAM-1, whereas CD44–biotin was used in combination with IL-2Rß, CD3 and Vß11. (B) CD44 expression as revealed with CD44–FITC or CD44–biotin and avidin–TriColor. With the markers shown, both CD44 stainings detected 91.2 ± 0.4, 5.5 ± 0.4 and 3.7 ± 0.2% of CD44low, CD44int and CD44high CD8+ spleen cells respectively.

 
Thus, CD8 T cells expressing high levels of CD44 show an activated phenotype in both F5 and Rag–/– F5 naive mice. Since T cells with dual TCR specificity cannot be generated in Rag–/– F5 mice, these results indicate that activation of these cells is independent of TCR engagement.

Primed CD8 CD44intT cells produce higher levels of IFN-{gamma} than naive cells
To investigate the cytokine profile of primed and naive cells following in vitro activation, cells were stimulated with peptide in the presence of irradiated spleen cells from C57Bl/10 mice. The mRNA coding for IL-2, IL-4, IL-5, TNF-{alpha} and IFN-{gamma} were analyzed by RT-PCR. RNA transcribed from the plasmid pMus3 (18) was included in the RT-PCR as a control for the reverse transcription and PCR reactions. At 48 h after peptide activation, naive and primed cells expressed mRNA coding for IL-2, TNF-{alpha} and IFN-{gamma} but undetectable to low levels of IL-4 and IL-5 mRNA (Fig. 5Go). The same cytokine profile was observed for both naive and primed cells 3 and 5 days after peptide challenge (data not shown). These results indicate that both naive and primed cells express mRNA coding for typical Tc1 cytokines.



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Fig. 5. Naive and primed cells express mRNA coding for IL-2, TNF-{alpha} and IFN-{gamma}. Spleen cells from two naive or two primed mice were pooled and the cells were stimulated with 30 nM peptide in the presence of irradiated B10 spleen cells for 48 h. RNA purification, cDNA synthesis and DNA amplification was performed as described in Methods. The expression of mRNA for IL-2, IL-4, IL-5, TNF-{alpha} and IFN-{gamma} were evaluated. One representative experiment out of three is shown.

 
To estimate the levels of mRNA coding for different cytokines, we performed semi-quantitative RT-PCR analysis by diluting the mRNA used for the reverse transcription reaction. Figure 6Go shows that primed cells express higher levels of mRNA coding for IFN-{gamma} compared to naive cells following peptide challenge in vitro. No significant difference in the levels of mRNA coding for TNF-{alpha} and IL-2 was observed. To further characterize the difference between naive and primed cells, the level of IFN-{gamma} mRNA in both subsets was measured over time following peptide stimulation in the presence or absence of IL-2. Results presented in Fig. 7Go show that the production of IFN-{gamma} mRNA by primed cells is more rapid and that at all time points the levels of IFN-{gamma} mRNA are higher in primed cells when compared to naive cells. This difference in IFN-{gamma} mRNA levels between naive and primed cells was independent of the presence of exogenous IL-2 (Fig. 7Go). To determine whether the differential IFN-{gamma} mRNA expression was correlated with protein secretion, we measured IFN-{gamma} production by ELISA. Cells were activated with different concentrations of peptide and supernatants were collected 4 days later. Significant levels of IFN-{gamma} were detected in supernatants from primed cells whereas only low amounts of IFN-{gamma} were present in supernatants from naive cells (Fig. 8Go). This difference in IFN-{gamma} production was observed when cells were stimulated in the presence or absence of exogenous IL-2. These results indicate that following in vitro activation by peptide, primed CD8 T cells produce more IFN-{gamma} than naive CD8 T cells.



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Fig. 6. Primed cells produce higher levels of mRNA coding for IFN-{gamma} than naive cells. Spleen cells from two naive or two primed mice were pooled and the cells were stimulated with 30 nM peptide in the presence of irradiated B10 spleen cells for 24 h. Different quantities of RNA from naive or primed cells were reverse transcribed, and the cDNA for IFN-{gamma}, TNF-{alpha} and IL-2 were amplified and analyzed. ß2-Microglobulin was used as a control to verify that similar quantities of RNA were present in the starting samples. One representative experiment out of three is shown.

 


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Fig. 7. Production of mRNA coding for IFN-{gamma} is more rapid in primed cells. Spleen cells from two naive or two primed mice were pooled and the cells were stimulated with 30 nM peptide in the absence of IL-2 (A) or 1 nM peptide in the presence of IL-2 (B) for 12, 24 and 48 h. Different quantities of RNA from naive or primed cells were reverse transcribed and the cDNA for IFN-{gamma} were amplified and analyzed. Three independent activation kinetics were performed. For each of them the RT-PCR was repeated at least twice. One representative kinetic experiment is shown.

 


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Fig. 8. Primed CD8 T cells expressing intermediate levels of CD44 secrete higher levels of IFN-{gamma} than naive cells. (A and B) Spleen cells from naive or primed mice were stimulated with different concentrations of peptide in the absence (A) or presence (B) of IL-2. Supernatants were collected after 4 days and the quantity of IFN-{gamma} was measured using an ELISA assay. One representative experiment out of three is shown. In all experiments cells were pooled from at least two naive or two primed mice respectively. (C) Lymph node cells from primed F5 mice were sorted for CD8 and low, intermediate or high levels of CD44, and activated by different concentrations of peptide in the presence of IL-2. Six days later supernatants were collected and IFN-{gamma} was measured by ELISA.

 
Finally, to confirm that the primed CD8 T cell subset expressing intermediate levels of CD44 was responsible for the increased IFN-{gamma} production, primed CD8 T cells were sorted on the basis of their expression of CD44. CD8 T cells expressing low, intermediate and high levels of CD44 were then stimulated with peptide, and the levels of IFN-{gamma} in the supernatant were determined 6 days later. As shown in Fig. 8Go(C), CD8 T cells expressing intermediate levels of CD44 produce higher levels of IFN-{gamma} than those expressing either low or high levels of this marker on the cell surface. These results demonstrate that primed CD8 T cells expressing intermediate levels of CD44 are responsible for the increased IFN-{gamma} production observed following in vitro stimulation of in vivo primed CD8 T cells.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have previously identified a population of primed CD8 cells in TCR transgenic mice that proliferate in response to lower doses of antigenic peptide in vitro in the presence of IL-2 (13). By directly sorting CD8 T cells expressing different levels of CD44, we have now shown that CD8 T cells expressing intermediate levels of CD44 are responsible for this hyper-reactivity. This may appear to contrast results by other groups showing that memory CD8 T cells express high levels of CD44 (2022). However, most studies do not discriminate between cells expressing intermediate and high levels of CD44, but only between low and high levels of CD44 expression. It is thus possible that memory CD8 T cells in other systems also express intermediate levels of CD44.

In this study, CD8 CD44high T cells fail to proliferate in response to peptide challenge in vitro. This finding excludes the possibility that primed hyper-reactive cells belong to the CD44high compartment. It also suggests that the CD8 CD44high subset is functionally compromised. However, in vivo, the CD8 CD44high subset is clearly cycling. We believe that these cells are not derived from cycling CD44low or CD44int CD8 T cells. Indeed, following activation by peptide CD44 up-regulation is gradual reaching maximum levels only after four to five divisions in vivo (data not shown). Hence, if CD44low or CD44int cells were cycling, BrdU+ cells should be detected in these subsets. These T cells do not appear to be a transgenic artefact since CD8 T cells with a similar phenotype have been described in non-transgenic mice (23). These cells can be generated in the thymus but also in the liver. Their number is increased in aging mice and following thymectomy (2325). This last point fits with our own observation that the proportion of CD44high CD8 T cells is increased in thymectomized animals whether they are naive or primed. We do not know what drives the proliferation of these cells. One could imagine that the expansion of these cells is driven by the recognition of an endogenous antigen by a TCR associating the Vß11 chain with an endogenous V{alpha}. However, the expansion of at least one subset of these cells is unlikely to depend on the expression of an endogenous {alpha} chain as they are also found in RAG–/– F5 mice. This would suggest that these cells are driven by some other mechanism. In this context it is interesting to note that CD8 T cells expressing high levels of CD44 proliferate in vivo in the absence of antigenic stimulation (26,27). Their proliferation is strongly increased in response to IFN-{alpha}/ß or IL-15. These CD8 CD44high cells expressed IL-2Rß and resemble the population of CD8 CD44high cells found in our system. It remains to be determined if the CD8 CD44high population in F5 transgenic mice proliferates in response to IFN- I or IL-15 in vivo.

In conclusion, our results suggest that at least some CD8 CD44 high T cells are not true memory T cells, and underlines the importance of using both phenotypic and functional criteria to identify memory CD8 T cells.

CD8 cells secreting different cytokine patterns have been named Tc1 and Tc2 cells with analogy to CD4 T cells secreting either Th1 or Th2 cytokines (28,29). Tc1 cells secrete IL-2 and IFN-{gamma}, whereas Tc2 cells secrete IL-4, IL-5, IL-6 and IL-10. In the current study, both naive and primed CD8 T cells showed a typical Tc1 cytokine pattern at the mRNA level following activation in vitro. This is in accordance with previous reports where exogenous IL-4 alone or in combination with anti-IFN-{gamma} was needed to induce CD8 T cells to secrete Tc2 cytokines (29). Thus, CD8 T cells in the F5 system show a strong bias towards Tc1.

In vivo activated primed cells showed higher levels of mRNA coding for IFN-{gamma} than naive cells following in vitro stimulation. A similar difference between naive and primed cells was observed when measuring IFN-{gamma} secretion by in vitro activated cells. This finding is in accordance with studies demonstrating that influenza-specific human memory CD8 T cells secreted IFN-{gamma} very rapidly (6 h) after in vitro stimulation with specific influenza peptide (30). Recently, similar results were obtained in another TCR transgenic model showing early production of IFN-{gamma} by memory CD8 T cells (31).

In conclusion, we show that resting primed CD8 T cells are functionally different from naive T cells in that they hyperproliferate to peptide stimulation and show an increased IFN-{gamma} response. Importantly, the increase in IFN-{gamma} production was observed when cells were stimulated in the presence or absence of exogenous IL-2. This is in contrast to the hyperproliferation of peptide-primed F5 CD8 T cells which is dependent on exogenous IL-2.


    Acknowledgments
 
We thank Dimitris Kioussis for the F5 and RAG–/– F5 mice. David Shire is acknowledged for giving permission to use the pMus3 plasmid and Marie-Thérèse Ducluzeau for the gift of RNA standards. We also thank Robin Buckland for critical reading of the manuscript. M. P. is supported by a fellowship from the Association pour la Recherche sur le Cancer, and P. M. D. is supported by a European Union Training and Mobility of Researchers fellowship. This work was supported by institutional grants from the Centre National de la Recherche Scientifique and the Ministère de l'Education et de la Recherche, and by additional supports from the Association pour la Recherche sur le Cancer and the Comité Départemental du Rhône de la Ligue Nationale Franciaise contre le Cancer.


    Abbreviations
 
PEphycoerythrin
TNFtumor necrosis factor

    Notes
 
Transmitting editor: A. Cooke

Received 22 September 1998, accepted 19 January 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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