Coexpression of granulocyte-macrophage colony-stimulating factor,

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Aug 8, 1988 - Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. & Bluestone,. J. A. (1987) Proc. Natl. Acad. Sci. USA 84, 1374-1378. 14. Kelso, A. & Owens, T.
Proc. Nati. Acad. Sci. USA Vol. 85, pp. 9189-9193, December 1988 Immunology

Coexpression of granulocyte-macrophage colony-stimulating factor, y interferon, and interleukins 3 and 4 is random in murine alloreactive T-lymphocyte clones (lymphokines/T-celi subsets)

ANNE KELSO AND NICHOLAS M. GOUGH The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050, Australia

Communicated by Jacques F. A. P. Miller, August 8, 1988 (received for review July 21, 1988)

ABSTRACT Lymphokine gene expression was examined in a panel of 116 short-term murine T-lymphocyte clones derived by single-cell micromanipulation from allogeneic mixed leukocyte cultures. About 30% of clonable T cells, including both CD41 CD8- and CD4- CD81 cells, could be expanded for assay at an average of 22 days after cloning. By RNA blot-hybridization analysis, most clones (85-96%) expressed detectable granulocyte-macrophage colony-stimulating factor, interleukin 3, and y interferon mRNAs, and 11% expressed interleukin 4 mRNA. Although no differences were noted between CD41 and CD81 clones in the combinations of lymphokines produced, CD4' clones on average transcribed and secreted higher levels. When the frequencies of coexpression of any pair of lymphokine mRNAs were determined, all were found to correspond to the values predicted for random assortment of the individual frequencies. For example, among 13 interleukin 4-positive clones, 11 also transcribed y interferon, giving the frequency of double-positive clones expected for random association (9.6% versus 10.8%). Therefore, expression of the four lymphokine genes segregated independently among the clones and did not allow the division of T cells into subsets with distinct patterns of lymphokine synthesis. T lymphocytes can produce at least 10 distinct lymphokines after activation in vitro, including interleukins 2 to 6 (IL-2 to IL-6), y interferon (IFN-y), granulocyte-macrophage colony-stimulating factor (GM-CSF), and lymphotoxin. These glycoproteins have diverse and sometimes opposing effects on the growth, differentiation, and activation of lymphoid, hemopoietic, and other cells. Their secretion by T cells may profoundly influence the development of an immune response-for example by stimulating the production and functional activities of monocytes, neutrophils, eosinophils, and mast cells (GM-CSF, IL-3, and IL-4); by enhancing H-2 antigen expression and the activation state of macrophages (IFN-y, lymphotoxin); and by affecting activation and isotype switching of B cells (IL-4, IL-5, IFN-y) (1-3). Therefore, it might be expected that mechanisms would exist for the selective production of appropriate lymphokines in the response to a

given immunogen.

For this reason, several groups have screened panels of monoclonal T-cell lines to determine whether subsets can be defined that differ in their patterns of lymphokine gene expression. Early studies showed that a single clone could synthesize several different lymphokines and that clones varied markedly in the quantities and types of lymphokines produced (4-7). Only recently has evidence been obtained for distinct lymphokine-secreting T-cell subsets. In particular, Mosmann and coworkers (8, 9), Killar et al. (10), and Janeway et al. (11) have reported that CD4+ T-cell clones can The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"

be classified into two groups: (i) THi or Tinf, which synthesize GM-CSF, IL-3, IFN-y, IL-2, and lymphotoxin; and (ii) TH2 or Th, which synthesize GM-CSF, IL-3, and IL-4. Some controversy exists concerning whether IL-S is produced by TH2 cells or by both subsets (9, 11). To date, however, this classification has been based on certain long-term T-cell clones, and it is unclear whether these are representative of T cells as a whole. Therefore, the present study was undertaken to screen a large, unselected panel of T-cell clones for production of several lymphokines as soon as possible after cloning. Clones were derived directly by micromanipulation from allogeneic mixed leukocyte cultures (MLC) because such cultures contain both CD4' and CD8' T cells and because alloreactive clones of both the THi and TH2 types have been described (8, 9). Expression of lymphokine genes was assessed by RNA blot hybridization (Northern analysis) to avoid the ambiguities of polyspecific lymphokine assays. In contrast with some studies of long-term lines, these experiments suggest that the production of GM-CSF, IL-3, IFN-y, and IL-4 is randomly assorted among T-cell clones of both the CD4' and CD8' phenotypes and, therefore, that there is no intrinsic preclusion to the expression of any combination of these four products.

MATERIALS AND METHODS T-Cell Clones. Individual blast cells from 5-day cultures of C57BL/6f/J WEHI (H_2b) spleen cells with 3000 R (1 R = 0.258 mC/kg)-irradiated DBA/2 wf WEHI (H-2d) spleen cells were transferred by micromanipulation into microtiter wells containing irradiated DBA/2 spleen cells and EL4 (mouse thymoma) cell culture supernatant as a source of IL-2 (7). In two experiments, CD4' blasts were isolated by fluorescence-activated cell sorting from MLC populations stained with the rat anti-CD4 antibody GK1.5 and fluorescein isothiocyanate-conjugated goat anti-rat Ig. After 7-14 days, clones were expanded and maintained by passage every 5-10 days with irradiated DBA/2 spleen cells and EL4 cell culture supernatant (7). CD4 and CD8 expression by the clones was assessed by flow cytometry using antibodies GK1.5 and 53-6.7, respectively. Stimulation of Clones. Clones were washed three times and cultured at 106 cells per ml with 5 ,ug of Con A (Pharmacia) per ml and 100 units of highly purified recombinant IL-2 from Escherichia coli (Cetus, Emeryville, CA) per ml (12) (experiments 1 and 2) or in wells coated with 5 ,ug of anti-CD3 antibody (145-2C11) per ml (13, 14). After 8 hr of incubation, culture supernatants were collected, and the cells were harvested by brief trypsinization. Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-y, y interferon; IL, interleukin; MLC, mixed leukocyte culture.

in accordance with 18 U.S.C. §1734 solely to indicate this fact.

9189

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Proc. Natl. Acad. Sci. USA 85

Immunology: Kelso and Gough

(1988)

Table 1. Derivation of T-cell clones Cells transferred, no. Exp. 192 1 288 2 3 288 280 4 5 192 192 6 7 200 200 8 1832 Total NT, not tested. *One CD4- CD8- clone.

Clones obtained, no. 31 34 75 68 67 59 37 46 417 (23%)

Lymphokine Assays. GM-CSF, IL-3, and IL-2 titers were determined in [3H]thymidine uptake assays with the factordependent cell lines FD clone 5/12 (15), 32D clone 3 (14), and CTLL (7), respectively. FD clone 5/12 responds to GM-CSF (detection limit, -0.4 unit/ml) and weakly to IL-3 (limit, -3 units/ml). 32D clone 3 responds only to IL-3 (limit, -0.2 unit/ml) (14). Neither line responds to IL-2 or IL-4 up to 103 units/ml. CTLL responds to IL-2 (limit, -0.1 unit/ml; maximum, 1-5 x 104 cpm) and weakly to IL-4 (limit, -300 units/ml; maximum -103 cpm) but not to GM-CSF or IL-3. Northern Blot Analysis. Total cytoplasmic RNA was extracted from up to 4 x 101 stimulated cells (16), and aliquots corresponding to 2.5-5 x 105 cells were fractionated on 1% formaldehyde/agarose gels, transferred to nitrocellulose, E9.D4

MLC clones 4 5 6 7 8 9 10 11 12 13 1415 1617 18 19 20 21

a:> ° c° 1 2 3

Clones analyzed, no. 8 7 3 17 19* 25 20 17 116 (28%)

CD4+

0 0 5 2 19 13 39

Phenotype CD8+ 2 11 8 16 1 2 40

NT 8 7 1 6 5 7 0 2 36

and hybridized as described (17). Riboprobes with a specific activity of 1-2 x 109 cpm/tug were derived by transcription of phage SP6 subclones for each lymphokine and used at =2 x 107 cpm/ml.

RESULTS Derivation of T-Cell Clones. T cells activated in primary MLC were cloned by single-cell micromanipulation with an average cloning efficiency of 23% (Table 1). In control experiments, transfer of single MLC cells into wells without filler cells indicated that the frequency of transferring >1 cell per well was