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Proliferation of human CD4+45R+ and CD4+45R- T helper cells is promoted by both IL-2 and IL-4 while interferon-gamma production is restricted to IL-2 activated CD4+45R- T cells
Immunology Letters, 20 (1989) 2 9 - 3 4 Elsevier IML 01147
Proliferation o f h u m a n C D 4 +45R + and C D 4 + 4 5 R - T helper cells is p r o m o t e d by both IL-2 and IL-4 while interferon-gamma production is restricted to IL-2 activated C D 4 + 4 5 R - T cells M. D o h l s t e n , G. H e d l u n d , H . Fischer, H . O. Sj6gren a n d R. C a r l s s o n l Department of TUmor Immunology, The Wallenberg Laboratory, University of Lund, Lund, Sweden; IBioinvent International AB, Lund, Sweden (Received 18 July 1988; revision received 23 September 1988; accepted 7 October 1988)
I. Summary Recombinant IL-2 (rlL-2) and IL-4 (rlL-4) promote proliferation of human CD4 + T cells activated in the presence o f PHA, TPA or OKT-3 monoclonal antibody (MAb), whereas the production o f interferon-gamma (IFN) can be induced only by rlL-2, rlL-4 induced strong proliferative responses both in accessory cell independent assays and in the presence of autologous monocytes, but has failed to induce IFN production in any of these systems. The ability o f rIL-2 to induce IFN production was strongly enhanced by the addition o f monocytes, although a similar proliferative response was recorded in the absence or presence of monocytes. The MAb anti-Tac inhibited the proliferative response and the production of IFN by CD4 + T cells ativated in the presence o f rIL-2, whereas the proliferative response to rIL-4 was unaffected. CD4+45R + and CD4+45R - T helper cell subsets proliferated in response to both IL-2 and IL-4. A kinetic analysis demonstrated that the production of IFN throughout a five day activation period was restricted to stimulation of CD4+45R - T cells with rIL-2. This report clearly demonstrates a dissociation o f IFN production and T cell proliferation in
Key words." 1L-2; IL-4; Interferon gramma production; T helper cell subsets
Correspondence to: M. Dohlsten, Dept. Tumor Immunology, The Wallenberg Laboratory, University of Lund, Lund, Sweden.
man. While proliferation can be induced by both IL2 and IL-4 in both the helper T cell subsets studied, IFN production was induced only in the CD4+45R - subsets and only in response to IL-2.
2. Introduction The activation of T cells leads to the production of several distinct lymphokines and to cell proliferation. Until recently, the interaction o f IL-2 with IL-2 receptor bearing T cells was considered to be required for induction o f DNA synthesis and production of IFN [1, 2]. However, recent studies have demonstrated that interleukin 4 (IL-4) can promote growth of both human and murine T cells [3-5]. Two different types of murine T helper cell clones (Trt) have been defined according to their profiles of lymphokine production [3-6]. The THI type produces IL-2, IFN and GM-CSF, whereas the T~a2 cell type produces IL-4, IL-5 and GM-CSF [3 - 5]. It has also been shown that TH2 cells can utilize IL-2 and IL-4 for cell proliferation, whereas TH1 cells only respond to IL-2 [5, 7]. The CD4+45R + cell subset has been reported to include cells with suppressor inducer function, whereas the CD4+45R cells possess helper inducer functions [8-10]. We recently reported that the human CD4+45R - cells are early II_-2 producers and the main producers of IFN after polyclonal activation, whereas CD4 + 45R + cells are late producers of IL-2 and lack the ability to produce IFN [11]. Recently, it has been implicated that human CD4 + 45R- T cells may represent mature antigen responding T helper inducer
0165-2478 / 89 / $ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
29
cells, whereas the CD4+45R ÷ cells appear to be an immature subset, which does not respond to recall antigens but may have the ability to differentiate into CD4÷45R - cells after activation [12, 13]. In this report we demonstrate that IL-2 and IL-4 promote proliferation of h u m a n CD4 + T helper cells. The proliferative response to IL-2 and IL-4 is about equal by both CD4+45R + and CD4+45R - cell subsets, but the production of IFN requires interaction of IL2 with IL-2 receptor bearing CD4+45R - T cells.
added to the T cells at a concentration of 1 x 105/ml.
3. Materials and methods
4. Results
3.3. Assays for IFN and DNA synthesis The IFN activity in culture supernatants was assessed in an antiviral microplaque reduction assay as described [15]. D N A synthesis was analyzed by [3H]thymidine incorporation as earlier described [15] after exposition of T lymphocytes for 2 4 - 96 h to various mitogens in 96-well microtiter plates.
3.1. Reagents. Phytohemagglutinin ( P H A ) and the phorbolester 4-phorbol-12-myristate 13 acetate (TPA) were purchased from Pharmacia (Uppsala, Sweden) and Sigma (St. Louis, MO, USA), respectively. Recombinant h u m a n II~2 and IL-4 were obtained from Sandoz (Vienna, Austria) and Genzyme (Boston, USA), respectively. MAbs Leu-3 PE (anti-CD4) and Leu-18-FITC (anti-CD45R) were obtained from Becton Dickinson Laboratories (Mountain View, CA, USA), whereas the MAb UCHL1 binding to the 180 kDa low molecular form of CD45 was obtained from Dakopatts (Copenhagen, Denmark). The antiTac monoclonal antibody, which reacts with and blocks the IL-2 receptor [14], was a kind gift from Dr. T. A. Waldman (NIH, Bethesda, Maryland, USA). 3.2. Cell separation and cultivation T lymphocytes were isolated from h u m a n blood mononuclear cells as earlier described [15]. For purification of CD4 ÷ T cells, the CD8 ÷ cells were removed by a negative panning selection technique [15]. The resulting CD4 ÷ cells were routinely about 95°70 pure. These cells could then be separated into CD45R + and C D 4 5 R - populations by the MAbs U C H L I and Leu-18 (anti-CD45R) employed in the negative panning procedure [11]. The purity of these CD45R ÷ and CD45R-- cells was analyzed in an E P I C S C cytofluorograph (Coulter Electronics, Hialeah, FL, USA) and was routinely about 95 °70. All tests on the purified CD4 + T-cells and T-cell subsets were performed at the concentration 1 × 106/ml in complete medium using 96-well plates (Nunc Roskilde, Denmark). Purified monocytes [15] were 30
Purified h u m a n CD4 + T helper ceils were stimulated with rlL-2 and rlL-4 in the presence of TPA and P H A , A strong proliferative response was induced by both rlL-2 and rlI_,4 (Fig. 1). Stimulating h u m a n CD4 ÷ T helper cells with rlL-2 and rlL-4 at concentrations 1- 300 U/ml in the absence o f TPA or PHA did not result in any significant D N A synthesis or production of IFN (data not shown). CD4 ÷ T helper cells stimulated with TPA responded to smaller amounts of rlL-4 than cells stimulated with P H A , whereas a reversed pattern was seen for rlI~2 (Fig. 1). A significant proliferative response was routinely detected with rlL-2 and rlL-4 at concentrations o f 1 0 - 30 U/ml, whereas a maximal proliferative response was observed at concentrations o f 100 U/ml. Activating CD4 ÷ T cells with TPA or P H A in the presence of rlL-2 resulted in a dosedependent induction of IFN whereas rlL-4 failed to induce IFN production (Fig. 1). rlL-2 induced significant amounts o f IFN at 10 U/ml and maximal amounts at 3 0 - I00 U/ml, whereas rlL-4 induced no IFN at concentrations of 1 0 - 5 0 0 U / m l (Fig. 1; Tables 1 and 2). We have earlier shown that the expression of CD45R distinguishes a functionally separate lymphokine producing CD4 + T helper cell subset [11]. The effects o f rlL-2 and rlL-4 on the two CD4 + subsets were further analyzed. TPA or P H A activated CD4÷45R ÷ and CD4÷45R - ceils responded with increased D N A synthesis to both rlL-2 and rlL4 (Table 1). The proliferative response of CD4÷45R + cells to rlL-2 and rlL-4 was routinely stronger than that o f CD4 + 4 5 R - cells. In contrast, the production of IFN was seen only in cultures of CD4+45R - cells stimulated with rlL-2 (Table 1). To elucidate whether the apparent inability of rlL-4 to
100
80
O
"=" PHA+rlL-2 "=- PHA+rlL-4
80 E
"~ TPA+dL-2
60
60
40 40 ~)
< z
20
20, 0
605O4O3OE
2O10-
'~
7
,
,
r
"•"
~30+
40.
PHA+rlL-2
i
0
0.1
i
30
"~ TPA+rlL-2
f
i
1
lO-1 i
1 Interleukin
10 (u/ml)
100
o!o
0.1
1
10
Interleukin
(u/ml)
100
Fig. I. D N A synthesis and IFN production of CD4 ÷ cells activated for 3 days with P H A (2.5 ~g/ml) or TPA (2 ng/ml) in the presence o f various concentrations o f rlL-2 and rlL-4. D N A synthesis was recorded in triplicate cultures by [3H]-thymidine incorporation and is expressed as m e a n c p m x 10 -3 +_ SEM. IFN production is expressed as units/ml. A representative experiment out of three performed is shown.
stimulate CD4+45R ÷ and C D 4 ÷ 4 5 R - cells to produce IFN was due to a difference in kinetics, we studied the production of IFN in supernatants obtained 24, 72 and 120 h after activation. The enhanced IFN activity in CD4 + and CD4+45R - cell cultures stimulated with OKT-3 and rIL-2 was seen already after 24 h and the peak o f I F N activity was seen after 72 h of culture, whereas cells stimulated with rIL-4 did not show any enhanced production of IFN during the entire culture period (Table 2). To analyze whether the proliferative effect of rlL-4 was related to a molecular interaction with IL-2 receptors or to the induction of an endogenous production of IL-2, we used the anti-Tac MAb to block functional IL-2 receptors on stimulated CD4 ÷ T cells. Anti-Tac completely blocked the proliferative response of 30 U / m l of rIL-2, whereas no effect was recorded on the proliferative response to 30 U / m l of rIL-4 (Table 3). Anti-Tac also blocked the production of IFN in cultures stimulated with
rlL-2 (data not shown). It has been demonstrated that monocyte derived accessory signals may be of importance in activation of murine T helper cells with rIL-4 [5, 7]. We therefore analyzed the effects of rIL-4 and rIL-2 on the proliferative response and the production of IFN by CD4 ÷ T helper cells activated in the presence or absence of monocytes, rIL-2 and rIL-4 induced a strong enhancement of the D N A synthesis by CD4 + T helper cells suboptimally activated with P H A and OKT-3 in the presence of monocytes or in the absence of monocytes by P H A and TPA. The ability of rlL-2 to induce production o f IFN was strongly enhanced in the presence of monocytes, whereas rIL-4 did not stimulate the production of IFN in the absence or presence of monocytes (Table 4). Addition of both rIL-2 and rIL-4 to activated T cells resulted in similar amounts of IFN as for stimulation with rIL-2 alone (Table 4).
31
TABLE 1
TABLE 2
D N A synthesis and IFN production by CD4+45R + and CD4 +45R - T cell subsets activated in the presence of IL-2 or IL-4.
Kinetics of IFN production by T cell subsets stimulated with rIL-2 or rlL-4. Additivea
Cells
CD4 +
CD4 + 45R +
CD4+45R -
Additivea
Days
Interferon production (U/ml)
IFN (U/ml)
DNA (cpm × 10- 3)
TPA +rlL-2 +rlL-4
< 10 75 <10
14 +_ l 95 ± 6 106 ± 8
OKT-3
PHA +rlL-2 +rlL-4
< 10 55 < 10
21 ± 2 229 ± 14 116 ± 9
+ rlL-2
TPA +rlL-2 +rlL-4
<10 <10 <10
13 ± 1 135 ± 6 114 _+ 5
+ rlL-4
PHA +rlL-2 +rIL-4
< 10 <10 < 10
1 ± 1 180 ± 5 72 +_ 5
aCD4 ÷ cells were stimulated with the OKT-3 MA b (dilution 10 -5) and 10% monocytes in the absence or presence of rlL-2 (250 U / m l ) and rlL-4 (500 U / ml ). One representative experiment out of two performed is shown.
TPA +rIL-2 +rIL-4
< 10 90 <10
3 ± 48 ± 42 ±
1 2 1
PHA +rlL-2 +rlL-4
< 10 85 < 10
17 ± 111 ± 61 ±
1 2 2
aThe cell subsets ( 2 × 1 0 5 / m l ) were stimulated with T P A (2 n g/ml), P H A (2.5/~g/ml), rlL-2 (100 U / m l ) and rlL-4 (100 U/ml). DNA synthesis ([3H]thymidine uptake, cpm ± SEM of triplicate determinants) and IFN production (U/ml) was measured after 72 h of culture. One representative experiment is presented out of three experiments performed.
5. Discussion
CD4 +
C D 4+45R +
1
11
< 10
3 5
20 12
< 10 < 10
10 60 17
10 36 26
48 350 170
1
100
3 5
200 150
1
5
3 5
14 5
<
<10
32
5
< 10 <10
25 10
4 even at concentrations of 1 0 0 - 5 0 0 U/ml, which provided optimal proliferation, did not induce any production of IFN. IL-4 failed to induce IFN production in accessory cell independent systems and in the presence of autologous monocytes as well. A further significant finding was that the I b 2 de-
TA B LE 3 Effect of anti-Tac MA b on rlL-2 and rlL-4 induced CD4 + T cell proliferation. Additivea
In this report we demonstrate that although both rlL-2 and rlL-4 can promote growth of h u m a n CD4 ÷ T helper cells, only rlL-2 can induce production of IFN. The inability of rlL-4 to stimulate IFN production by CD4 ÷ T cells was not due to any interference of rlL-4 with the biological activity of 1FN, since rlL-4 neither influenced the anti-viral activity of a standard preparation of recombinant IFN (data not shown), nor inhibited the production of IFN by CD4 ÷ T ceils activated by polyclonal activators and rlL-2. The production of IFN was enhanced by rlL-2 at concentrations as low as 1 0 - 30 U/ml, whereas rlL-
CD4+45R
TPA
DNA synthesis c p m x 10 3 Interleukin
anti-Tac
-
-
-
0.7
+ + + + +
rlL-2 rlL-2 rlL-4 rlL-4
+ +
3.6 16.0 2.1 15.2 14.9
±
_+ +_ _+ ± ±
0.2 0.5 2.0 0.2 0.6 0.2
aCD4 + T cells were stimulated with TP A (2 ng/ml) in the presence or absence of 30 U / m l of rlL-2 or rlL-4 and anti-Tac (ascites dilution 1/1000). DNA synthesis, recorded as [3HIthymidine uptake in triplicate cultures, was measured after 3 days of culture. Mean ± SEM in one representative experiment is shown out of three performed.
TABLE 4 Proliferation and IFN production of CD4 + T cells activated with rlL-2 and rlL-4 in the presence or absence of monocytes. Additivea
IFN (U/ml)
Inducer
Interleukin
P H A 2.5
+rlL-2 +rlL-4 +rlL-2 +rlL-4 + rlL-2 +rlL-4 +rlL-2/rlL-4 +rlL-2 +rlL-4 +rlL-2 +rlL-4 +rlL-2 +rlL-4 +rlL-2/rlL-4 +rlL-2 +rlL-4 +rlL-2/rlL-4
P H A 0.2 + Mo
P H A 0.2 + Mo
TPA
OKT-3 + Mo
OKT-3 + Mo
OKT-3 + Mo
<10 60 < 10 240 5150 170 335 2670 285 2750 < 10 42 <10 153 4064 115 160 1090 150 2750 55 380 50 390
DNA synthesis (cpm x 10- 3)
27 91 76 29 90 60 26 84 52 104 4 60 40 22 122 50 8 109 55 104 15 122 98 133
+ 5 +_ 7 ± 4 ± 2 + 2 + 1 ± 1 ___ 1 ± 1 ± 3 ± 1 ± 6 ± 9 ± 1 ±_ 3 ± 1 ± 1 ± 6 ± 1 ± 3 ± 1 ± 6 _+ 5 ± 3
aCD4+ T cells were stimulated in the presence of 10% monocytes (Mo) with P H A (2.5/~g/ml or 0.2 # g / m l ) , T P A (2 ng / ml ), OKT-3 MAb (10 -5 dilution), rlL-2 ( 1 0 0 - 2 5 0 U/ml) and rlL-4 ( 1 7 5 - 250 U / m l ) as indicated. D N A synthesis was recorded as [3H]thymidine incorporation (mean +_ SEM) in triplicate cultures after three days of culture.
pendent production o f IFN was strongly enhanced by addition of autologous monocytes, although the proliferative response o f CD4 ÷ T cells was stimulated by IL-2 to a similar extent in the presence or absence of monocytes. The accessory activity of the monocytes on IFN production could not be replaced by soluble IL-1 (data not shown), indicating that either cell membrane interactions or the presence of other monokines (e.g. TNF) are o f importance for the production o f IFN [16, 17]. It has been suggested that analysis o f IFN production is more sensitive than a proliferation assay as an indication o f human T cell activation [18]. However, in this report we clearly demonstrate a dissociation of
IFN production and T cell proliferation in man. We have earlier demonstrated a direct relationship between IL-2 stimulation and IFN production by human CD8 ÷ T cells [15]. This relationship is, however, not seen for IL-2 stimulated CD4+45R ÷ cells. A similar dissociation of IFN production and T cell proliferation has also been suggested in the murine system [5, 7, 19]. Both T u l and TH2 murine cell clones proliferate in response to exogenous IL-2, whereas IFN can only be induced in TH1 clones. In man, the expression of the CD45R molecule distinguishes a functionally discrete subset of T helper cells [8-11]. The CD4+45R - T cells are the main producers of IFN [11], which suggests that they include an analagous subset to the murine TH1 cells. In man both CD4+45R - and CD4+45R + cells proliferate in response to rlL-2 and rlL-4, whereas TH2 but not TH1 murine cell clones proliferate in response to exogenous IL-4 [5, 7]. The CD4+45R ÷ cells proliferate more efficiently to the two interleukins than the CD4+45R - cells, but the production of IFN is seen only in cultures of CD4÷45R - cells in response to IL-2. The inability o f IL-4 to induce IFN production in CD4 + T cell cultures despite its activating effects on proliferation suggests differences in the signal transduction system utilized by the two interleukins. An important role for phospholipase A 2 and arachidonic acid metabolites as possible second messengers for IFN production has recently been implicated [20]. It is tempting to speculate that the different responses of CD4 + T cells to IL-2 and IL-4 may reflect differences in the turnover of such second messengers. This possibility is presently being investigated in our laboratory.
Acknowledgements We thank Ms. Lena Hultman and Ms. Ingar Nilsson for excellent technical assistance and Ms. Inger Boketoft for skillful secretarial assistance. This work was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Society, the John and Augusta Persson's Foundation, the Medical Faculty University of Lund and the Swedish Society of Medicine.
References [1] CantreU, D. A. and Smith, K. A. (1984) Science 224, 1312.
33
[2] Lipkowitz, S., Greene, W. C., Rubin, A. L., Novogrodsky, A. and Stenzel, K. H. (1984) J. Immunol. 132, 31. [3] Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. and Coffman, R. L. (1986) J. Immunol. 136, 2348. [4] Lichtman, A. H., Kurt-Jones, E. A. and Abbas, A. K. (1987) Proc. Natl. Acad. Sci. USA 84, 824. [5] Janeway, C. A., Carding, S., Jones, B., Murray, J., Portolis, P., Rasmussen, R., Rojo, J., Saizawa, K., West, J. and Bottomly, K. (1988) Immunol. Rev. 101, 39. [6] Spits, H., Yssel, H., Takebe, Y., Arai, N., Yokota, T., Lee, E, Arai, K. I., Banchereau, J. and Devries, J. E. (1987) J. Immunol. 139, 1142. [7] Kurt-Jones, E. A., Hamberg, S., Ohara, J., Paul, W. E. and Abbas, A. K. (1987) J. Exp. Med. 166, 1774. [8] Morimoto, C., Letvin, N. L., Distaso, J. A., Aldrich, W. R. and Schlossman, S. E (1985a) J. Immunol. 134, 1508. [9] Takeuchi, T., DiMaggio, M., Levine, H., Schlossman, S. E and Morimoto, C. (1988) Cell. Immunol. 111, 398. [10] Kalish, R. S., Morimoto, C., and Schlossman, S. E (1988) Cell. Immunol. 111, 379.
34
[11] Dohlsten, M., Hedlund, G., Sj6gren, H. O. and Carlsson, R. (1988) Eur. J. lmmunol., in press. [12] Akbar, A. N., Terry, L., Timms, A., Beverly, E C. L. and Janossy, G. (1988) J. Immunol. 140, 2171. [13] Serra, H. M., Krowka, J. E, Ledbetter, J. A. and Pilarski, L. M. (1988) J. Immunol. 140, 1435. [14] Depper, J., Leonard, W., Robb, R., Waldman, T. and Greene, W. (1983) J. Immunol., 131, 690. [15] Dohlsten, M., Sj6gren, H. O. and Carlsson, R. (1986) Cell. lmmunol. 101, 493-501. [16] Scheurich, E, Thoma, B., Uzer, U. and Pfizenmaier, K. (1987) J. Immunol. 138, 1786. [17] Akiyama, Y., Zicht, R., Ferrone, S., Bonnard, G. D. and Herberrnann, R. B. (1985) Cell. Immunol. 91,477. [18] Hao, X.-S., Le, J., Vileck, J. and Chang, T. W. (1986) J. Immunol. Methods 92, 59-63. [19] Hecht, T. T., Longo, D. L. and Marls, L. A. (1983) J. lmmunol. 131, 1049. [20] Russel, J. K., Torres, B. A. and Johnson, H. M. (1987) J. Immunol. 139, 3442.
Proliferation o f h u m a n C D 4 +45R + and C D 4 + 4 5 R - T helper cells is p r o m o t e d by both IL-2 and IL-4 while interferon-gamma production is restricted to IL-2 activated C D 4 + 4 5 R - T cells M. D o h l s t e n , G. H e d l u n d , H . Fischer, H . O. Sj6gren a n d R. C a r l s s o n l Department of TUmor Immunology, The Wallenberg Laboratory, University of Lund, Lund, Sweden; IBioinvent International AB, Lund, Sweden (Received 18 July 1988; revision received 23 September 1988; accepted 7 October 1988)
I. Summary Recombinant IL-2 (rlL-2) and IL-4 (rlL-4) promote proliferation of human CD4 + T cells activated in the presence o f PHA, TPA or OKT-3 monoclonal antibody (MAb), whereas the production o f interferon-gamma (IFN) can be induced only by rlL-2, rlL-4 induced strong proliferative responses both in accessory cell independent assays and in the presence of autologous monocytes, but has failed to induce IFN production in any of these systems. The ability o f rIL-2 to induce IFN production was strongly enhanced by the addition o f monocytes, although a similar proliferative response was recorded in the absence or presence of monocytes. The MAb anti-Tac inhibited the proliferative response and the production of IFN by CD4 + T cells ativated in the presence o f rIL-2, whereas the proliferative response to rIL-4 was unaffected. CD4+45R + and CD4+45R - T helper cell subsets proliferated in response to both IL-2 and IL-4. A kinetic analysis demonstrated that the production of IFN throughout a five day activation period was restricted to stimulation of CD4+45R - T cells with rIL-2. This report clearly demonstrates a dissociation o f IFN production and T cell proliferation in
Key words." 1L-2; IL-4; Interferon gramma production; T helper cell subsets
Correspondence to: M. Dohlsten, Dept. Tumor Immunology, The Wallenberg Laboratory, University of Lund, Lund, Sweden.
man. While proliferation can be induced by both IL2 and IL-4 in both the helper T cell subsets studied, IFN production was induced only in the CD4+45R - subsets and only in response to IL-2.
2. Introduction The activation of T cells leads to the production of several distinct lymphokines and to cell proliferation. Until recently, the interaction o f IL-2 with IL-2 receptor bearing T cells was considered to be required for induction o f DNA synthesis and production of IFN [1, 2]. However, recent studies have demonstrated that interleukin 4 (IL-4) can promote growth of both human and murine T cells [3-5]. Two different types of murine T helper cell clones (Trt) have been defined according to their profiles of lymphokine production [3-6]. The THI type produces IL-2, IFN and GM-CSF, whereas the T~a2 cell type produces IL-4, IL-5 and GM-CSF [3 - 5]. It has also been shown that TH2 cells can utilize IL-2 and IL-4 for cell proliferation, whereas TH1 cells only respond to IL-2 [5, 7]. The CD4+45R + cell subset has been reported to include cells with suppressor inducer function, whereas the CD4+45R cells possess helper inducer functions [8-10]. We recently reported that the human CD4+45R - cells are early II_-2 producers and the main producers of IFN after polyclonal activation, whereas CD4 + 45R + cells are late producers of IL-2 and lack the ability to produce IFN [11]. Recently, it has been implicated that human CD4 + 45R- T cells may represent mature antigen responding T helper inducer
0165-2478 / 89 / $ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
29
cells, whereas the CD4+45R ÷ cells appear to be an immature subset, which does not respond to recall antigens but may have the ability to differentiate into CD4÷45R - cells after activation [12, 13]. In this report we demonstrate that IL-2 and IL-4 promote proliferation of h u m a n CD4 + T helper cells. The proliferative response to IL-2 and IL-4 is about equal by both CD4+45R + and CD4+45R - cell subsets, but the production of IFN requires interaction of IL2 with IL-2 receptor bearing CD4+45R - T cells.
added to the T cells at a concentration of 1 x 105/ml.
3. Materials and methods
4. Results
3.3. Assays for IFN and DNA synthesis The IFN activity in culture supernatants was assessed in an antiviral microplaque reduction assay as described [15]. D N A synthesis was analyzed by [3H]thymidine incorporation as earlier described [15] after exposition of T lymphocytes for 2 4 - 96 h to various mitogens in 96-well microtiter plates.
3.1. Reagents. Phytohemagglutinin ( P H A ) and the phorbolester 4-phorbol-12-myristate 13 acetate (TPA) were purchased from Pharmacia (Uppsala, Sweden) and Sigma (St. Louis, MO, USA), respectively. Recombinant h u m a n II~2 and IL-4 were obtained from Sandoz (Vienna, Austria) and Genzyme (Boston, USA), respectively. MAbs Leu-3 PE (anti-CD4) and Leu-18-FITC (anti-CD45R) were obtained from Becton Dickinson Laboratories (Mountain View, CA, USA), whereas the MAb UCHL1 binding to the 180 kDa low molecular form of CD45 was obtained from Dakopatts (Copenhagen, Denmark). The antiTac monoclonal antibody, which reacts with and blocks the IL-2 receptor [14], was a kind gift from Dr. T. A. Waldman (NIH, Bethesda, Maryland, USA). 3.2. Cell separation and cultivation T lymphocytes were isolated from h u m a n blood mononuclear cells as earlier described [15]. For purification of CD4 ÷ T cells, the CD8 ÷ cells were removed by a negative panning selection technique [15]. The resulting CD4 ÷ cells were routinely about 95°70 pure. These cells could then be separated into CD45R + and C D 4 5 R - populations by the MAbs U C H L I and Leu-18 (anti-CD45R) employed in the negative panning procedure [11]. The purity of these CD45R ÷ and CD45R-- cells was analyzed in an E P I C S C cytofluorograph (Coulter Electronics, Hialeah, FL, USA) and was routinely about 95 °70. All tests on the purified CD4 + T-cells and T-cell subsets were performed at the concentration 1 × 106/ml in complete medium using 96-well plates (Nunc Roskilde, Denmark). Purified monocytes [15] were 30
Purified h u m a n CD4 + T helper ceils were stimulated with rlL-2 and rlL-4 in the presence of TPA and P H A , A strong proliferative response was induced by both rlL-2 and rlI_,4 (Fig. 1). Stimulating h u m a n CD4 ÷ T helper cells with rlL-2 and rlL-4 at concentrations 1- 300 U/ml in the absence o f TPA or PHA did not result in any significant D N A synthesis or production of IFN (data not shown). CD4 ÷ T helper cells stimulated with TPA responded to smaller amounts of rlL-4 than cells stimulated with P H A , whereas a reversed pattern was seen for rlI~2 (Fig. 1). A significant proliferative response was routinely detected with rlL-2 and rlL-4 at concentrations o f 1 0 - 30 U/ml, whereas a maximal proliferative response was observed at concentrations o f 100 U/ml. Activating CD4 ÷ T cells with TPA or P H A in the presence of rlL-2 resulted in a dosedependent induction of IFN whereas rlL-4 failed to induce IFN production (Fig. 1). rlL-2 induced significant amounts o f IFN at 10 U/ml and maximal amounts at 3 0 - I00 U/ml, whereas rlL-4 induced no IFN at concentrations of 1 0 - 5 0 0 U / m l (Fig. 1; Tables 1 and 2). We have earlier shown that the expression of CD45R distinguishes a functionally separate lymphokine producing CD4 + T helper cell subset [11]. The effects o f rlL-2 and rlL-4 on the two CD4 + subsets were further analyzed. TPA or P H A activated CD4÷45R ÷ and CD4÷45R - ceils responded with increased D N A synthesis to both rlL-2 and rlL4 (Table 1). The proliferative response of CD4÷45R + cells to rlL-2 and rlL-4 was routinely stronger than that o f CD4 + 4 5 R - cells. In contrast, the production of IFN was seen only in cultures of CD4+45R - cells stimulated with rlL-2 (Table 1). To elucidate whether the apparent inability of rlL-4 to
100
80
O
"=" PHA+rlL-2 "=- PHA+rlL-4
80 E
"~ TPA+dL-2
60
60
40 40 ~)
< z
20
20, 0
605O4O3OE
2O10-
'~
7
,
,
r
"•"
~30+
40.
PHA+rlL-2
i
0
0.1
i
30
"~ TPA+rlL-2
f
i
1
lO-1 i
1 Interleukin
10 (u/ml)
100
o!o
0.1
1
10
Interleukin
(u/ml)
100
Fig. I. D N A synthesis and IFN production of CD4 ÷ cells activated for 3 days with P H A (2.5 ~g/ml) or TPA (2 ng/ml) in the presence o f various concentrations o f rlL-2 and rlL-4. D N A synthesis was recorded in triplicate cultures by [3H]-thymidine incorporation and is expressed as m e a n c p m x 10 -3 +_ SEM. IFN production is expressed as units/ml. A representative experiment out of three performed is shown.
stimulate CD4+45R ÷ and C D 4 ÷ 4 5 R - cells to produce IFN was due to a difference in kinetics, we studied the production of IFN in supernatants obtained 24, 72 and 120 h after activation. The enhanced IFN activity in CD4 + and CD4+45R - cell cultures stimulated with OKT-3 and rIL-2 was seen already after 24 h and the peak o f I F N activity was seen after 72 h of culture, whereas cells stimulated with rIL-4 did not show any enhanced production of IFN during the entire culture period (Table 2). To analyze whether the proliferative effect of rlL-4 was related to a molecular interaction with IL-2 receptors or to the induction of an endogenous production of IL-2, we used the anti-Tac MAb to block functional IL-2 receptors on stimulated CD4 ÷ T cells. Anti-Tac completely blocked the proliferative response of 30 U / m l of rIL-2, whereas no effect was recorded on the proliferative response to 30 U / m l of rIL-4 (Table 3). Anti-Tac also blocked the production of IFN in cultures stimulated with
rlL-2 (data not shown). It has been demonstrated that monocyte derived accessory signals may be of importance in activation of murine T helper cells with rIL-4 [5, 7]. We therefore analyzed the effects of rIL-4 and rIL-2 on the proliferative response and the production of IFN by CD4 ÷ T helper cells activated in the presence or absence of monocytes, rIL-2 and rIL-4 induced a strong enhancement of the D N A synthesis by CD4 + T helper cells suboptimally activated with P H A and OKT-3 in the presence of monocytes or in the absence of monocytes by P H A and TPA. The ability of rlL-2 to induce production o f IFN was strongly enhanced in the presence of monocytes, whereas rIL-4 did not stimulate the production of IFN in the absence or presence of monocytes (Table 4). Addition of both rIL-2 and rIL-4 to activated T cells resulted in similar amounts of IFN as for stimulation with rIL-2 alone (Table 4).
31
TABLE 1
TABLE 2
D N A synthesis and IFN production by CD4+45R + and CD4 +45R - T cell subsets activated in the presence of IL-2 or IL-4.
Kinetics of IFN production by T cell subsets stimulated with rIL-2 or rlL-4. Additivea
Cells
CD4 +
CD4 + 45R +
CD4+45R -
Additivea
Days
Interferon production (U/ml)
IFN (U/ml)
DNA (cpm × 10- 3)
TPA +rlL-2 +rlL-4
< 10 75 <10
14 +_ l 95 ± 6 106 ± 8
OKT-3
PHA +rlL-2 +rlL-4
< 10 55 < 10
21 ± 2 229 ± 14 116 ± 9
+ rlL-2
TPA +rlL-2 +rlL-4
<10 <10 <10
13 ± 1 135 ± 6 114 _+ 5
+ rlL-4
PHA +rlL-2 +rIL-4
< 10 <10 < 10
1 ± 1 180 ± 5 72 +_ 5
aCD4 ÷ cells were stimulated with the OKT-3 MA b (dilution 10 -5) and 10% monocytes in the absence or presence of rlL-2 (250 U / m l ) and rlL-4 (500 U / ml ). One representative experiment out of two performed is shown.
TPA +rIL-2 +rIL-4
< 10 90 <10
3 ± 48 ± 42 ±
1 2 1
PHA +rlL-2 +rlL-4
< 10 85 < 10
17 ± 111 ± 61 ±
1 2 2
aThe cell subsets ( 2 × 1 0 5 / m l ) were stimulated with T P A (2 n g/ml), P H A (2.5/~g/ml), rlL-2 (100 U / m l ) and rlL-4 (100 U/ml). DNA synthesis ([3H]thymidine uptake, cpm ± SEM of triplicate determinants) and IFN production (U/ml) was measured after 72 h of culture. One representative experiment is presented out of three experiments performed.
5. Discussion
CD4 +
C D 4+45R +
1
11
< 10
3 5
20 12
< 10 < 10
10 60 17
10 36 26
48 350 170
1
100
3 5
200 150
1
5
3 5
14 5
<
<10
32
5
< 10 <10
25 10
4 even at concentrations of 1 0 0 - 5 0 0 U/ml, which provided optimal proliferation, did not induce any production of IFN. IL-4 failed to induce IFN production in accessory cell independent systems and in the presence of autologous monocytes as well. A further significant finding was that the I b 2 de-
TA B LE 3 Effect of anti-Tac MA b on rlL-2 and rlL-4 induced CD4 + T cell proliferation. Additivea
In this report we demonstrate that although both rlL-2 and rlL-4 can promote growth of h u m a n CD4 ÷ T helper cells, only rlL-2 can induce production of IFN. The inability of rlL-4 to stimulate IFN production by CD4 ÷ T cells was not due to any interference of rlL-4 with the biological activity of 1FN, since rlL-4 neither influenced the anti-viral activity of a standard preparation of recombinant IFN (data not shown), nor inhibited the production of IFN by CD4 ÷ T ceils activated by polyclonal activators and rlL-2. The production of IFN was enhanced by rlL-2 at concentrations as low as 1 0 - 30 U/ml, whereas rlL-
CD4+45R
TPA
DNA synthesis c p m x 10 3 Interleukin
anti-Tac
-
-
-
0.7
+ + + + +
rlL-2 rlL-2 rlL-4 rlL-4
+ +
3.6 16.0 2.1 15.2 14.9
±
_+ +_ _+ ± ±
0.2 0.5 2.0 0.2 0.6 0.2
aCD4 + T cells were stimulated with TP A (2 ng/ml) in the presence or absence of 30 U / m l of rlL-2 or rlL-4 and anti-Tac (ascites dilution 1/1000). DNA synthesis, recorded as [3HIthymidine uptake in triplicate cultures, was measured after 3 days of culture. Mean ± SEM in one representative experiment is shown out of three performed.
TABLE 4 Proliferation and IFN production of CD4 + T cells activated with rlL-2 and rlL-4 in the presence or absence of monocytes. Additivea
IFN (U/ml)
Inducer
Interleukin
P H A 2.5
+rlL-2 +rlL-4 +rlL-2 +rlL-4 + rlL-2 +rlL-4 +rlL-2/rlL-4 +rlL-2 +rlL-4 +rlL-2 +rlL-4 +rlL-2 +rlL-4 +rlL-2/rlL-4 +rlL-2 +rlL-4 +rlL-2/rlL-4
P H A 0.2 + Mo
P H A 0.2 + Mo
TPA
OKT-3 + Mo
OKT-3 + Mo
OKT-3 + Mo
<10 60 < 10 240 5150 170 335 2670 285 2750 < 10 42 <10 153 4064 115 160 1090 150 2750 55 380 50 390
DNA synthesis (cpm x 10- 3)
27 91 76 29 90 60 26 84 52 104 4 60 40 22 122 50 8 109 55 104 15 122 98 133
+ 5 +_ 7 ± 4 ± 2 + 2 + 1 ± 1 ___ 1 ± 1 ± 3 ± 1 ± 6 ± 9 ± 1 ±_ 3 ± 1 ± 1 ± 6 ± 1 ± 3 ± 1 ± 6 _+ 5 ± 3
aCD4+ T cells were stimulated in the presence of 10% monocytes (Mo) with P H A (2.5/~g/ml or 0.2 # g / m l ) , T P A (2 ng / ml ), OKT-3 MAb (10 -5 dilution), rlL-2 ( 1 0 0 - 2 5 0 U/ml) and rlL-4 ( 1 7 5 - 250 U / m l ) as indicated. D N A synthesis was recorded as [3H]thymidine incorporation (mean +_ SEM) in triplicate cultures after three days of culture.
pendent production o f IFN was strongly enhanced by addition of autologous monocytes, although the proliferative response o f CD4 ÷ T cells was stimulated by IL-2 to a similar extent in the presence or absence of monocytes. The accessory activity of the monocytes on IFN production could not be replaced by soluble IL-1 (data not shown), indicating that either cell membrane interactions or the presence of other monokines (e.g. TNF) are o f importance for the production o f IFN [16, 17]. It has been suggested that analysis o f IFN production is more sensitive than a proliferation assay as an indication o f human T cell activation [18]. However, in this report we clearly demonstrate a dissociation of
IFN production and T cell proliferation in man. We have earlier demonstrated a direct relationship between IL-2 stimulation and IFN production by human CD8 ÷ T cells [15]. This relationship is, however, not seen for IL-2 stimulated CD4+45R ÷ cells. A similar dissociation of IFN production and T cell proliferation has also been suggested in the murine system [5, 7, 19]. Both T u l and TH2 murine cell clones proliferate in response to exogenous IL-2, whereas IFN can only be induced in TH1 clones. In man, the expression of the CD45R molecule distinguishes a functionally discrete subset of T helper cells [8-11]. The CD4+45R - T cells are the main producers of IFN [11], which suggests that they include an analagous subset to the murine TH1 cells. In man both CD4+45R - and CD4+45R + cells proliferate in response to rlL-2 and rlL-4, whereas TH2 but not TH1 murine cell clones proliferate in response to exogenous IL-4 [5, 7]. The CD4+45R ÷ cells proliferate more efficiently to the two interleukins than the CD4+45R - cells, but the production of IFN is seen only in cultures of CD4÷45R - cells in response to IL-2. The inability o f IL-4 to induce IFN production in CD4 + T cell cultures despite its activating effects on proliferation suggests differences in the signal transduction system utilized by the two interleukins. An important role for phospholipase A 2 and arachidonic acid metabolites as possible second messengers for IFN production has recently been implicated [20]. It is tempting to speculate that the different responses of CD4 + T cells to IL-2 and IL-4 may reflect differences in the turnover of such second messengers. This possibility is presently being investigated in our laboratory.
Acknowledgements We thank Ms. Lena Hultman and Ms. Ingar Nilsson for excellent technical assistance and Ms. Inger Boketoft for skillful secretarial assistance. This work was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Society, the John and Augusta Persson's Foundation, the Medical Faculty University of Lund and the Swedish Society of Medicine.
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33
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