Regulation of the development of distinct subsets of CD4+ T cells

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35th FORU,'d I N IMJ'vlUNOLOGY

Regulation of the development of distinct subsets of CD4 + T cells S.L. Swain Department o f Biology and the Cancer Center-O063, University o f Cah'fornia, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0063 (USA)

Introduction The initial description of two patterns of lymphokine secretion by 2 subsets of long-term helper T-cell lines (reviewed in Coffman et al., 1988) was greeted with considerable enthusiasm. The analysis, however, of the development of equivalent subsets of normal T cells and the factors which regulate them has proceeded slowly, and indeed there has been some controversy about whether any "normal" helper T cells correspond to Thl and Th2 subsets. We would like to discuss here our own experiments on the development of CD4 + T cells secreting different patterns of lymphokines. We can identify 3 developmentally distinct subsets of helper T cells: precursors, effectors and memory cells, and can subdivide the effector cells into at least two predominant phenotypes based on their pattern of lymphokine secretion. The precursors, effectors and memory cells can each be identified by patterns of surface marker expression and by functional characteristics, and we have defined some of the lymphokines whose presence in culture directs the development of the different phenotypes o f lymphokinesecreting cells. Developmentally subsets

of the 3 distinct developmental stages (Swain et al., 1988, 1990; Weinberg et al., 1990). Precursors We have operationally defined precursor helper T cells as those short-lived CD4 + cells which otisappear after adult thymectomy and which can be driven to produce an effector population by in vitro culture for 4 days. The effector population can be restimulated to synthesize and secrete high levels of lymphokines. Phenotypically, precursors are small resting CD4+ T cells which are enriched in young animals, in thymus and in spleen and which are low in Pgpl (Swain et ai., 1990). We have also found that MEL14 ÷, an enriched population from resting CD4+ T cells, contains virtually all the precursor activity (Bradley, Atkins, and ;wain, unpublished). The prec~ rsor population (enriched MELt4+ cells) proliferates vigorously in response to polyclonal stimulation (ConA, antiCD3), secretes IL2 but no other lymphokine we have assayed (IL3, IL4, ILS, IL6, IFN T, GM-CSF), and after 2-7 days of culture with ConA and APC, proliferates in response to both IL2 or IL4 (Swain, unpublished).

defined T-cell

Our published studies have characterized some of '+hefeatures

Memory cells The other major population found among resting CD4+ T

cells is the memory population. This is operationally defined by its long lifespan (adult thymectomy leaves this population intact), and the rapid secretion of moderate titres of IL2 and low titres of IL3, GM-CSF, IFNT and IL4 in response to polyclonal stimulation. The population remaining after adult thymectomy, which includes the memory cells, is high in Pgpl expression and low in expression of CD45RB and MELI4- (Swain, Bradley, Atkins and Weinberg, unpublished). In immunized animals, the MELI4- population contains the cells which proliferate, secrete lymphokines and pro. . . . . . . . v sp~.iv~, to Ag (Atkins and Swain, unpublished). The memory population develops fairly rapidly into lymphokine-secreting cells after stimulation (12-18 h), and both purified CD4+ MEL14- cells from normal mice and CD4+ cells from adult thymectomized animals proliferate in response to polyclonal stimulation, with ConA or CD3 plus PMA. (This is in contrast to precursors which proliferate well in the absence of PMA.) In the absence of PMA costimulation, both the cell recovery and proliferation in response to ConA or anti-CD3 of T cells from thymectomized animals falls off rapidly after 2-3 days of culture and no substantial effector population is recovered under our culture conditions at 4-14 days after stimulation (Swain et al., 1990; Swain et al., in press). Thus, the precursor and memory populations can be clearly distin-

C D 4 + T-CELL SUBSETS: DIFFERENTIA TION A N D F U N C T I O N

guished by phenotype, lymphokine secretion and response to stimulation.

Effector cells

The effector population is operationally defined as those larger, activated CD4+ T cells recovered after 4-7 days in mitogen-stimulated cultures of either isolated precursors or mixed resting CD4+ T ceils (including both precursors and memory cells) supplemented with APC and lymphokines (IL2 plus IL4) (Swain et al., 1988, 1990; Weinberg et ai., 1990). Effector helper cells are rather uniformly large, CD4+, IL2R +, Pgpl high. Effectors can be induced to secrete very high titres of lymphokines IL3, IL4, IL5, GM-CSF and IFNy. This population expresses high levels of CD45RA, B and C (Weinberg and Swain, unpublished), and also expresses MEL14 heterogeneously. That the effector population is derived from precursors is suggested from the fact that CD4+ T cells from adult thymectomized animals and MEL14- T cells from normal animals give rise to few CD4 cells that can be restimulated to secrete lymphokines (Swain et al., 1990) while MEL14+ populations contain enriched precursor activity (Bradley, Atkins apd Swain, unpublished).

Regulation of effector cell developme~tt by lymphokines A major focus of our investigations has been to define the requirements for development of effector cells. The most clear cut requirements include a population e f precursor cells, an appropriate activation signal which can be a mitogen (ConA, PHA, etc.), antiCD3 or antigens (alloantigen or conventional Ag), lymphokines, and probably a source of APC. Lymphokines are not only required for the generation of effectors developed, but influence the

15

kind of effectors that are generated (Swain e t a ' . , in press).

variable, but production.

IL2 promotes effector development in general regardless of other lymphokines present. Since some IL2 is invaria ~ !y produced during culture, the effect of IL2 is seen most clearly in blocking experiments, in our case using anti-IL2R reagents. When IL4 is present, however, some effectors develop even when IL2 usage is blocked, suggesting that IL4 can serve as an alternate helper growth factor in these cultures. Indeed, by two days following polyclonal activation, the proliferative response of cells in our cultures to IL4 was nearly as great as to IL2 (Swain, unpublished).

IFNy added to cultures promotes effectors able to secrete IFN'r, and, to a lesser extent and with slightly delayed kinetics, IL2. In our experiments so far, the effects of IFNy are somewhat weaker than those of IL4 and require fairly lfigh IFNy amounts (1,000 or more units as defined by Genentech for rIFN3,). Furthermore, the effects of IFNy are rather easily overcome by IL4 (Swain et al., 1990b).

IL4, in addition, exerts dramatic effects on the phenotype of effectors which develop (Swain et al., in press). Added at the initiation of cultures, IL4 promotes developn ent of effectors which can be restimulated to secrete IL4/IL5 (TH2-1ike) and strongly suppresses development of IL2- secreting effectors. At a doses two times the dose required for optimum development of IL-4/IL-5-secreting IIA dramatically suppresses the development of IFN~.-secreting cells. Only the higher dose of IL4 added at the beginning of culture leads to a "pure" Th2-1ike phenotype. Lower doses of IL4, or addition delayed by 1-2 days, leads to the development of a population with high secretion of IFN~,, as well as IL4 and IL5 (a possible TH0 phenotype, Firestein et aL, 1989; or a mixed population of cells). IL3 and GM-CSF (Swain et al., 1990) are produced in high titres by all effector populations we have examined. When memory cells have not been removed from the starting population containing precursors, there are often low levels of IL4 produced during the cultures. Addition of anti-IL4 to cultures of CD4-enriched normal T cells leads to a greater degree of IFNy production by effectors and to

still low,

IL2

When cultures grown in IL4 (plus IL2) versus those grown in IL2 either with or without IFNy are compared over two weeks, the lymphokine production pattern in the populations of T cells which grow out, persists even though the lymphokines present in culture removed by washing at day 4 are replaced by IL2 alone (Swain et al., in press). Despite the clear cut differences in lymphokine secretion profiles of cells grown with and without IL4 or with IFN~', the effector populations recovered after culture with IL4 versus IFN3, at days 4-8 differ little in any phenot ~ i c marker we have exazTfined including Pgpl, MEL14, CD45RB, 3G11, 6C10, IL2R (Swain, unpublished). These results are consistant with a model in which the initial presence of IL4 directs developing CD4 precursors down one of several alternate pathways of differentiation. It is hard to characterize the effectors produced in the absence of IL4 which secrete high levels of IFNy, IL3 and GMCSF as Thl-like because of the low to moderate IL2 production. In examining the CD4 + T cells which develop at low or negligible levels of IL4 it is clear that while little IL2 is produced by effectors on day 4, substantially more IL2 is produced at days 6-8 and sometimes IFN)" production also increases. Studies by Fitch and colleagues (Gajewski et al., 1988) and recent studies from Mosmann (Street et aL, 1990) and from Fox's

16 laboratories (Betz and Fox, 1990) suggest that lymphokines can also regulate helper T-cell development when added later in the developmental pathway. Thus, IFN T promotes cells with a TH 1 phenotype to develop from cells that secrete multiple lymphokines, and IL4 influences cells taken 7 days after in vivo stimulation to b~.come more committed to IL4 secretion (Betz and Fox, 1990). In addition CSIF, a product of Th2, also known as ILl0, causes a transient, immediate suppression of IFN)" cells which are already committed to secreting IFN-r (Fiorentino et aL, 1989).

Physiological relevance of in vitro pathways Several experiments lead us to believe that the basic model of response we are studying in vitro reflects pathways of response that are real options in vivo. First, although many of our studies have used ConA or antiCD3 as polyclonal mitogens, we have repeated all the basic observations with alloantigen stimulation (Swain et al., 1990; Swain et al., in press). The alloantigenstimulated cells behave very similarly except that in the absence of IL4 more IL2 is produced by the effector population generated at day 4. One difference between the two situations is the 5-fold higher number of APC added to the alloantigen-stimulated culture. We think such APC may release an additional lymphokine which influences helper T cell differentiation. Nonetheless, the effects of IL4 in suppressing IL2 and IFN~, are equally dramatic in the alloantigen model. Second, ~ve have recently set up the basic model using CD4+ T from TCR a,~ transgenic mice which bear a TCR specific for IE k +cytochrome C (Kaye et al., 1990). The memory T cells initially produce high IL2 in response to Ag + APC. Effectors are generat-

35th F O R U M I N I M M U N O L O G Y

ed from precursors after 4 days of culture with Ag/APC plus IL2 and IL4 secrete a Th2 pattern of lymphokines while those grown in the absence of added IL4 secrete IL4 and IFN~. (Weinberg, Kaye, Hedrick and Swain, unpublished). Thus the subsets of T cells generated by stimulation with Ag/APC appear to be equivalent to those generated in the polyclonal model. Our third line of argument derives from studies of the in vivo development of lymphokinesecreting cells. We have followed the in vivo kinetics of development of cells that will release lymphokines after restimulation with specific Ag. Using either a particulate Ag, SRBC or a classic soluble protein, KLH injected in FCA, we see the emergence of effectorlike cells 4-7 days following Ag stimulation (Bradley et al., 1990). The in vivo effector cells secrete predominantly IL2 and IL3 in the case of SRBC injection and IL2, IL3 and IL4 in the case of KLH injection. As in the in vitro model, both populations are transient and largely gone by 14 days. Also as in vitro, the effector populations are probably generated from shortlived T cells since adult thymectomy interferes with their development.

Hypothetical model of helper T-cell subset development Taken together, these observation and those of others suggest to us the pathway shown in figure 1. We postulate a single uncommitted precursor cell capable of alternate pathways of development. In limiting dilution experiments, more than 50 % of c,dtures with a single precursor can ~,e driven to develop into IL4-secreting clones (Muralidba.-, Bradley and Swain, unpublished). This argues that virtually every cell can develop into this effec~or phenotype, and we suggest that IL4 added early drives most cell ~ to be committed to a Th2-1ik~ .,dhway. IFN~, is shown

driving cells down a pathway leading to high production of IFN~,. In the absence of sufficient IL4 (and perhaps IFN~') a mixed or multipotential population of ceils develops and IL4 and IFN't are both made after stimulation. We have no evidence at this point about the lymphokine secretion of individual cells, but we knew that TH0 cells can exist from the experiments of Glasebrook and colleagues (Firestein et aL, 1990; Torbett et al., 1990) so we have depicted a population capable of multiple secretion which would have the potential for future commitment. We have also included the effects of TGF~1 which drives, the development of cells which can be restimulated to secrete markedly enhanced levels of IL2 and iFN-f and which suppresses the IL4 pathway (Swain and Weinberg, unpublished). Needless to say, these may not be the only lymphokines which determine the pathways or helper T-cell development, and it is also very likely that other variables such as the nature of the antigen, the kind of APC involved, etc., contribute important influences regulating effector development. One subject which we find particularly interesting but relatively unexplored is the regulation of development of memory helper T cells. We imagine we can define long-lived "resting" memory cells in vivo and that they can be distinguished from effector cells by their persistance (long lifespan following adult thymectomy) and by phenotypic markers (see earlier discussion). The phenotype of recently derived memory cells is less clear. We would expect recently developed memory cells to be a proliferating population that will later become more quiescent (if not fully resting). Recently, we found that TGF.~ present during in vitro cultures reduces development of a distinct population of cells. This population has lost CD45RB (in marked contrast to "effectors" described above which develop in response to IL4 or IFN~, and ex-

C D 4 ÷ T-CELL SUBSETS: DIFFERENTIA TION A N D F U N C I I O N

press high levels of CD45RB). This population proliferates extremely well to IL2 and can be propagated efficiently in IL2, secretes high titres of IL2 (as mentioned above). We postulate that this could be an emerging population of memory cells. We are currently further examining the characteristics of this cell population to see if it has other prope~ies suggesting a memory phenotype especially an ability to persist in adoptive hosts. This potential pathway is also included in the model with the designation "memo r y ? " to indicate that its description as a memory cell is based on quite circumstantial evidence. We were initially surprised by the apparent expression of MEL 14 (the putative lymph node homing receptor) on precursor but not memory cells. However, recent studies in the sheep suggest memory cells enter the lymph node by a different non-HEV dependent pathway (Mackay et al., 1990).

Major issues and uncertainties A somewhat unexpected aspect of our model is that IL2 is made in low or negligible amounts by

most effectors we generate in vitro. IL4 efficiently suppresses generation of IL2-secreting helpers, but even when IL4 is not added and endogenous IL4 is blocked by antiIL4, only modest titres of IL2 are made by effectors. This is in contrast to the lymphokine secretion we see in in vivo generated effectors which secrete abundant IL2 (Bradley et ai., submitted) and to TH 1 cell lines. Thus, we hypothesize that some lymphokine(s) or signal(s) available in vitro is not being provided in our model. We do not think that the nature of the stimulus to the CD4 cells is per se responsible since the pattern of low IL2 production by effectors developed in IL4 is found when ConA, anti-CD3, alloantigen or Agbearing APC are used to trigger T cells in vitro. However, it is likely that APC populations may play a key role since somewhat higher (Ag/APC) levels of IL2 secretion are obtained in alloantigen models where higher numbers of APC are added to culture. Another unexpected observation is that under our culture conditions, memory cells do not proliferate, persist and secrete lyre-

phokines after 3 days or so of culture. We think we are beginning to understand this phenomenon since our recent studies suggest that memory cells may require additional signals to proliferate. Thus, we find ConA or CD3 plus PMA, but not without PMA, stimulated proliferation of enriched memory populations, whereas precursors do not require the addition of PMA (Atkins and Swain, unpublished). The concept of a more stringent requirement for accessory signals in memory ceils runs counter to prevailing paradigms which tend to stress t[~'e ease with which memory cells respond. Nonetheless, one can reasonably postulate that memory response could well be tightly regu'~ated to prevent inappropriate re,:ponses. Obviously, more careful stt~dies are needed to further establish the requirements of memory and precursor helper cells. A number of questions are still unsolved. 1) Is there heterogeneity in the precursor population which leads to different phenotypes of effectots and/or to effector versus memory cells?

EFFECTORS I

CD45RB++

PRECURSOR(S) [

IFN T

IL-2R++

"~ IL-2 Pgp-l++

IL-2R-(~CD45RB+

CD45RB++ . @ IL-4 - IL-2R++ "~ IL-5

Pgp-I Io ~ , ~

IFN y

Pgp-I++ CD45RB++

IL-2R-~ D 4 5 R B -

~

MEL-14-X~..~pgp.l++

IL-4

IL-2P.++

lv~ }--~ IL-2 [ MEMORY? ]

17

-¢" IL-5 Pgp-l++

Fig. I. Role of IL in helper-T-cell development.

18 2) W h a t a d d i t i o n a l lymphokines regulate the generation of effector and memory cells ? 3) What is the source of the lymphokines which regulate helper T-cell development in vivo ? Do different Ag preferentially stimulate production of such lymphokines so that different helper T-cell populations develop ? 4) What are the roles of different APC populations in generating different helper effectors/memory cells ? 5) Are there memory cells which have distinct Thl and Th2 phenotypes or do all memory cells make one pattern of lymphokine ? 6) What functional role(s) have the different population of CD4 helpers in B-cell responses, CTL responses, DTH, etc. This list, though by no means exhaustive, highlights the considerable gaps in our understanding of the regulation of helper T cells. This is a fertile field for investigation aimed at discovering the mechanisms governing the regulation of immune response.

References

Betz, M. & Fox, B.S. (1990), Regulation and development of cytochrome c specific IL-4-producing T cells. J. Immunol., 145, 1046-1052. Bottomly, K. (1988), A functional dichotomy in CD4 + T iymphocytes. Iraraunol. Today, 9, 268-274. Bradigy, L.M., Duncan, D.D. &

35th F O R U M I N I M M U N O L O G Y

Swain, S.L. (1990), Induction of antigen-specific CD4 ÷ lymphokine-secreting effector cells in vivo. Fed. Proc., 4, 206. Coffman, R.L., Seymour, B.W.P., Lebman, D.A., Hiraki, D.D., Christiansen J.A., Shrader, B., Cherwinsk,. H., Savelkoul, H.F.J., Finkelman, F.D., Bond, M.W. & Mosmann, T.R. (1988), The role of helper T-ceU products in mouse B-cell differentiation and isotype regulation. Immunol. Rev., i02, 5-28. Fiorentino, D.F., Bond, M.W. & Mosmann, T.R. (1989), Two types of mouse T helper cell. IV. -- Th2 clones secrete a factor that inhibits cytokine production by Thl clones. J. exp. Med., 170, 2081-2095. Firestein, G.S., Roeder, W.D., Laxer, J.A., Townsend, K.S., Weaver, C.T., Horn, J.T., Linton, H.J., Torbett, B.E. & Glasebrook, A.L. (1989), A new murine CD4 + T-cell subset with an unrestricted cytokine profile. J. Immunol., 143, 518-525. Ga]ewski, T.F., Joyce, J. & Fitch, J.W. (1988), Anti-proliferative effect of IFNq" in immune regulation. III. -- Differential selection of Thl and Th2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-v.. J. Immunol.. 140, 4245-4252. Kaye, J., Hsu, M.-L., Sauron, M.E., Jameson, S.C., Gascoigne, N.R.J. & Hedrick, S.M. (1989), Selective development of CD4 T cells in transgenie mice expressing a class II MHC-restricted antigen receptor. Nature (Lond.), 341,746-749. Mackay, C.R., Marston, W.L. & Dudley, L. (1990), Naive and memory T cells show distinct pathways of lymphocyte recirculation. J. exp. Med., 171,801-817.

Street, N.E., Shumacher, J.H., Fong, A.T., Bass, H., Fiorentino, D.F., Leverah, J.A. & Mosmann, T.R. (1990), Heterogeneity of mouse helper T cells. Evidence from bulk cultures and limiting dilution cloning for precursors of Thl and Th2 cells. J. lmmunol., 144, 1629-1639. Swain, S.L., McKenzie, D.T., Weinberg, A.D. & Hancock, W. (1988), Characterization of T helper I and 2 cell subsets in normal mice. Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells. J. lrnmunol., 141, 3445-3455. Swain, S.L., Weinberg, A.D. & English, M. (1990), CD4 + T-cell subsets: lymphokine secretion of memory cells and of effector cells which develop from precursors in vitro. J. l m m u n o l . , 144, 1788-1799. Swain, S.L., Weinberg, A.D., English, M. & Huston, G. (1990a), IL-4 directs the development of Th2-like helper effectors. J. lmmunol. (in press). Swain. S.L., Weinherg, A.D. & Huston, G. (1990b), IL-4 and IFN-y direct the development of distinci subsets of helper T cells. Fed. Proc., 4, 2020. Torbett, B.E., Laxer, J.A. & Glasebrook, A.L. (1990), Frequencies ~,fT cells secreting IL-2 and/or IL-4 among unprimed CD4 + populations. Evidence that clones secreting IL-2 and IL-4 give rise to clones which secrete only IL-4. lmmunol. Letter, 23,227-234. Weinberg, A.D., English, M.E. & Swain, S.L. (1990), Distinct regulation of lymphokine production is found in fresh versus in vitro primed murine helper cells. J. lmmunol., 144, 1800-1807.