Immunology and Cell Biology (2014) 92, 825–836 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14 www.nature.com/icb

ORIGINAL ARTICLE

The glucocorticoid receptor 1A3 promoter correlates with high sensitivity to glucocorticoid-induced apoptosis in human lymphocytes Douglas R Liddicoat1, Konstantinos Kyparissoudis1, Stuart P Berzins1,2, Timothy J Cole3,5 and Dale I Godfrey1,4,5 Glucocorticoids (GCs) are powerful inhibitors of inflammation and immunity. Although glucocorticoid-induced cell death (GICD) is an important part of GCs actions, the cell types and molecular mechanisms involved are not well understood. Untranslated exon 1A3 of the human glucocorticoid receptor (GR) gene is a major determinant of GICD in GICD-sensitive human cancer cell lines, operating to dynamically upregulate GR levels in response to GCs. We measured the GICD sensitivity of freshly isolated peripheral blood mononuclear cells and thymocytes to dexamethasone in vitro, relating this to GR exon 1A3 expression. A clear GICD sensitivity hierarchy was detected: B cells4thymocytes/natural killer (NK) cells4peripheral T cells. Within thymocyte populations, GICD sensitivity decreased with maturation. Interestingly, NK cell subsets were differentially sensitive to GICD, with CD16 þ CD56int (cytotoxic) NK cells being highly resistant to GICD, whereas CD16 CD56hi (cytokine producing) NK cells were highly sensitive (similar to B cells). B-cell GICD was rescued by co-culture with interleukin-4. Strikingly, although no significant increases in GR protein were observed during 48 h of culture of GICD-sensitive and -resistant cells alike, GR 1A3 expression was increased over pre-culture levels in a manner directly proportional to the GICD sensitivity of each cell type. Accordingly, this is the first evidence that the GR exon 1A3 promoter is differentially regulated during thymic development and maturation of human T cells. Furthermore, human peripheral blood B cells are exquisitely GICD-sensitive in vitro, giving new insight into how GCs may downregulate immunity. Collectively, these data show that GR 1A3 expression is tied with GICD sensitivity in human lymphocytes, underscoring the potential for GR 1A3 expression to be used as a biomarker for sensitivity to GICD. Immunology and Cell Biology (2014) 92, 825–836; doi:10.1038/icb.2014.57; published online 22 July 2014

Glucocorticoids (GCs) are powerful inhibitors of activated immunity, acting by downregulation of cytokine production, immune cell trafficking, and induction of immune cell death. Owing to these actions, synthetic GCs have become a mainstay long-term treatment for a wide range of chronic inflammatory diseases, including autoimmune disorders, asthma, dermatitis and transplant rejection.1 However, GCs also induce a range of clinically important side effects such as osteoporosis, hyperglycaemia, depression and secondary infection or malignancy, and thus judicious consideration to dose is required to gain the optimal response.1 Furthermore, although most patients are initially responsive to GCs, many develop reduced sensitivity, or complete resistance during treatment, and thus development of methods to accurately monitor resistance would greatly improve disease management.2 Although glucocorticoidinduced cell death (GICD) is a cornerstone of chemotherapy for

leukaemia and lymphoma, our knowledge of its role in the treatment of chronic inflammatory disease, is far less complete. While the GICD sensitivity of non-malignant thymocytes and immature bone marrow B cells is well known in mice,3–5 the relative GICD sensitivity of developing and mature lymphocyte populations in human remains largely unknown, but may hold important ramifications for immunosuppression in the clinic. GICD is mediated by the glucocorticoid receptor (GR), a cytosolic receptor that, upon ligand binding, translocates to the nucleus to act as a transcription factor and modulate the activity of a large array of genes.6–8 GR null mice are refractory to GICD, and a strong correlation exists between GICD susceptibility and GR levels.9–14 A large number of studies have shown that GICD sensitivity is also linked with the ability of GCs to upregulate expression of the GR itself in a positive regulatory loop known as ‘auto-upregulation’.15–20 In

1Department of Microbiology and Immunology, The Peter Doherty Institute, University of Melbourne, Parkville, Victoria, Australia; 2Fiona Elsey Cancer Research Institute; and School of Health Sciences, Federation University, Ballarat, Victoria, Australia; 3Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia and 4Australian Research Council Centre of Excellence for Advanced Molecular Imaging at the University of Melbourne, Parkville, Victoria, Australia 5Co-chief investigators. Correspondence: Professor DI Godfrey, Department of Microbiology and Immunology, The Peter Doherty Institute, University of Melbourne, Grattan Street, Parkville, Victoria 3010, Australia. E-mail: [email protected] or Associate Professor TJ Cole, Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia. E-mail: [email protected] Received 18 April 2014; revised 10 June 2014; accepted 11 June 2014; published online 22 July 2014

Glucocorticoid-induced cell death of human B cells DR Liddicoat et al 826

contrast, GICD-resistant cell types were found to ‘auto-downregulate’ GR levels in response to GCs.21–23 Exon 1A3 of the human GR gene is a major determinant of autoregulation and GICD in haemopoetic cells.24–27 In humans and rodents, the GR gene has multiple untranslated first exons, each with a unique promoter region and splice donor site that confers splicing to a single second exon containing the translation start sequence. All first exons discovered to date belong to either the proximal or distal gene regulatory region, B5 or 30 kb upstream of exon 2, respectively. In the human GR gene, the proximal region contains exons 1B, 1C, 1D, 1E, 1F, 1H and 1J, which are generally active in all cell types.28 In contrast, the distal region contains exons 1A (which has three major splice variants 1A1, 1A2 and 1A3) and 1I, which are expressed exclusively in haemopoetic cells and the brain.17,24,28,29 Expression studies in primary lymphoid cells and leukaemic cell lines show that, of these distal exons, exon 1A3 is by far the predominant transcriptional product (expression of the other distal variants is at least 10–100-fold lower) and the only one that may significantly contribute to overall GR protein levels.29,30 Subsequently, multiple studies have shown that exon 1A3 expression is markedly autoupregulated relative to proximal exons in GICD-sensitive cell lines, such as T-cell ALL line CEM-C7.25 Conversely, exon 1A3 expression was markedly auto-downregulated in GICD-resistant lines, such as B lymphoblastoid line IM-9, suggesting that the 1A3 promoter is a means by which overall GR levels are dynamically regulated.24,26,29,30 Furthermore, studies of the 1A3 promoter sequence revealed a halfglucocorticoid response element that mediated auto-regulation in concert with adjacent overlapping response elements for c-Myb and c-Ets proteins.24,27 These response elements were shown to constitute a ‘molecular switch’, whereby c-myb, which was shown to predominate in GICD-sensitive cells, paired with GR to upregulate 1A3 promoter usage. Conversely, Ets proteins PU.1 and Spi-B, which were shown to predominate in GICD-resistant cells, paired with GR to downregulate 1A3 promoter usage.27,31 GR exon 1A has also been suggested to be a key determinant of GICD in mice.17,32 GC treatment of GICD-sensitive mouse thymocytes, but not GICD-resistant splenocytes, results in increased GR1A exon levels as a percentage of total GR transcripts, concomitant with increases in GR protein.17 Furthermore, expression of exon 1A as a percentage of total GR transcripts has been shown to correlate highly with GICD sensitivity in mouse thymocyte populations.17 It is currently unknown whether the human GR 1A3 promoter is similarly regulated in human T-lymphocyte development and maturation. In this study, we explored the GICD sensitivity of primary human lymphocytes from human thymus and blood, and identified peripheral blood B cells as a new, highly GICD-sensitive cell population. Furthermore, by analysis of 1A3 promoter usage in these cell types, we identified a correlation between GICD sensitivity and preferential upregulation of 1A3 transcripts relative to total GR transcripts, reinforcing the association between exon 1A3 usage and GICD established in cancer cell lines and mouse studies. This constitutes the first direct evidence to suggest that exon 1A3 usage is an important determinant of GICD in normal primary human lymphocytes, underscoring its potential for future use as a biomarker of sensitivity to GICD. RESULTS Peripheral B cells are highly susceptible to GICD in vitro Although resting peripheral blood T cells are known to be resistant to GICD, the relative GICD sensitivity of other primary peripheral blood mononucleated cell (PBMC) subsets is currently unknown.33–36 To address this, we cultured PBMCs in vitro for 48 h with Immunology and Cell Biology

10 7 M dexamethasone (DEX), a concentration corresponding to pharmacological/psychological stress levels in vivo.37 To avoid changes in gene expression associated with storage of blood at room temperature, PBMCs were extracted from freshly taken blood from a cohort of healthy human donors. After 48 h of culture, the sensitivity of each cell population to GICD was analysed by 7-aminoactinomycin D (7AAD) staining and analysis by flow cytometry (Figure 1a). Consistent with previous studies33,34 at 48 h, whole PBMCs were completely resistant to DEX treatment; percentage viability by trypan blue exclusion was 90±1% for both VEH and DEX cultures. In line with a previous study of GICD sensitivity in mice,17 the ratio of viable cell recovery for each lymphocyte population from DEX versus VEH cultures was then used to obtain a quantitative value, termed the survival index (SI), indicating sensitivity to GICD (Figure 1b). The populations investigated include CD19 þ B cells, CD3 þ CD4 þ T cells, CD3 þ CD8 þ T cells and CD3 CD19 CD56 þ natural killer (NK) cells. T cells, which comprise the majority of PBMCs, were GICD resistant, with an SI of 0.98. CD4 þ and CD8 þ T cells were also examined in terms of CD45RA þ CD45RO  naive, and CD45RA  CD45RO þ memory subsets and all were found to be GICD resistant (Supplementary Figure 1a). To our surprise, peripheral blood B cells had an SI of 0.41, indicating they were very sensitive to GICD. CD56 þ NK cells were also clearly sensitive (SI ¼ 0.60). This hierarchy of GICD sensitivity was also consistently seen at 24 h of culture (albeit the spread of SI values were smaller), as well as in preliminary DEX dose-response experiments ranging up to 10 6 M DEX (not shown). Another surprising trend observed for all donors was downregulation of CD56 on NK cells. As shown in Supplementary Figure 1b, fresh CD3  PBMCs exhibited a clear distinction between CD56  and CD56 þ peaks. However, at 12 h this gap started to close, and by 48 h expression of CD56 decreased substantially. Human NK cells consist of two main subsets that differ by surface phenotype and effector function. The majority of NK cells are CD16 þ CD56int ‘cytotoxic’ NK cells, which specialize in target cell killing, whereas the remaining minor NK cell population is CD16 CD56hi and produces the majority of NK cell cytokines.38 Because CD56 downregulation will complicate identification of the CD56 þ NK cell subset, we co-stained with another marker for this sub-population, CD94 (Figure 1a). Subsequent examination of CD56 mean fluorescence intensities showed that both NK cell populations clearly downregulated CD56 in response to DEX (Figure 1c; Supplementary Figure 1b). SI values for the NK sub-populations defined by CD16 and CD94 expression were calculated and revealed that, although CD16 þ NK cells were relatively resistant to GICD, CD16 CD94 þ (CD56hi) NK cells were highly GICD sensitive. When these NK subsets were not separately examined (total NK cells), this gave the misleading appearance that NK cells had an intermediate GICD sensitivity (Figure 1d). As an independent measure of GICD sensitivity, Annexin V staining for early apoptotic cells was performed on PBMC samples at 12 h of culture. The difference between the percentage of Annexin V-positive cells in DEX culture and that in VEH was calculated, providing a preview of GICD sensitivity (Figures 2a and b). In line with the SI value calculations at 48 h, B cells showed the greatest increase in Annexin V staining, followed by NK cells and then CD4 þ and CD8 þ T cells. NK cells were not divided into CD16 þ /  subsets in these experiments. B cells undergo GICD when cultured alone with DEX and are rescued by IL-4 The high GICD sensitivity of peripheral blood B cells is of potential clinical relevance and thus deserves further characterization. To

Glucocorticoid-induced cell death of human B cells DR Liddicoat et al 827

GICD sensitivity decreases during early human thymocyte development and maturation In the mouse thymus, a clear correlation exists between GICD sensitivity and developmental state, with immature populations such as CD4 þ CD8 þ double positive (DP) cells being highly GICD sensitive, and mature CD24lo/int cells being highly resistant.17,39 To investigate whether a similar hierarchy exists in the human thymus, human thymocytes were cultured with 10 7 M DEX. The thymic populations investigated include DP cells, as well as CD3hiCD4 þ CD8  and CD3hiCD4 CD8 þ single positive (SP) cells, which were further split into CD1a þ (early) and CD1a  (mature) subpopulations.40,41 Also, prompted by studies in mice where GR expression was found to peak in double negative (DN)3 thymocytes,39 in addition to investigating the CD4 CD8  DN population as a whole (which contains a variety of non-T lineage-committed cells), the CD4 CD8 CD34 þ CD1a þ ‘Pre-T1’ population was investigated. The Pre-T1 stage could be considered roughly equivalent to the mouse DN2/DN3 stage, based on

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address whether high GICD sensitivity is an intrinsic quality of peripheral B cells or whether it involves other DEX-induced factors derived from co-cultured PBMCs, cell sorting was used to isolate CD19 þ B cells from fresh PBMC samples. These sorted B cells were then cultured for 48 h with or without DEX, and SI values were calculated (ratio of viable cell recovery from each culture condition to VEH) as in Figure 1. In addition, to gain insight into whether GICD of B cells could be expected in vivo or whether it is contingent upon the presence of survival factors not present in culture, the sorted B cells were also cultured in the presence of key B-cell survival factor, interleukin (IL)-4. Consistent with earlier results, sorted peripheral B cells were also clearly sensitive to GICD with a SI of 0.64 (Figure 2c), although this is not as sensitive as seen in whole-PBMC culture (SI of 0.41). Nonetheless, high GICD sensitivity primarily appears to be a characteristic intrinsic to B cells. Interestingly, addition of human IL-4 (100 ng ml 1) resulted in a strong rescue of B cells from DEX-induced apoptosis, giving a SI value of 1.28.

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Figure 1 Peripheral blood B cells, as well as CD16 CD94 þ NK cells, are highly sensitive to GICD. Human PBMCs were isolated from fresh blood samples and cultured in vitro for 48 h with 10 7 M DEX or VEH (1/1000 ethanol in culture medium v/v). Following culture, the PBMCs were counted and stained with Abs specific for CD3, CD4, CD8, CD16, CD19, CD56, CD94, CD45RA and CD45RO and analysed by flow cytometry. Non-viable cells were removed by 7AAD staining. (a) Representative dot plots showing frequency of parent for each population of interest pre-culture (fresh), as well as at 48 h for VEH and DEX cultures. (b) SI of PBMC populations at 48 h, as gated in a. B cells and T cells: n ¼ 8 donors, NK cells: n ¼ 5 donors. ***Po0.001, different to T cells by the Kruskal–Wallis test. Data were collected over five experiments. Error bars: s.e.m. (c) Average of CD56 geometric mean fluorescence intensities (MFIs) for CD16 þ NK cells (n ¼ 8 donors) and CD94 þ CD16  NK cells (n ¼ 5 donors) at 48 h. (d) SI for these populations as well as total NK cells (n ¼ 5 donors) at 48 h. (c,d) Data are derived from five experiments. *Po0.05; **Po0.01 by the Mann–Whitney test. Error bars: s.e.m. Immunology and Cell Biology

Glucocorticoid-induced cell death of human B cells DR Liddicoat et al 828

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Survival Index Following 10-7M DEX Figure 2 Relative levels of GICD amongst peripheral blood lymphocyte populations by annexin V staining. Human PBMCs were isolated from fresh blood samples and cultured in vitro for 12 h with 10 7 M DEX or VEH. Following culture, the PBMCs were counted and stained with Abs specific for CD3, CD4, CD8, CD19, CD56, as well as annexin V, and analysed by flow cytometry. (a) Representative histograms indicating the percentage of annexin V-positive cells within each lymphocyte population for both VEH and DEX treatment. (b) Percentage of DEX-induced annexin V-positive cells within each population (background subtracted). n ¼ 5 donors collected from three experiments. Error bars: s.e.m. *Po0.05; **Po0.01, different to B cells by the Kruskal–Wallis test. (c) Human peripheral blood B cells are sensitive to GICD and can be rescued by IL-4. CD19 þ B cells were separated from the peripheral blood of healthy donors by cell sorting and cultured in vitro for 48 h under the conditions indicated (DEX 10 7 M, hIL-4 100 ng ml 1). Following culture, the B cells were counted and stained with 7AAD and analysed by flow cytometry, allowing viable cell recovery to be calculated for each condition. These data were used to calculate the SI of B cells for all treatments, relative to vehicle. n ¼ 5 donors collected from three experiments. Error bars: s.e.m. *Po0.05 by the Kruskal–Wallis test.

recent T-cell-lineage commitment and TCRb gene segment recombination.40,41 The percentage viability of total thymocytes for VEH and DEX cultures by trypan blue staining at 48 h was 76±2% and 44±4%, respectively, which is consistent with a previous study.33 SI values were then calculated, using the same method as described for PBMC cultures (Figure 3). In line with the above-mentioned mouse studies, populations corresponding to early stages in T-cell development, including Pre-T1, DP and CD4 þ CD1a þ SP thymocytes were found to be GICD sensitive (although not as sensitive as peripheral blood B cells), whereas the more mature CD4 þ CD1a  SP and CD8 þ CD1a  SP thymocyte populations were found to be GICD resistant with SI values of B1.0. This overall hierarchy of GICD sensitivity in the thymus was consistently seen at 24 h of culture, as well as in preliminary DEX dose-response experiments ranging up to 10 6 M DEX (not shown). To further check the viability of 7AAD  cells incorporated into the SI calculation, each population was checked for increased side scatter (SSC), a morphological change associated with GICD in thymocytes and PBMC (Figure 3c).42,43 Although the 7AAD  fraction of other cell populations showed negligible changes between VEH and DEX treatment, Pre-T1 and DP cells consistently exhibited a distinct increase in SSC with DEX treatment across all donors. This suggests that these cells have increased granularity and were in the early stages of apoptosis, thus providing further evidence of the high GICD sensitivity of these cells. As an independent measure of GICD sensitivity, Annexin V staining for early apoptotic cells was performed on thymocyte cultures at 12 h Immunology and Cell Biology

(Figure 3d; supplementary Figure 2). In line with 7AAD staining at 48 h, Pre-T1, DP and CD4 þ CD1a þ SP thymocytes exhibited relatively high GICD sensitivity, whereas mature CD1a  SP populations were relatively resistant. For all populations examined, the percentage of annexin V-positive cells in VEH cultures was markedly lower (10–20%). Basal 1A3 promoter usage and GR expression peaks at the Pre-T1 stage in the human thymus Prompted by previous studies suggesting the importance of the GR 1A promoter for GICD in human leukaemic cells lines and mouse lymphocytes,17,25,26 we investigated the relative levels of GR 1A promoter-driven transcripts in freshly prepared human thymocyte and peripheral blood lymphocyte populations isolated by fluorescence-activated cell sorting (Figure 4). Quantitative PCR (qPCR) amplicons for human 1A exon splice variants 1A1, 1A2 and 1A3, as well as total GR transcripts were employed to investigate GR 1A promoter usage as a percentage of total GR messenger RNA (mRNA). Previous studies on a wide range of human tissue biopsies and primary cell types, as well as leukaemic cell lines, have shown that GR 1A1 and 1A2 transcripts are essentially undetectable.30,44 We tested our complete panel of freshly sorted cells for GR 1A1 and 1A2 expression. Although GR 1A1 and 1A2 transcripts were detectable in 5–10% of these samples, they were always below the lower threshold of the dynamic range for the qPCR assay (data not shown). In contrast, GR 1A3 transcripts were expressed robustly, with very

Glucocorticoid-induced cell death of human B cells DR Liddicoat et al 829 CD4+ 83

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Figure 3 The early stages of human thymocyte development are sensitive to GICD. Thymocytes were isolated from fresh human thymus biopsies and cultured in vitro for 48 h with 10 7 M DEX or VEH. Following culture, thymocytes were counted and stained with Abs specific for CD1a, CD3, CD4, CD8 and CD34, and analysed by flow cytometry. Non-viable cells were removed by 7AAD staining. (a) Representative dot plots indicating the frequencies of parent for each population of interest at each time point including freshly isolated cells (fresh), as well as VEH and DEX cultures at 48 h. (b) Survival index (SI) of the thymocyte populations at 48 h. Data are derived from n ¼ 4 donors collected over four experiments. Error bars: s.e.m. (c) The side-scatter profile of viable (7AAD ) cells in each population at 48 h. (d) Relative levels of GICD among thymocyte populations by annexin V staining after 12 h culture with 10 7 M DEX or VEH. Following culture, cells were counted and stained with Abs specific for CD1a, CD3, CD4, CD8 and CD34, as well as annexin V, and analysed by flow cytometry. Average percentage of DEX-induced annexin V-positive cells within each population are shown. n ¼ 4 donors collected over four separate experiments. Error bars: s.e.m.

consistent expression profiles for the cell types examined between individual donors. The only exception to this was peripheral blood NK cells; of eight donors, four were completely negative, whereas the other four were below the dynamic range of the qPCR assay. Interestingly, amongst thymocyte populations, the percentage GR 1A3 promoter usage was found to be higher in Pre-T1 cells (Po0.05, with the exception of the CD4 þ CD1a þ cells: P ¼ 0.07, the Kruskal– Wallis test) (Figure 4b). In the periphery, we also noted that the CD4 þ CD45RA  memory population showed increased GR 1A3 expression compared with peripheral B cells (Po0.001) and CD8 þ CD45RA  memory T cells (Po0.05) (Figure 4a). Given our observation of high GR 1A3 expression and GICD sensitivity at the Pre-T1 stage in human thymus, we investigated GR 1A expression in FACS-sorted B6 mouse DN thymocytes to determine whether this promoter was differentially expressed within the welldefined subsets of these cells. The populations investigated include:

CD44 þ CD25 þ (DN2), CD44 CD25 þ (DN3) and CD44 CD25  (DN4) subsets within the CD4 CD8  double-negative population, the CD8 þ CD24 þ abTCR  immature CD8 single-positive (ISP8) subset, abTCR  and abTCR þ subsets within the CD4 þ CD8 þ double-positive population and CD24 þ (early) and CD24  (late) subsets within the abTCR þ CD4 þ and abTCR þ CD8 þ singlepositive populations (Supplementary Figure 3). Every subset of sorted thymocytes examined expressed GR and GR 1A transcripts, and very similar expression profiles were seen comparing individual mice. In line with prior RNase protection assays, GR 1A usage in DP thymocytes was higher than that in SP populations.17 Interestingly, DN3 and DN4 thymocytes had very high GR 1A usages of B40%, approximately twofold above levels seen in DP cells. This is consistent with the elevated GR 1A3 expression seen in the human DN Pre-T1 population, suggesting a conserved role for the GR 1A promoter at this point in T-cell development. Immunology and Cell Biology

Glucocorticoid-induced cell death of human B cells DR Liddicoat et al 830

Figure 4 Basal GR 1A3 promoter usage in the human thymus and PBMCs measured by Taqman qPCR. (a) PBMCs were isolated from fresh blood samples and sorted for the populations indicated. Data are derived from three sort experiments. B cells/NK cells: n ¼ 8 donors, CD4 þ T cells: n ¼ 7 donors, CD8 þ T cells: n ¼ 6 donors. NQ, not quantifiable (signal below dynamic range). Error bars: s.e.m. *Po0.05; ***Po0.001, lower than CD4 þ CD45RA  memory T cells by the Kruskal–Wallis test. (b) Thymocytes were isolated from fresh thymus biopsies and sorted for the populations indicated. Note: scale for GR 1A3 mRNA is different to a. Data are derived from three experiments comprising one donor each. Error bars: s.e.m. *Po0.05, higher than all other thymocyte subsets, with the exception of CD3 þ CD4 þ CD1a þ thymocytes (P ¼ 0.07), by the Kruskal–Wallis test.

Culture of human peripheral blood PBMCs with DEX does not result in robust increases in GR protein levels Many previous studies suggest that auto-upregulation of GR protein is required for GICD, whereas GR levels remain constant, or are downregulated in GICD-resistant populations.15–23 Given the high GICD sensitivity of peripheral blood B cells and complete GICD resistance of peripheral blood T cells in this study, we investigated auto-regulation in PBMCs using a technique developed to measure GR protein levels in individual cells by FACS45–47 (Figure 5). Fresh PBMC samples were cultured in vitro with DEX, and GR protein levels were measured in the viable (7AAD ) subset of each population. Interestingly, all cell types exhibited a clear single peak for GR protein at each time point, with the exception of B cells and CD16  CD94 þ NK cells at 48 h, where a sub-population of each of these cell types reduced GR protein levels by approximately twofold. Further investigation of these cells showed the GRhi cells were mostly 7AAD  SSClo, whereas the cells that had downregulated GR were mostly 7AAD SSChi. Inclusion of this population in our analysis resulted in an apparent decline in GR mean fluorescence intensity readings, which was reversed when these cells were excluded by tighter forwardscatter/SSC gating before analysis of GR expression (dotted line) (Figures 5b and c). As increased SSC in lymphocytes is a hallmark of early apoptosis preceding membrane permeability to 7AAD,42,43 this suggests GR protein levels are decreased during early apoptosis. In line with this observation, cell lineage markers (CD19 and CD56) were also downregulated in their respective SSChiGRlo subpopulations (data not shown). GR protein levels for all cell types including SSClow and SSChi cells, are summarized in Figure 5c. Immunology and Cell Biology

Overall, no significant change in GR protein was observed in any cell type, including GICD-sensitive B cells and CD94 þ NK cells (even when the GRloSSChi populations were excluded), suggesting that auto-upregulation was not required for GICD in these cultures. GR 1A3 promoter usage is upregulated in human peripheral blood B cells and whole thymocytes in the presence of DEX Given the large spread of GICD sensitivities observed for the major lymphocyte populations examined in this study, from highly sensitive B cells (SI ¼ 0.41), GICD-sensitive whole thymocytes (SI ¼ 0.62), through to GICD-resistant T cells (SI ¼ 0.98), we used these populations to investigate the potential relationship between regulation of GR transcripts and GICD sensitivity (Figure 6). Whole thymocytes, as well as sorted peripheral blood T and B cells, were separately cultured with DEX, and GR 1A1, 1A2, 1A3 and total GR transcript levels were assessed by qPCR. As described previously, although GR 1A3 mRNA was easily detectable, GR 1A1 and 1A2 mRNAs in all samples were again below the dynamic range of the qPCR assay (data not shown). GR 1A3 mRNA expression was generally synchronized with changes in total GR mRNA across the culture conditions investigated, although GR 1A3 levels generally changed in a far more pronounced manner. DEX treatment of whole thymocytes resulted in a more than twofold induction in percentage GR 1A3 promoter usage over freshly isolated, or VEH-treated cells (Figure 6a), which is consistent with equivalent studies with mouse thymocytes.17 Surprisingly, culture of human B cells in VEH (without DEX) resulted in a 39.3- and 3.0-fold increase in GR 1A3 and total GR mRNA, respectively, culminating in a 12.1-fold increase in percentage GR 1A3 usage over that in freshly

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Figure 5 Flow cytometric analysis of intracellular GR expression levels in PBMC populations during 48 h culture in VEH or DEX (10 7 M). PBMCs were counted and stained with Abs specific for CD3, CD4, CD8, CD16, CD19, CD56, CD94, viability stain 7AAD, and then subjected to intracellular staining with anti-GR monoclonal antibody. (a) (i) Representative dot plots showing the gating hierarchy used to identify T cells, NK cells and B cells. Figures beside each gate indicate frequency of parent for each population. (ii) Analysis of GR expression (open) vs isotype control (filled) for each of these populations. Histogram peaks are displayed as percentage of maximum. (b) Representative side scatter vs GR-staining profile of viable (7AAD ) cells in each PBMC population at 0 and 48 h. (c) GR expression (average geometric mean fluorescence intensities (MFIs)) for each PBMC subset at various time points post culture. GR expression is displayed as a ratio to basal (fresh) levels. n ¼ 5 donors, derived from three experiments. Error bars: s.e.m. Dotted lines show the impact of exclusion of the SSChi sub-population for DEX-treated B cells and CD16 CD94 þ NK cells (shown in b). No significant change in GR levels were observed relative to fresh controls by the Mann–Whitney test with Bonferroni correction, using raw MFIs.

isolated cells. In DEX-treated cultures, percentage GR 1A3 usage was 7.5-fold increased over freshly isolated levels. Vehicle treatment also increased GR 1A3 and total GR transcript levels in sorted T cells, although the changes in expression (particularly for GR 1A3) were far more modest compared with B cells, and in the presence of DEX, the percentage GR 1A3 usage was not significantly increased over basal levels. The induction of GR 1A3 and total GR mRNA in sorted B cells and T cells by VEH treatment cannot be attributed to the 1/1000 (v/v) ethanol in vehicle, as the same increases were seen in medium-alone controls (data not shown). Overall, percentage GR 1A3 promoter usage remained higher than basal levels during DEX treatment in B cells (Po0.01) and thymocytes (Po0.05), but not GICD-resistant T cells (Figure 6a). However, the GR 1A3 promoter induction in B cells appeared to be a consequence of the cell culture itself, rather than the presence of added DEX. In direct contrast to the percentage 1A3 usage, no significant change in total GR mRNA was observed in the presence of DEX for any cell type tested. As changes in GR mRNA species will only affect apoptosis when steroid is present, we next

compared the net effect of in vitro culture and DEX treatment on total GR mRNA as well as GR 1A3 mRNA levels in T cells, thymocytes and B cells by normalizing DEX-treated samples to basal (pre-culture) levels (Figure 6b). Importantly, the degree of upregulation of percentage GR 1A3 usage in GICD-sensitive B cells and thymocytes compared with GICD-resistant T-cell controls (which were nonsignificantly upregulating) was proportional to GICD sensitivity. Taken together, for the primary human lymphocyte populations tested, GICD best correlated with upregulation of percentage GR 1A3 usage above basal levels.

DISCUSSION Although GICD constitutes an important effect of GCs in the clinic, our understanding of what cell types are affected, as well as the underlying mechanism behind GICD, is incomplete. In this study, we show for the first time that the strong correlation between distal GR 1A promoter usage and GICD sensitivity, previously established in Immunology and Cell Biology

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Figure 6 Changes in GR 1A3 promoter usage by human lymphocyte populations in response to 4 h in vitro culture with VEH or DEX (10 7 M). (a) Before (fresh) and following culture, percentage GR 1A3 promoter usage was measured by Taqman qPCR. Sorted CD19 þ peripheral blood B cells: n ¼ 6 donors, derived from four experiments. Sorted CD3 þ peripheral blood T cells: n ¼ 6 donors, derived from three experiments. Whole thymocytes: n ¼ 4 donors, derived from four separate experiments. *Po0.05; **Po0.01, different to fresh controls by the Mann–Whitney test with Bonferroni correction. Error bars: s.e.m. (b) Summary of GR transcript expression for the lymphocyte subsets shown in a after 4 h culture in 10 7 M DEX, expressed as average fold induction (DEX/FRESH). *Po0.05; **Po0.01, significantly different to GICD-resistant T-cell controls by the Mann–Whitney test with Bonferroni correction. Error bars: s.e.m.

human cancer cell lines and mouse primary cells, is also present in primary human lymphocyte populations. Administration of GCs to humans results in a well-documented transient lymphopenia for 4–6 h, followed by lymphocytosis beyond 24 h. These effects have primarily been attributed to cell trafficking between lymphoid organs and blood.48–51 In addition, apoptotic cells are rapidly removed by phagocytes in vivo. Thus, in vitro culture was the best approach to ensure accurate assessment of GICD sensitivity. We opted for a two-pronged analysis using the apoptotic marker Annexin V at 12 h and cell viability as measured by 7AAD at 48 h. This gave consistent hierarchies of GICD sensitivity among the lymphocyte populations examined. The most striking result was the Immunology and Cell Biology

exquisite GICD sensitivity of peripheral blood B cells relative to other lymphocyte subsets, including thymocytes. This may be of particular relevance for rheumatic/autoimmune diseases such as systemic lupus erythematous and rheumatoid arthritis, where recently developed B-cell depletion strategies have demonstrated therapeutic effects.52,53 GCs are a traditional mainstay therapy for these diseases, despite the often severe, long-term side effects they induce, and a paucity of knowledge regarding their specific mechanisms of efficacy.1,54 To our knowledge, the sensitivity of non-malignant human B cells to GICD has not been previously reported. Our data suggest that GC therapy may induce wide-spread peripheral B-cell death in humans—a possibility that is supported by previous studies in mice both in vitro and in vivo.55,56 Given our observation that primary B-cell GICD can be blocked by high dose IL-4 in vitro, B cell sensitivity to GICD in vivo may also depend on the presence of this cytokine and/ or other survival factors. In line with this possibility, IL-4 was recently shown to protect from restraint-stress-induced depletion of B-cell numbers in the mouse.57 Another interesting observation from the GICD assays with potential clinical relevance was the clear GC-induced downregulation of the lineage marker CD56 on NK cells. The ability of GCs to downregulate NK cell-mediated cytotoxicity is well demonstrated both in vitro and in vivo, and has important implications in cancer therapy, transplantation and prevention of infection and malignancy during immunosuppression.58–61 Our observation of GC-induced CD56 downregulation on primary NK cells has been previously observed in an NK cell line (NK3.3) and, as CD56 is an adhesion molecule demonstrated to have a role in lysis of allogeneic and malignant targets, it may be that GC-mediated CD56 downregulation is a mechanism by which GCs reduce NK cytolytic activity.62–64 In this study, DEX treatment induced strong CD56 downregulation in both CD16  and CD16 þ NK populations, prompting the use of an alternative NK cell marker CD94 to ensure detection of the CD16  sub-population. This in turn suggests NK numbers could be significantly underestimated in the many clinical studies that use CD56 alone as an NK cell marker following GC treatment.65–68 Given the need to gate on CD16  NK cells and CD16 þ NK cells separately, we also took the opportunity to analyse their respective sensitivities to GICD and observed that, although CD16 CD94 þ NK cells were exquisitely sensitive to GICD, CD16 þ NK cells were far more GICD resistant, highlighting a novel functional distinction between these cell populations. As the CD16 CD94 þ NK population is the primary cytokine-producing population of NK cells, this strongly suggests NK cell cytokine production would be severely decreased as a consequence of GICD, in addition to suppression of cytokine production at the cellular level, at least in assays of primary NK activity following GC treatment in vitro, but potentially also in vivo. Interestingly, despite their high sensitivity to GICD, GR 1A3 expression was uniquely absent in freshly isolated CD3 CD19 CD56 þ NK cells (Figure 4a). It is possible that in the NK cell lineage, factors other than GR 1A3 expression determine GICD sensitivity. In studying human thymocytes, our observations that changes in total GR and especially GR 1A3 expression levels were synchronized across each developmental stage, suggested GR 1A3 is a means to dynamically regulate overall GR levels during thymocyte development. A very similar pattern of expression in T-cell development was also observed in mice. This synchronization may be attributed to the very similar architectures shared between proximal GR gene promoters and the GR 1A3 promoter.31 Of particular note, expression of total GR transcripts, as well as GR 1A3 transcripts and percentage GR 1A3 usage, peaked highly at the point of TCRb selection in both

Glucocorticoid-induced cell death of human B cells DR Liddicoat et al 833

human (PT1) and mouse (TN3/TN4), suggesting an evolutionarily conserved role for GR 1A transcripts at this stage in development. Although no similarities between basal GR and GR 1A3 mRNA expression profiles were seen across the different human PBMC populations investigated (Figure 4a), sorted peripheral blood T cells and B cells exhibited synchronized changes in total GR mRNA and GR 1A3 mRNA levels in response to in vitro culture and DEX treatment (Figure 6a). As seen across the different stages of thymic development, far more pronounced changes were seen in GR 1A3 mRNA levels relative to total GR mRNA, suggesting the GR 1A3 promoter may also have a role in dynamic regulation of overall GR levels in the periphery. Unexpectedly, a transcriptional upregulation of total GR, and in particular, GR 1A3 transcripts was observed in both sorted B cells and T cells in response to culture in vehicle (or medium-alone without Dex) (Figure 6a). To our knowledge this effect has not been reported previously, probably because PBMCs usually undergo at least one round of pre-incubation in vitro before GR transcript levels are measured (for example, culture-based monocyte removal, growth factor-based expansion and blood banking).69,70 The stimulus behind these increases is currently uncertain. It appears as a deductive process involving modulation of GCs and other culture media elements that may selectively alter the activity of transcriptional regulators of the GR gene, such as Myb, PU.1 and Spi-B,24,27 will be required to identify the signal. In line with a study of GR transcript expression in primary human B and T-ALL cells, our results show no correlation between baseline percentage GR 1A3 usage and GICD sensitivity.71 For example, although B cells were at the opposite end of the GICD sensitivity scale to T cells, there was no difference in basal percentage GR 1A3 usage between these populations. The only exception to this was CD4 þ CD45RA  T cells, which had higher percentage GR 1A3 usage than B cells (Po0.001) (Figure 4a). These observations are in contrast with our previous study in mice, which showed a strong correlation between basal percentage GR 1A usage and GICD sensitivity in thymocyte subsets.17 However, it is notable that the percentage GR 1A usage levels in mouse thymus are considerably higher than those seen in the human thymus and PBMCs. Upon establishment of a clear hierarchy of GICD sensitivity among primary human lymphocytes, we investigated these populations for correlates between GICD and regulation of GR protein and mRNA during GC treatment. Studies in mice and human cancer cell lines have long suggested the ability to auto-upregulate GR levels in response to GC stimulation is a key mechanism by which GICD sensitivity is conferred.16,17,20,24,27,30,31,72 However, some recent studies suggest auto-upregulation per se may not be essential for GICD, and instead maintenance of a critical threshold level of GR protein may be the more critical factor.13,73–76 In this study, although total GR mRNA was upregulated in B cells (threefold) and T cells (twofold) in response to in vitro culture in the absence of added DEX, these increases were not as marked in the presence of DEX, which if anything, appeared to dampen the culture-induced upregulation of GR mRNA. Indeed, it appears these overall transcriptional responses were of insufficient magnitude to markedly impact on GR protein, because overall GR protein levels did not increase in T cells and B cells in response to VEH or DEX across 48 h of culture. We considered the possibility that the intracellular GR staining by flow cytometry was misleading as a result of loss of a fraction of GR protein during the fixation and permeabilisation process; however, this seems very unlikely because both control and dexamethasone-treated samples were stained in the same way. Because no increases in Dex-treated cells over VEH were seen at the GR protein, or total GR mRNA level,

it appears that auto-upregulation in response to GC stimulation is not required for GICD in primary human lymphocytes. This result is in line with two recent studies on primary acute lymphoblastic leukaemia cells, where maintenance, or auto-downregulation of GR levels was observed in response to GC treatment in both GICDsensitive and -resistant isolates.75,76 It is also important to point out that increased GR levels are not the only determinant of sensitivity to GC, which is also regulated by the balance of pro- and anti-apoptotic members of the Bcl-2 family.77,78 Indeed, GC sensitivity is generally dissociated from GR expression in mouse thymocyte populations.39 In contrast to the stability in overall GR levels, a striking induction of GR 1A3 mRNA was observed in sorted peripheral blood B cells and, to a lesser extent, T cells, in response to in vitro culture. Although the addition of DEX appeared, if anything, to dampen this effect, the percentage GR 1A3 usage in B cells remained 7.5-fold higher than pre-culture levels, whereas in T cells no obvious induction was observed. Indeed, by comparing percentage GR 1A3 usage levels after culture with DEX, to original basal levels for T cells, thymocytes and B cells, a clear association between GICD sensitivity and increased GR 1A3 usage in the presence of ligand was evident. These results suggest that GR 1A3 transcript usage may be a key determinant of GICD in primary human lymphocytes, and in particular peripheral B cells, in turn highlighting the potential utility of GR 1A3 transcript usage as a biomarker of GICD sensitivity. Up to one-third of patients with rheumatoid arthritis, lupus, asthma and ulcerative colitis develop reduced GC sensitivity and GC resistance during GC treatment, presenting a major challenge to efficacy.2 In turn, a diagnostic system that would allow stratification of patient sensitivity to GC therapy represents a major aim in the field as it may permit more tailored treatment.2,79,80 Alternative treatments could be sought for resistant individuals, whereas GC dose may be limited in very sensitive individuals, allowing minimization of side effects. Multiplex gene quantification of 1A3 mRNA in combination with other markers of GC-responsiveness such as BIM and GILZ81,82 may provide more robust profiles.13 Such approaches to monitor GC sensitivity are already under development in the context of GC resistance in leukaemia.72 The results of this study highlight the association between GR exon 1A3 upregulation and GICD sensitivity. To date, two unique mechanisms have been suggested that may explain this phenomenon. First, compared with other first exons, 1A3 exon usage has been shown to favour translation of GR protein from the second start codon in exon 2 of the GR gene, resulting in increased production of GR protein isoform GR-B, which has been shown to be more efficient at transactivation than full-length isoform, GR-A.26,83 In turn, this may assist in the transcription of pro-apoptotic genes.26 However, the ability of GR 1A3 transcripts to drive translation of other GR isoforms such as GR-C and GR-D, the former of which is known to activate pro-apoptotic genes,84,85 remains to be investigated, and further studies are required. Second, GR 1A3 usage may lead to expression of GR at the cell membrane (mGR). Transfection of mouse GR1A complementary DNA into mGR-negative cell lines is known to impart expression of mGR (detectable on the cell surface with anti-cytosolic GR antibodies) and mGR expression has been shown to correlate with GICD sensitivity in multiple studies.86–89 Given the human GR 1A3 exon has high homology with the mouse GR 1A exon, and furthermore, mGRs are have been detected on primary human B cells and monocytes, investigation of the potential link between human exon GR 1A3 and mGR expression is a compelling area of further study.17,90 Ultimate proof of the role of human GR 1A3 promoter in determining GICD sensitivity will require inactivation of Immunology and Cell Biology

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the GR 1A3 gene. Nonetheless, the data herein demonstrate a clear association between GR 1A3 promoter expression and GICD in primary human lymphocytes, particularly B cells. This provides a valuable step towards understanding GICD and underscores the potential use of GR 1A3 expression as a biomarker of GICD sensitivity, with the ultimate goal of manipulating this process in disease settings. METHODS Ethics statement These studies were reviewed and approved by the University of Melbourne Health Sciences Human Ethics sub-committee. For use of human thymus tissue approval was also obtained from the Ethics in Human Research Committee at the Royal Children’s Hospital (Melbourne, Australia).

Human tissue and blood samples Blood samples were collected in heparinised tubes. To avoid changes in gene expression associated with storage of blood at room temperature, PBMCs were obtained by Ficoll-mediated density centrifugation of freshly taken blood from a cohort of healthy human donors. All blood samples were taken with written, informed consent from each donor. Thymic samples were obtained from young patients undergoing corrective cardiac surgery where thymus fragments are routinely removed. To minimize changes in gene expression associated with storage, thymic biopsies were immediately stored in saline at 4 1C and used for in vitro culture or cell sorting within 12 h of surgery. Thymocyte suspensions were prepared by gently pushing thymus fragments through a metal sieve. Written, informed consent was obtained from the legal guardian of each donor.

Primary cell culture and reagents Cells were cultured in flat bottom plates in RPMI-1640 medium (Invitrogen Life Technologies, Mulgrave, VIC, Australia), 10% fetal calf serum (CSL, Melbourne, VIC, Australia), 2 mM GlutaMax (Invitrogen Life Technologies) and 50 mg ml 1 penicillin/streptomycin (Invitrogen Life Technologies) at a cell density of 2.5  106 cells ml 1. The only exception to this was sorted peripheral blood B cells, which were cultured in round bottom plates at 1.0  106 cells ml 1. DEX (Sigma, Castle Hill, NSW, Australia) was diluted from a 10 2-M stock stored in ethanol, with a (1/1000) dilution of ethanol alone as a vehicle control (VEH). Cell viability was assessed post culture by trypan blue stain and counted on a haemocytometer.

Flow cytometry Cell suspensions were prepared in cold phosphate-buffered saline containing 2% fetal calf serum, and counted and stained with antibodies for 20 min at 4 1C, before acquisition on an LSR-II cytometer (Becton Dickinson, North Ryde, NSW, Australia). Cell doublets were gated out by doublet discrimination. Apoptotic cells were determined by staining with 7AAD during standard antibody staining. Owing to slight granularity and size differences between lymphocyte populations, forward and side scatter were not used to exclude apoptotic cells. All antibodies used for cell surface staining were from Becton Dickinson, including anti-human abTCR (T10B9.1A-31), CD1a (HI149), CD3e (UCHT1), CD4 (SK3), CD8a(RPA-T8), CD16 (3G8), CD19 (HIB19), CD34 (My10), CD45RA (HI100), CD45RO (UCHL1), CD56 (NCAM16.2) and CD94 (HP-3D9); anti-mouse abTCR (H57-597), CD3e (145-2C11), CD4 (RM4-5), CD8a (53-6.7) and CD44 (IM7). Early apoptotic cells were determined by Annexin V staining. Cells were washed in phosphate-buffered saline and then antibody stained (including Annexin V, Becton Dickinson) in Annexin V-binding buffer for 10 min, at room temperature (21 1C) in the dark. Cells were then washed once in Annexin V-binding buffer before analysis. Intracellular GR levels were determined following standard surface staining by flow cytometry using anti-GR monoclonal antibody 5E4 (AbD serotec, Raleigh, NC, USA) and intracellular staining reagents from the FoxP3 Staining Buffer Set (eBiosciences, San Diego, CA, USA). Fixation and permeabilization was performed according to the manufacturer’s instructions, including the optional block step with 2% normal rat serum. Immunology and Cell Biology

Cell sorting and complement-mediated lysis Following antibody staining, cells were sorted using a FACSAria cell sorter (Becton Dickinson). A sample of sorted cells was always analysed to assess purity, which was always 497%. Sorted samples were snap frozen at 80 1C until RNA extraction. For the sorting of CD4 CD8  populations of the mouse thymus, CD8 þ cells were removed from thymocyte cell suspensions by complement-mediated lysis before sorting. Briefly, thymocyte suspensions containing 0.02 g ml 1 DNase I (Roche, Castle Hill, NSW, Australia) were stained with anti-mouse CD8 (clone 3.155, grown in-house) for 10 min at 4 1C, then incubated with 2 ml of rabbit complement (GTI, Waukesha, WI, USA) for 30 min at 37 1C. Viable cells were isolated on a Histopaque 1.083 g ml 1 gradient (Sigma-Aldrich, Castle Hill, NSW, Australia) and subsequently counted and stained with Abs for cell sorting.

Quantitative real-time PCR Total RNA was isolated using the RNeasy kit (Qiagen, Venlo, Netherlands), and complementary DNA was synthesized by using the Omniscript kit (Qiagen) according to the manufacturer’s instructions with random hexamers (Invitrogen Life Technologies). Primers were designed using Primer Express software (Applied Biosystems, Paisley, UK). Samples were analysed on an ABI7700 RealTime PCR machine (Applied Biosystems) and results were assessed using Sequence Detector software (Applied Biosystems). Human cell populations were analysed using the Taqman chemistry with normalization by 18S rRNA, which was detected using Applied Biosystems predeveloped Taqman assay reagents. The PCR conditions were 50 1C for 2 min, 95 1C for 10 min, followed by 45 cycles of 95 1C for 15 s and 60 1C for 1 min. Specific detection of each of the three human GR 1A mRNA transcripts containing alternatively spliced exons 1A1, 1A2 or 1A3, was achieved using three separate amplicons, each including a forward primer in each respective exon 1, a probe spanning each respective splice site and a reverse primer in the common exon 2. The primer pairs and their respective probes were: total GR transcripts (spans exons 5/6, F: 50 -TAGGAGGGCGGCAAGTGAT-30 , R: 50 -TGCAGTAGGGTCATTTGGTCAT-30 , P: FAM-50 -CAAAGGCAATACCAGG TTTCAGGAACTTACACCT-30 -TAMRA), GR 1A3 splice variant (spans exons 1A3/2, F: 50 -GCCTGGCTCCTTTCCTCAA-30 , R: 50 -ACCAGGAGTTAATGATT CTTTGGAGT-30 , P: FAM-50 -CATCAGTGAATATCAACTTCCTTCTCAGAC ACTTT-30 -TAMRA), GR 1A2 splice variant (spans exons 1A2/2, F: 50 -CGCATGTGTCCAACGGAA-30 , R: 50 -TCCTGAGCAAGCACACTGCT-30 , P: FAM-50 -CTTTGGAGTCCATCAGTGAATATCAACCTCT-30 -TAMRA), GR 1A1 splice variant: (spans exons 1A1/2, F: 50 -TTTAAATGGCAGAGAGAAGGAGAAA-30 , R: 50 -TCTACCAGGAGTTAATGATTCTTTGGA-30 , P: FAM-50 TCCATCAGTGAATATCAACTTCTAAGGTCCAGTGA-30 -TAMRA). Mouse cell populations were analysed using the SYBR green chemistry (Applied Biosystems) with levels normalized to 18S rRNA. The PCR conditions were 95 1C for 10 min, followed by 45 cycles of 95 1C for 15 s and 60 1C for 1 min, ending with a melting curve to validate product specificity. The primer pairs used to detect all mRNA species spanned at least one exon boundary. 18S rRNA (F: 50 -GTAACCCGTTGAACCCCATT-30 , R: 50 -CCATCCAATCGGTAGTAGCG-30 ), GR1A (exon 1/2, F: 50 -CGTTAAGATGT CTGGGAGGAAGTT-30 , R: 50 -GGTTTTATACAAGTCCATCACGCTT-30 ), total GR transcripts (spanning exon 2/3, F: 50 -GCAGGCCGCTCAGTGTTTT-30 , R: 50 -CCATAATGGCATACCGAAGCTT-30 ).

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Daniel Pellicci and Mark Veitch for technical assistance. We are grateful to David Ritchie, Royal Melbourne Hospital, for critically appraising this manuscript. We also thank the Picchi Brothers Foundation for generously supporting our flow cytometry facility and Ken Field for assisting with FACS sorting. This work was supported by a National Health and Medical Research Council of Australia (NHMRC) Project Grant (350302). We also acknowledge the following support: DRL, Monash University Graduate Scholarship; DIG, NHMRC Senior Principal Research Fellowship (1020770); NHMRC Program

Glucocorticoid-induced cell death of human B cells DR Liddicoat et al 835 Grants (454569 and 1013667); SPB, NHMRC project grant (454363) and RD Wright Fellowship (454731).

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