IL-2 secretion by soluble antigen-reactive human T-cell clones

CELLULAR

IMMUNOLOGY

IL-2 Secretion

77, 363-371 (1983)

by Soluble Antigen-Reactive

Human T-Cell Clones

JAMES BURNS,’ ANTHONY ROSENZWEIG,’ BURTON ZWEIMAN, AND ROBERT P. LEAK Department of Neurology and the Allergy and Immunology Section of the Department of Medicine, University of Pennsylvania School of Medicine, Multiple Sclerosis Research Center of the University of PennsylvaniaWistar Institute, Philadelphia, Pennsylvania 19104 Received

October

26, 1982; accepted

January

27, 1983

Humantetanus toxoid specific T-cell lines and clones capable of producing IL-2 were established. IL-2 production occurred only when the antigen-specific T cells were cultured with both tetanus toxoid antigen and an autologous, irradiated adherent cell population. The T-cell lines and clones remained strictly dependent on exogenous IL-2 for proliferation at all other times. Phenotypic characterization with monoclonal antibodies recognizing T-cell subsets revealed that the antigen-specific lines and clones bore predominantly OKT3 and OKT4 markers with essentially no OKT8 positive cells present. T-cell clones which were demonstrated to secrete IL-2 activity could also partially deplete media of IL-2 if cultured in the absence of soluble antigen and irradiated adherent cells. INTRODUCTION

The ability of T-cell growth factor, also called interleukin 2 (1L-2),3 to maintain long-term cultures of proliferating human and murine T cells provides a method for obtaining homogeneous populations of cloned, antigen-activated T cells for study of phenotypic and functional characteristics ( l- 12). Cloned IL-Zdependent human Tcell lines as previously described by other investigators often have maintained the antigen-specific responses of the parent cell line during the long-term cultures (612). Most investigators have found that antigen-reactive human T-cell lines or clones continue to require MHC-restricted antigen presenting cells (APC) in addition to the appropriate antigenic stimulus to maintain proliferation when cultured in the absence of added IL-2 (10, 12- 14). Autologous, T-cell-depleted blood mononuclear cells can serve as APC in such reactions (12, 14, 15). Since IL-2 is secreted by T cells (1618), it may be that the antigen-induced responses of these sensitized T-cell clones include the production of sufficient IL-2 to maintain their proliferation. This report presents the results of experiments designed to determine whether antigen-induced proliferation of human T-cell lines and clones results in IL-2 pro’ Address all correspondence to Dr. James B. Bums, Department of Neurology, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, Penn. 19 104. * Current address: Harvard School of Medicine, Boston, Mass. 3 Abbreviations: IL-2, interleukin 2; APC, antigen presenting cells; TT, tetanus toxoid, SKSD, streptokinase-streptodomasese;PHA, phytohemagglutinin; Adh, adherent cells from peripheral blood mononuclear cells; PBM, peripheral blood mononuclear cells; CTC, cultured T cells; MHC, major bistocompatibility. 363 0008-8749183 $3.00 Copyright D 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.

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duction. We have confirmed prior observations that antigen-specific T-cell lines and T-cell clones will proliferate when the proper antigen is presented by autologous Tcell-depleted APC. In addition, we have found that the supernatant from cultures of antigen-stimulated T cells does contain IL-2 activity. MATERIALS

AND

METHODS

Lymphocyte and adherent cell preparation. Peripheral blood mononuclear cells (PBM) were isolated from a normal donor by Ficoll/Hypaque gradients (Pharmacia Fine Chemicals, Piscataway, N.J.) in the standard manner (19) and washed three times. For isolation of adherent cells, the method of Kumagai et al. was followed (20). The average yield of adherent cells was 4 to 5% of the initial PBM sample with the adherent cells consisting of less than 1% E-rosetting lymphocytes. In immunofluorescent studies, less than 1% of the adherent cells bore the T-cell markers OKT3 and OKT4, while 93% bore the OKMI marker thought to label predominantly monocyte/macrophage (2 1, 22) (mean of three experiments). IL-2 production. A mitogen-depleted IL-2-containing media was prepared by a modification of the method previously reported (23). For convenience, this is referred to as reference IL-2. Briefly, PBM were cultured in supplemented RPM1 1640 (GIBCO, Grand Island, N.Y.) (with penicillin (100 units/ml), streptomycin ( 100 pg/ml), 2 mA4 glutamine, 1% nonessential amino acids (GIBCO), and 1 mM sodium pyruvate (GIBCO)) containing 1% pooled human serum at 2.5 X 106/ml at 37°C in upright flasks (Falcon, Division of Becton Dickinson Co., Oxnard, Calif.) containing 100 ml cell suspensions. The flasks were incubated 18 to 24 hr at 37°C in 5% COz/air without mitogen. The cells were then irradiated (1800 R) and pulsed with phytohemagglutinin (PHA) (Lot K8000, Burroughs-Wellcome, Beckenham, England) in a final concentration of 2 pg/ml for 6 hr. The cells were then washed two times and resuspended at approximately 2 X 106/ml in supplemented RPM1 with 1% pooled human serum for an additional 48 hr prior to harvesting of supernatants. Each batch of supernatants was assayed for the optimal concentration which yielded maximal IL-2 activity using the method of Gillis et al. (24). IL-2 assay. IL-2 activity was functionally defined as the ability of a test supernatant to maintain the proliferation of T cells which were otherwise dependent for growth upon the IL-2 activity of our reference IL-2. The assay method is similar to that reported by Gillis et al. (24). Ten thousand IL-Zdependent T cells were incubated in 100 ~1 of media containing 50% of the test supernatant or 10% of the reference IL-2. After 44 hr, tritiated thymidine ([3H]Tdr; New England Nuclear, Boston, Mass.), 1 &i/well was added for the final 4 hr of culture and isotope incorporation measured by liquid scintillation spectroscopy (Beckman Instruments LSSOOO). A quantitative assay of IL-2 activity was also performed by assessment of the [3H]Tdr incorporation by IL-2-dependent cultured T cells incubated in serial dilutions of test supematants or reference IL-2 (24). The isotope incorporation induced by reference IL-2 was assigned an activity of 10 units. In preliminary studies it was determined that as little as 0. I units of IL-2 activity induced statistically significant proliferation. Induction of antigen-specijc T-cell lines and clones. Preliminary experiments with PBM from a normal donor revealed a significant in vitro proliferative response to both partially purified tetanus toxoid (TT) (Lot LP445PR, Commonwealth of Massachusetts Department of Public Health, Boston, Mass.) and streptokinase-strepto-

T-CELL

CLONES

PRODUCING

IL-2

365

dornase (SKSD) (Varidase, Lederle Lab, Pearl River, N.Y.). PBM from this donor were cultured for 5 days at 1.5 X 106/ml in supplemented RPM1 with 10% autologous serum and tetanus toxoid at a final concentration of 0.5 Lf units/ml. Following this culture the proliferating lymphoblasts were recovered on a Ficoll/Hypaque gradient, washed, and resuspended at 1 X lo5 cells/ml in supplemented RPM1 with 10% fetal calf serum (FCS) (Microbiological Associates, Walkersville, Md.) and an optimal concentration of reference IL-2. Proliferating cultures were maintained for approximately 2 weeks before cloning was performed. After this initial 2 weeks, all antigenspecific cell lines and clones were maintained in culture for up to 4 months by expansion in two sequential steps similar to those described by Sredni et al. ( 10). Briefly, antigen-specific cell lines were maintained in IL-2 containing media alone for approximately 2 weeks with frequent refeeding and subculture as needed. When proliferation under these conditions slowed, the cultured T cells (CTC) were washed and 2 X lo5 CTC were placed in 2.5 ml of supplemented RPM1 containing (a) 10% autologous serum, (b) 5 X lo6 autologous, irradiated (2500 R) PBM, and, (c) an optimal concentration of tetanus toxoid. Cultures were continued for 3 days and responding cells were placed again in culture with IL-2 until it was necessary to repeat the above procedure. After approximately 4 months in culture the T-cell lines and clones became unresponsive to both IL-2 and antigen stimulation and could not be continued. Cloning was performed by limiting dilution of TT responsive lymphoblasts with (a) 0.2 ml of supplemented RPM1 + 10% autologous serum, (b) 2 X 10’ freshly isolated, 2500 R irradiated autologous PBM, and (c) TT at 0.5 Lf units/ml. No exogenous IL-2 was provided for the first 3 days of culture. Ninety-six wells were seeded with an average of 0.5 cells per well. At 3-day intervals, all wells were refed with IL-2-containing media until cell growth was noted, generally after 7 to 10 days. Clones were expanded from this time by culture with irradiated feeder cells and antigen or IL-2-containing media alone as described above. Subcloning was performed in an identical manner. Two subclones, 4E and 45, were chosen for study. Antigen specijicity assay of cultured T cells. T-cell lines and clones were examined for antigen-specific proliferation in a 48-hr assay by measurement of [3H]Tdr incorporation. Before use in these antigen-specific proliferation assays, cultures of T cells were maintained in IL-2 alone, without additional feeder cells or antigen, for 7 to 10 days. Fifteen thousand cultured T cells were incubated in 0.2 ml of supplemented RPM1 with 10% autologous serum, 1 X 1O5irradiated fresh PBM, or 2 X 1O4irradiated adherent cells, and antigen or mitogen. [3H]Tdr (1 &i/well) was added for the final 4 hr of culture and isotope incorporation measured by liquid scintillation spectroscopy. Supernatant production. Supernatants were obtained for assay of IL-2 activity by culture of 1 X lo6 antigen-specific cultured T cells (lines, clones, or subclones) in 1 ml of supplemented RPM1 with 10% autologous serum and adherent cells obtained from 5 X lo6 irradiated, freshly collected PBM. Antigen was added at optimal concentrations and supernatants collected for assay after 24 and/or 48 hr culture at 37°C and 5% COz/air. Cells were removed by centrifugation and supernatants stored at 4°C until use. Supematants were assayed for IL-2 activity as described above. Depletion of IL-2 activity by T-cell clones. Cloned T cells recovered from the 24 hr cultures used to study IL-2 production were washed two times and resuspended at 1 X 106/ml in supplemented RPM1 containing a known amount of reference IL2. Control cultures containing no cells were maintained in parallel. Following culture

366

BURNS ET AL.

for 24 hr at 37°C and 5% COz, the supernatant was collected and the IL-2 activity remaining in the media was measured as described above. Surface marker phenotype of cultured cells. Surface marker characteristics of cultured T cells and adherent cell populations were determined as previously described by rosetting with neuraminidase-treated sheep RBC (23) for T cells and by indirect immunofluorescence for T-cell subsets using monoclonal antibodies OKT3, OKT4, OKT8, OKIa, and OKMI (Ortho Pharmaceutical Co., Raritan, N.J.) as previously described (21, 22, 25). RESULTS Antigen-SpeciJic T Cells and IL-2 Production

Uncloned T-cell lines responding to TT were isolated from PBM. Cloning and subcloning of the TT-reactive T cells was performed by limiting dilution with a cloning efficiency of approximately 20%. This two step limiting dilution cloning provides a probability of approximately 95%, by Poisson distribution analysis, that each subclone represents the progeny of a single T cell. Subclones designated 4E and 45 were chosen for study. Table 1 shows the antigenic specificity of these cloned and uncloned cells following l-3 months of culture. Proliferation occurred only when the cultured T cells were incubated with irradiated antigen presenting cells (APC) and tetanus toxoid. The T-cell clones responded equally well whether “unseparated” PBM or T-cell-depleted adherent cells were present as the irradiated APC. Since this proliferation occurred in the absence of added IL-2, we have assayed supernatants for IL-2 activity from cultures containing TT-reactive T cells, tetanus toxoid antigen, and irradiated adherent cells. A T-cell-depleted adherent cell population was chosen for use as antigen presenting cells in order to lessen the possibility of any participation by IL-2-producing T cells in the freshly obtained whole PBM. TABLE 1 Antigen Specificity of T-Cell Lines and T-Cell Clones” Culture conditions Medium alone TT alone PBM’ alone Adh d alone Adh + SKSD Adh + TT PBM + TT

TT-CTC 137 111 4,313 4,Ol I 3,606 78,950 97,042

Subclone 4E

Subclone 45

141 NDb 449 394 191 14,271 15,378

112 ND 177 462 207 37,648 34,703

a A T-cell line (TT-CTC) and two T-cell clones (4E and 45) were maintained for 3 months in continuous culture as described under Methods. Ten thousand T cells were cultured with (I) tetanus toxoid (TT), (2) autologous irradiated PBM (1 X lO’/microwell) or irradiated Adh (2 X 104/microwell) as APC, (3) PBM or Adh with either TT or streptokinase-streptodomase (SKSD). Stimulation was assessedby incorporation of [‘Hlthymidine. Values represent mean counts per minute (cpm) of triplicate cultures. SEM were omitted for simplicity but rarely were greater than 15% of the mean cpm. b ND, Not determined. ’ PBM, Irradiated peripheral blood mononuclear cells (1 X 105/well). d Adh, Irradiated adherent cells (2 X 104/well).

T-CELL

l&e~tyupernotont

Medium

CLONES PRODUCING

IL-2

Actwityo(3H-;$ 5

367

IL-2

Inc;;poro~~;

CP;

x I”;:)

Alone

Adh

+

TT

CTC

+

Adh

CTC

+

Adh

+ TT-

24hrs.

CTC

+

Adh

+ TT-

48hrs.

Reference

I L- 2

FIG. I. IL-2 production by a tetanus toxoid (TT) reactive uncloned T-cell line. Test supematants were generated by 24 or 48 hr culture of I X IO6 TT-reactive T cells (CTC) in 1 ml of supplemented RPM1 1640 with 10% autologous serum and (I) irradiated adherent cells (Adh) alone, or (2) adherent cells with TT antigen. Supematant from adherent cells cultured with TT alone without CTC present was also tested. IL-2 activity was determined by assessing [‘Hlthymidine incorporation by an IL-2-dependent T-cell line (1 x lo4 T cells/microwell) following 48 hr culture in (1) medium alone, (2) 50% (v/v) of the test supernatants, or (3) reference IL-2. SKSD-reactive, IL-Zdependent T cells were employed as target cells for IL2 activity. Values represent mean counts per minute + SEM of triplicate cultures.

Figure 1 shows the results of preliminary kinetic and antigen specificity studies. The TT reactive cell line used in this experiment was not a cloned cell line. Supematants taken after 24 and 48 hr from cultures containing TT-reactive T cells, irradiated adherent cells, and tetanus toxoid antigen were able to support the proliferation of IL-2-dependent T cells. No IL-2 activity was detected in control cultures containing either the cultured T cells and APC without antigen, or the APC plus antigen without cultured T cells. The IL-2 activity was greater after 24 than 48 hr of culture. Preliminary observations confirmed that thymidine incorporation paralleled cell growth for both reference IL-2 and antigen-induced supematants (data not shown). This cell line was maintained for 3 months before being lost due to contamination. A second tetanus-reactive uncloned population was initiated and displayed a similar IL-2-producing capability (data not shown). This second tetanus-reactive T-cell line was then cloned and subcloned yielding subclones 4E and 45. These subclones were assayed for antigen response (Table 1) and the ability to produce supematant IL-2 activity. Table 2 shows the results of assays for IL-2 activity of supematants generated by these two subclones in the presence of APC and either an inappropriate soluble antigen (SKSD), or tetanus toxoid. The target cell population used to assay IL-2 activity was an IL-2 dependent, PHA-initiated T-cell line from a second unrelated normal donor (HLA-D nonidentity was confirmed by MLR, data not shown). Each subclone yielded supernatants with IL-2 activity as documented by proliferation of the T cells used to assay IL-2 activity. In this culture system, subclone 45 displayed both greater IL-2 production and greater proliferation than 4E. Supematants from parallel cultures containing APC, tetanus reactive T cells, and SKSD showed no IL2 activity.

368

BURNS ET AL. TABLE 2 IL-2 Production by Cloned T-Cell Lines Test supematant source”

IL-2 activityb

Medium alone Reference IL-2 4E + TT + Adh 4E + SKSD + Adh 45 + TT + Adh 45 + SKSD + Adh TT + Adh

2,679 16,432 10,448 2,366 20,119 3,081 2,921

+ 155 f 2,118 f 502 + 163 f 959 f 126 k 154

Units of IL-2 activity’ 0 10 1.1 0 1.6 NDd 0

’ Supematants were generated by 24 hr culture of 1 X lo6 tetanus toxoid (TT) reactive subclones 4E and 45 in I ml of supplemented RPM1 with 10% autologous serum and irradiated adherent cells (Adh) and either (I) SKSD antigen, or (2) tetanus toxoid antigen. Supematant from adherent cell cultures with antigen alone without cloned T cells present was also tested. * IL-2 activity was determined as in Fig. 1. The IL-2-dependent T cells were PHA initiated T cells from a normal donor. T cells (1 X 104) were cultured in (I) 50% of test supematant, or (2) medium alone, or (3) 10% reference IL-2. Values represent mean cpm of triplicate cultures f SEM. cUnits of IL-2 activity were determined by comparing the IL-2 activity of serial dilutions of test supernatants and reference IL-2. Reference IL-2 is arbitrarily assigned an activity of 10 units. d ND, Not determined.

Depletion of IL-2 Activity by Cloned T Cells In order to confirm our impression that these subclones which produced IL-2 could also deplete media of IL-2, the subcloned cells taken from the cultures generating IL-2 were placed in media with a known amount of reference IL-2 activity present. After 24 hr of culture, the IL-2 activity remaining in the supernatant was measured. Table 3 demonstrates the partial depletion of IL-2 activity by those subclones which in the prior 24 hr had produced IL-2. TABLE 3 IL-2 Depletion by Cloned Cell Lines Supematant source’

IL-2 activity *

1. Medium alone 2. Reference IL-2 3. Control well containing Reference IL-2 without cloned cells present 4. Reference IL-2 + clone 45 5. Reference IL-2 + clone 4E

2,791 + 348 10,044 * 451 11,138 f 96 4,312 + 167 4,767 f 145

’ Supplemented RPM1 containing 10% reference IL-2 was incubated for 24 hr with clone 4E or 45 (1 X lo6 cells/ml) at 37°C in 5% CO*. Cells were removed by centrifugation and the supematant was assayed for remaining IL-2 activity. A control well was maintained in parallel under identical conditions at 37°C but without any T cells present (Line 3). Freshly thawed Reference IL-2 was simultaneously assayed (Line 2). * IL-2 activity was determined as described in Fig. 1. The IL-2-dependent T cells used were PHA-initiated T cells as described in Table 2. Values represent mean cpm + SEM for duplicate cultures.

T-CELL

CLONES

PRODUCING

369

IL-2

OKT Phenotype of Cultured T Cells As reported by other investigators (9, 14) we found that all T-cell lines and clones responsive to a soluble antigen were composed predominantly of OKT4+ cells with no OKT8+ cells noted in the cloned populations (Table 4). The percentage of these cloned cells bearing the DR marker varied, but was generally greater than 50%. DISCUSSION The results presented in this report indicate that cloned, tetanus toxoid-reactive, human T cells produce IL-2 when cultured with tetanus toxoid antigen and autologous antigen-presenting cells. Irradiated adherent cells, consisting of less than 1% T cells, were used as antigen-presenting cells for IL-2 production. Since IL-2 has been shown to be a T-cell product (16- 18) we believe that the TT-reactive T cells are the source of the IL-2 activity recovered from these cultures. Neither IL-2 production nor proliferation occurred unless the cloned T cells were cultured with both the correct soluble antigen and antigen-presenting cells. The T-cell clones examined were OKT3+, 4+, and 8-, consistent with previous reports that T cells responding to soluble antigens are primarily OKT4+ (26) and that the OKT3+, 4+, 8- T-cell subset is capable of IL2 production (27). Our findings that IL-2-dependent T cells will also produce IL-2 differ somewhat from results of studies using mitogen-stimulated mm-me spleen cells (28) or neoplastic human T-lymphocytes (29). These previous studies concluded that cells producing IL-2 in response to mitogen stimulation do not themselves respond to IL-2 and thus are not present in long-term cultures of T cells (28), or die after being stimulated by mitogen to produce IL-2 (29). This finding may be valid for some IL-2-producing T-cell subpopulations. However, our T-cell lines may be different in several respects: (a) the T-cell lines were selected initially by their response to antigen and not mitogen; (b) our culture methods may have selected those T cells which proliferate in media containing either IL-2 or, alternately, antigen with APC, and (c) our reference IL-2 used for propagation of the T cells was mitogen depleted. It has been suggested that long-term exposure to mitogen may have deleterious effects on cultured T cells (30). However, Palacio (27) has recently described a spontaneously proliferating, and thus IL-2-independent, T-cell line derived from normal PBM which will produce IL-2 TABLE OKT

Tetanus-CTC Subclones 4E 45

Phenotype

4

of Antigen-Reactive

Cell Lines

and Clones”

OKT3 (%)

OKT4 @)

OKT8 (%I

DR (%)

99

100

2

NDb

83 98

89 94

0 0

90 14

’ Cells (1 X 106) were stained with mouse anti-T-cell subset hybridoma conjugated anti-mouse IgG. Between 100 and 200 cells were examined. ’ ND, Not determined.

antibodies

and fluorescein-

370

BURNS ET AL.

repeatedly after Con A or PHA stimulation. This suggests that factors other than lack of mitogen toxicity may be responsible for the different findings we report here. A second conclusion to be drawn from these experiments is that the progeny of a cloned T cell may be sufficient to provide the necessary T-cell/macrophage interactions during antigen recognition which lead to IL-2 production. This is in agreement with the findings of several other groups of investigators who have reported that cloned murine T-cell lines are capable of secreting IL-2 and/or other soluble lymphokines with immunoregulatory activities (31-33). In addition, cloned human Tcell hybridomas, produced by fusion of human antigen specific T cells and selected, mutant Jurkat cells have been reported by DeFreitas et al. to produce IL-2 (34). Also, a clone of IL-2-dependent alloantigen reactive T cells has been described recently by Kaieda et al. which produces not only IL-2 but a killer helper factor and T-cell replacing factor as well (7). This current study is the first, as far as we are aware, to demonstrate that human IL-2-dependent, soluble antigen-reactive T-cell clones will also generate IL-2 activity when cultured with APC and the sensitizing soluble antigen. The definition of IL-2 used for our studies was purely functional and further characterization of this growth-promoting activity by biochemical means is required for a more complete comparison with the IL-2 obtained from mitogen-stimulated PBM. Further study will also be required to determine whether or not other lymphokines with immunoregulatory activities are also produced by cloned, solubleantigen-specific human T cells. If these cloned cells do prove to be multipotential in their lymphokine secretions, then culture methods of this type will prove useful for further dissecting the process of antigen-induced lymphocyte activation and control of lympholcine production. ACKNOWLEDGMENTS This work was supported by a Harry Weaver Neuroscience Scholarship Award and Grants U-60-A-1 and RG 894-C-4 from the National Multiple Sclerosis Society and USPHS NS 11037. The authors wish to thank Dr. Peter Nowell and Gary Koretzky for their helpful discussions and Tracy Rhodes for invaluable help with manuscript preparation.

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