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© 2000 Nature America Inc. • http://immunol.nature.com A RTICLES Response of naïve and memory CD8+ T cells to antigen stimulation in vivo © 2000 Nature America Inc. • http://immunol.nature.com Henrique Veiga-Fernandes1, Ulrich Walter2, Christine Bourgeois1, Angela McLean3 and Benedita Rocha1 We studied the influence of memory T cell properties on the efficiency of secondary immune responses by comparing the in vivo immune response of the same numbers of T cell receptor (TCR) transgenic (Tg) naïve and memory T cells. Compared to naïve Tg cells, memory cells divided after a shorter lag time; had an increased division rate; a lower loss rate; and showed more rapid and efficient differentiation to effector functions.We found that initial naïve T cell priming resulted in cells expressing mutually exclusive effector functions, whereas memory T cells were multifunctional after reactivation, with each individual cell expressing two to three different effector functions simultaneously.These special properties of memory T cells ensure the immediate control of reinfection. The efficiency of secondary immune responses is at least partially due to modifications of the primed cell repertoire, including an increased frequency of antigen-specific T cells1–5 and the selection of high affinity clones6–8. These modifications, however, may be transient, and insufficient to ensure long-term memory in the absence of antigen restimulation. It has been shown that in a normal environment, where individuals are subjected to successive infections by different pathogens, the number of memory cells specific to a first antigen declines, a phenomenon called attrition9. The maintenance of memory in such circumstances would require either repeated exposure to specific antigen, or rely on novel biological capacities of memory cells10–18. The impact of memory T cell properties on the efficiency of secondary immune responses cannot be studied in normal mice. First, the frequency of antigen-specific naïve cells is too low to identify them with peptideMHC (major histocompatibility complex) tetramer complexes before they expand in vivo4,5. As a result, the early in vivo events of naïve and memory activation cannot be compared. Second, T cell repertoires are modified after priming: the frequency of antigen-specific T cells increases1–5 and high affinity clones are selected6–8. These phenomena themselves facilitate secondary immune responses and hinder studies aimed at establishing whether memory cells are more efficient than naïve cells on a “per cell basis” at dealing with antigen in vivo. Because of these limitations, previous comparative studies between naïve and primed populations were made with T cell receptor-transgenic (TCR-Tg) populations, usually after in vitro activation. It was shown that under these conditions memory cells are more efficient and more precocious cytokine secretors14–18 but that they proliferate poorly15,17,18. It was concluded that the influence of memory cell properties on secondary immune responses relied on improved cytokine production only14,15,17,18. If this were so, memory responses would require the continuous presence of high frequencies of antigen-specific T cells and should disappear in a normal environment, where multiple infection by different pathogens leads to the progressive decay of the number of memory cells specific to a first antigen9. A few studies also analyzed naïve and memory TCR-Tg cells behavior in vivo after their transfer into normal15,17 or immunodeficient hosts16. In normal hosts no differences were found between the accumulation of both CD8+ cell types. In immunodeficient hosts it was shown that memory CD4+ cells incorporate BrdU preferentially, suggesting that they divide more. These discrepancies could be due to differences in the donor cells (as either CD8+ or CD4+ cells were used) or in the type of recipient mice. In particular, the use of immune-deficient hosts could be associated with antigen-independent cell division that could affect naïve and memory cells differently19. Finally, the study of cell populations in blood16,17 is restrictive, as antigen stimulation occurs in lymphoid organs, and lymphocyte migration to the blood varies during the course of the immune response. To examine the intrinsic properties of naïve and memory cell types, we compared RAG-2–deficient T cells expressing the same transgenic TCR, recovered from naïve or in vivo immunized mice long after antigen elimination. We studied proliferation and differentiation of individual naïve and memory cells into effector functions, during primary and secondary immune responses in vivo. Results We studied naïve and memory T cells expressing the same TCR. The naïve cells were from RAG-2–deficient female mice20, expressing a Tg αβ T cell receptor, specific for the male antigen21. It was a pure population of naïve cells, as the male antigen was absent, and cross-reactivity with environmental antigen could not be detected: these cells were CD44+, and did not divide or express mRNA coding for cytokines12. To obtain memory cells, these naïve cells were stimulated in vivo with relatively low doses of male cells12. During antigen stimulation, all naïve cells became activated, expressed CD44, expanded, and eliminated the male cells. From these mice we recovered a genuine memory population, because all Tg cells were antigen-experienced and functionally competent, and because we took great care to ensure they persisted in vivo in the absence of antigen12,14,22. As with CD8+ CD44+ T cells from normal mice, these memory cells showed a panoply of surface markers (in preparation) and similar patterns of lymphokine expression12. The same number of naïve and memory Tg cells (expressing the T3.70+ INSERM U.345, 2U373, Institut Necker, Paris, France. 3Institute for Animal Health, Compton, UK. Correspondence should be addressed to H.V.-F. ([email protected]). 1 http://immunol.nature.com • july 2000 • volume 1 no 1 • nature immunology 47 © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES cells (polyclonal populations in particular) modified Tg cell behavior. Naïve cell proliferation was most 40 103 affected. We concluded that the intrinsic properties of naïve and memory cells were best studied when no 102 30 other antigen-specific T cells were present. 101 To study naïve and memory cell proliferation, Tg cells were followed in separate recipients (Fig. 1a, 100 20 100 101 102 103 right panel). Memory cells expanded more than naïve Thy 1.1 cells and they accumulated much faster. Twenty-four 10 hours after transfer the total yields of naïve and memory cells were similar. In contrast, memory cells 0 yields were four-fold higher on day 4 and ten-fold 0 5 10 15 0 10 20 50 0 10 20 50 Days after transfer higher on days 5 to 7. Memory cell expansion peaked by day 7, whereas naïve cell expansion peaked 1 Figure 1. Expansion of naïve and memory cells. (a) Naïve and memory Tg cells (0.5×106) and CD3ε- week later. Even then, the naïve cell population was deficient male bone marrow cells (0.5×106) were injected into female hosts. Left panel, naïve and memory less than half that of the memory cells. The early cells injected into B6 RAG-2+/+ mice. Middle panel, naïve and memory cells injected into RAG-2–/– mice. Right –/– panel, naïve and memory cells injected separately into RAG-2 mice. Tg cells were then followed in the kinetics and higher amplitude of memory cells divilymph nodes, spleen, bone marrow and blood.Tg cells did not accumulate in the blood. Results taken from sion was found in all individual organs studied. one of the six experiments carried out show the total number of Tg cells recovered from the spleen plus To understand the differences in proliferation the lymph nodes and bone marrow, at different time points after transfer.Tg cell proliferation occurred in behavior we compared the early events of cell activaall lymphoid organs, but predominated in the bone marrow and spleen.This was probably due to preferention (Fig. 2). The kinetics of CD69 induction were tial homing of bone marrow male cells in these organs59. (b) Origin of T3.70+ T cells in immunized B6 hosts similar for both naïve and memory cells, but both the 2 weeks after priming.The injected naïve Tg cells were Thy1.1+; endogenous T cells were Thy1.1-. frequency of CD69+ cells (Fig. 2a) and the intensity of CD69 expression (Fig. 2b) were higher in naïve TCR and a different Thy1 allotype marker) were injected into female cells. These results rule out the possibility that naïve cells were excluded immunized hosts, and followed in all host’s lymphoid organs (lymph from antigen contact. The consequences of antigen encounter, however, nodes, spleen, bone marrow and blood) (Fig. 1). Memory cells proliferat- were very different (Fig. 2c). Blastogenesis occurred early in memory ed extensively in normal B6 hosts, whereas naïve cells did not (Fig. 1a, cells: increased cell size was detected by 8 h and all cells had become left panel). In mice injected with naïve cells we also saw the emergence of blasts 24 h after in vivo transfer. In contrast, only a minority of naïve cells a large cohort of T3.70+ CD8+ host T cells (Fig. 1b). We could not detect increased in size during this period; the size of most naïve cells remainthese cells before immunization, but they appeared four days later and ing unchanged up to two days after immunization. Next we studied the progression through cell division of naïve and accumulated with time. By day 15, 75% of T3.70+ cells were of host origin. These results demonstrated the participation of host T cells in the ongoing a c immune response. The impact of this host Memory Naïve 80 immune response on Tg cell growth cannot be fully appreciated, as T3.70 male-specific host T cells were likely also present. This 60 5h polyclonal population might compete with donor Tg cells for antigen binding, and/or 40 contribute to more efficient antigen elimination. This competition might differently 8h 20 affect naïve and memory Tg cells. To avoid a concomitant polyclonal pro0 liferation of host T cells, we used T cell0 24 48 72 96 120 Time (h) deficient immunized hosts. Naïve and 12 h memory cells were coinjected into the same b e( ) mice (Fig. 1a, middle panel) or injected into separate mice (Fig. 1a, right panel). Under both circumstances memory cells 24 h expanded more than naïve cells. In the early days after transfer, the presence of 100 200 300 100 200 300 memory cells did not modify the behavior Forward light scatter (channel number) 100 101 102 103 104 of naïve cells. From day 7, the presence of CD69 fluorescence intensity memory cells inhibited naïve cell accumulation. This may have been due to early elimination of antigen by memory cells or Figure 2. Early events of cell activation. Naïve and memory cells were followed from 1 h to 5 days after transfer into immunized hosts. Results show: (a) the kinetics of CD69 expression in naïve (closed symbols) and memory (open to other forms of competition between the symbols) cells; (b) the intensity of CD69 expression in naïve (thin lines) memory cells (broad lines); (c) the cell size of 23,24 two populations . These studies showed Tg populations recovered at different time points after immunization (broad lines) as compared to the initial size of the that the presence of other antigen-specific T same population, before antigen stimulation (thin lines). b 104 24.4 Relative cell number % CD69+ cells T3.70 Naïve Memory Relative cell number © 2000 Nature America Inc. • http://immunol.nature.com Number of Tg cells per mouse (10-6) a 48 nature immunology • volume 1 no 1 • july 2000 • http://immunol.nature.com © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES Naïve Memory © 2000 Nature America Inc. • http://immunol.nature.com Female 3.5 days ell number Relative cell number Female 2 days Female immunized 3.5 days 100 101 102 103 104 100 101 102 103 104 CFSE Figure 3. The role of antigen stimulation in Tg cell division in T cell-deficient hosts. Immunized and non-immunized female RAG-2-deficient hosts were injected with CFSE-labeled naïve (left panel) or memory (right panel) Tg cells and female or male CD3ε-deficient bone marrow cells. Results are taken from a pool of three mice per time point and show the intensity of CFSE-labeling of Tg cells recovered from the spleen at different time points after transfer. Similar patterns were found in cells recovered from the bone marrow. memory cells. Before injection these cells were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and the dilution of the label was followed. Because we were using T cell–deficient hosts in which transferred T cells may divide without intentional immunization19, we first compared Tg cell division in immunized and non-immunized mice (Fig. 3). In the absence of antigen stimulation, naïve Tg cells do not divide12, and we did not detect memory T cell division in the first days after transfer. By day 3.5 most of the memory cells that we detected were in the first round of division. The same memory cells transferred to an immunized host were then virtually all CSFE-negative. These results show that the rapid cycling of Tg cells in T cell–deficient hosts required antigen stimulation. In immunized hosts, the memory population always progressed faster than the naïve population (Fig. 3, bottom panel and Fig. 4). In the experiment shown in Fig. 4 and b, 60% of memory cells had undergone two or more divisions 44 h after immunization, whereas 56% of naïve cells had not yet divided. One day later 70% of memory cells had undergone six or more divisions whereas the bulk of the naïve population was still at division 4 to 5. The majority of naïve and memory cells were distributed across cell divisions in a single bell-shaped curve. In addition, we always found memory cells that apparently lost CFSE more rapidly, forming a population of outlying cells. In the experiment shown in Fig. 4, CFSE-negative cells represented 7% of memory cells 44 h after transfer and 20% 1 day later. Perhaps these cells divide faster, but we think they actually start dividing earlier, as we obtained evidence supporting the early division of a minority of memory cells. We found that 20 h after immunization, most memory cells had divided once, but a distinct 1% subpopulation divided four times (not shown). These cells could be the progeny of “steady-state” memory cells in synthesis/gap-2 (S/G2) and mitosis (M) phases of the cell cycle25,26. If so, the rare cycling cells that are always present in memory cell populations may have the potential to generate a sizeable cohort of T cells during secondary immune responses, as they require no lag time to start dividing—growing exponentially immediately after immunization. Because the distribution of cell numbers across divisions is known at each time point, our data provide estimates of lag times, rates of division and loss during these primary and secondary immune responses. Indeed, and infinite number of combinations of division and loss rates would be compatible with the overall T cell growth curves, as shown in Fig. 1. A unique combination of one lag time, one loss rate and one division rate can be identified as giving a best fit simultaneously: with cell numbers in each and every division and at each and every time point (as shown in a c Figure 4. Progression through the division of naïve and memory T cells. CFSE-labeled naïve and memory T cells were injected into immunized hosts and followed from 12 h to 5 days after transfer. Results are taken from a pool of three mice per time point in one out of three experiments. (a) Tg cells recovered from the spleen (similar results were obtained in other organs). (b) Number of Tg cells present in each division in the spleen plus bone marrow. (Naïve, closed symbols; memory, open symbols.) (c) Mathematical analysis of naïve (upper panel) and memory (lower panel) T cell demography. Data (dots) at 67 h (open symbols) and 115 h (closed symbols) and model (lines) showing predicted distributions for the best fitting parameters of cell loss and division rates for each population. Note: later points (115 h) can be studied, as long as some CFSE-positive cells yet remain. In this case, only the lower part of the left slope of the single bell shaped curve can be fitted to the data.At 115 h many naïve T cells still express CFSE, but most memory T cells are CFSE-negative. (d) Predicted parameter values fitting to the observed naïve and memory distribution through cell divisions. b d http://immunol.nature.com • july 2000 • volume 1 no 1 • nature immunology 49 © 2000 Nature America Inc. • http://immunol.nature.com © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES Fig. 4). We used a simple mathematical Table 1. mRNA expression ex vivo model taking a standard age-structured form similar to some models applied to Cell source % IL-2 % IFN-γ % Perforin data on human lymphocyte population S BM S BM S BM dynamics27,28. These models only allow Naïve donor cells the calculation of a limited number of Starting 0 0 2.1 (0.3) average parameters of behavior. We population selected the average transit time from 0.5 (0.2) Positive (a) 0.7 (0.2) Positive (a) 39 30 division 0 to 1 (lag time), the average 7 h in recipient division rates as cells progress throughout all other divisions, and the average loss 4 d 0.7 (0.6) 2.3 (2) 7.2 (4) 10.2 (4.8) 35 60.5 rate in all divisions. The theoretical in recipient growth curves we obtained using best-fit parameters fitted well to the actual exper- Memory donor cells imental data (Fig. 4c). These results sug- Starting 0.07 (0.03) 9.5 (3.7) 74 gested that the early kinetics and higher population amplitude of secondary immune respons7h 2.3 (0.08) 5.3 (2.1) 20.5 (5.8) 34.1 (12) 76 81 es were due to a 15 h reduced lag time, a in recipient 2 h reduced division rate and 20 h reduced loss rate in the memory cell population TCR Tg naïve and memory cells were injected into immunized mice. Individual cells were sorted and tested for mRNA coding for Hprt, Il2, Ifng and Pfp 7 h and 4 days later.When the frequency of mRNA expression was low, cells were also (Fig. 4d). sorted in limiting dilution at four to six different cell concentrations per well (24 wells per concentration).The freTo explain this different behavior, we quency estimates were calculated according to the Poisson distribution58 (95% confidence limits are shown in brackexamined the cycle status of “resting” ets). In naïve cells recovered from the bone marrow 7 h after transfer, (a) the number of negative wells did not follow naïve and memory cells, surviving in vivo a single hit Poisson distribution so accurate frequency estimates could not be determined. In secondary immune in the absence of antigen (Fig. 5). All responses, the frequency of lymphokine expressers did not increase from 7 h to 4 days after immunization, so data for 4 days is not shown. (S, spleen; BM, bone marrow). naïve cells contained 2N DNA; their RNA content was distributed in a low intensity peak. We confirmed that 1 to 2% of resting memory cells were in the S/G2+M phases of the cell cycle25,26. sion of all three genes by each individual cell. We studied naïve and However, when we analyzed the RNA content of 2N DNA Gap-0/Gap-1 memory cells at various stages: surviving in vivo in the absence of anti(G0/G1) memory cells, we found a high RNA expression. About half of gen (“resting” naïve and memory cells); 7 h after antigen stimulation, the 2N cells contained as much RNA as the S/G2+M population, sug- when no cells had yet divided; and on day 4, during the exponential phase of both naïve and memory cell growth, at which time most cells had gesting that they may be at a late G1 phase. To characterize T cell differentiation, we studied gene expression by divided at least ten times. Resting naïve cells do not express lymphokine mRNA12. After antigen individual T cells stimulated in vivo (Table 1 and Fig. 6) or in vitro (Table 2) by means of a single cell reverse transcription polymerase stimulation, Pfp mRNA was expressed rapidly by a large fraction of Tg chain reaction (RT-PCR) technique. The characteristics and sensitivity of cells: 30–40% cells were positive by 7 h, and this figure increased to 70% this assay are described in detail in the methods section. We focused on in time. In contrast, only a minor fraction of primed cells ever expressed perforin (Pfp), interleukin 2 (IL-2) and interferon γ (IFN-γ) because these lymphokine mRNA after in vivo immunization. Even after substantial Tg cells (as well as CD8+ CD44+ peripheral T cells from normal mice) do division, on day 4 after antigen stimulation only ≈2% expressed Il2 not produce T helper cell subset 2 cytokines12. We determined the expres- mRNA and ≈8% Ifng mRNA (Table 1). These results suggested that most CD8+ cells differentiated into cytotoxic cells, lymphokine producers being rare throughout the primary immune response. When Tg cells stopped Table 2. Expression of mRNA 7 h after in vitro antigen stimulation expanding and reverted to a resting memory state, certain effector functions were not lost: the frequency of the cells expressing Ifng or Pfp % IL-2 % IFN-γ % Perforin mRNA did not change, and only Il2 mRNA-positive cells became rare. Naïve T cells We also determined the coexpression of these three mRNAs in ≈8,000 3 1.4 37.3 cells in the course of the primary immune response and in resting mem(1.5) (1.3) (9.6) ory cells. This cohort contained 28 cells expressing Il2 mRNA, and a Memory T cells larger number expressing Ifng mRNA. We found that Il2 and Ifng mRNA 17.7 87 73 expression was always mutually exclusive in individual cells (Fig. 6). In addition, soon after stimulation each cell expressed only a single mRNA Naïve T cells and resting memory T cells (recovered 4 months after immunization) were incubated in vitro for 7 h with female spleen cells from CD3ε-deficient mice type; Pfp+ cells did not express cytokine mRNA. These results indicate and 10-7 M specific peptide49. Stimulated cells were sorted and analyzed as that naïve cells differentiated directly to individual effector functions in described in Table 1. Frequency estimates were calculated in naïve cells by limiting vivo. Perhaps similarly, the majority of CD4+ cells restimulated in vitro dilution using the Poisson distribution (95% confidence limits are shown in brackshortly after in vivo priming secrete a single cytokine type29. ets): in memory T cells: in 50 individual cells. In naïve activated T cells, each individMemory cell differentiation during secondary immune responses folual cell expressed a single mRNA type. ≈70% of memory cells coexpressed two or more mRNAs as follows: 15.5%: Il2 + Ifng + Pfp; 2.2%: Il2 + Ifng; 51% Ifng + Pfp; of lowed different kinetics and was quantitatively different from naïve T cell the 20% single expressing cells 17.8% expressed Ifng and 2.2% Pfp.We did not priming. After in vivo antigen restimulation, memory cells expressed detect lymphokine or Pfp mRNAs in 11% of HPRT+ cells. cytokine mRNA earlier and at a higher frequency than naïve cells: ≈35% 50 nature immunology • volume 1 no 1 • july 2000 • http://immunol.nature.com © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES a b © 2000 Nature America Inc. • http://immunol.nature.com Figure 5. RNA content of naïve and memory cells. Resting naïve and memory cells (recovered 4 months after priming) were stained with acridine orange. (a) The RNA and DNA content of naïve and memory cells. (b) The RNA content of 2N (G0/G1) gated cells. Naïve (thin line); memory (broad line).The bar shows the level of RNA staining in S/G2+M memory T cells. of memory T cells were Ifng mRNA-positive, ≈5% expressed Il2 mRNA 7 h after in vivo stimulation. These frequencies did not increase later (not shown). The study of gene coexpression by antigen-stimulated individual memory cells in vivo also showed remarkable qualitative differences: all Il2 mRNA-positive cells were Ifng mRNA-positive (Fig. 6). When the same memory cells were activated in vitro, 80% expressed Ifng mRNA and 20% were Il2 mRNA-positive (Table 2), a frequency much higher than that ever found in in vivo activated T cells. Like in vivo activated memory cells, all Il2 mRNA+ cells coexpressed Ifng mRNA after in vitro activation. As the frequency of cytokine mRNA expressers was known, we could also determine the amount of cytokines secreted by each individual T cell during the 7 h culture time period. For naïve cells this was: IL-2, 0.5 fgr; IFN-γ, 3.5 fgr. For memory cells: IL-2, 14 fgr; IFN-γ, 190 fgr. On a per cell basis, memory cell secretion of IL-2 was 28-fold higher and IFN-γ 54-fold higher relative to naïve cells. One of the reasons for such a difference should be the early kinetics of cytokine secretion by in vitro activated memory cells12–18, but it could also involve other mechanisms of more efficient secretion14. Discussion To study the influence of memory T cell properties on the efficiency of secondary immune responses it is tempting to compare naïve and memory Tg cells transferred into the physiological environment of a normal mouse. Our results demonstrate that this strategy has a major disadvantage: the concomitant polyclonal immune response of the host T cells to antigen stimulation. This polyclonal response cannot be fully appreciated as it is likely to be complex, including heterogeneous clones responding to various antigen epitopes. Its impact on donor Tg cell growth may vary in different systems, depending on Tg cell affinity and on the relative representation of the Tg and other epitopes. Because of this limitation, it was predictable that the studies of Tg T cell function after transfer to normal hosts yields contradictory findings15–17. Naïve and memory Tg cells also competed when cotransferred to the same immune-deficient host, and so we concluded that the intrinsic properties of naïve and memory cells should be studied when no other antigen-specific T cells were present. Naïve and memory cell behavior after in vitro antigen stimulation has been studied previously12–18, and it has been shown that memory cells are efficient cytokine secretors, but that they proliferate poorly. We also found reduced in vitro proliferation of our Tg memory cells (C. Tanchot & B. Rocha, unpublished data), which contrasts with their very efficient in vivo growth. We also found other major differences between in vitro and in vivo responses. In vitro, memory cell TCR is down-modulated http://immunol.nature.com • july 2000 rapidly, within the first 12 h post-activation15,30,31 (data not shown). Despite analyzing cells every 4 h, we observed no TCR down-modulation during the first 24 h after in vivo immunization. In addition, when memory cells started cycling, TCR surface expression actually increased with each division, reaching a maximum in cells that had divided more than six times. In a representative experiment (Fig. 4), the mean fluorescence intensity from division 0 to division 6 was: 1604, 1789, 1858, 1933, 2086, 2252, 2401. The other major difference was the frequency of lymphokine mRNA expressers. Ex vivo, cytokine producers were rare, even in secondary immune responses. When primed cells were further activated in vitro by peptide, however, the frequency of lymphokine mRNA expressers became very high, confirming the conclusions of other reports where only in vitro differentiation was studied4,5,15–18. These results suggest that in vivo activation favors strong division and moderate differentiation, whereas in vitro activation predominantly induces differentiation. The mechanisms underlining these differences are so far unknown, but in vitro data cannot be extrapolated to in vivo behavior. In naïve and memory CD4+ T cells cotransferred into the same host it was found that memory cells in the blood incorporated BrdU preferentially suggesting that the memory cells divided more16. By analyzing dividing cells in all lymphoid organs—we studied lag time, progression throughout divisions, and cell accumulation—we found that memory cell proliferated earlier and with much higher amplitude. This was dependent on a very short lag time before division, a moderate increase in division rates, but also on a very efficient accumulation. We found a very reduced loss of memory cells. We can exclude the possibility that this was caused by different migration patterns within lymphoid organs because we studied all these organs. Thus the increased accumulation may be due either to reduced cell death or to an absence of migration to non-lymphoid tissues. The latter alternative is not compatible with studies in which memory T cells (rather than naïve T cells) were found to migrate preferentially to non-lymphoid tissues32. Our data suggest that the differences in proliferation may be due to a difference in the cycle status of naïve and memory populations before antigen stimulation. Naïve cells had a low RNA content, suggesting that they were resting. Their DNA may not be licensed33, so it requires epigenetic modifications before initiation of DNA synthesis and cell division34–36. In contrast, most resting memory cells had a high RNA expression, perhaps in late G1 phase of the cell cycle, and thus able to rapidly engage in cell division after antigen restimulation. The cycle status of resting naïve and memory cells requires further detailed characterization. However, these results indicate that after antigen stimulation primed cells never revert to a resting state comparable to that of naïve cells, challenging the existence of a G0 resting memory T cell37. Next we studied differentiation of individual cells during primary and Hprt Il2 Ifng Hprt Il2 Ifng Figure 6. Expression of lymphokine mRNA by individual cells ex vivo. Results show the expression of Hprt, Il2 and Ifng by 27 individual Tg cells during the primary and secondary immune response. The observed frequencies of cytokine coexpressing memory cells were compared with the expected values calculated for random coincidence of the two independent variables using a χ2 test. Random gene association in memory T cells was statistically excluded (P < 0.002). • volume 1 no 1 • nature immunology 51 © 2000 Nature America Inc. • http://immunol.nature.com © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES secondary immune responses. The results differ from existing published data on in vitro T cell differentiation. In in vitro studies, cell differentiation was correlated to cell division with cytokine expression increasing as cells progressed through different cell cycles38–41. We therefore expected that early on post-activation, naïve cells would differentiate poorly, but that all would eventually express cytokine mRNA during the primary immune response, as by then they have undergone extensive division. In fact, we observed very different behavior. Naïve cells differentiated very efficiently into Pfp+ cells in the absence of cell division and only a small minority expressed cytokine mRNA even after extensive division. These results suggest an immediate and relatively efficient differentiation of CD8+ naïve cells into cytotoxic cells; they also demonstrate that lymphokine expressers are very rare throughout this primary immune response. The differential pattern of cytokine expression by individual cells is also in conflict with previous models of T cell differentiation. The frequency and kinetics of gene expression exclude the possibility that all CD8+ effector functions must originate from a common IL-2–producing precursor37,42. Differential gene expression cannot result from different activation thresholds (IL-2 being expressed at higher thresholds)43–46 because if this were true, all IL-2-expressing cells would coexpress IFNγ and perforin. The single effector functions of individual cells that we found are best explained by acquisition of a certain effector function being associated with some type of inhibition of alternative differentiation pathways. It was recently suggested that such behavior guided the differentiation of immature bone marrow precursors into mature B cells47. With respect to memory cell generation, it is assumed that antigen elimination is followed by reversion of effector cells to a resting memory state. Our results demonstrate, however, that the Pfp and Ifng mRNA expression acquired during the primary immune response is not lost. Similarly, it was clearly demonstrated that resting memory T cells (surviving for long time periods after antigen elimination, as we also showed) are capable of killing target cells directly ex vivo48,49. Based on our findings and these previous reports definitions of effector versus memory cell types clearly require revision, and at the single cell level, may be more quantitative than qualitative. Regarding memory cell differentiation, we observed an early kinetics of lymphokine mRNA expression after in vivo activation, predictable from previous in vitro activation data12–18. However, most memory cells did not express cytokine mRNA in secondary immune responses, despite very extensive division. Although most naïve cells expressed mutually exclusive effector functions, reactivated memory cells became multifunctional. Random gene association cannot explain why all the yet rare Il2 mRNA+ cells coexpressed Ifng mRNA. The finding that none of the Il2 mRNA expressers coexpressed Ifng mRNA in primary immune responses whereas during secondary immune responses all Il2 mRNA expressers also coexpressed Ifng mRNA is puzzling. There are two possible explanations. Either a higher efficiency of signal transduction may render memory cells capable of transcribing many genes simultaneously, or coexpression of these genes reflects epigenetic modifications induced during the primary immune response. If the latter were true, either some genes would undergo epigenetic modifications34–36 without being transcribed, or transcription may vary in each individual cell throughout the immune response. Information on these alternative mechanisms could be obtained by monitoring lymphokine gene transcription with reporter genes, but results based on this approach are contradictory50,51. Most pathogens grow exponentially in infected hosts. In this context, the long lag time that is required for naïve cell proliferation, the increased loss rate during cell division and the heterogeneity of func52 nature immunology • volume 1 no 1 • tions of each individual primed cell are major handicaps for control of infection. Conversely, the immediate and extensive proliferation of memory cells, their reduced loss rate and the capacity of each cell to mediate several effector functions simultaneously has a major impact in the control of infection before any pathogenic effects have time to develop immune responses. Finally, the inhibitory effect of memory cells on naïve cell expansion we described ensures reinforcement of clonal dominance during secondary immune responses4–8. In a normal environment individuals are subjected to successive infection by different pathogens, and the number of memory cells specific to a first antigen declines. The properties of memory cells we describe here ensure the persistence of immunological memory even in these circumstances, as very efficient secondary immune responses can be generated from rare memory cells. Methods Mice. C57BL/6 mice were: Ly 5.1; RAG-2-deficient20; CD3ε-deficient52; or RAG-2-deficient transgenic (Tg) for the TCRαβ Tg receptor specific for the male antigen21 expressing either the Thy 1.1 or Thy 1.2 allotype marker. All mice were bred at the Center for Development of Advanced Experimentation Techniques, Orleans, France. Naïve and memory monoclonal Tg cells. Naïve Tg cells were recovered from RAG-2deficient Tg female mice. Memory T cells were obtained as described elsewhere12,14. 0.5×106 naïve Tg lymph-node cells were injected intravenously (i.v.) into RAG-2-deficient female mice, and immunized with 0.5×106 male CD3ε-deficent bone marrow cells. Primed cells were recovered 2 to 6 months after immunization. These cells are all antigen-experienced, functionally competent and persist in vivo in the apparent absence of antigen12,14. We failed to detect male cells in any lymphoid organs, including bone marrow, by FACS analysis (the male cells used for immunization express an allotype marker) or by a very sensitive PCR method, that identified male cells at a frequency of 10-6 22. We also injected i.v. a new set of naïve T cells in mice that had previously received Tg cells and had eliminated male cells. Over two weeks, these naïve T cells were given the opportunity to recirculate and contact the antigen, but we found they remained CD44- and did not divide12. To obtain pure monoclonal populations, cell suspensions were depleted of platelets, macrophages, granulocytes, erythrocytes and B cells by using a mixture of monoclonal antibodies and magnetic sorting with coated Dynabeads (Dynal, A. S., Oslo, Norway). Antibodies and immunofluorescence analysis. Four-color immunofluorescence analysis was performed using a FACS-Calibur system (Becton Dickinson, San Jose). The antibodies used were: PE-labeled: anti-CD69, anti-CD44, anti-Thy-1.2 (Pharmingen) and anti-CD25 (Caltag, San Francisco). Biotin-labeled T3.7053 and anti-Ly-5.2 (104-2.1). Red 613-labeled anti-CD8α (GibcoBRL, Grand Island, NY); Cy-Chrome-labeled anti-CD8α (Pharmingen). Fluoroscein isothiocyanate- (FITC)-labeled anti-Thy-1.1 (HO22.1). Biotinylated antibodies were revealed with Streptavidine-Allophycocyanin (APC) (Molecular Probes, Eugene, OR). Cell size was determined by forward light scattering. Cell division. 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes) labeling was performed as previously described54. The intensity of labeling of resting T cells was established in naïve T cells transferred to female mice, in which they do not divide. The cell cycle analysis was determined by acridine orange cytometric analysis55 using the Modfit LT Verity program. Evaluation of division and loss rates. The difference equation model27,28 developed to describe the number of T cells in each division at each hour after transfer describes the number of cells that have been through i divisions at time t hours, N(t,i), in terms of the distribution of cells 1 h previously. The model has three parameters and two equations. Parameters are: loss rate m, and division rates b0 for the first division, and b1 for all subsequent divisions. Of the equations, the first applies to cells that have never divided—N(t,0)—that can either persist or be lost, either through loss (at per cell rate m h-1) or through division (to produce two daughter cells that have divided once) at per cell rate b0 h-1. So N(t+1,0) = N(t,0) − (m+b0) N(t,0). The second equation applies to cells that have divided i > 0 times. They obey the same rules of cell loss as non-dividing cells, but they also have a source term, representing the arrival of two daughter cells that have divided for the i + 1th time, progeny of a cell that had previously divided i times. N(t+1,i+1) = N(t,i+1) (1−(m+b1)) + 2bjN(t,i) where bj = b0 if i = 1 and bj = b1 if i > 1. To fit this model to the data, the model was initiated at t = 20 h using the 20 h data, solved through time 115 h and the parameter values m and b that give the best fit between model and data determined. The goodness of fit criterion was minimization of the squared differences between the natural logarithm of model and data. This procedure gives a conservative estimate of the difference between naïve and memory cells as it ignores the difference between the two populations that is already apparent at t = 20 h. The results obtained describe the average behavior of whole populations, and thus cannot be directly extrapolated to individual cells cycling time. Single cell RT-PCR analysis. Individual Tg cells were sorted using a FACS Vantage equipped with an automatic cell deposition unit (Becton Dickinson). Cells were lysed by july 2000 • http://immunol.nature.com © 2000 Nature America Inc. • http://immunol.nature.com © 2000 Nature America Inc. • http://immunol.nature.com A RTICLES cooling at –80 °C followed by heating to 65 °C for 2 min. After cooling to 4 °C, RNA was reverse-transcribed for 1 h at 37 °C using 0.13 µM specific 3′ primers in a 10 µl volume also containing 5x first-strand buffer (GibcoBRL), dNTP (1mM) (Pharmacia Biotech, Piscataway, NY), DTT (10mM) (GibcoBRL), RNase block (2.6 U/µl) (Stratagene, La Jolla, CA) and M-MLV (4.6 U/µl) (GibcoBRL). The reaction was stopped by 3′ incubation at 95 °C. Il2, Ifng and Hprt cDNAs were amplified by modified nested two-step PCR56. The first PCR round consisted of 30 cycles of amplification (45′′ at 94 °C; 1′ at 58 °C; 1′ 30′′ at 72 °C) with 0.2 µM dNTP, 0.035 U/µl Taq polymerase (Perkin Elmer, Brauchburg, NJ) and 0.44 µM specific primers in a volume of 85 µl. The first PCR products were split and each gene was amplified separately for 38 cycles (30′′ at 94 °C; 45’’ at 58 °C; 1′ at 72 °C) in 20 µl containing 0.25 µM dNTP, 0.05 U/µl Taq polymerase (Perkin Elmer) and 1.25 µM specific primers. The 3′ primers (the same as used for reverse-transcription) were: (Il2: 5′ TCAATTCTGTGGCCTGCTTG-3′; Ifng: 5′-AAAGAGATAATCTGGCTCTGC-3′; Hprt: 5′-TCCAACACTTCGAGAGGTCC-3′). The 5′ primers were: Hprt: 5′-GGGGGCTATAAGTTCTTTGC-3′, 5′-GTTCTTTGCTGACCTGCTGG-3′ (nested); Il2: 5′GACACTTGTGCTCCTTGTCA-3′, 5′-CTCTACAGCGGAAGCACAGC-3′ (nested); Ifng: 5′-GCTCTGAGACAATGAACGCT-3′, 5′-TGTTTCTGGCTGTTACTGCC-3′ (nested). None of the primer combinations amplify genomic DNA. The interpretation of our data depends on the sensitivity of our PCR system. Single-cell RT-PCRs are not quantitative, but we attempted to characterize the sensitivity of our assay by using several approaches. First, we always tested plating efficiency, by simultaneous amplification of Hprt. Second, we compared the relative efficiency of Il2, Ifng and Hprt amplification in bulk populations by studying the slopes of PCR product accumulation with different cycle numbers in non-saturating conditions. We found the three slopes were parallel, indicating that the three PCRs amplifications were similarly efficient. These results support the notion that lymphokine cDNA in each cell could be amplified when present at the same frequency as Hprt (the calculated number of Hprt mRNA copies/cell is of the order of 5–1057. Finally, the efficiency of Il2 and Ifng mRNA amplification was tested in T cell clones (98–100% positive) and also in in vitro stimulated memory T cells (90% Ifng-positive). It must be recalled that the mRNA from each cell is reversed transcribed with a specific primer, and amplified for 68 cycles. It is therefore unlikely that lymphokine mRNA expressing cells would be scored as negative in this test. Cell culture and cytokine secretion. To evaluate cytokine production after in vitro antigen stimulation, 106 Tg cells were incubated for 7 h with 2×106 CD3ε-deficient female spleen cells and 10-7 M specific peptide49. Secretion of lymphokines was evaluated by enzymelinked immunosorbent assay14. Acknowledgements We thank C. Garcia for cell sorting; A.M. Joret and S. Leaument for technical assistance; A. Le Campion for statistics; J. Di Santo, A.A. Freitas, D. Guy-Grand, A. Sarukhan and H. von Boehmer for reviewing the manuscript.This work was supported by grants from the National Association of AIDS Research, France. H.V.-F. was supported by a grant from Technology and Science Foundation, Praxis XXI, Portugal. Received 23 March 2000; accepted 10 May 2000. 1. Owen, J.A., Allouche, M. & Doherty, P.C. Limiting dilution analysis of the specificity of influenzaimmune cytotoxic T cells. Cell. Immunol. 67, 49–59 (1982). 2. Ahmed, R. & Gray, D. Immunological memory and protective immunity: understanding their relation. Science 272, 54–60 (1996). 3. Doherty, P.C.,Topham, D.J. & Tripp, R.A. Establishment and persistence of virus-specific CD4+ and CD8+ T cells memory. Immunol. Rev. 150, 23–44 (1996). 4. Flynn, K. J. et al. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 8, 683–691 (1998). 5. Murali-Krishna, K. et al. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8, 177–187 (1998). 6. Busch, D.H. & Pamer, E.G.T cell affinity maturation by selective expansion during infection. J. Exp. Med. 189, 701–10 (1999). 7. McHeyzer-Williams, L.J., Panus, J.F., Mikszta, J.A. & McHeyzer-Williams, M.G. Evolution of antigenspecific T cell receptors in vivo: preimmune and antigen-driven selection of preferred complementarity-determining region 3 (CDR3) motifs. J. Exp. Med. 189, 1823–38 (1999). 8. Savage, P.A., Boniface, J.J. & Davis, M.M. A kinetic basis for T cell receptor repertoire selection during an immune response. Immunity 10, 485–921 (1999). 9. Selin, L. K. et al. Attrition of T cell memory: Selective loss of LCMV epitope-specific memory CD8 T cells following infections with heterologous viruses. Immunity 11, 733–742 (1999). 10. Budd, R.C. et al. Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation. J. Immunol. 138, 3120–3129 (1987). 11. Bruno, L., Kirberg J. & von Boehmer, H. On the cellular basis of immunological T cell memory. Immunity 2, 37–43 (1995). 12. Tanchot, C. et al. Differential requirements for survival and proliferation of CD8 naïve or memory T cells. Science 276, 2057–2062 (1997). 13. Curtsinger, J.M., Lins, D.C. & Mescher, M.F. CD8+ memory T cells (CD44high, Ly-6C+) are more sensitive than naive cells (CD44low, Ly-6C-) to TcR/CD8 signaling in response to antigen. J. Immunol. 160, 3236–3243 (1998). 14. Tanchot, C. et al. Modifications of CD8+ T cell function during in vivo memory or tolerance induction. Immunity 8, 581–590 (1998). 15. Cho, B. K. et al. Functional differences between memory and naive CD8 T cells. Proc. Natl Acad. Sci. USA 96, 2976–2981 (1999). 16. Garcia, S., DiSanto, J. & Stockinger, B. Following the Development of a CD4 T cell response in vivo: from activation to memory formation. Immunity 11, 163–171 (1999). http://immunol.nature.com • july 2000 17. Zimmermann, C., Prevost-Blondel, A., Blaser, C. & Pircher, H. Kinetics of the response of naive and memory CD8 T cells to antigen: similarities and differences. Eur. J. Immunol. 29, 284–290 (1999). 18. Bachmann, M.F., Barner, M.,Viola, A. & Kopf, M. Distinct kinetics of cytokine production and cytolysis in effector and memory T cells after viral infection. Eur. J. Immunol. 29, 291–299 (1999). 19. Tanchot, C. & Rocha, B.The organization of mature T cell pools. Immunol.Today 19, 575–579 (1998). 20. Shinkai,Y. et al. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855–867 (1992). 21. Kisielow, P et al. Tolerance in T cell receptor transgenic mice involves deletion of nonmature CD4+CD8+ thymocytes. Nature 333, 742–746 (1988). 22. Rocha, B., Grandien, A. & Freitas, A. A. Anergy and exhaustion are independent mechanisms of peripheral tolerance. J. Exp. Med. 181, 993–1003 (1995). 23. McLean, A. R. et al. Resourse competition as a mechanism for B cell homeostasis. Proc. Natl Acad. Sci. USA 94, 5792–5797 (1997). 24. Borghans, J.A.M.,Taams, L.S.,Wauben, M.H.M. & De Boer, R. Competition for antigenic sites during T cell proliferation: a mathematical interpretation of in vitro data. Proc. Natl Acad. Sci. USA 96, 10782–10787 (1999). 25. Tripp, R.A., Lahti, J.M. & Doherty, P.C. Laser light suicide of proliferating virus-specific CD8+ T cells in an in vivo response. J. Immunol. 155, 3719–3721 (1995). 26. Sprent, J.,Tough, D.F. & Sun,S. Factors controlling the turnover of T memory cells. Immunol. Rev. 156, 79–85 (1997). 27. Leslie, P.H. Some further notes on the use of matrices in population mathematics. Biometrika 35, 213–245 (1948). 28. De Boer R.J. & Noest A.J.T cell renewal rates, telomerase and telomere length shortening. J. Immunol. 160, 5832–5837 (1998). 29. Karulin, A.Y., Hesse, M.D.,Tary-Lehmann, M. & Lehmann, P.V. Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity. J. Immunol. 164, 1862–1872 (2000). 30. Valitutti, S. et al. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature 11, 148–151 (1995). 31. Bachmann, M.F. et al. Developmental regulation of Lck targeting to the CD8 coreceptor controls signaling in naive and memory T cells. J. Exp. Med. 189, 1521–1529 (1999). 32. Mackay, C.R. Migration pathways and immunologic memory among T lymphocytes. Semin. Immunol. 4, 51–58 (1992). 33. Brown, K.E. et al. Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division. Mol. Cell. 3, 207–217 (1999). 34. Agarwal, S. & Rao, A. Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation. Immunity 9, 765–775 (1998). 35. Lewin, B.The mystique of epigenetics. Cell 93, 301–303 (1998). 36. Fitzpatrick, D.R., Shirley, K.M. & Kelso, A. Stable epigenetic inheritance of regional IFN-gamma promoter demethylation in CD44 high CD8+ T lymphocytes. J. Immunol. 162, 5053–5057 (1999). 37. Swain, S.L. et al. From naive to memory T cells. Immunol Rev. 150, 143–167 (1996). 38. Gett, A. & Hodgkin, P.D. Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation. Proc. Natl Acad. Sci. USA 95, 9488–9493 (1998). 39. Bird, J.J. et al. Helper T cell differentiation is controlled by the cell cycle. Immunity 9, 229–237 (1998). 40. Richter, A., Lohning, M. & Radbruch, A. Instruction for cytokine expression in T helper lymphocytes in relation to proliferation and cell progression. J. Exp. Med. 190, 1439–1450 (1999). 41. Gudmundsdottir, H.,Wells A.D. & Turka, L.A. Dynamics and requirements of T cell clonal expansion in vivo at the single-cell level: effector function is linked to proliferative capacity. J. Immunol. 162, 5212–5223 (1999). 42. Sad, S. & Mosmann,T.R. Single IL-2 secreting precursor CD4 T cell can develop into either Th1 or Th2 cytokine secretion phenotype. J. Immunol. 153, 3514–3522 (1994). 43. Viola, A. & Lanzavecchia, A.T cell activation determined by T cell receptor number and tunable thresholds. Science 273, 104–106 (1996). 44. Itoh,Y. & Germain, R.N. Single cell analysis reveals regulated hierarchical T cell antigen receptor signaling thresholds and intraclonal heterogenety for individual cytokine responses of CD4+ T cells. J. Exp. Med. 186, 757–766 (1997). 45. Waldorp, S.L., Davis, K.A., Maino,V.C. & Picker, L.J. Normal human CD4+ memory cells display broad heterogenety in their activation threshold for cytokine synthesis. J. Immunol. 161, 5282–5295 (1998). 46. Weaver, C.T. Heterogeneity in the clonal T cell response: implications for models of T cell activation and cytokine phenotype development. Immunol. Res. 17, 279–302 (1998). 47. Nutt, S.L., Heavey, B., Rolink, A.G. & Busslinger, M. Commitment to B-lymphoid lineage depends on the transcription factor Pax5. Nature 401, 556–562 (1999). 48. Selin, L.K. & Welsh, R.M. Cytolytically active memory CTL present in lymphocytic choriomeningitis virus-immune mice after clearance of virus infection. J. Immunol. 158, 5366–5373 (1997). 49. Opferman, J.T., Ober, B.T. & Ashton-Rickardt, P.G. Linear differentiation of cytotoxic effectors into memory T lymphocytes. Science 283, 1745–1748 (1999). 50. Naramura, M., Hu, R. & Gu, H. Mice with a fluorescent marker for interleukin 2 gene activation. Immunity 9, 209–216 (1998). 51. Saparov, A. et al. Interleukin-2 expression by a subpopulation of primary T cells is linked to enhanced memory/effector function. Immunity 11, 271–280 (1999). 52. Malissen, M. et al. Altered T cell development in mice with a targeted mutation of the CD3 ε gene. EMBO J. 14, 4641–4653 (1995). 53. Teh, H.S. et al. Thymic major histocompatibility complex antigens and the αβ T-cell receptor determine the CD4/CD8 phenotype of T cells. Nature 335, 229–233 (1988). 54. Lyons, A.B. & Parish, C.R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods. 171, 131–137 (1994). 55. Tafuri, A. et al. Combination of hematopoietic growth factors containing IL-3 induce acute myeloid leukemia cell sensitization to cycle specific and cycle non-specific drugs. Leukemia 8, 749–757 (1994). 56. Loffert, D., Ehlich, A., Muller,W. & Rajewsky, K. Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development. Immunity 4, 133–144 (1996). 57. Pannetier, C. et al. Quantitative titration of nucleic acids by enymatic amplification reactions run to saturation. Nucleic Acids Res. 21, 577–583 (1993). 58. Taswell, C. Limiting dilution assays for the determination of immunocompetent cell frequencies. III. Validity tests for the single-hit Poisson model. J. Immunol. Methods 72, 29–40 (1984). 59. Siminovitch, L., McCulloch, E.A. & Till, J E.The distribution of colony forming cells among spleen colonies. J. Cell Comp. Physiol. 62, 327–336 (1963). • volume 1 no 1 • nature immunology 53