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Deciphering the relationship between central and effector memory CD8+ T cells
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| Research Focus
Deciphering the relationship between central and effector memory CD81 T cells David F. Tough The Edward Jenner Institute for Vaccine Research, Compton, Newbury, Berkshire, RG20 7NN, UK
Memory CD81 T cells can be divided into subpopulations termed central memory cells and effector memory cells based on their phenotype and potential to home to lymph nodes versus non-lymphoid tissues, respectively. Two recent studies, one in humans and one in mice, provide new insight into the lineage relationship between these subpopulations. In response to infection, T cells recognizing epitopes within the invading pathogen become activated and undergo massive expansion. Once the infection is cleared, most of the activated cells die, but a small proportion survives, resulting in the persistence of pathogen-specific T cells at much higher numbers than were present in the preimmune (naı¨ve) state. This numerical rise, together with altered functional properties of primed T cells establishes a state of immunological memory – an ability to respond faster and more effectively on a subsequent encounter with the same pathogen [1,2]. Understanding the basis of memory, however, is complicated by the fact that memory T cells are phenotypically and functionally diverse. One way in which memory T cells can be partitioned is by their expression of the chemokine receptor CCR7 and the lymph node homing receptor CD62L, which together allow for T-cell entry into lymph nodes through high endothelial venules [3,4]. CCR7þCD62Lþ memory cells have been termed central memory cells (TCM), whereas cells lacking these molecules are referred to as effector memory cells (TEM) [5]. In addition to possessing the potential to home to different tissues, TCM and TEM appear to be functionally distinct, with TEM but not TCM exhibiting direct (ex vivo) effector activity [5,6]. Because both TCM and TEM can be generated during the same immune response, a key question is how these subpopulations relate to each other; that is, are TCM and TEM stable, independent subpopulations, or does one represent an intermediate stage of differentiation that can convert to the other following the input (or removal) of a specific signal? For CD4 cells, previous work showed that TCM could acquire characteristics of TEM in vitro following stimulation through the T-cell receptor (TCR) [5] or with cytokines [7]. These results implied that these subpopulations are not independent but rather can interconvert and led to the suggestion that increasing signal strength is associated with progressive differentiation from TCM to TEM (progression model). This notion was further supported by the finding that CD4 TEM had significantly Corresponding author: David F. Tough ([email protected]). http://treimm.trends.com
shorter telomeres than TCM, an indication that TEM have undergone more rounds of cell division [5].
…these subpopulations are not independent but rather can interconvert … suggestion that increasing signal strength is associated with progressive differentiation from TCM to TEM… For CD8 T cells, there is some evidence suggesting that TCM and TEM might be generated differentially during an immune response, depending on the conditions of activation. This comes from a report showing that murine CD8 cells activated in vitro can acquire the characteristics of either TCM or TEM after injection into mice, depending on the particular cytokines added during the culture period; activated CD8 cells cultured in a high dose of interleukin-2 (IL-2) differentiated into TEM, and those cultured in IL-15 or low dose IL-2 became TCM [8,9]. These findings are consistent with a progression model, if one accepts that high and low dose IL-2 treatment differ only in the strength of the signal delivered, but would also fit a model in which TCM and TEM are independent endpoints and arise from qualitatively different signals (i.e. IL-2 versus IL-15). However, as for CD4 cells, human TCM CD8 cells acquire markers of TEM after exposure to cytokines in vitro, indicating that conversion between CD8 memory subpopulations can occur, at least in tissue culture [10]. Does this happen in vivo? Two groups, Baron et al. [11] and Wherry et al. [12], have now investigated the relationship between TCM and TEM CD8 cells generated in vivo. The two groups studied different species, used different approaches and reached slightly different conclusions. Relationship between TCM and TEM in humans Baron et al., studying CD8 cells in human peripheral blood, took the approach of examining the TCR repertoire among TCM and TEM cells, using elegant molecular techniques to identify T cells expressing TCRs with specific CDR3 (complementarity-determining region 3) sequences in their Vb chains [11]. Their rationale was that if TCM and TEM represent closely related, interconvertible cells, the same clones should be prominent in both populations. Furthermore, fluctuations over time in the size of a clone
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present in one population should be apparent in the other population. Contrary to these predictions, the authors found that the number of clonotypes shared by CD8 TCM and TEM within an individual was very low, specifically 6.2% and 5.6% of the total number of clonotypes detected within the two Vb families analysed. When several clonotypes (both shared and unique) were followed within one individual over a seven month period, some clones displayed a stable frequency within the subpopulations although others varied by as much as 10-fold. Significantly, however, when changes in the frequency of a clone were observed in one memory population, no corresponding alterations were observed in the other. These authors also investigated directly the distribution of T-cell clones specific for one particular antigen, a peptide within the matrix protein of influenza A virus. In this experiment, influenza-specific CD8 cells were purified using MHC-tetramers and six clonotypes were identified in the antigen-specific population. All six clonotypes were detected within the TCM subset in this individual and two were also found among TEM cells. Although the incidence of shared clonotypes was higher than for the randomly analysed Vb families, it was notable that changes in the frequencies of these clones over a nine month period again revealed no evidence for transition between TCM and TEM subpopulations. Based on their overall results, the authors proposed that CD8 TCM and TEM are largely independent subpopulations.
conversion occurred more quickly after infection of mice with a low dose of L. monocytogenes, where antigen could not be detected for .2 – 3 days after infection, than after infection with high dose L. monocytogenes or with LCMV, where antigen can be detected for 5– 7 days. These results are reminiscent of those reported in an earlier study, in which virus-specific CD8 cells reverted to a CD62Lhi phenotype at different rates when mice were infected with influenza versus Sendai virus [13]. To determine whether the different tempo of TEM ! TCM conversion was due to differences in the level of persisting antigen or other environmental differences in the differently infected mice, Wherry et al. obtained primed CD8 cells (taken at day 8 post-infection) from mice that had been infected separately with low dose L. monocytogenes or with LCMV, and injected a mixture of these cells into secondary hosts. If the environment of the memory cells was the primary determinant of the rate of TEM ! TCM conversion, memory cells obtained from both types of mice should change phenotype at the same rate in the secondary recipients. In fact, what was observed in the adoptive hosts was that TEM derived from low dose infected mice still converted to TCM faster than TEM from high dose infected mice. These results indicated that the rate at which TEM convert to TCM was determined very early in the immune response – during the first week after infection – and that this timing could be linked to the initial antigen dose.
Relationship between TCM and TEM in mice Wherry et al. investigated the appearance of CD8 TCM and TEM after infection of mice with a virus, lymphocytic choriomeningitis virus (LCMV), or an intracellular bacterium, Listeria monocytogenes [12]. Antigen-specific CD8 cells were followed over time using either TCR transgenic cells specific for an epitope expressed in both agents (the L. monocytogenes used was a recombinant expressing an LCMV epitope) or using MHC tetramers containing this epitope. In initial experiments studying LCMV infection, the authors found that the total number of antigen-specific CD8 memory cells remained constant once the memory pool had been established, confirming many other reports in this system. By contrast, however, the numbers of TEM and TCM changed substantially over time, and did so in a reciprocal manner, with TCM increasing and TEM decreasing. Because one explanation for these results was that TEM were converting into TCM, the authors went on to investigate this possibility directly by transferring purified populations of CD8 TEM or TCM into secondary recipients and examining the fate of the transferred cells. The striking finding was that TCM retained their phenotype for at least 30 days after transfer while approximately half of the TEM cells acquired the phenotype of TCM during this time. Importantly, the authors demonstrated that this phenotypic conversion occurred in the absence of cell division, ruling out the possibility that the results stemmed from outgrowth of a small number of contaminating TCM in the transferred population. The data therefore provided strong evidence that CD8 TEM can convert to TCM under appropriate in vivo conditions. Interestingly, Wherry et al. observed that TEM ! TCM
…the rate at which TEM convert to TCM was determined very early in the immune response – during the first week after infection – and that this timing could be linked to the initial antigen dose.
http://treimm.trends.com
Based on these observations, Wherry et al. proposed a linear differentiation model for the generation of CD8 TCM and TEM, in which TEM are derived directly from effector cells and TCM are derived from TEM [12]. In contrast to the model proposed previously for CD4 memory cells, they found little evidence that CD8 TCM convert to TEM under ‘stable’ in vivo conditions, that is, in the absence of re-challenge with antigen. On this point, their conclusions are in agreement with those of Baron et al. [11]; whether this represents a real difference between CD4 and CD8 memory subpopulations will await similar in vivo studies for CD4 cells. TEM ! TCM conversion: mouse versus humans As described earlier, Baron et al. concluded that there was little movement of T-cell clones in either direction between TCM and TEM. Therefore, at face value these two studies yield conflicting messages about whether TEM ! TCM conversion takes place; what is the reason for this discrepancy? One possibility is that there are fundamental differences between mouse and human
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CD8 memory cells, or at least in the subpopulations analysed. In this respect, it should be borne in mind that human CD8 cells can be subdivided into four populations on the basis of CD62L (CCR7) and CD45 isoform expression. Thus, in addition to CD62L (CCR7)þ CD45R0þ TCM, CD62L (CCR7)2 CD45R0þ TEM and CD62L (CCR7)þ CD45RAþ naı¨ve cells, there is a population of CD62L (CCR7)2 CD45RAþ ‘effector memory RA’ cells, and considerable numbers of antigen-specific cells can be identified in this population after certain virus infections [14 – 16]. This fourth population was not studied by Baron et al. and hence any movement of clones in or out of this subset would not have been detected. By contrast, because no markers have been described that identify this subpopulation in the mouse (if it exists), these cells could have influenced the findings of Wherry et al. Furthermore, it is also worth noting that, unlike what has been described for human CD8 TEM [5], Wherry et al. did not detect direct (ex vivo) effector activity among murine CD8 TEM [12], although this has been observed in other studies [6]. Another factor that might have had a bearing on the contradictory results is that a different relationship between TCM and TEM would be observed depending on the time point in the immune response that these cells were examined. For the mouse experiments, the kinetics of the responses were known precisely because cells responding to infections given at defined time points were examined. This was not the case for the study of human cells by Baron et al., where, for the most part, the antigenic specificity of the CD8 cells examined was unknown. Interestingly, when influenzaspecific CD8 cells were analysed, two of six clones found among TCM were also present in the TEM population. One interpretation of these data, based on the results of Wherry et al., is that these cells were studied at a relatively late time point after infection, when the majority of TEM have converted to TCM. If this were the case, however, one might have expected to see evidence of TEM ! TCM conversion among the remaining two shared influenza-specific clones over the nine months of analysis, or, by chance, among the thirteen clones of unknown specificity that were studied over a seven month period.
Another factor that might have had a bearing on the contradictory results is that a different relationship between TCM and TEM would be observed depending on the time point in the immune response that these cells were examined. Evidence of such phenotypic conversion could be obscured by the nature of the infectious agent to which the CD8 cells react. For example, different results might be seen with http://treimm.trends.com
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pathogens that are cleared versus those that persist because the latter will provide a source of antigen for periodic restimulation of memory cells. This could occur in a stochastic manner, causing a proportion of cells in the memory population to acquire or retain a more activated phenotype at any one time, yet giving the appearance that there are stable numbers of cells in distinct subpopulations. In this respect, it should be noted that triggering through the TCR has been shown to both downregulate CCR7 on CD8 TCM [10] and upregulate CCR7 on CD8 TEM [14]. Similar effects might result from exposure to previously seen and cross-reactive antigens [17], something that does not complicate experiments with mice under controlled conditions. One clear indication that contact with antigen during persistent infection can modulate the phenotype of memory cells has come from studies of Epstein–Barr (EBV) virus infection, in which CD8 cells specific for epitopes expressed during the lytic cycle of the virus became CD45RAþ CD45R02 CCR72 with time after infection, although those reactive with latent epitopes remained CD45RA2R0þ [18]. Concluding remarks Overall, these papers provide several new insights into the relationship between TCM and TEM CD8 cells in humans and mice. They also raise several important questions. For example, how is the kinetics of TEM ! TCM conversion ‘programmed’ during the first week of the immune response and is this altered if the infectious agent is able to persist at some level? Does the relationship between TEM and TCM cells observed in the systemic infection models used by Wherry et al. also hold true for CD8 memory cells generated after infections at mucosal sites? Is there interconversion between memory subpopulations and ‘effector memory RA’ cells and does an equivalent population exist in mice? Answering these questions will contribute to our growing understanding of how immunological memory is generated and maintained. Acknowledgements This is publication number 67 from the Edward Jenner Institute for Vaccine Research. I thank Persephone Borrow for critical reading of this manuscript and helpful comments.
References 1 Dutton, R.W. et al. (1998) T cell memory. Annu. Rev. Immunol. 16, 201 – 223 2 Berard, M. and Tough, D.F. (2002) Qualitative differences between naı¨ve and memory T cells. Immunology 106, 1 – 12 3 Berg, E.L. et al. (1991) The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor. J. Cell Biol. 114, 343 – 349 4 Gunn, M.D. et al. (1998) A chemokine expressed in lymphoid high endothelial benules promotes the adhesion and chemotaxis of naı¨ve T lymphocytes. Proc. Natl. Acad. Sci. U. S. A. 95, 258 – 263 5 Sallusto, F. et al. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708 – 712 6 Masopust, D. et al. (2001) Preferential localization of effector memory cells in nonlymphoid tissue. Science 291, 2413– 2416 7 Geginat, J. et al. (2001) Cytokine-driven proliferation and differentiation of human naı¨ve, central memory, and effector memory CD4þ T cells. J. Exp. Med. 194, 1711 – 1719 8 Manjunath, N. et al. (2001) Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J. Clin. Invest. 108, 871 – 878
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9 Weninger, W. et al. (2001) Migratory properties of naı¨ve, effector, and memory CD8þ T cells. J. Exp. Med. 194, 953 – 966 10 Geginat, J. et al. (2003) Proliferation and differentiation potential of human CD8þ memory T cell subsets in response to antigen or homeostatic cytokines. Blood 101, 4260 – 4266 11 Baron, V. et al. (2003) The repertoires of circulating human CD8þ central and effector memory T cell subsets are largely distinct. Immunity 18, 193 – 204 12 Wherry, E.J. et al. (2003) Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4, 225– 234 13 Tripp, R.A. et al. (1995) Temporal loss of the activated L-selectin-low phenotype for virus-specific CD8þ memory T cells. J. Immunol. 154, 5870–5875 14 Champagne, P. et al. (2001) Skewed maturation of memory HIVspecific CD8 T lymphocytes. Nature 410, 106 – 111 15 Hislop, A.D. et al. (2001) EBV-specific CD8þ T cell memory:
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relationships between epitope specificity, cell phenotype, and immediate effector function. J. Immunol. 167, 2019 – 2029 16 Wills, M.R. et al. (2002) Identification of naı¨ve or antigen-experienced human CD8þ T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8þ T cell response. J. Immunol. 168, 5455– 5464 17 Selin, L.K. et al. (1999) Attrition of T cell memory: selective loss of LCMV epitope-specific memory CD8 T cells following infections with heterologous viruses. Immunity 11, 733– 742 18 Hislop, A.D. et al. (2002) Epitope-specific evolution of human CD8þ T cell responses from primary to persistent phases of Epstein – Barr virus infection. J. Exp. Med. 195, 893 – 905
1471-4906/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1471-4906(03)00169-8
| Letters
A genetic basis for human gd T-cell reactivity towards microbial pathogens Matthias Eberl and Hassan Jomaa Biochemisches Institut, Justus-Liebig-Universita¨t Giessen, Friedrichstrasse 24, 35392 Giessen, Germany
Elevated levels of Vg9Vd2 T cells in the peripheral blood of patients are well documented in a variety of bacterial infections, such as brucellosis, ehrlichiosis, listeriosis, salmonellosis, tuberculosis or tularaemia [1]. However, as recently addressed by Chen and Letvin, the investigation of Vg9Vd2 T-cell responses in infectious diseases has been greatly hindered by the absence of relevant animal models, with this population being unique for humans and nonhuman primates and by their unconventional reactivity towards low molecular weight antigens [2]. Over the past decade, a number of natural compounds have been postulated to be responsible for the specific expansion of the Vg9Vd2 T-cell subset in these diseases, including 2,3-diphosphoglycerate, isopentenyl pyrophosphate (IPP), isobutylamine and other non-peptidic antigens. Yet, the poor in vitro bioactivities of these molecules make it unlikely that they have a pivotal role under physiological conditions in vivo. Recently, the metabolite (E)-4-hydroxy3-methyl-but-2-enyl pyrophosphate (HMB-PP), an intermediate of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis, was isolated from Escherichia coli and shown to display a bioactivity of 0.1 nM; thus, HMB-PP is approximately four orders of magnitude more potent than IPP, and 6-7 magnitudes more potent than isobutylamine [3]. Molecules with the biochemical characteristics of HMB-PP have been detected in lysates from various mycobacteria species, Brucella suis, Francisella tularensis, Pseudomonas aeruginosa and other bacteria [4]. Meanwhile, the genes of the MEP pathway have been identified in nearly every bacterial species linked with elevated Corresponding author: Matthias Eberl ([email protected]). http://treimm.trends.com
Vg9Vd2 T-cell levels, whereas they are apparently absent in the genome of non-Vg9Vd2 T-cell-inducing bacteria, such as streptococci and staphylococci, as well as in humans [5]. Therefore, it can be assumed that HMB-PP greatly accounts for the described Vg9Vd2 T-cell reactivity towards many pathogenic bacteria. Yet, Vg9Vd2 T cells also expand in protozoan diseases, and patients suffering from malaria, leishmaniasis or toxoplasmosis show increased Vg9Vd2 T-cell numbers in the peripheral blood [6– 10]. In the case of malaria, this observation could recently be explained by the demonstration that green algae and higher plants use the MEP pathway in their chloroplasts, whereas isoprenoid biosynthesis in the cytoplasm proceeds through the classical mevalonate pathway. Similarly, Plasmodium falciparum actually harbours the MEP pathway in a specialized subcellular organelle, the so-called apicoplast, which is evolutionarily derived from the choloroplast of an ancient endosymbiont [11]. Consequently, malaria parasites stimulate Vg9Vd2 T cells in vitro and a non-peptidic antigen likely to be identical with HMB-PP can be isolated from parasite extracts [12]. Because the presence of an apicoplast is a distinct feature of all apicomplexan protozoa, we wondered whether other members of this phylum also possess the genes of the MEP pathway. In fact, most of the respective genes could be identified in the preliminary genomic sequences of the human pathogens Cryptosporidium parvum, Plasmodium vivax, and Toxoplasma gondii [5], as well as in the related species Eimeria tenella, Plasmodium yoelii, Theileria annulata and Theileria parva (Fig. 1). Thus, in analogy to the case of bacterial infection, the production of HMB-PP in the apicoplasts of parasites might also be responsible for the
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| Research Focus
Deciphering the relationship between central and effector memory CD81 T cells David F. Tough The Edward Jenner Institute for Vaccine Research, Compton, Newbury, Berkshire, RG20 7NN, UK
Memory CD81 T cells can be divided into subpopulations termed central memory cells and effector memory cells based on their phenotype and potential to home to lymph nodes versus non-lymphoid tissues, respectively. Two recent studies, one in humans and one in mice, provide new insight into the lineage relationship between these subpopulations. In response to infection, T cells recognizing epitopes within the invading pathogen become activated and undergo massive expansion. Once the infection is cleared, most of the activated cells die, but a small proportion survives, resulting in the persistence of pathogen-specific T cells at much higher numbers than were present in the preimmune (naı¨ve) state. This numerical rise, together with altered functional properties of primed T cells establishes a state of immunological memory – an ability to respond faster and more effectively on a subsequent encounter with the same pathogen [1,2]. Understanding the basis of memory, however, is complicated by the fact that memory T cells are phenotypically and functionally diverse. One way in which memory T cells can be partitioned is by their expression of the chemokine receptor CCR7 and the lymph node homing receptor CD62L, which together allow for T-cell entry into lymph nodes through high endothelial venules [3,4]. CCR7þCD62Lþ memory cells have been termed central memory cells (TCM), whereas cells lacking these molecules are referred to as effector memory cells (TEM) [5]. In addition to possessing the potential to home to different tissues, TCM and TEM appear to be functionally distinct, with TEM but not TCM exhibiting direct (ex vivo) effector activity [5,6]. Because both TCM and TEM can be generated during the same immune response, a key question is how these subpopulations relate to each other; that is, are TCM and TEM stable, independent subpopulations, or does one represent an intermediate stage of differentiation that can convert to the other following the input (or removal) of a specific signal? For CD4 cells, previous work showed that TCM could acquire characteristics of TEM in vitro following stimulation through the T-cell receptor (TCR) [5] or with cytokines [7]. These results implied that these subpopulations are not independent but rather can interconvert and led to the suggestion that increasing signal strength is associated with progressive differentiation from TCM to TEM (progression model). This notion was further supported by the finding that CD4 TEM had significantly Corresponding author: David F. Tough ([email protected]). http://treimm.trends.com
shorter telomeres than TCM, an indication that TEM have undergone more rounds of cell division [5].
…these subpopulations are not independent but rather can interconvert … suggestion that increasing signal strength is associated with progressive differentiation from TCM to TEM… For CD8 T cells, there is some evidence suggesting that TCM and TEM might be generated differentially during an immune response, depending on the conditions of activation. This comes from a report showing that murine CD8 cells activated in vitro can acquire the characteristics of either TCM or TEM after injection into mice, depending on the particular cytokines added during the culture period; activated CD8 cells cultured in a high dose of interleukin-2 (IL-2) differentiated into TEM, and those cultured in IL-15 or low dose IL-2 became TCM [8,9]. These findings are consistent with a progression model, if one accepts that high and low dose IL-2 treatment differ only in the strength of the signal delivered, but would also fit a model in which TCM and TEM are independent endpoints and arise from qualitatively different signals (i.e. IL-2 versus IL-15). However, as for CD4 cells, human TCM CD8 cells acquire markers of TEM after exposure to cytokines in vitro, indicating that conversion between CD8 memory subpopulations can occur, at least in tissue culture [10]. Does this happen in vivo? Two groups, Baron et al. [11] and Wherry et al. [12], have now investigated the relationship between TCM and TEM CD8 cells generated in vivo. The two groups studied different species, used different approaches and reached slightly different conclusions. Relationship between TCM and TEM in humans Baron et al., studying CD8 cells in human peripheral blood, took the approach of examining the TCR repertoire among TCM and TEM cells, using elegant molecular techniques to identify T cells expressing TCRs with specific CDR3 (complementarity-determining region 3) sequences in their Vb chains [11]. Their rationale was that if TCM and TEM represent closely related, interconvertible cells, the same clones should be prominent in both populations. Furthermore, fluctuations over time in the size of a clone
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present in one population should be apparent in the other population. Contrary to these predictions, the authors found that the number of clonotypes shared by CD8 TCM and TEM within an individual was very low, specifically 6.2% and 5.6% of the total number of clonotypes detected within the two Vb families analysed. When several clonotypes (both shared and unique) were followed within one individual over a seven month period, some clones displayed a stable frequency within the subpopulations although others varied by as much as 10-fold. Significantly, however, when changes in the frequency of a clone were observed in one memory population, no corresponding alterations were observed in the other. These authors also investigated directly the distribution of T-cell clones specific for one particular antigen, a peptide within the matrix protein of influenza A virus. In this experiment, influenza-specific CD8 cells were purified using MHC-tetramers and six clonotypes were identified in the antigen-specific population. All six clonotypes were detected within the TCM subset in this individual and two were also found among TEM cells. Although the incidence of shared clonotypes was higher than for the randomly analysed Vb families, it was notable that changes in the frequencies of these clones over a nine month period again revealed no evidence for transition between TCM and TEM subpopulations. Based on their overall results, the authors proposed that CD8 TCM and TEM are largely independent subpopulations.
conversion occurred more quickly after infection of mice with a low dose of L. monocytogenes, where antigen could not be detected for .2 – 3 days after infection, than after infection with high dose L. monocytogenes or with LCMV, where antigen can be detected for 5– 7 days. These results are reminiscent of those reported in an earlier study, in which virus-specific CD8 cells reverted to a CD62Lhi phenotype at different rates when mice were infected with influenza versus Sendai virus [13]. To determine whether the different tempo of TEM ! TCM conversion was due to differences in the level of persisting antigen or other environmental differences in the differently infected mice, Wherry et al. obtained primed CD8 cells (taken at day 8 post-infection) from mice that had been infected separately with low dose L. monocytogenes or with LCMV, and injected a mixture of these cells into secondary hosts. If the environment of the memory cells was the primary determinant of the rate of TEM ! TCM conversion, memory cells obtained from both types of mice should change phenotype at the same rate in the secondary recipients. In fact, what was observed in the adoptive hosts was that TEM derived from low dose infected mice still converted to TCM faster than TEM from high dose infected mice. These results indicated that the rate at which TEM convert to TCM was determined very early in the immune response – during the first week after infection – and that this timing could be linked to the initial antigen dose.
Relationship between TCM and TEM in mice Wherry et al. investigated the appearance of CD8 TCM and TEM after infection of mice with a virus, lymphocytic choriomeningitis virus (LCMV), or an intracellular bacterium, Listeria monocytogenes [12]. Antigen-specific CD8 cells were followed over time using either TCR transgenic cells specific for an epitope expressed in both agents (the L. monocytogenes used was a recombinant expressing an LCMV epitope) or using MHC tetramers containing this epitope. In initial experiments studying LCMV infection, the authors found that the total number of antigen-specific CD8 memory cells remained constant once the memory pool had been established, confirming many other reports in this system. By contrast, however, the numbers of TEM and TCM changed substantially over time, and did so in a reciprocal manner, with TCM increasing and TEM decreasing. Because one explanation for these results was that TEM were converting into TCM, the authors went on to investigate this possibility directly by transferring purified populations of CD8 TEM or TCM into secondary recipients and examining the fate of the transferred cells. The striking finding was that TCM retained their phenotype for at least 30 days after transfer while approximately half of the TEM cells acquired the phenotype of TCM during this time. Importantly, the authors demonstrated that this phenotypic conversion occurred in the absence of cell division, ruling out the possibility that the results stemmed from outgrowth of a small number of contaminating TCM in the transferred population. The data therefore provided strong evidence that CD8 TEM can convert to TCM under appropriate in vivo conditions. Interestingly, Wherry et al. observed that TEM ! TCM
…the rate at which TEM convert to TCM was determined very early in the immune response – during the first week after infection – and that this timing could be linked to the initial antigen dose.
http://treimm.trends.com
Based on these observations, Wherry et al. proposed a linear differentiation model for the generation of CD8 TCM and TEM, in which TEM are derived directly from effector cells and TCM are derived from TEM [12]. In contrast to the model proposed previously for CD4 memory cells, they found little evidence that CD8 TCM convert to TEM under ‘stable’ in vivo conditions, that is, in the absence of re-challenge with antigen. On this point, their conclusions are in agreement with those of Baron et al. [11]; whether this represents a real difference between CD4 and CD8 memory subpopulations will await similar in vivo studies for CD4 cells. TEM ! TCM conversion: mouse versus humans As described earlier, Baron et al. concluded that there was little movement of T-cell clones in either direction between TCM and TEM. Therefore, at face value these two studies yield conflicting messages about whether TEM ! TCM conversion takes place; what is the reason for this discrepancy? One possibility is that there are fundamental differences between mouse and human
406
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CD8 memory cells, or at least in the subpopulations analysed. In this respect, it should be borne in mind that human CD8 cells can be subdivided into four populations on the basis of CD62L (CCR7) and CD45 isoform expression. Thus, in addition to CD62L (CCR7)þ CD45R0þ TCM, CD62L (CCR7)2 CD45R0þ TEM and CD62L (CCR7)þ CD45RAþ naı¨ve cells, there is a population of CD62L (CCR7)2 CD45RAþ ‘effector memory RA’ cells, and considerable numbers of antigen-specific cells can be identified in this population after certain virus infections [14 – 16]. This fourth population was not studied by Baron et al. and hence any movement of clones in or out of this subset would not have been detected. By contrast, because no markers have been described that identify this subpopulation in the mouse (if it exists), these cells could have influenced the findings of Wherry et al. Furthermore, it is also worth noting that, unlike what has been described for human CD8 TEM [5], Wherry et al. did not detect direct (ex vivo) effector activity among murine CD8 TEM [12], although this has been observed in other studies [6]. Another factor that might have had a bearing on the contradictory results is that a different relationship between TCM and TEM would be observed depending on the time point in the immune response that these cells were examined. For the mouse experiments, the kinetics of the responses were known precisely because cells responding to infections given at defined time points were examined. This was not the case for the study of human cells by Baron et al., where, for the most part, the antigenic specificity of the CD8 cells examined was unknown. Interestingly, when influenzaspecific CD8 cells were analysed, two of six clones found among TCM were also present in the TEM population. One interpretation of these data, based on the results of Wherry et al., is that these cells were studied at a relatively late time point after infection, when the majority of TEM have converted to TCM. If this were the case, however, one might have expected to see evidence of TEM ! TCM conversion among the remaining two shared influenza-specific clones over the nine months of analysis, or, by chance, among the thirteen clones of unknown specificity that were studied over a seven month period.
Another factor that might have had a bearing on the contradictory results is that a different relationship between TCM and TEM would be observed depending on the time point in the immune response that these cells were examined. Evidence of such phenotypic conversion could be obscured by the nature of the infectious agent to which the CD8 cells react. For example, different results might be seen with http://treimm.trends.com
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pathogens that are cleared versus those that persist because the latter will provide a source of antigen for periodic restimulation of memory cells. This could occur in a stochastic manner, causing a proportion of cells in the memory population to acquire or retain a more activated phenotype at any one time, yet giving the appearance that there are stable numbers of cells in distinct subpopulations. In this respect, it should be noted that triggering through the TCR has been shown to both downregulate CCR7 on CD8 TCM [10] and upregulate CCR7 on CD8 TEM [14]. Similar effects might result from exposure to previously seen and cross-reactive antigens [17], something that does not complicate experiments with mice under controlled conditions. One clear indication that contact with antigen during persistent infection can modulate the phenotype of memory cells has come from studies of Epstein–Barr (EBV) virus infection, in which CD8 cells specific for epitopes expressed during the lytic cycle of the virus became CD45RAþ CD45R02 CCR72 with time after infection, although those reactive with latent epitopes remained CD45RA2R0þ [18]. Concluding remarks Overall, these papers provide several new insights into the relationship between TCM and TEM CD8 cells in humans and mice. They also raise several important questions. For example, how is the kinetics of TEM ! TCM conversion ‘programmed’ during the first week of the immune response and is this altered if the infectious agent is able to persist at some level? Does the relationship between TEM and TCM cells observed in the systemic infection models used by Wherry et al. also hold true for CD8 memory cells generated after infections at mucosal sites? Is there interconversion between memory subpopulations and ‘effector memory RA’ cells and does an equivalent population exist in mice? Answering these questions will contribute to our growing understanding of how immunological memory is generated and maintained. Acknowledgements This is publication number 67 from the Edward Jenner Institute for Vaccine Research. I thank Persephone Borrow for critical reading of this manuscript and helpful comments.
References 1 Dutton, R.W. et al. (1998) T cell memory. Annu. Rev. Immunol. 16, 201 – 223 2 Berard, M. and Tough, D.F. (2002) Qualitative differences between naı¨ve and memory T cells. Immunology 106, 1 – 12 3 Berg, E.L. et al. (1991) The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor. J. Cell Biol. 114, 343 – 349 4 Gunn, M.D. et al. (1998) A chemokine expressed in lymphoid high endothelial benules promotes the adhesion and chemotaxis of naı¨ve T lymphocytes. Proc. Natl. Acad. Sci. U. S. A. 95, 258 – 263 5 Sallusto, F. et al. (1999) Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708 – 712 6 Masopust, D. et al. (2001) Preferential localization of effector memory cells in nonlymphoid tissue. Science 291, 2413– 2416 7 Geginat, J. et al. (2001) Cytokine-driven proliferation and differentiation of human naı¨ve, central memory, and effector memory CD4þ T cells. J. Exp. Med. 194, 1711 – 1719 8 Manjunath, N. et al. (2001) Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J. Clin. Invest. 108, 871 – 878
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9 Weninger, W. et al. (2001) Migratory properties of naı¨ve, effector, and memory CD8þ T cells. J. Exp. Med. 194, 953 – 966 10 Geginat, J. et al. (2003) Proliferation and differentiation potential of human CD8þ memory T cell subsets in response to antigen or homeostatic cytokines. Blood 101, 4260 – 4266 11 Baron, V. et al. (2003) The repertoires of circulating human CD8þ central and effector memory T cell subsets are largely distinct. Immunity 18, 193 – 204 12 Wherry, E.J. et al. (2003) Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4, 225– 234 13 Tripp, R.A. et al. (1995) Temporal loss of the activated L-selectin-low phenotype for virus-specific CD8þ memory T cells. J. Immunol. 154, 5870–5875 14 Champagne, P. et al. (2001) Skewed maturation of memory HIVspecific CD8 T lymphocytes. Nature 410, 106 – 111 15 Hislop, A.D. et al. (2001) EBV-specific CD8þ T cell memory:
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relationships between epitope specificity, cell phenotype, and immediate effector function. J. Immunol. 167, 2019 – 2029 16 Wills, M.R. et al. (2002) Identification of naı¨ve or antigen-experienced human CD8þ T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8þ T cell response. J. Immunol. 168, 5455– 5464 17 Selin, L.K. et al. (1999) Attrition of T cell memory: selective loss of LCMV epitope-specific memory CD8 T cells following infections with heterologous viruses. Immunity 11, 733– 742 18 Hislop, A.D. et al. (2002) Epitope-specific evolution of human CD8þ T cell responses from primary to persistent phases of Epstein – Barr virus infection. J. Exp. Med. 195, 893 – 905
1471-4906/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1471-4906(03)00169-8
| Letters
A genetic basis for human gd T-cell reactivity towards microbial pathogens Matthias Eberl and Hassan Jomaa Biochemisches Institut, Justus-Liebig-Universita¨t Giessen, Friedrichstrasse 24, 35392 Giessen, Germany
Elevated levels of Vg9Vd2 T cells in the peripheral blood of patients are well documented in a variety of bacterial infections, such as brucellosis, ehrlichiosis, listeriosis, salmonellosis, tuberculosis or tularaemia [1]. However, as recently addressed by Chen and Letvin, the investigation of Vg9Vd2 T-cell responses in infectious diseases has been greatly hindered by the absence of relevant animal models, with this population being unique for humans and nonhuman primates and by their unconventional reactivity towards low molecular weight antigens [2]. Over the past decade, a number of natural compounds have been postulated to be responsible for the specific expansion of the Vg9Vd2 T-cell subset in these diseases, including 2,3-diphosphoglycerate, isopentenyl pyrophosphate (IPP), isobutylamine and other non-peptidic antigens. Yet, the poor in vitro bioactivities of these molecules make it unlikely that they have a pivotal role under physiological conditions in vivo. Recently, the metabolite (E)-4-hydroxy3-methyl-but-2-enyl pyrophosphate (HMB-PP), an intermediate of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis, was isolated from Escherichia coli and shown to display a bioactivity of 0.1 nM; thus, HMB-PP is approximately four orders of magnitude more potent than IPP, and 6-7 magnitudes more potent than isobutylamine [3]. Molecules with the biochemical characteristics of HMB-PP have been detected in lysates from various mycobacteria species, Brucella suis, Francisella tularensis, Pseudomonas aeruginosa and other bacteria [4]. Meanwhile, the genes of the MEP pathway have been identified in nearly every bacterial species linked with elevated Corresponding author: Matthias Eberl ([email protected]). http://treimm.trends.com
Vg9Vd2 T-cell levels, whereas they are apparently absent in the genome of non-Vg9Vd2 T-cell-inducing bacteria, such as streptococci and staphylococci, as well as in humans [5]. Therefore, it can be assumed that HMB-PP greatly accounts for the described Vg9Vd2 T-cell reactivity towards many pathogenic bacteria. Yet, Vg9Vd2 T cells also expand in protozoan diseases, and patients suffering from malaria, leishmaniasis or toxoplasmosis show increased Vg9Vd2 T-cell numbers in the peripheral blood [6– 10]. In the case of malaria, this observation could recently be explained by the demonstration that green algae and higher plants use the MEP pathway in their chloroplasts, whereas isoprenoid biosynthesis in the cytoplasm proceeds through the classical mevalonate pathway. Similarly, Plasmodium falciparum actually harbours the MEP pathway in a specialized subcellular organelle, the so-called apicoplast, which is evolutionarily derived from the choloroplast of an ancient endosymbiont [11]. Consequently, malaria parasites stimulate Vg9Vd2 T cells in vitro and a non-peptidic antigen likely to be identical with HMB-PP can be isolated from parasite extracts [12]. Because the presence of an apicoplast is a distinct feature of all apicomplexan protozoa, we wondered whether other members of this phylum also possess the genes of the MEP pathway. In fact, most of the respective genes could be identified in the preliminary genomic sequences of the human pathogens Cryptosporidium parvum, Plasmodium vivax, and Toxoplasma gondii [5], as well as in the related species Eimeria tenella, Plasmodium yoelii, Theileria annulata and Theileria parva (Fig. 1). Thus, in analogy to the case of bacterial infection, the production of HMB-PP in the apicoplasts of parasites might also be responsible for the