International Immunology

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International Immunology, Vol. 11, No. 4, 617-621, April 1999
© 1999 Japanese Society for Immunology

Single-cell analysis by RT-PCR reveals differential expression of multiple type 1 and 2 cytokine genes among cells within polarized CD4⁺ T cell populations

Anne Kelso, Penny Groves, Louise Ramm and Anthony G. Doyle¹

Leukocyte Biology Unit, Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Queensland 4029, Australia

Correspondence to: A. Kelso

	Abstract

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RT-PCR was used to examine the expression of IFN-, IL-2, IL-4, IL-5, IL-6 and IL-10 mRNAs by single murine CD4⁺ T cells activated either in a strongly type 1-polarized mixed lymphocyte reaction (MLR) or in the type 2-polarized response to immunization with keyhole limpet hemocyanin (KLH) in alum. The frequencies of expression of each cytokine differed markedly between the two responses, consistent with their polarization at the population level. However, most cells expressed only none to three of the six cytokines assayed, few displayed the canonical type 1 profile and none in either response expressed a full type 2 or type 0 profile. A significant fraction of cells co-expressed IFN- with IL-4 and/or other type 2 cytokines at frequencies that suggested that most of these genes were independently regulated. Collectively, these single-cell expression patterns indicate that polarization at the population level can mask substantial intercellular heterogeneity, and show directly that multiple type 1 and 2 cytokines can be expressed simultaneously in an individual T cell.

Keywords: IFN-, IL-2, IL-4, IL-5, IL-6, IL-10, mixed lymphocyte reaction, keyhole limpet hemocyanin, T_h1, T_h2, T_h0

	Introduction

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Now that some of the signals controlling the activation of type 1 and 2 cytokine gene expression have been identified (1,2), there is growing interest in defining the stages through which naive T cells pass in developing into effector cells with polarized cytokine profiles. However, individual T cells can be highly heterogeneous in the combinations of type 1 and 2 cytokines they express (3–6), and it is therefore difficult to analyze this differentiative process in bulk cultures of polyclonal, and even monoclonal, T cell populations in which cell-to-cell variation is masked.

Significant advances have been made in addressing this problem through the development of single-cell cytokine detection methods. In particular, in situ mRNA hybridization, ELISPOT assays and immunofluorescent detection of trapped intracellular or surface-bound cytokines have been useful in allowing frequencies of cytokine-producing cells to be estimated in activated T cell populations in a wide variety of circumstances (3,4,6–11). These technical approaches are well-suited to screening large numbers of cells but, since none of these methods can readily measure more than two cytokine species per cell, they are unable to provide complete information on individual T cell cytokine profiles.

As a single-cell detection method, RT-PCR is unsuitable for screening large cell numbers but offers the unique ability to measure multiple mRNA species in individual activated T cells (5,12,13). Using this approach, it should therefore be possible to determine whether individual T cells in a polarized population display the full type 1 or 2 pattern, and whether there are significant associations or dissociations of expression of type 1 and 2 cytokines, or their various upstream regulatory molecules, at the single-cell level. A significant concern, however, has been the extreme sensitivity of PCR, raising the possibility of measuring low-level, biologically insignificant, transcriptional `noise'. It would therefore be an important test of this approach to determine whether single-cell cytokine profiles revealed by RT-PCR correspond to those predicted by more conventional methods.

To perform such a test, we have used RT-PCR to assay individual cells from two T cell populations which we previously found by clonal studies of cytokine mRNA and protein to be polarized in opposite directions. Consistent with other biological features of these responses, CD4⁺ T cell clones derived from allogeneic mixed lymphocyte reactions (MLR) were strongly skewed towards type 1 cytokine synthesis (high IFN- and IL-2; low IL-4) (14,15), while populations and clones activated against keyhole limpet hemocyanin (KLH) were more mixed with skewing towards type 2 cytokine synthesis (high IL-2, IL-4 and IL-6; low to intermediate IFN-) (5,16,17).

C57BL/6 T cells were activated in a conventional one-way MLR by culture of lymph node cells with irradiated DBA/2 spleen cells. After 5 days, CD4⁺CD44^high cells were purified by flow cytometry and recultured overnight with solid-phase anti-CD3 mAb to induce cytokine mRNA expression prior to isolation of single cells. In separate experiments, C57BL/6 T cells were activated by s.c. immunization with alum-precipitated KLH. CD4⁺CD44^high cells were purified from the draining lymph nodes after 7 days, cultured with KLH and antigen-presenting cells for 5 days, then recultured overnight with solid-phase anti-CD3 mAb prior to single-cell isolation. Assays of supernatants collected after anti-CD3 re-stimulation showed that cells activated in MLR secreted IFN- without detectable IL-4, whereas those activated in response to KLH secreted both cytokines (Fig. 1). Previous experiments had shown that clonogenic KLH-reactive CD4⁺ T cells were enriched in the CD44^high fraction (16) and that the frequency of these cells was increased ~20-fold by one cycle of in vitro re-stimulation with KLH (A. Kelso and P. Groves, unpublished observations).

View larger version (26K): [in this window] [in a new window]   Fig. 1. Cytokine secretion by CD4+ T cells activated in MLR or in response to KLH. MLR were established by culturing 5x106 pooled brachial, axillary, inguinal and para-aortic lymph node cells of female C57BL/6 (H-2b) mice with 5x106 3000 rad-irradiated DBA/2 (H-2d) spleen cells in supplemented DMEM (5) with 5% FCS (DMEM-FCS) in 2 cm2 tissue culture wells. After 5 days, cells were enriched for CD4+CD44high cells by flow cytometry as described below then re-stimulated at 5x105 cells/ml in DMEM-FCS for 24 h in wells previously coated with 10 µg/ml anti-CD3 mAb (145–2C11). The KLH response was induced by s.c. injection of female C57BL/6 mice at the base of the tail with 100 µg KLH (Calbiochem, La Jolla, CA) in alum (16). After 7 days, CD4+CD44high cells were purified from the enlarged inguinal and para-aortic lymph nodes and cultured at 5x104 cells/ml in DMEM-FCS with 40 µg/ml KLH, 5x106 3000 rad-irradiated C57BL/6 spleen cells for 5 days, then re-stimulated at 5x105 cells/ml in anti-CD3 mAb-coated wells for 24 h. Supernatants from anti-CD3 mAb re-stimulated cultures were serially diluted in duplicate in assays for IFN- using WEHI-279 cells (open bars) and for IL-4 by ELISA (hatched bars), and their titers estimated by reference to standard curves obtained using recombinant IFN- and IL-4 as previously described (17). Flow cytometry. Cells were incubated sequentially with phycoerythrin-conjugated anti-CD4 mAb (GK1.5; Becton Dickinson, San Jose, CA), biotinylated anti-CD44 mAb (IM7.8; PharMingen, San Diego, CA) and fluorescein-conjugated streptavidin (Caltag, South San Francisco, CA), then resuspended in balanced salt solution with 5% FCS and 1 µg/ml propidium iodide (Calbiochem). CD4+ CD44high cells were purified using a FACS Vantage (Becton Dickinson FACS Systems, Sunnyvale, CA) with Lysys II software by exclusion of dead cells based on forward and 90° scatter properties and propidium iodide uptake, and selection of cells ranging from small lymphocyte to blast size which were positive for CD4 and in the upper 30% of the CD44 distribution.

 
Single cells were lysed, and the mRNA was reverse-transcribed and amplified by PCR with primers for the constitutively expressed T cell-specific product CD3, the type 1 cytokines IFN- and IL-2, and the type 2 cytokines IL-4, IL-5, IL-6 and IL-10, then analyxed by Southern hybridization. CD3 cDNA was successfully amplified from 89 of the 196 MLR cells (45%) and from 52 of the 140 KLH-primed cells (37%) assayed in two experiments with each response. Results are presented only for these CD3⁺ samples.

Figure 2 shows representative results of Southern hybridization of PCR products obtained from single cells activated in the two responses. The full set of data for expression of six cytokine mRNA species by single cells is summarized in Fig. 3. As shown in the latter figure, 60% of CD⁺ samples from the MLR and 85% from the KLH response yielded one or more cytokine PCR products. Amplification of cDNA from pools of at least 10⁴ cells in each experiment reproducibly revealed expression of all cytokines except IL-6 in MLR cells and all six cytokines in KLH cells (Fig. 2 and data not shown), consistent with the sum of patterns shown in Fig. 3. Reproducibility of single-cell mRNA detection ranged from 83 to 100% (mean 94%, n = 7 tests) when assessed by repetition of one or both rounds of PCR amplification from groups of 23–43 single-cell cDNAs.

View larger version (104K): [in this window] [in a new window]   Fig. 2. Cytokine gene expression by single cells activated in MLR or in response to KLH. Cells were activated in MLR (A) or by immunization and re-stimulation with KLH (B) as described in the legend to Fig. 1, then re-stimulated by culture at 1–5x105 cells/ml in plates previously coated with 10 µg/ml anti-CD3 mAb. After 16 h, cells were transferred individually into lysis buffer by micromanipulation with a fine capillary pipette or using the single-cell deposition unit attached to the FACS Vantage. RNA was isolated using either guanidinium thiocyanate followed by phenol–chloroform or lysis with Nonidet P-40 (28). Subsequent steps in reverse transcription, cDNA amplification by nested rounds of 40-cycle then 30-cycle PCR and Southern hybridization of PCR products were performed as described (5,12) using previously described primers (28). The figure shows autoradiographs of PCR products obtained from 10 single cells from each response, and from positive control cDNA (+), bulk cDNA obtained from at least 104 anti-CD3 mAb-stimulated MLR or KLH cells (B) and six negative control samples to which cDNA was not added.

 

View larger version (34K): [in this window] [in a new window]   Fig. 3. Summary of cytokine expression patterns among single cells activated in MLR (A) and in response to KLH (B). The figure shows data obtained for all CD3+ samples derived from single cells in two experiments each for the MLR and KLH responses. Detection of a cytokine PCR product is indicated by shading of the boxes. The number of samples with each pattern is indicated at the right and the frequency of expression of each cytokine is indicated below the panels. The mean threshold of detection of cytokine cDNA in these experiments was 10–16 g for IFN-, IL-4, IL-5 and IL-10, and 10–17 g for IL-2 and IL-6 determined from serial 10-fold dilutions of cloned cDNA included in each set of PCR reactions.

 
As shown in Fig. 3, most MLR cells expressed none or one of the six cytokines and most KLH cells expressed only one, two or three cytokines. However, the frequencies and patterns of cytokine expression among these positive cells differed markedly between the MLR and KLH responses. MLR cells most commonly expressed IFN- without any other cytokine (44%). Although MLR cells expressing one or more type 2 cytokines were relatively rare (10%), only two cells displayed the canonical type 1 profile (positive for IFN- and IL-2; negative for IL-4, IL-5, IL-6 and IL-10), and IL-10 was expressed as frequently as IL-2. By contrast, 52% of KLH cells expressed one or more type 2 cytokines without either IFN- or IL-2; none displayed the full type 2 profile and one displayed a full type 1 profile. No cells expressing all six cytokines (T_h0 cells) were found in either panel.

No absolute associations or dissociations of expression were observed between any pair of cytokines, suggesting that expression of one cytokine was neither contingent on nor precluded by expression of any other. As shown in Table 1, however, the pairs IFN-/IL-6 and IL-2/IL-6 were co-expressed by KLH cells at higher frequencies than expected by chance, while IL-2/IL-5 and IL-5/IL-6 were co-expressed at lower frequency than expected by chance. No positive or negative associations were detected in the co-expression of any other cytokine pairs among either the MLR or the KLH cells.

View this table: [in this window] [in a new window]   Table 1. Cytokine co-expression patterns among single CD4+ cells activated in MLR or in response to KLH immunization

 
Several significant conclusions can be drawn from this analysis. First, the patterns of cytokine gene expression revealed by PCR in the two populations studied here are consistent with those determined by more conventional bulk and clonal methods. This is clearest for IFN- and IL-4 where the strong bias towards IFN- synthesis by the MLR population in bulk culture (Fig. 1) and in previously reported clonal studies was reflected in the high ratio of IFN- producers to IL-4 producers detected at the single-cell level (Figs 2 and 3). Similarly, the less marked bias towards IL-4 synthesis by T cell populations and clones activated in response to KLH was reflected in the modest excess of IL-4 producers over IFN- producers detected by single-cell PCR. Therefore the extreme sensitivity of RT-PCR apparently does not result in detection of background transcriptional activity that might mask biologically important differences in the cytokine profiles of the two populations. The estimation that background transcription of several `silent' genes in mammalian cells yields about one transcript/10⁴ cells (18) also suggests that such activity should have negligible effect in single-cell assays.

Second, most cells expressed mRNA for only a small number (none to three) of cytokines. A similar observation was made less directly by Bucy et al. using double-label in situ hybridization to detect expression of various cytokine pairs (6). Flow cytometric assays of intracellular cytokine accumulated over several hours in the presence of Brefeldin A have often also revealed a large fraction of negative cells in activated polyclonal T cell populations (10,19). Since these methods do not allow sequential assays of the same cell, it is not known whether these mRNA `snap-shots' or the intervals sampled by flow cytometry reveal the full cytokine repertoire that might be expressed by a cell during one cycle of response to TCR cross-linking or whether one cell necessarily produces the same cytokine(s) when activated a second time. The development of a method for live sorting of cytokine-producing cells (11) should now allow these questions to be answered. There are many properties that might vary between cells, either transiently or stably, to alter the strength and character of their cytokine response to CD3 ligation. These include TCR expression levels, pool sizes of signaling intermediates, methylation and chromatin-dependent accessibility of cytokine promoters, expression of transcription factors, activation thresholds for cytokine synthesis, and kinetics of mRNA production and decay (20–24). Under physiological conditions where these variables combine with variation in the strength of signal delivery by antigen-presenting cells and other influences, heterogeneity in the cytokine responses of individual T cells is probably even greater than that seen under controlled conditions in vitro. This heterogeneity might serve an important role by maintaining diversity in target cell responses, even under strongly polarizing type 1 or type 2 conditions.

Third, although significant numbers of cells from these two populations only expressed cytokines from the type 1 or type 2 category, few cells met the formal definition of T_h1 cells and none expressed the full T_h2 phenotype. Others expressed mRNAs from both categories, such as IFN- and IL-4, but again none expressed the full T_h0 phenotype. Although statistical analyses of co-expression frequencies revealed positive or negative relationships between some cytokines, they did not demonstrate a clear dissociation between type 1 and 2 cytokine expression (such as IFN- and IL-4) or association within types (such as IL-4 and IL-5) at the single-cell level. These and other single-cell data support the idea that polarization is a population phenomenon achieved by altering the frequencies of cells expressing each cytokine, rather than by activating discrete subsets (25). They also add to the body of evidence that expression of each cytokine gene, and perhaps even each allele, can be regulated independently in individual T cells and clones (3–6,14,23,26). Finally, the frequent co-expression of type 1 and 2 cytokines is consistent with T_h1–T_h2 heterokaryon experiments (27) showing that the distinct signaling pathways that activate expression of these genes are not mutually repressive.

In conclusion, by exploiting the power of RT-PCR to detect expression of multiple cytokine genes in single activated T cells, we show here that individual cells within polarized populations can vary markedly in their co-expression of cytokines both within and between the type 1 and 2 groups. The use of a single indicator cytokine of each type is therefore insufficient to identify a cell as T_h1 or T_h2.

	Acknowledgments

 
We thank Dr David Fitzpatrick for helpful discussion and Grace Chojnowski for expert assistance with flow cytometry. This work was supported by the National Health and Medical Research Council of Australia and The Queensland Institute of Medical Research Trust.

	Abbreviations

 

KLH	keyhole limpet hemocyanin
MLR	mixed lymphocyte reaction

	Notes

 
¹ Present address: Peptech Ltd, Locked Bag 2053, North Ryde, NSW 2113, Australia

Transmitting editor: D. Tarlinton

Received 20 November 1998, accepted 28 December 1998.

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