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The synergy between naive and memory T cells during activation
The synergy between naive and memory T cells during activation Arne N. Akbar, Michael Salmon and George Janossy Naive and memory T-cell subsets differ in their ability to synthesize and respond to a variety of cytokines in vitro and each subset can produce cytokines that amplify the response of the other subset. The significance of these interactions to antigen responsiveness has, until now, been unclear. In this article Arne Akbar and colleagues point out that both subsets are activated to the same extent by alloantigen and suggest that synergy may be an important event in initiating potent responses against transplanted allografts.
Recent technological advances have enabled naive and memory T cells to be identified and isolated 1,2. CD4 + and CD8 + T-cell subsets can now be distinguished from each other on this basis, and studies on their cytokine production and responsiveness performed. Consequently, it is now feasible to analyse cytokine production during primary and secondary responses to antigen. In this article the hypothesis that synergy between such naive and memory T cells may occur is discussed; this is fuelled by differences in their ability to synthesize and respond to cytokines. In particular, the interaction between the subsets may be efficiently amplified during transplant rejection as a result of the synergy between alloactivated T cells, which may lead to a particularly strong and destructive response against the graft.
Table 1. Phenotypic differences between naive and memory T cells Surface antigena
Naive
Memory
Refs
CD45RA CD45RO CD7 b CD44 (Pgp-1) CD29 CD18/CDlla (LFA-1) CD2 Leukocyte adhesion molecule 1 (Leu8,TQ1) CD58 (LFA-3) b CD25 (IL-2R) b MHC class IIb CD54 (ICAM-1) b CD26 (TA1) b p75 (IL-2R) b
+++ ++ ++ ++ ++
+++ + +++ +++ +++
1,2 (reviews) 1,2 5,6 2 1,2 1,2
++ ++
+++ +++
1,2 7
+ -
++ + + + + +
1,4 1,4,5,8 4,8 4,8 1 9
aThe antigen expression of resting human naive or memory subsets from peripheral blood; bexpressionof these antigenshas been shown to transiently increase after activationof both subsets.
The phenotypic and functional diversity of naive and memory T cells The study of memory T cells in humans and other animals has been greatly facilitated by the identification of unique surface antigens (Refs 1-4 and Table 1). The antibodies most widely used to discriminate between naive and memory T cells are those directed to different isoforms of CD45 (Ref. 1). These isoforms differ only in the extracellular portion of the molecule, which is encoded by three exons - A, B and C. In humans, naive T cells express the A isoform of CD45 and are recognized by anti-CD45RA antibodies2. Memory T cells do not express any of the three exons and are recognized by antiCD45RO antibodies 1,4. Antibodies against CD45RA and CD45RO mainly recognize reciprocal populations of resting T cells. However after activation, there is a unidirectional transition from CD45RA to CD45RO reactivity, which is now considered to parallel the differentiation from naive to memory T cells 1,2,4.Although the reversal from a CD45RO + to a CD45RA + phenotype has not been shown in vitro, the possibility that this may occur in vivo cannot be ruled out. A close appraisal of the phenotype of memory cells identified by CD45RO expression shows that they express low levels of a variety of activation markers (Table 1), which suggests that memory T cells in the circulation may have been recently activated. This has led to the hypothesis that the longevity of memory T cells may be a result of continuous low-grade stimulation by persistent or crossreactive antigen4. Some functional differences between naive and memory T cells are summarized in Table 2. In rodents, anti-CD45RB antibodies that recognize sequences encoded by the B exon have been used to identify naive T cells3,2°; memory T cells do not react with anti-CD45RB antibodies (CD45RB-). It should be noted that anti-CD45RB antibodies may identify an overlapping but not completely identical population to that recognized by anti-CD45RA antibodies in humans and direct comparisons between human and rodent data should therefore be performed with caution 2I.
© 1991, ElsevierSciencePublishers Ltd, UK. 0167--4919191/$02.00
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Table 2. Functional differences between naive and memory T cells Function
Naive
Memory
Refs
Proliferation/recall antigen Proliferation/CD3 and CD2 mAbs Helper function/Ig production (CD4 + cells) Cytotoxic precursor cells (antigen-specific) Cytotoxic precursor cells (alloantigen-specific) Cytotoxic effector cells (alloantigen-specific) Adherence to endothelium/augments vascular permeability Response to chemotactic stimuli Inhibition of proliferation by CD25 mAb Proliferation to allogeneic cells Proliferation to mitogens Proliferation to autologous cells Induction of suppressor function Inhibition of proliferation by CD7 antibodies
-+--++ + + ++++ + + ++ ++++ ++++ ++++ ++++ +++
++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ + + +
1 1,4 1,3,10 11 12,13 11,13 14,15 16 5,17,18 1,9,19 19 19 19 5
Anti-CD44 (Pgp-1) antibodies have been used to positively identify memory T cells in rodents 2. Important information on the patterns of recirculation of naive and memory T cells in vivo has been deduced in sheep, where anti-CD45RA antibodies have been used to identify naive T cells, in an analogous way to humans =. An anti-CD45RO antibody equivalent is not currently available in this species 22. An interesting and potentially important observation is that alloantigen, unlike recall antigen, activates naive and memory T cells to the same extent and with similar kinetics of proliferation in vitro. This may have implications for the nature of immune responsiveness against transplanted grafts; specifically, naive and memory T cells may synergize with each other during activation, as recently suggested23-2s.
The synthesis of cytokines by naive and memory T cells A number of studies have examined the ability of naive and memory CD4 + T cells to synthesize cytokines after activation (Table 3). Similar observations on CD8 + T cells have been rare but our preliminary observations suggest that naive and memory CD8 + populations can synthesize cytokines, albeit to a lesser extent than the CD4 + population. When data on CD4 + T-cell cytokine production from different laboratories are compared, it is apparent that there is some variability between different, and in some cases even the same, stimuli. However, the tendency, in both humans and rodents, is that the naive cells synthesize greater amounts of interleukin 2 (IL-2) than memory T cells do. The synthesis of IL-4 after activation is more restricted, with the memory CD4 + T-cell population producing virtually all of this cytokine after activation. Gamma-interferon (IFN-y), IL-3 and IL-6 are also preferentially synthesized by the memory T-cell population rather than by the naive cells. An interesting question is whether the synthesis of cytokines by naive and memory CD4 + T cells falls into a TH1 and TH2 pattern as demonstrated for mouse CD4 + clones 33. Whereas TH1 clones synthesize IL-2 and IFN-y, TH2 clones synthesize IL-4 and IL-5 (Refs 30,33). It does not appear at present that the profile of cytokine synthesis by human naive and memory CD4 ÷ T cells or
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CD4+CD45RO + T-cell clones fits into this pattern 26. It has been proposed that TH1 and TH2 patterns of cytokine production may represent a further differentiation or specialization within human memory cells 3,33 but this issue remains to be resolved. An important observation is that the proliferation of naive T cells in response to various stimuli, including anti-CD2 and anti-CD3 antibodies, can be substantially increased by the addition of IL-4 (Ref. 34), whereas, under similar conditions, memory T cells show only a minimal enhancement of proliferation. These results have been confirmed using phytohemagglutinin (PHA) or alloantigens as the original stimulus in humans 9, which suggests that a cytokine produced predominantly by memory T cells may amplify the response of naive T cells in a paracrine manner. In contrast, although memory CD4 ÷ T cells can synthesize IL-2, the amounts synthesized are insufficient for optimal proliferation while naive CD4 + T cells synthesize an excess of IL-2. This phenomenon is substantiated by studies that show that exogenous IL-2, when added to cultures of activated memory T cells, further enhances proliferative activity but has little, if any, effect on naive T cells 9,24. The preferential inhibition of memory T-cell proliferation with anti-CD25 (i.e. anti-IL-2R) antibodies and cyclosporin A (CsA) compared with naive T cells underlines the greater dependence of memory T cells on IL-2 as a signal for proliferation5. On the basis that memory T cells are larger than naive T cells 8,2s, identical observations have been demonstrated in the mouse31; this again suggests that the proliferation of memory T cells may be amplified by IL-2-secreting naive T cells. All of these results taken together indicate that, during activation, synergy may indeed occur between naive and memory T cells; each subset produces a cytokine that amplifies the response of the other (Fig. 1). The subsets show similar levels of expression of IL-2R (p55 and p75) and IL-4R (assessed using biotinyllated IL-4) after activation, pointing to the synergy at the level of cytokine production, rather than at the level of receptor expression (authors' unpublished observations). In addition to IL-2 and IL-4, other cytokines may also be important in this synergy. Memory, but not naive, CD4 + T cells synthesize IL-6 after activation with anti-CD2
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Table 3. Lymphokine production by naive and memory T cells Lymphokine
Species
Stimulusa
Naive b
Memory b
Refs
IL-2
Humans
PHA PHA PMA/calcium ionophore PWM Allogeneic cells CD3 CD3 CD3 SEAc PHA/ConA/PWM CD3 Allogeneic cells ConA PMA/calcium ionophore PHA,CD3 Allogeneic cells CD3, PHA, ConA, PWM PHA, CD3, allogeneic cells PWM, SEA CD3 Mitogens and alloantigen ConA Mitogens and alloantigen CD2
++++ ++++ ++++ ++++ ++++ ++++ ++++ + ++++ ++++ ++++ ++++ ++++ -
++ ++++ ++ ++ ++ ++ ++++ ++++ ++++ + + ++++ + ++++
+ -
++++ ++++ ++++
9,24,26 1 26 10 9 9,27 10 28 29 20,30 31 b 2 3 26 9,26,27 9 2,20,30 1,9,26,10,29
++++ + ++++ + -
++++ ++++ ++ ++++ ++++
10,27 2 3 2 32
Mice
IL-4
Rats Humans
IFN-~/
Mice Humans
IL-3 IL-6
Mice Rats Mice Humans
aPMA: phorbol myristateacetate; SEA: staphylococcolenterotoxin A; ConA: concanavalinA; PWM: pokeweed mitogen; bnaive and memoryT cells distinguishedon the basis of cell size (see text); csubsetsshow differentkineticsof IL-2 production. antibodies, while both subsets require IL-6 for this form of activation32. It is likely, therefore, that IL-6 synthesized by memory T cells may represent part of the synergy that has been demonstrated between naive and memory T cells upon activation with anti-CD2 antibody 2s. The obvious question that arises is whether this scheme may help to explain the cytokine production in response to antigen in vivo.
Antigenic activation of naive and memory T cells By definition, a primary response to an antigen occurs if it has not previously been encountered in vivo; response to a primary antigen should mainly involve naive T cells. A secondary response occurs if the host has previously been exposed to the antigen, and memory T cells will form the major responding population. This has recently been substantiated in a mouse model where naive and memory T cells were separated on the basis of CD45RB expression 2°. These definitions oversimplify real events as many studies show that crossreactivity occurs among some antigens after their processing and partial degradation by macrophages 4,35. Nevertheless we would predict that, upon activation by any one particular antigen in vivo, simultaneous activation of both naive and memory T cells will not normally occur as the antigen will preferentially recruit either one or other of these subsets. From the observations of cytokine synthesis in vitro, it would be expected that the response to a primary antigen by naive T cells will initially result in IL-2 production, followed by IL-4 production after these cells have become activated and have undergone a functional transition to memory cells. This has been con-
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firmed in mice, where it has been shown that, after activation with a primary antigen, IL-2 is synthesized earlier than IL-4 (Ref. 36). We predict, from the in vitro data, that IL-4 is synthesized earlier and in larger amounts by memory T cells during secondary responses, but they may remain starved of IL-2. In situations involving extensive injury or infection, high concentrations of more than one antigen (primary and secondary) may be present and drain into lymph nodes or the spleen. Powerful synergy between the naive and memory T-cell subsets may occur as they may become activated simultaneously. In addition, a secondary response occurring at a given period after the primary response may involve both memory T cells and newlygenerated naive T cells that are reactive with the same antigen. Synergy between naive and memory T cells may again occur, especially if there is a high concentration of antigen present that precludes the preferential activation of the memory subset. Functional data derived in vitro must be analysed together with histological information on the distribution of naive and memory T-cell subsets since it may not be feasible, from an anatomical standpoint, for synergy between these cells to occur. Histological studies indicate that naive and memory T cells do reside in close proximity in the paracortex of lymph nodes 23. It is, therefore, possible for the draining antigens in afferent lymph to activate both naive and memory T cells in close proximity. As the naive T cells differentiate into memory T cells after activation, the synergy may be self limiting, with only IL-4 synthetic capacity present in the latter part of such a response. Indeed, the extent of synergy may be proportional
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ii!iii!ii!i!iiiii!ii!iiiil
iiiiiiiii!iii!iiiil IL-2
to the strength and diversity of the antigenic challenge. The simultaneous synthesis of IL-2 and IL-4 by naive and memory T cells may have synergistic effects on T-cell activation yet restrict the nonspecific recruitment of other cells within lymphoid tissue. For example, while IL-2 and IL-4 are synergistic for T-cell proliferation, IL-4 counteracts the stimulatory effects of IL-2 on B-cell function 37and also inhibits the IL-2-mediated induction of lymphokine-activatedkiller activity38. Obviously the contribution of other cytokines such as IFN-y must eventually be included in these schemes of cytokine interaction. However, the synergism between naive and memory T cells may provide clues that explain the innate logic of cytokine pleiotropy during immune activation in vivo.
The response of naive and memory T cells to alloantigen Conventional antigen and alloantigen responsive T cells are not separate lineages; the same cell may respond to both challenges 39. The precursor cell frequency of alloresponsive T cells is far higher than the frequency of antigen-responsive T cells4°, suggesting that a multiplicity of separate T-cell populations with different antigenic specificities may be activated by alloantigen. While the precursor cell frequency for recall antigen is much higher within the memory T-cell subset than in the naive population, the same rule does not apply for alloantigens. Both naive and memory populations contain similar high levels of alloresponsive precursors TM and respond with the same proliferative kinetics 17,18.Possible explanations for alloantigen recruitment of such a large number of T cells have been reviewed in detail elsewhere 39~1. If synergy between naive and memory T cells does occur, then the collaboration between these populations during activation may add further vigour to the alloantigenic response. The immune response against transplanted tissue is often so intense that it leads to graft rejection, even in the presence of potent immunosuppression. T cells activated by donor alloantigens during transplant rejection in vivo can be isolated from both the graft itself and from the circulation42. When investigated for the expression of CD45 determinants, both naive and memory T cells have been shown within rejecting renal grafts, and a significant proportion of these are activated, proliferating and switching from a naive to a memory phenotype in situ 13,43. The profile of cytokine production by naive and memory T cells in response to alloantigen in vitro is similar to that observed with mitogens and anti-CD3 antibodies (Table 3). Furthermore, when both subsets are separated and subsequently reconstituted in varying proportions before alloactivation in vitro, the subset synergy in the induction of proliferation can be clearly demonstrated, even when cell number is accounted for 9. The in vitro data is therefore compatible with the hypothesis that interactions between naive and memory T-cell subsets may amplify the responses to graft alloantigens. A further important point is that, whereas in antigen-specific responses the effector mechanisms eventually lead to the removal of the original antigenic stimulus, during the allogeneic response in the graft, the generation of cytokines such as IFN-~ increases the expression of major histocompatibility complex (MHC) antigens, not only on passenger leukocytes but also on epithelial and endoImmunology Today
Naive
~
Memory 11_-4
Fig. 1. Synergy will occur when naive and memory T cells are activated in close proximity (a) when primary and secondary antigens are both present and (b) in response to alloantigens. thelial cells44. This will increase the original stimulus for the alloresponsive cells and may in turn further amplify the anti-graft response. In view of the fact that memory T cells have an inherently greater capacity to respond to activation stimuli than naive cells do, it may be surprising that the kinetics of proliferation to alloantigen in vitro is similar in both subsets. Naive T cells have been shown to have very Stringent requirements for antigen presentation compared with memory T cells45. This may be due in part to their low level of expression of adhesion molecules; strong T-cell receptor interactions are therefore required for activation. In naive T-cell responses to primary antigen, a small number of MHC molecules on the antigenpresenting cell may contain fragments of the primary antigen in relatively low concentrations. In contrast, the high density and uniformity of foreign MHC protein on allostimulatory cells may lead to T-cell activation even when the interaction with T-cell receptors is relatively weak. Once activated, the capacity of the naive T cells to synthesize IL-2 would enable them to undergo clonal expansion at least as efficiently as the activated memory T cells. The high density of MHC molecules on the allogeneic cells thus serves to lower the threshold of activation and, therefore, to bypass the stringent activation requirements of the naive subset. In an analogous situation, low concentrations of anti-CD3 antibodies preferentially activate memory T cells yet high concentrations of this antibody activate both subsets equally1.
Implications for immunosuppressive therapy during transplantation Recent clinical trials have used monoclonal antibodies together with CsA to achieve optimal immunosuppression during organ transplantation 4s. Antibodies, for example against the CD3 determinant, show particularly powerful immunosuppressive action when used in conjunction with CsA46. These strategies not only allow the dose of CsA to be reduced, but may also lead to more selective regimes of immunosuppression. Two antibodies that may be of use in this regard are directed against CD7 and CD25 molecules. These antibodies react strongly with activated T cells but may spare resting T cellss,6. It is important, therefore, that anti-
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•iii•i2ii•iiiiiiiiii!•ii•!ii•i•!i•iiii!i!i!i!i!!iiiii•ii•••ii•i!iii•iiii•i•iii!ii•ii•i•i!•iii•iiiiii•i!iiii•i•ii•i•i•iii•i•ii••ii!•i
iiiiiiii!iiiiiiiiiiiiiiiiiiii!iiii!ii!iii!i ii!iiii!iii!iiiii!i!iii!iiiiiiiiiii!i!!i i CD7 antibodies preferentially inhibit alloactivated naive T cells whereas anti-CD25 antibodies preferentially inhibit memory T cellss. These antibodies show additive inhibition of isolated naive and memory T-cell subsets and also show additive inhibition when combined with CsAs. Thus a combination of anti-CD7 and anti-CD25 antibodies may interfere with the activation of naive and memory T cells individually and with the synergy between them. A chimeric form of the anti-CD7 antibody6 is currently being assessed for its immunosuppressive potential in renal rejection. In future, the selection of antibodies or other drugs for use in the prophylaxis of graft rejection may be assessed on their ability to reduce the alloactivation of either naive or memory T cells and serve as a basis for a more selective approach to immunosuppressive regimes. Conclusions It is of interest that naive and memory T cells, which are at different stages of maturity, may interact with each other during activation. The scheme of synergy proposed here is simple yet may explain much of the conflicting data on the action of cytokines, especially IL-2 and IL-4. Undoubtedly other cytokines will be involved in the interaction between naive and memory T cells and also between these and other cell types. An interacting cascade of cytokine synthesis, as outlined here, will help to place the pleiotropy and redundancy of cytokine production into perspective.
The authors are grateful to our colleagues Kamal Ivory and Darrell Pilling who have contributed to the work and to P.L. Amlot and P.C.L. Beverleyfor critical reviewof the manuscript. This work was supported by Sandoz Pharma Ltd. Arne N. Akbar and George Janossy are at the Dept of Clinical Immunology, Royal Free Hospital School of Medicine, Pond Street, London NW3 2QG, UK and Michael Salmon is at the Dept of Rheumatology, The Medical School, Birmingham University, Birmingham B15 2TJ, UK. References 1 Sanders, M.E., Makgoba, M.W. and Shaw, S. (1988) Immunol. Today 9, 195-199 2 Cerottini, J.C. and Robson MacDonald, H. (1989) Annu. Rev. Immunol. 7, 77-89 3 Powrie, F. and Mason, D. (1988) Immunol. Today 9, 274-277 4 Beverley,P.C.L. (1990) Immunol. Today 11,203-205 5 Akbar, A.N., Amlot, P.L., Ivory, K. et al. (1990) Transplantation 50, 823-829 6 Heinrich, G., Gram, H., Kocher, H.P. et al. (1989) J. Immunol. 143, 3589-3597 7 Tedder, T.F., Matsuyama, T., Rothstein, D. et al. (1990) Eur. J. Immunol. 20, 1351-1357 8 Buckle, A.M. and Hogg, N. (1990) Eur. J. Immunol. 20, 337-341 9 Akbar, A.N., Salmon, M., Ivory, K. et al. Int. ImmunoI. (in press) 10 Sleasman,J.W., Morimoto, C., Schlossman,S.F. et al. (1990) Eur. J. Immunol. 20, 1357-1366 11 Merkenschlager, M. and Beverley,P.C.L. (1989) Int. Immunol. 1,450-456 12 Yamashita, N. and Clement, L.T. (1989) J. Immunol. 143, 1518-1523
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13 Akbar, A.N., Amlot, P.L., Timms, A. et al. (1990) Clin. Exp. Immunol. 81,225-231 14 Pitzalis, C., Kingsley,G., Haskard, D. et al. (1988) Eur. J. Immunol. 18, 1397-1404 15 Damle, N.K. and Doyle, L.V. (1990) J. Immunol. 144, 1233-1240 16 Schall, T.J., Bacon, K., Toy, K.J. et al. (1990) Nature 347, 669-673 17 Akbar, A.N., Terry, L., Timms, A. et aI. (1988) J. Immunol. 140, 2171-2178 18 Merkenschlager, M., Terry, L., Edwards, R. et al. (1988) Eur. J: Immunol. 18, 1653-1661 19 Morimoto, C., Letvin, N.L., Distaso, J.A. et al. (1985) J. Immunol. 134, 1508-1515 20 Lee,W.T. and Vitetta, E.S. (1990) Cell. Immunol. 130, 459-471 21 Mason, D. and Powrie, F. (1990) Immunology 70, 427-433 22 Mackay, C.R., Marston, W.L. and Dudler, L. (1990) J. Exp. Med. 171,801-817 23 Janossy, G., Campana, D. and Akbar, A. (1989) in Current Topics in Pathology (Cell Kinetics of the Inflammatory Reactions, Vol. 79) (Iversen, O.H., ed.), pp. 59-99, Springer-Verlag 24 Salmon, M., Kitas, G.D., Hill Gaston, J.S. et al. (1988) Immunology 65, 81-85 25 Wallace, D.L. and Beverley,P.C.L. (1990) Immunology 69, 460-467 26 Salmon, M., Kitas, G.D. and Bacon, P.A. (1989) J. Immunol. 143,907-912 27 Bettens, F., Walker, C., Gauchat, J.F. et al. (1989) Eur. J. Immunol. 19, 1569-1575 28 Byrne, J.A., Butler, J.L., Reinherz, E.L. et al. (1989) Int. Immunol. 1, 29-35 29 Dohlsten, M., Hedlund, G., Sjogren, H.O. et al. (1988) Eur. J. Immunol. 18, 1173-1178 30 Bottomly, K. (1988) Immunol. Today 9, 268-277 31 Ben-Sasson,S.Z., Le Gros, G., Conrad, D.H. et al. (1990) J. Immunol. 145, 1127-1136 32 Kasahara, Y., Miyawaki, T., Kato, K. et al. (1990) J. Exp. Med. 172, 1419-1424 33 Mossman, T.R. and Coffman, R.L. (1989) Annu. Rev. Immunol. 7, 145-173 34 Wasik, M. and Morimoto, C. (1990) J. Immunol. 144, 3334-3340 35 Jones, K.R., Hickling, J.K., Targett, G.A.T. et al. (1990) Eur. J. Immunol. 20, 307-315 36 Mohler, K.M. and Butler, L.D. (1990) J. Immunol. 145, 1734-1739 37 Llorente, L., Mitjavila, F., Crevon, M.C. et al. (1990) Eur. J. Immunol. 20, 1887-1892 38 Spits, H., Yssel, H., Paliard, X. et al. (1988) J. Immunol. 141, 29-36 39 Lechler, R.I., Lombardi, G., Batchelor, J.R. et al. (1990) Immunol. Today 11, 83-88 40 Simonsen, M. (1990) Scand. J. Immunol. 32, 565-575 41 Sharrock, C.E.M., Man, S., Wanachiwanawin, W. et al. (1987) Transplantation 43, 699-703 42 Preffer, F.I., Colvin, R.B., Leary, C.P. et al. (1986) J. Immunol. 137, 2823-2830 43 Stein-Oakley, A.N., Kerr, P.G., Kraft, N.E. et al. (1989) Transplantation 48,787-790 44 Dallman, M.J. and Morris, P.J. (1988) in Kidney Transplantation: Principles and Practice (Morris, P.J., ed.), pp. 15-37, W.B. Saunders 45 Inaba, K. and Steinman, R.M. (1986) J. Exp. Med. 163, 247-261 46 Cosimi, A.B. (1988) in Kidney Transplantation, Principles and Practice (Morris, P.J., ed.), pp. 343-371, W.B. Saunders
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Recent technological advances have enabled naive and memory T cells to be identified and isolated 1,2. CD4 + and CD8 + T-cell subsets can now be distinguished from each other on this basis, and studies on their cytokine production and responsiveness performed. Consequently, it is now feasible to analyse cytokine production during primary and secondary responses to antigen. In this article the hypothesis that synergy between such naive and memory T cells may occur is discussed; this is fuelled by differences in their ability to synthesize and respond to cytokines. In particular, the interaction between the subsets may be efficiently amplified during transplant rejection as a result of the synergy between alloactivated T cells, which may lead to a particularly strong and destructive response against the graft.
Table 1. Phenotypic differences between naive and memory T cells Surface antigena
Naive
Memory
Refs
CD45RA CD45RO CD7 b CD44 (Pgp-1) CD29 CD18/CDlla (LFA-1) CD2 Leukocyte adhesion molecule 1 (Leu8,TQ1) CD58 (LFA-3) b CD25 (IL-2R) b MHC class IIb CD54 (ICAM-1) b CD26 (TA1) b p75 (IL-2R) b
+++ ++ ++ ++ ++
+++ + +++ +++ +++
1,2 (reviews) 1,2 5,6 2 1,2 1,2
++ ++
+++ +++
1,2 7
+ -
++ + + + + +
1,4 1,4,5,8 4,8 4,8 1 9
aThe antigen expression of resting human naive or memory subsets from peripheral blood; bexpressionof these antigenshas been shown to transiently increase after activationof both subsets.
The phenotypic and functional diversity of naive and memory T cells The study of memory T cells in humans and other animals has been greatly facilitated by the identification of unique surface antigens (Refs 1-4 and Table 1). The antibodies most widely used to discriminate between naive and memory T cells are those directed to different isoforms of CD45 (Ref. 1). These isoforms differ only in the extracellular portion of the molecule, which is encoded by three exons - A, B and C. In humans, naive T cells express the A isoform of CD45 and are recognized by anti-CD45RA antibodies2. Memory T cells do not express any of the three exons and are recognized by antiCD45RO antibodies 1,4. Antibodies against CD45RA and CD45RO mainly recognize reciprocal populations of resting T cells. However after activation, there is a unidirectional transition from CD45RA to CD45RO reactivity, which is now considered to parallel the differentiation from naive to memory T cells 1,2,4.Although the reversal from a CD45RO + to a CD45RA + phenotype has not been shown in vitro, the possibility that this may occur in vivo cannot be ruled out. A close appraisal of the phenotype of memory cells identified by CD45RO expression shows that they express low levels of a variety of activation markers (Table 1), which suggests that memory T cells in the circulation may have been recently activated. This has led to the hypothesis that the longevity of memory T cells may be a result of continuous low-grade stimulation by persistent or crossreactive antigen4. Some functional differences between naive and memory T cells are summarized in Table 2. In rodents, anti-CD45RB antibodies that recognize sequences encoded by the B exon have been used to identify naive T cells3,2°; memory T cells do not react with anti-CD45RB antibodies (CD45RB-). It should be noted that anti-CD45RB antibodies may identify an overlapping but not completely identical population to that recognized by anti-CD45RA antibodies in humans and direct comparisons between human and rodent data should therefore be performed with caution 2I.
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Table 2. Functional differences between naive and memory T cells Function
Naive
Memory
Refs
Proliferation/recall antigen Proliferation/CD3 and CD2 mAbs Helper function/Ig production (CD4 + cells) Cytotoxic precursor cells (antigen-specific) Cytotoxic precursor cells (alloantigen-specific) Cytotoxic effector cells (alloantigen-specific) Adherence to endothelium/augments vascular permeability Response to chemotactic stimuli Inhibition of proliferation by CD25 mAb Proliferation to allogeneic cells Proliferation to mitogens Proliferation to autologous cells Induction of suppressor function Inhibition of proliferation by CD7 antibodies
-+--++ + + ++++ + + ++ ++++ ++++ ++++ ++++ +++
++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++ + + +
1 1,4 1,3,10 11 12,13 11,13 14,15 16 5,17,18 1,9,19 19 19 19 5
Anti-CD44 (Pgp-1) antibodies have been used to positively identify memory T cells in rodents 2. Important information on the patterns of recirculation of naive and memory T cells in vivo has been deduced in sheep, where anti-CD45RA antibodies have been used to identify naive T cells, in an analogous way to humans =. An anti-CD45RO antibody equivalent is not currently available in this species 22. An interesting and potentially important observation is that alloantigen, unlike recall antigen, activates naive and memory T cells to the same extent and with similar kinetics of proliferation in vitro. This may have implications for the nature of immune responsiveness against transplanted grafts; specifically, naive and memory T cells may synergize with each other during activation, as recently suggested23-2s.
The synthesis of cytokines by naive and memory T cells A number of studies have examined the ability of naive and memory CD4 + T cells to synthesize cytokines after activation (Table 3). Similar observations on CD8 + T cells have been rare but our preliminary observations suggest that naive and memory CD8 + populations can synthesize cytokines, albeit to a lesser extent than the CD4 + population. When data on CD4 + T-cell cytokine production from different laboratories are compared, it is apparent that there is some variability between different, and in some cases even the same, stimuli. However, the tendency, in both humans and rodents, is that the naive cells synthesize greater amounts of interleukin 2 (IL-2) than memory T cells do. The synthesis of IL-4 after activation is more restricted, with the memory CD4 + T-cell population producing virtually all of this cytokine after activation. Gamma-interferon (IFN-y), IL-3 and IL-6 are also preferentially synthesized by the memory T-cell population rather than by the naive cells. An interesting question is whether the synthesis of cytokines by naive and memory CD4 + T cells falls into a TH1 and TH2 pattern as demonstrated for mouse CD4 + clones 33. Whereas TH1 clones synthesize IL-2 and IFN-y, TH2 clones synthesize IL-4 and IL-5 (Refs 30,33). It does not appear at present that the profile of cytokine synthesis by human naive and memory CD4 ÷ T cells or
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CD4+CD45RO + T-cell clones fits into this pattern 26. It has been proposed that TH1 and TH2 patterns of cytokine production may represent a further differentiation or specialization within human memory cells 3,33 but this issue remains to be resolved. An important observation is that the proliferation of naive T cells in response to various stimuli, including anti-CD2 and anti-CD3 antibodies, can be substantially increased by the addition of IL-4 (Ref. 34), whereas, under similar conditions, memory T cells show only a minimal enhancement of proliferation. These results have been confirmed using phytohemagglutinin (PHA) or alloantigens as the original stimulus in humans 9, which suggests that a cytokine produced predominantly by memory T cells may amplify the response of naive T cells in a paracrine manner. In contrast, although memory CD4 ÷ T cells can synthesize IL-2, the amounts synthesized are insufficient for optimal proliferation while naive CD4 + T cells synthesize an excess of IL-2. This phenomenon is substantiated by studies that show that exogenous IL-2, when added to cultures of activated memory T cells, further enhances proliferative activity but has little, if any, effect on naive T cells 9,24. The preferential inhibition of memory T-cell proliferation with anti-CD25 (i.e. anti-IL-2R) antibodies and cyclosporin A (CsA) compared with naive T cells underlines the greater dependence of memory T cells on IL-2 as a signal for proliferation5. On the basis that memory T cells are larger than naive T cells 8,2s, identical observations have been demonstrated in the mouse31; this again suggests that the proliferation of memory T cells may be amplified by IL-2-secreting naive T cells. All of these results taken together indicate that, during activation, synergy may indeed occur between naive and memory T cells; each subset produces a cytokine that amplifies the response of the other (Fig. 1). The subsets show similar levels of expression of IL-2R (p55 and p75) and IL-4R (assessed using biotinyllated IL-4) after activation, pointing to the synergy at the level of cytokine production, rather than at the level of receptor expression (authors' unpublished observations). In addition to IL-2 and IL-4, other cytokines may also be important in this synergy. Memory, but not naive, CD4 + T cells synthesize IL-6 after activation with anti-CD2
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Table 3. Lymphokine production by naive and memory T cells Lymphokine
Species
Stimulusa
Naive b
Memory b
Refs
IL-2
Humans
PHA PHA PMA/calcium ionophore PWM Allogeneic cells CD3 CD3 CD3 SEAc PHA/ConA/PWM CD3 Allogeneic cells ConA PMA/calcium ionophore PHA,CD3 Allogeneic cells CD3, PHA, ConA, PWM PHA, CD3, allogeneic cells PWM, SEA CD3 Mitogens and alloantigen ConA Mitogens and alloantigen CD2
++++ ++++ ++++ ++++ ++++ ++++ ++++ + ++++ ++++ ++++ ++++ ++++ -
++ ++++ ++ ++ ++ ++ ++++ ++++ ++++ + + ++++ + ++++
+ -
++++ ++++ ++++
9,24,26 1 26 10 9 9,27 10 28 29 20,30 31 b 2 3 26 9,26,27 9 2,20,30 1,9,26,10,29
++++ + ++++ + -
++++ ++++ ++ ++++ ++++
10,27 2 3 2 32
Mice
IL-4
Rats Humans
IFN-~/
Mice Humans
IL-3 IL-6
Mice Rats Mice Humans
aPMA: phorbol myristateacetate; SEA: staphylococcolenterotoxin A; ConA: concanavalinA; PWM: pokeweed mitogen; bnaive and memoryT cells distinguishedon the basis of cell size (see text); csubsetsshow differentkineticsof IL-2 production. antibodies, while both subsets require IL-6 for this form of activation32. It is likely, therefore, that IL-6 synthesized by memory T cells may represent part of the synergy that has been demonstrated between naive and memory T cells upon activation with anti-CD2 antibody 2s. The obvious question that arises is whether this scheme may help to explain the cytokine production in response to antigen in vivo.
Antigenic activation of naive and memory T cells By definition, a primary response to an antigen occurs if it has not previously been encountered in vivo; response to a primary antigen should mainly involve naive T cells. A secondary response occurs if the host has previously been exposed to the antigen, and memory T cells will form the major responding population. This has recently been substantiated in a mouse model where naive and memory T cells were separated on the basis of CD45RB expression 2°. These definitions oversimplify real events as many studies show that crossreactivity occurs among some antigens after their processing and partial degradation by macrophages 4,35. Nevertheless we would predict that, upon activation by any one particular antigen in vivo, simultaneous activation of both naive and memory T cells will not normally occur as the antigen will preferentially recruit either one or other of these subsets. From the observations of cytokine synthesis in vitro, it would be expected that the response to a primary antigen by naive T cells will initially result in IL-2 production, followed by IL-4 production after these cells have become activated and have undergone a functional transition to memory cells. This has been con-
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firmed in mice, where it has been shown that, after activation with a primary antigen, IL-2 is synthesized earlier than IL-4 (Ref. 36). We predict, from the in vitro data, that IL-4 is synthesized earlier and in larger amounts by memory T cells during secondary responses, but they may remain starved of IL-2. In situations involving extensive injury or infection, high concentrations of more than one antigen (primary and secondary) may be present and drain into lymph nodes or the spleen. Powerful synergy between the naive and memory T-cell subsets may occur as they may become activated simultaneously. In addition, a secondary response occurring at a given period after the primary response may involve both memory T cells and newlygenerated naive T cells that are reactive with the same antigen. Synergy between naive and memory T cells may again occur, especially if there is a high concentration of antigen present that precludes the preferential activation of the memory subset. Functional data derived in vitro must be analysed together with histological information on the distribution of naive and memory T-cell subsets since it may not be feasible, from an anatomical standpoint, for synergy between these cells to occur. Histological studies indicate that naive and memory T cells do reside in close proximity in the paracortex of lymph nodes 23. It is, therefore, possible for the draining antigens in afferent lymph to activate both naive and memory T cells in close proximity. As the naive T cells differentiate into memory T cells after activation, the synergy may be self limiting, with only IL-4 synthetic capacity present in the latter part of such a response. Indeed, the extent of synergy may be proportional
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ii!iii!ii!i!iiiii!ii!iiiil
iiiiiiiii!iii!iiiil IL-2
to the strength and diversity of the antigenic challenge. The simultaneous synthesis of IL-2 and IL-4 by naive and memory T cells may have synergistic effects on T-cell activation yet restrict the nonspecific recruitment of other cells within lymphoid tissue. For example, while IL-2 and IL-4 are synergistic for T-cell proliferation, IL-4 counteracts the stimulatory effects of IL-2 on B-cell function 37and also inhibits the IL-2-mediated induction of lymphokine-activatedkiller activity38. Obviously the contribution of other cytokines such as IFN-y must eventually be included in these schemes of cytokine interaction. However, the synergism between naive and memory T cells may provide clues that explain the innate logic of cytokine pleiotropy during immune activation in vivo.
The response of naive and memory T cells to alloantigen Conventional antigen and alloantigen responsive T cells are not separate lineages; the same cell may respond to both challenges 39. The precursor cell frequency of alloresponsive T cells is far higher than the frequency of antigen-responsive T cells4°, suggesting that a multiplicity of separate T-cell populations with different antigenic specificities may be activated by alloantigen. While the precursor cell frequency for recall antigen is much higher within the memory T-cell subset than in the naive population, the same rule does not apply for alloantigens. Both naive and memory populations contain similar high levels of alloresponsive precursors TM and respond with the same proliferative kinetics 17,18.Possible explanations for alloantigen recruitment of such a large number of T cells have been reviewed in detail elsewhere 39~1. If synergy between naive and memory T cells does occur, then the collaboration between these populations during activation may add further vigour to the alloantigenic response. The immune response against transplanted tissue is often so intense that it leads to graft rejection, even in the presence of potent immunosuppression. T cells activated by donor alloantigens during transplant rejection in vivo can be isolated from both the graft itself and from the circulation42. When investigated for the expression of CD45 determinants, both naive and memory T cells have been shown within rejecting renal grafts, and a significant proportion of these are activated, proliferating and switching from a naive to a memory phenotype in situ 13,43. The profile of cytokine production by naive and memory T cells in response to alloantigen in vitro is similar to that observed with mitogens and anti-CD3 antibodies (Table 3). Furthermore, when both subsets are separated and subsequently reconstituted in varying proportions before alloactivation in vitro, the subset synergy in the induction of proliferation can be clearly demonstrated, even when cell number is accounted for 9. The in vitro data is therefore compatible with the hypothesis that interactions between naive and memory T-cell subsets may amplify the responses to graft alloantigens. A further important point is that, whereas in antigen-specific responses the effector mechanisms eventually lead to the removal of the original antigenic stimulus, during the allogeneic response in the graft, the generation of cytokines such as IFN-~ increases the expression of major histocompatibility complex (MHC) antigens, not only on passenger leukocytes but also on epithelial and endoImmunology Today
Naive
~
Memory 11_-4
Fig. 1. Synergy will occur when naive and memory T cells are activated in close proximity (a) when primary and secondary antigens are both present and (b) in response to alloantigens. thelial cells44. This will increase the original stimulus for the alloresponsive cells and may in turn further amplify the anti-graft response. In view of the fact that memory T cells have an inherently greater capacity to respond to activation stimuli than naive cells do, it may be surprising that the kinetics of proliferation to alloantigen in vitro is similar in both subsets. Naive T cells have been shown to have very Stringent requirements for antigen presentation compared with memory T cells45. This may be due in part to their low level of expression of adhesion molecules; strong T-cell receptor interactions are therefore required for activation. In naive T-cell responses to primary antigen, a small number of MHC molecules on the antigenpresenting cell may contain fragments of the primary antigen in relatively low concentrations. In contrast, the high density and uniformity of foreign MHC protein on allostimulatory cells may lead to T-cell activation even when the interaction with T-cell receptors is relatively weak. Once activated, the capacity of the naive T cells to synthesize IL-2 would enable them to undergo clonal expansion at least as efficiently as the activated memory T cells. The high density of MHC molecules on the allogeneic cells thus serves to lower the threshold of activation and, therefore, to bypass the stringent activation requirements of the naive subset. In an analogous situation, low concentrations of anti-CD3 antibodies preferentially activate memory T cells yet high concentrations of this antibody activate both subsets equally1.
Implications for immunosuppressive therapy during transplantation Recent clinical trials have used monoclonal antibodies together with CsA to achieve optimal immunosuppression during organ transplantation 4s. Antibodies, for example against the CD3 determinant, show particularly powerful immunosuppressive action when used in conjunction with CsA46. These strategies not only allow the dose of CsA to be reduced, but may also lead to more selective regimes of immunosuppression. Two antibodies that may be of use in this regard are directed against CD7 and CD25 molecules. These antibodies react strongly with activated T cells but may spare resting T cellss,6. It is important, therefore, that anti-
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•iii•i2ii•iiiiiiiiii!•ii•!ii•i•!i•iiii!i!i!i!i!!iiiii•ii•••ii•i!iii•iiii•i•iii!ii•ii•i•i!•iii•iiiiii•i!iiii•i•ii•i•i•iii•i•ii••ii!•i
iiiiiiii!iiiiiiiiiiiiiiiiiiii!iiii!ii!iii!i ii!iiii!iii!iiiii!i!iii!iiiiiiiiiii!i!!i i CD7 antibodies preferentially inhibit alloactivated naive T cells whereas anti-CD25 antibodies preferentially inhibit memory T cellss. These antibodies show additive inhibition of isolated naive and memory T-cell subsets and also show additive inhibition when combined with CsAs. Thus a combination of anti-CD7 and anti-CD25 antibodies may interfere with the activation of naive and memory T cells individually and with the synergy between them. A chimeric form of the anti-CD7 antibody6 is currently being assessed for its immunosuppressive potential in renal rejection. In future, the selection of antibodies or other drugs for use in the prophylaxis of graft rejection may be assessed on their ability to reduce the alloactivation of either naive or memory T cells and serve as a basis for a more selective approach to immunosuppressive regimes. Conclusions It is of interest that naive and memory T cells, which are at different stages of maturity, may interact with each other during activation. The scheme of synergy proposed here is simple yet may explain much of the conflicting data on the action of cytokines, especially IL-2 and IL-4. Undoubtedly other cytokines will be involved in the interaction between naive and memory T cells and also between these and other cell types. An interacting cascade of cytokine synthesis, as outlined here, will help to place the pleiotropy and redundancy of cytokine production into perspective.
The authors are grateful to our colleagues Kamal Ivory and Darrell Pilling who have contributed to the work and to P.L. Amlot and P.C.L. Beverleyfor critical reviewof the manuscript. This work was supported by Sandoz Pharma Ltd. Arne N. Akbar and George Janossy are at the Dept of Clinical Immunology, Royal Free Hospital School of Medicine, Pond Street, London NW3 2QG, UK and Michael Salmon is at the Dept of Rheumatology, The Medical School, Birmingham University, Birmingham B15 2TJ, UK. References 1 Sanders, M.E., Makgoba, M.W. and Shaw, S. (1988) Immunol. Today 9, 195-199 2 Cerottini, J.C. and Robson MacDonald, H. (1989) Annu. Rev. Immunol. 7, 77-89 3 Powrie, F. and Mason, D. (1988) Immunol. Today 9, 274-277 4 Beverley,P.C.L. (1990) Immunol. Today 11,203-205 5 Akbar, A.N., Amlot, P.L., Ivory, K. et al. (1990) Transplantation 50, 823-829 6 Heinrich, G., Gram, H., Kocher, H.P. et al. (1989) J. Immunol. 143, 3589-3597 7 Tedder, T.F., Matsuyama, T., Rothstein, D. et al. (1990) Eur. J. Immunol. 20, 1351-1357 8 Buckle, A.M. and Hogg, N. (1990) Eur. J. Immunol. 20, 337-341 9 Akbar, A.N., Salmon, M., Ivory, K. et al. Int. ImmunoI. (in press) 10 Sleasman,J.W., Morimoto, C., Schlossman,S.F. et al. (1990) Eur. J. Immunol. 20, 1357-1366 11 Merkenschlager, M. and Beverley,P.C.L. (1989) Int. Immunol. 1,450-456 12 Yamashita, N. and Clement, L.T. (1989) J. Immunol. 143, 1518-1523
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13 Akbar, A.N., Amlot, P.L., Timms, A. et al. (1990) Clin. Exp. Immunol. 81,225-231 14 Pitzalis, C., Kingsley,G., Haskard, D. et al. (1988) Eur. J. Immunol. 18, 1397-1404 15 Damle, N.K. and Doyle, L.V. (1990) J. Immunol. 144, 1233-1240 16 Schall, T.J., Bacon, K., Toy, K.J. et al. (1990) Nature 347, 669-673 17 Akbar, A.N., Terry, L., Timms, A. et aI. (1988) J. Immunol. 140, 2171-2178 18 Merkenschlager, M., Terry, L., Edwards, R. et al. (1988) Eur. J: Immunol. 18, 1653-1661 19 Morimoto, C., Letvin, N.L., Distaso, J.A. et al. (1985) J. Immunol. 134, 1508-1515 20 Lee,W.T. and Vitetta, E.S. (1990) Cell. Immunol. 130, 459-471 21 Mason, D. and Powrie, F. (1990) Immunology 70, 427-433 22 Mackay, C.R., Marston, W.L. and Dudler, L. (1990) J. Exp. Med. 171,801-817 23 Janossy, G., Campana, D. and Akbar, A. (1989) in Current Topics in Pathology (Cell Kinetics of the Inflammatory Reactions, Vol. 79) (Iversen, O.H., ed.), pp. 59-99, Springer-Verlag 24 Salmon, M., Kitas, G.D., Hill Gaston, J.S. et al. (1988) Immunology 65, 81-85 25 Wallace, D.L. and Beverley,P.C.L. (1990) Immunology 69, 460-467 26 Salmon, M., Kitas, G.D. and Bacon, P.A. (1989) J. Immunol. 143,907-912 27 Bettens, F., Walker, C., Gauchat, J.F. et al. (1989) Eur. J. Immunol. 19, 1569-1575 28 Byrne, J.A., Butler, J.L., Reinherz, E.L. et al. (1989) Int. Immunol. 1, 29-35 29 Dohlsten, M., Hedlund, G., Sjogren, H.O. et al. (1988) Eur. J. Immunol. 18, 1173-1178 30 Bottomly, K. (1988) Immunol. Today 9, 268-277 31 Ben-Sasson,S.Z., Le Gros, G., Conrad, D.H. et al. (1990) J. Immunol. 145, 1127-1136 32 Kasahara, Y., Miyawaki, T., Kato, K. et al. (1990) J. Exp. Med. 172, 1419-1424 33 Mossman, T.R. and Coffman, R.L. (1989) Annu. Rev. Immunol. 7, 145-173 34 Wasik, M. and Morimoto, C. (1990) J. Immunol. 144, 3334-3340 35 Jones, K.R., Hickling, J.K., Targett, G.A.T. et al. (1990) Eur. J. Immunol. 20, 307-315 36 Mohler, K.M. and Butler, L.D. (1990) J. Immunol. 145, 1734-1739 37 Llorente, L., Mitjavila, F., Crevon, M.C. et al. (1990) Eur. J. Immunol. 20, 1887-1892 38 Spits, H., Yssel, H., Paliard, X. et al. (1988) J. Immunol. 141, 29-36 39 Lechler, R.I., Lombardi, G., Batchelor, J.R. et al. (1990) Immunol. Today 11, 83-88 40 Simonsen, M. (1990) Scand. J. Immunol. 32, 565-575 41 Sharrock, C.E.M., Man, S., Wanachiwanawin, W. et al. (1987) Transplantation 43, 699-703 42 Preffer, F.I., Colvin, R.B., Leary, C.P. et al. (1986) J. Immunol. 137, 2823-2830 43 Stein-Oakley, A.N., Kerr, P.G., Kraft, N.E. et al. (1989) Transplantation 48,787-790 44 Dallman, M.J. and Morris, P.J. (1988) in Kidney Transplantation: Principles and Practice (Morris, P.J., ed.), pp. 15-37, W.B. Saunders 45 Inaba, K. and Steinman, R.M. (1986) J. Exp. Med. 163, 247-261 46 Cosimi, A.B. (1988) in Kidney Transplantation, Principles and Practice (Morris, P.J., ed.), pp. 343-371, W.B. Saunders
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