Activation of functionally distinct subsets of CD4+ T lymphocytes

23

CD4 + T-CELL SUBSETS: DIFFERENTIA TION A N D FUNCTION

Gajewski, T.F., Pinnas, M., Wong, T. & Fitch, F.W. (1991), Murine Tal and Ta2 clones proliferate optimally response to distinct antigen presenting cell populations. J. Immunol. (in press). Gajewski, T.F., Schell, S.R., Nau, G. & Fitch, F.W. (1989b), Regulation of T-cell activation: differences among T-cell subsets. Immunol. Rev., 111, 79-110. Gajewski, T.F., Schell, S.R. & Fitch, F.W. (1990), Evidence implicating utilization of different T cell receptor-associated signalling pathways by TH1 and TH2 clones. J. I m m u n o l . , 144, 4110-4120. Greenbaum, L.A., Horowitz, J.B., Woods, A., Pasqualini, T., Reich, E.-P. & Bottomly, K. (1988), Autocrine growth of CD4 + T cells. Differential effects of IL-1 on helper and inflammatory T cells. J. Immunol., 140, 1555-1560. Heinzel, F.P., Sadick, M.D., Holaday, B.J., Coffman, R.L. & Locksley, RoM. (1989), Reciprocal expression of interferon V or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets. J. exp. Med., 169, 59-72. Hu-Li, J., Shevach, E.M., MizuguchJ, J., Ohara, J., Mosmann, T. & raul, w .~. [lYS/), b cell sumulatory factor 1 (interleukin 4) is a potent costimulant for normal

resting T lymphocytes. J. exp. Med., 165, 157-172. Lichtman, A.H., Kart-Jones, E.A. & Abbas, A.K. (1987), B-cell stimulatory factor 1 and not interleukin 2 is the autocrine growth factor for some helper T lymphocytes. Proc. nat. Acad. Sci. (Wash.), 84, 824-827. Lowenthal, J.W., Castle, B.E., Christiansen, J., Schreurs, J., Rennick, D., Arai, N., Hoy, P., Takebe, Y. & Howard, M. (1988), Expression of high affinity receptors for murine interleukin 4 (BSF-I) on hemopoietic and nonhemopoietic cells. J. Immunol., 140, 456-464. Magilavy, D.B., Fitch, F.W. & Gajewski, T.F. (1989), Murine hepatic accessory cells support the proliferation of THI but not TN2 helper T lymphocyte clones. J. exp. Med., 170, 985-990. Meuer, S.C., Hussey, R.E., Cantrell, D.A., Hodgdon, J.C., Schlossman, S.F., Smith, K . A . & Reinherz, E.L. (1984), Triggering of the T3-Ti antigen-receptor complex results in clonal T-cell proliferation through an interleukin 2-dependent autocrine pathway. Proc. nat. Acad. Sci. (Wash.), 81, 1509-1513. Mosmann, T.R. & Coffman, R.L. (1989), THI and TH2 cells: different patterns of lymphokine . . . . . .

--__

t

J

J

z~z,

S C C I ' I ~ [ i O B If~BI,I L U I [ . l l I l ¢ [ g l l L

,t'. . . .

iUll~-

tional properties. Ann. Rev. lmmunol., 7, 145-173.

Mueller, D.L., Jenkins, M.K. & Schwartz, R.H. (1989), Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann. Rev. lmmunol., 7, 445-480. Rock, K.L., Haber, S.I., Liano, D., Benacerraf, B. & Abbas, A.K. (1986), Antigen presentation by hapten-specific B lymphocytes. -III. Analysis of the immunog!obulin-dependent pathway of antigen presentation to interleukin 1-dependent T lymphocytes° Europ. J. Immunol., 16, 1407-1412. Scott, P., Natovitz, P., Coffman, R.L., Pearce, E. & Sher, A. (1988), Immunoregnlation of cutaneous leishmaniasis. T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens. J. exp. Med., 168, 1675-1684. 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. Immunol., 144, 1800-1807. Williams, M.E., Lichtman, A.H. & Abbas, A.K. (1990), Anti-CD3 antibody induces unresponsiveness t o ~ " " : - -lrlkl ~l ~^ ~| f^f l- J~~ 3 , . . . . . ^llqJ~. , in Th2 clones. J. Immunol., 144, 1208-1214. l ~ - ~

|ll

ffUL

Activation of functionally distinct subsets of CD4 + T lymphocytes M.E. Williams, T.L. Chang, S.K. Burke, A.H. Lichtman and A.K. Abbas Departments o f Pathology, Brigham & W o m e n ' s Hospital and Harvard Medical School, Boston, M A 02115 ( U S A )

Introduction

CD4+ T l y m p h o c y t e s play central roles in the induction and regulation o f both cell-mediated

and humoral immune responses to protein antigens. The activation o f CD4+ T cells is initiated by the recognition o f processed fra[~ments o f protein antigens associated with

class II M H C molecules on the surfaces o f antigen-presenting cells (APC). In addition, T-cell activation is dependent on secreted or membrane-associated "costimula-

24 tors" produced by the APC. The response of CD4 ÷ T cells consists of: (1) the secretion of cytokines which stimulate autocrine growth and act on other cells, including B lymphocytes and macrophages, to mediate many of the effector functions of the T cells, and (2) proliferation, leading to preferential expansion o f the antigenrecognizing clone(s). This general scheme is thought to apply to most or all CD4 ÷ T cells. It has, however, become apparent that CD4+ T cells are a heterogeneous population that may consist of multiple subsets that can be distinguished on the basis of phenotype and function. Perhaps the clearest demonstration of functionally distinct subsets is based on analyses of cloned lines, first reported by Mosmann and subsequently by several other investigators (Mosmann and Coffman, 1989; Bottomly, 1988). Murine " T h l " clones produce IL2, IFN~., TNF and high levels of lymphotoxin, whereas " T h 2 " clones produce IL4, IL5 and IL6. The available evidence indicates that only Thl cells mediate delayed-type hypersensitivity reactions. In contrast, Th2 cells are the most efficient helper cells for resting B lymphocytes, and are the only T cells capable of inducing antibody class switching to certain isotypes, particularly IgE. The division of CD4+ T cells into these subpopulations is by no means universal, because many T cells in humans, rats and even mice produce mixtures of cytokines and cannot be classified into Thl and Th2 subsets. In fact, it is possible that naive T cells are capable of producing multiple cytokines, and as they differentiate in response to antigenic stimulation they may develop an increasingly restricted cytokine profile (Street et al., 1990). T lymphocytes can also be classified into resting and memory cells. In humans, resting T cells are CD45RA+, Mell4-high, and express low levels of CD2, LFAI and

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

CD44, whereas memory cells are CD45RO +, Me114-1ow and express higher a.mounts of CD2, LFA1 and CD44 (Cerottini and MacDonald, 1989). These differences in surface protein expression may influence the responses and the recirculation or migration patterns of the cells.

crine growth factor (Williams et al., 1990; Gajewski et aL, 1989; Williams and Unanue, 1990). Similarly, exposure of Thl but not Th2 clones to MHC-associated processed antigens in the absence of costimulators leads to a longlived state of anergy or tolerance (Schwartz, 1990).

The significance of T-ceU subsets is that different populations may participate in different physiologic immune responses or may be altered in different pathologic conditions. For instance, it is likely that Th2 cells play important roles in helminthic infections and allergic reactions, and Th 1 cells in granulomatous inflammation. Different conditions of antigenic exposure may also result in the preferential or even selective stimulation of one or the other T-cell subset. It is, therefore, important to define the conditions that lead to the differentiation of resting T cells into phenotypically or functionally distinct subsets, and the stimuli that trigger the proliferation and effector functions of these subsets. In this review, we will summarize our own work addressing these questions.

These results suggest that the biologic consequences of ligation of antigen receptors are fundamentally different in Thl and Th2 clones. The molecular basis of this difference is not yet known. Some studies have shown that only Thl clones show changes in inositol phospholipid metabolism upon receptor cross-linking and are dependent on extracellular Ca+ + for functional responses (Gajewski et al., 1990). All the Thl clones we have examined show increases in intracellular Ca + + in response to anti-CD3 antibody or the lectin, concanavalin A (ConA). Th2 clones are much more variable in this response (table I). Nevertheless, the induction of receptormediated unresponsiveness is unlikely to be due to activation of the Ca+ +-inositol phospholipidprotein kinase C pathway, because: (1)some Th2 clones do show increases in intracellular Ca ++ in response to ConA or anti-CD3 but are not rendered unresponsive to IL2 or IL4, and (2) anti-CD3 antibody-mediated unresponsiveness in the clones we have studied cannot consistently be mimicked by Ca + + ionophores with or without activators of protein kinase C (unpublished results).

Antigen-receptor-mediated activation of CD4+ T-cell clones The simplest experimental system for analysing the responses of T cells to stimuli delivered via the antigen receptor is to stimulate cloned T cells, devoid of accessory cells, with antibodies against the T-cell receptor or the CD3 complex. Both Th 1 and Th2 clones cultured with immobilized anti-CD3 antibody secrete their respective cytokines. Th2 clones also show good proliferative responses which are dependent on or augmented by the costimulatory cytokine, ILl (see below). In striking contrast, Thl clones exposed to anti-CD3 antibody not only fail to proliferate but are rendered incapable of proliferating in response to exogenously added IL2, their auto-

We are currently attempting to define in more detail the intracellular second messengers activated in these clones by receptormediated stimulation. In any event, these results suggest that exposure of the immune system to high concentrations of protein antigens presented by APC deficient in costimulators may lead to inactivation of IL2-producing cells and relative expansion of IL4-producing cells.

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

Responses of CD4 + T-cell clones to antigens presented by different APC Two o f the principal A P C for CD4+ T cells are macrophages and B lymphocytes. We have compared the ability o f these A P C populations to present antigens to established T h l and Th2 clones (T.L. Chang, C.M. Shea, R.C. Thompson, W . H . Boom and A.K. Abbas, submitted). Several features o f the T-cell responses are noteworthy. First, b o t h sets o f clones produce cytokines when cultured with specific antigen and either A P C population. In general, both T-cell types respond comparably to macrophages, but Th2 clones respond better to B cells than T h l clones. This may be because o f differences in costimulators be-

tween the two A P C populations. Alternatively, and more probably, T h l cells produce IFN T and lymphotoxin, which inhibit B-cell proliferation and may even lyse the B cells. Therefore, when 3timulated by B cells, T H 1 clones tend to kill the A P C , and this may limit T-cell activation (Tite and Janeway, 1984). Second, the two types o f T cells d i f f e r in t h e i r p r o l i f e r a t i v e respooses to antigens presented by different APC. In particular, Th2 clones proliferate weakly or not at all when stimulated with purified B cells, unless ILl is added. This result further supports the idea that ILl is a costimulator for the proliferation o f Th2 cells. As expected, a competitive antagonist o f I L l blocks the p r o l i f e r a t i v e responses o f Th2 clones only (fig. 1). The costimulator for T h l

Table I. ConA-stimulated changes in intracellular Ca + + in T-cell clones.

Clones

No.

ConA-induced increase in intracellular Ca + +

Thl

D1.1 D1.5 D1.6 B8 Gl1

11 7 11 I1 8

226 345 287 482 520

Th2

CDC25

10 3

14 429

CDC35

2 6

16 194

D10

6 2

18 171

4.7

9 2

41 244

Cloned T cells were loaded with Fura-2, stimulated at 37°C with ConA (5-10 ~tg/ml), and intracellular Ca ~÷ concentrations were measured in a fluorimeter. Results are expressed as arithmetic means of all determinations; No. = number of assays. ConA consistently induces an increase in intracellular Ca +* in all the Thl clones we have examined. In contrast, the responsesof Th2 clones are much more variable, showing no significant change in some experimentsand increasescomparable to the Th I clones in other experiments. The reason for this variability is unknown; it does not correlate with a detectable variability in the biologic response patterns of the T-cell clones. In all experiments, control antibodies (anti-CD4 or anti-CD45R) did not stimulate a change in intracellular Ca ++.

25

clones is not ILl ; nevertheless, it is clear that resvonses o f T h l clones are also dependent on costimulators that are expressed by both B lymphocytes and macrophages but whose nature is largely undefined (Schwartz, 1990). These results with cloned lines suggest that the expansion o f IL4-producing helper T cells in vivo may require I L l , which is secreted by activated m a c r o phages. This may be the reason why optimal primary antibody responses to protein antigens require immunization with adjuvants, which are potent macrophage-activating agents. On the other hand, some types o f immunization, such as contact sensitization with chemicals, may stimulate lower levels o f ILl p r o d u c t i o n and, consequently, relative expansion o f IL2-producing T cells that mediate delayed hypersensitivity reactions. These studies with established cloned lines have demonstrated numerous differences between Thl and Th2 clones with respect to activation requirement~ and response patterns (table II). We have already mentioned some o f the possible physiologic implications o f our findings. Such experimental analyses are feasible with cloned cell lines, because the cells can be purified and studied in the complete absence o f contaminating cell populations or in the presence o f particular types o f exogenously added accessory cells. However, results with cloned cell lines can be extrapolated to physiologic immune responses only to the extent that established clones resemble uncloned, heterogeneous lymphocytes. Moreover, it is not clear if cell populations equivalent to the T h l and Th2 clones exist in species other than mice, or in uncloned T cells even in mice. We have, therefore, initiated a series o f studies to define the cytokine profiles o f resting T lymphocytes and the conditions that induce their differentiation into p o p u l a t i o n s that produce distinct sets o f cytokines.

26

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

Differentiation of resting T lymphocytes T lymphocytes isolated from the spleens, lymph nodes or Peyer's patches of normal, unimmunized adult mice produce IL2 and IFNy but low or undetectable amounts of IL4 in response to receptor-mediated stimulation in vitro (Powers et al., 1988). In fact, IL4 mRNA can be detected using sensitive RNase protection assays (Tepper et al., 1990). If these T cells are repeatedly stL-nulated with antigens, they differentiate into cells that produce mostly IL2 or mostly IL4 (Powers et al., 1988).

Furthermore, if splenic T cells from unimmunized mice are fractionated into small (resting) and large (activated) populations, the small cells produce biologically active IL2 and IFNy but only the large cells produce IL4 as ,veil. Thus, IL4-producing T cells may arise or be expanded to detectable levels as a consequence of antigenic stimulation. Antigenic stimulation in vivo can be mimicked, at least partially, by activation with immobilized anti-CD3 antibody in vitro (Swain et al., 1988; S.K. Burke, P. Bossu, R.A. Miller, A.H. Lichtman and A.K. Abbas, in preparation).

Resting T cells stimulated with anti-CD3 antibody alone or with IL2 for 4 to 5 days develop into cells that produce IL2 and IFN-t" upon restimulation. In contrast, stimulation with anti-CD3 antibody plus IL4 promotes the development of IL4-producing cells. This same cell population also secretes IFNy. Thus, this in vitro stimulated T-cell population may consist of at least two subsets, one IL4-producing and the other IFN~,producing, or there may be only one population that secretes both cytokines and, therefore, does not belong to either the Thl or Th2 subset. Furthermore, the develop-

Table ]I. Patterns of lymphokine secretion and proliferation of T-cell clones. Thl Lymphokine secretion

Anti-CD3/ConA Anti-CD3/ConA+IL1 Inhibition by anti-CD3 Resting B cells + Ag Resting B cells+Ag+IL1 pl~t'~ =.,.. a_ T ,A.~ ~

Stimulus

Th2 Proliferation

Lymphokine secretion

Proliferation

+ + + + No

Yes

+ + + + No

+/+ + No

+ + + + + +

+ + + +

+ + + + +

+/+ + +

A. IL-2/IL-4S e c r e t i o n

B.

Proliferation

4.7 C

~- AR

D

C

3

5

~

1.. 91 D1.1/

o

2'0

4'0

6~

.

1~o Percentage

120 of

i

z

i

z

i

0

20

40

60

80

/

I O0

120

ControlResponse

Fig. 1. Inhibition of T-cell responses by an ILl receptor antagonist. Cloned T cells were stimulated with their specific antigens and irradiated syngeneic splenocytes as APC, with and without an ILl receptor antagonist that competitively inhibits binding of ILl to its receptor (Eisenberg et aL, 1990). Results are pooled from 4 experiments, and are expressed as responses with the antagonist as a percent of responses without the antagonist. Note that the proliferation of Th2 clones (hatched bars) but not of Th I clones (solid bars) is inhibited by the IL 1 receptor antagonist.

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

ment of IL4-producing T cells is not dependent on ILl (Burke et al., in preparation), indicating that ILl is a costimulator for established Th2 clones but may not be required for the differentiation of resting T cells to the Th2 phenotype. Taken together, these results suggest that IL4 itself is a powerful stimulus for the development of IL4-secreting CD4+ T cells. It is possible that in helminthic infections, IL4 produced by T cells or by mast cells or their precursors may provide an amplification mechanism for the expansion of this T-cell subset (Conrad et al., 1990). The relative frequencies of CD4 + T cells producing different cytokines may, therefore, reflect the types and quantities of antigens to which an individual is exposed, the duration of antigen exposure and the levels of cytokines at the sites of lymphocyte activation. Summary The studies summarized in this review have established that cloned

lines of CD4 ÷ T cells that produce distinct cytokines differ markedly in their responses to different forms of antigenic stimulation. Furthermore, we are beginning to develop experimental systems for better defining the signals that stimulate the differentiation of resting T ceils into functionally distinct subsets. From these studies it is possible to construct the following hypothetical model for the differentiation of mature CD4+ T cells (fig. 2). Resting cells produce IL2 as the principal growth factor, IFNy, and little or no IL4. Antigenic stimulation in the presence of IL4 (which may be produced by nonT cells) leads to the preferential expansion of IL4-producing cells. These cells secrete their cytokines maximally when stimulated with antigens presented by B cells, which are also the principal targets of these cytokines. Continued expansion of IL4-producing T cells may require antigen exposures that also stimulate the production of ILl by macrophages. In the absence of IL4 and ILl (and in the presence of costimulators that are

not yet defined) the T cells that are preferentially expanded belong to the IL2-producing subset. In addition, each subset may produce cytokines that stimulate the expansion of that subset and inhibit the other (Fiorentino et al., 1989). It is apparent that a number of assays and reagents need to be developed if these results are to be extended to physiologic immune responses First, it is important to identify surface molecules that may serve as phenotypic markers for functionally distinct subsets of CD4+ cells. Second, sensitive assays for detecting cytokine mRNA or production from single cells would be invaluable for defining the extent of heterogeneity in lymphocyte populations. Finally, techniques for detecting cytokineproducing cells in situ, Le. in lymphoid tissues, are essential for in vivo analyses. The application of such methods, based on the information gained from in vitro systems and cell lines, should provide a much clearer picture of the regulation of CD4 + T cells in physiologic and pathologic immune responses.

Thl

Antigen

/ /

.."~SIF - , ~(IL-IO) ,

/ 11.-4

\

~

Pml~ra~m

F..'Y~.~..

~ n

Mature T cell, ~ ('l"hO')

TM

27

IL-!

Fig. 2. Hypothetical model for the differentiation of resting T cells into Thl and Th2 subsets. Solid lines indicate stimulatory signals, and dashed lines indicate possible inhibitory pathways.

35th FORUM IN IMMUNOLOG Y

28 References

Bottomly, K. (1988), A functional dichotomy in CD4 ÷ T lymphocytes. hnmunoL Today, 9, 268-274. Cerottini, J.-C. & MacDonald H.R. (1989), The cellular basis of T-cell memory. Ann. Rev. lmmunoL, 7, 77-89. Conrad, D.H., Ben-Sasson, S.Z., LeGros, G., Finkelman, F.D. & Paul, W.E. (1990), Infection with Nippostrongylus brasiliensis or injection of anti-IgD antibodies markedl~ enhances Fc receptormediated interleukin 4 production by non-B, non-T cells. J. exp. Med., 171, 1497-1508. Eisenberg, S.P., Evans, R.J., Arend, W.P., Verderber, E., Brewer, M.T., Hannum, C.H. & Thompson, R.C. (1990), Primary structure and functional expression from complementary DNA of a human interleukin-I receptor antagonist. Nat,ire (Lond.), 343, 341-346. Fiorentino, D.F., Bond, M.W. & Mosmann, T.R. (1989), Two types of mouse helper T cell.-IV. Th2 clones secrete a factor that inhibits cytokine production by Thl clones. J. exp. Med., 170, 2081-2095. Gajewski, T.F., Schell, S.R., Nau, G.

& Fitch, F.W. (1989), Regulation of T-cell activation: differences among T cell subsets, lmmunol. Rev., 111, 79-110. Gajewski, T.F., Schell, S.R. & Fitch, F.W. (1990), Evidenc~ implicating utilization of different T cell receptor-associated signalling pathways by Th 1 and Th2 clones. J. Immunol., 144, 4110-4120. Mosmann, T.R. & Coffman, R.L. (1989), Thl and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann. Rev. lmmunoL, 7, 145-173. Powers, G.D., Abbas, A.K. & Miller R.A. (1988), Frequencies of IL-2 and IL-4 secreting T cells in naive and antigen-stimulated lymphocyte populations. J. lmmunol., 140, 3352-3357. Schwartz, R.H. (1990), A cell culture model for lymphocyte clonal anergy. Science, 248, 1349-1356. Street, N.E., Schumacher, J.H., Fong, T.A., Bass, H., Fiorentino, D.F., Levevah, 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., Wein-

berg, S.D. & Hancock, W. (1988), Characterization of T helper 1 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. lmmunoL, 141, 3445-3455. Tepper, R.I., Levinson, D.A., Stanger, B.Z., Campos-Torres, J., Abbas, A.K. & Leder, P. (1990), IL-4 induces ailergic-like inflammatory disease and alters T cell development in transgenic mice. Cell (in press). Tite, J.P. & Janeway, C.A., Jr. (1984), Cloned helper T cells can kill B lymphoma cells in the presence of specific antigen: Ia restriction and cognate vs. non-cognate interactions in cytolysis. Europ J. Immunom., 14, 878-883. Williams, I.R. & Unanue, E.R. (1990), Costimulatory requirements of murine Thl clones. The role of accessory cell-derived signals in responses to anti-CD3 antibody. J. ImmunoL, 145, 85-93. Williams, M.E., Lictman, A.H. & Abbas, A.K. (1990), Anti-CD3 antibody induces unresponsiveness to IL-2 in Thl clones but not in Th2 clones. J. ImmunoL, i44, 1208-1214.

Induction of Thl and Th2 CD4 + subsets during murine Leishmania major infection R . M . L o c k s l e y , F . P . H e i n z e l , B . J . H o l a d a y , S.S. M u t h a , S.L. Reiner and M . D . Sadick

University o f California San Francisco Medical Center, San Francisco, CA 94143 (USA)

Infection o f inbred strains o f mice with Leishmania major results in strain-specific reproduction of the spectrum of disease that occurs in humans with L. donova-

ni infections; self-limited cure occurs in most strains, such as C57BL/6 and C 3 H / H e N , whereas the B A L B / c mouse is unable to control infection and dies o f

progressive visceral disease that mimics human kala-azar. Infection in both resistant and susceptible strains o f mice is associated with activation o f CD4+ T lympho-