Multiple differences in gene expression in regulatory V␣24J␣Q T cells from identical twins discordant for type I diabetes S. Brian Wilson*†, Sally C. Kent‡, Heidi F. Horton§, Andrew A. Hill§, Paul L. Bollyky¶, David A. Hafler‡, Jack L. Stromingerʈ, and Michael C. Byrne§† *Cancer Immunology and AIDS, Dana Farber Cancer Institute, Boston, MA 02115; ‡Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA 02115; §Genetics Institute, Cambridge, MA 02140; ¶Harvard Medical School, Boston, MA 02115; and ʈDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 Contributed by Jack L. Strominger, April 10, 2000 Quantitative and qualitative defects in CD1d-restricted T cells calcium flux determination was performed by using a MoFlo have been demonstrated in human and murine autoimmune cytometer (Cytomation, Fort Collins, NJ) as described (8). diseases. To investigate the transcriptional consequences of T cell receptor activation in human V␣24J␣Q T cell clones, DNA Cell Culture. Single V␣24-positive, CD4͞8-negative single-cell microarrays were used to quantitate changes in mRNA levels sorts were grown on irradiated allogeneic feeders at 50,000 after anti-CD3 stimulation of clones derived from identical twins cells per well with 5,000 cells per well irradiated (5,000 rads) discordant for type 1 diabetes and IL-4 secretion. Activation 721.221 lymphoblastoid cells with 1 ␮g͞ml PHA-P, IL-2, and resulted in significant modulation of 226 transcripts in the IL-4 IL-7 each at 10 units͞ml (Boehringer Mannheim) and prop- secreting clone and 86 in the IL-4-null clone. Only 28 of these agated as described (8). Clones positive for V␣24 and NKR- genes were in common. The differences observed suggest both P1A by flow cytometry and a V␣24J␣Q CDR3 T cell antigen ineffective differentiation of diabetic V␣24J␣Q T cells and a role receptor sequence were assayed for cytokine secretion in ͞ for invariant T cells in the recruitment and activation of cells C1R CD1d restriction experiments. For cytokine secretion IMMUNOLOGY ␣ ␣ from the myeloid lineage. and inhibitor studies, V 24J Q T cell clones GW4 (nondia- betic) and ME10 (diabetic) at 5 ϫ 104 cells per well were activated with plate-bound anti-CD3 or Ig control at 1 ␮g͞ml. nvariant CD161ϩ T cells are reported to be important in the Secreted IL-4 and IFN-␥ were assayed by ELISA after4hof Iregulation of T helper cell (Th) Th1͞Th2 bias (1). In several ϩ ␣ ␣ activation as described (8). Optimal concentrations of inhib- murine models of autoimmunity, CD161 V 14J 281 T cells itors previously were determined by inhibitor dose–response were shown to be present in diminished numbers and to further experiments. The concentrations of inhibitors used were 10 decrease in frequency before the onset of disease (2–4). When nM wortmannin; 10 ␮M LY294002; 50 ␮M PD98059, a this population of cells was transferred from either nonobese ␮ ͞ ␣ ␣ mitogen-activated protein kinase kinase inhibitor; and 50 M nondiabetic (NOD) or nonobese diabetic V 14J 281- SB203580, a p38 kinase inhibitor. The concentrations of transgenic donors to prediabetic animals, the recipients were phorbol ester and calcium ionophore used were 1 ng͞ml protected from diabetes (4, 5). This transfer of protection was phorbol 12-myristate 13-acetate (PMA) and 1 ␮g͞ml ionomy- significantly inhibited by the coadministration of anti-IL-4 an- cin. Cyclosporin A (CsA) was used at 5 ng͞ml. Calcium flux tibodies (6). was determined by loading cells with indo-1 as per the Humans have a homologous invariant (i.e., with no N region manufacturer’s specifications (Molecular Probes), followed by ϩ ␣ ␣ additions) CD161 V 24J Q T cell population whose restriction activation with anti-CD3 at 10 ␮g͞ml. Maximal calcium flux element, like that for the murine CD161ϩV␣14J␣281 T cells, is the was determined by the addition of ionomycin. nonpolymorphic class Ib molecule CD1d (7). We recently demon- strated that in five sets of monozygotic twins and triplets discordant Messenger RNA Expression. V␣24J␣Q T cell clones GW4 and for type 1 diabetes, invariant V␣24J␣Q T cells were present at ME10 (1 ϫ 107 cells) were activated for4hwith10␮g͞ml significantly higher frequencies in the nondiabetic siblings (8). soluble anti-CD3 or control IgG. Optimal concentrations of Moreover, V␣24J␣Q T-cell clones from the nondiabetic siblings anti-CD3 previously were determined by dose–response ex- secreted both IL-4 and IFN-␥, whereas those derived from the periments measuring cytokine secretion. Total RNA was diabetic siblings had an extreme impairment in the ability to secrete isolated with Qiagen RNeasy kits. Total RNA then was IL-4. To delineate differences in gene expression that might ac- converted to double-stranded cDNA by priming with an count for the discordant phenotype and to ask whether the loss of oligo(dT) primer that included a T7 RNA polymerase pro- IL-4 secretion was the only defect, a comprehensive analysis of T moter site at the 5Ј end (11). The cDNA was used directly in cell activation was undertaken in a representative clone pair derived an in vitro transcription reaction in the presence of biotinylated from these disease-discordant twins. nucleotides to produce labeled cRNA (antisense RNA), which Methods Abbreviations: Th, T helper; PI3-kinase, phosphoinositide-3-OH kinase; PMA, phorbol Antibodies. Anti-V␣24, -V␤11, and -␣␤TCR were purchased 12-myristate 13-acetate. from Immunotech (Westbrook, ME). Anti-CD4, -CD8, and See commentary on page 6933. -CD161 were purchased from PharMingen. Anti-CD3, clone †To whom reprint requests should be addressed. E-mail: [email protected] (M.C.B.) or [email protected] (S.B.W.). UCHT1, was purchased from Ancell (Bayport, MN), and IgG1 The publication costs of this article were defrayed in part by page charge payment. This control was purchased from Sigma. article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Flow Cytometry. Stained cells were analyzed on a FACScan Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.120161297. cytometer (Beckton Dickinson), and single-cell sorting and Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.120161297 PNAS ͉ June 20, 2000 ͉ vol. 97 ͉ no. 13 ͉ 7411–7416 Downloaded by guest on September 25, 2021 Fig. 1. Discordant expression of PI3-kinase-regulated events differentiates IL-4ϩ from IL-4-null V␣24J␣Q T cell clones. (A)V␣24J␣Q T cell clones GW4 (nondiabetic) and ME10 (diabetic) were activated with plate-bound anti-CD3 or Ig control. Levels of secreted IL-4 and IFN-␥ were assayed by ELISA. The concentrations of inhibitors used were 10 nM wortmannin (wort.); 10 ␮M LY294002 (LY); 50 ␮M PD98059 (PD), a mitogen-activated protein kinase kinase inhibitor; and 50 ␮M SB203580 (SB), a p38 kinase inhibitor. Data points were collected in triplicate, and the Fig. is representative of four independent experiments. The concentrations of phorbol ester and calcium ionophore used were 1 ng͞ml PMA and 1 ␮g͞ml ionomycin (Iono). Cyclosporin A (CsA) was used at 5 ng͞ml. (B)V␣24J␣Q T cell clones GW4 (IL-4ϩ) and ME10 (IL-4-null), 1 ϫ 106 cells each, were loaded with Indo-1 (10 ␮M) for 45 min, then stimulated with 10 ␮g͞ml anti-CD3 (open arrowhead) and analyzed on a Cytomation MoFlo instrument. At the end of the experiment, ionomycin was added to a final concentration of 1 ␮g͞ml (filled arrowhead) to determine maximal flux. The ratio of Indo-1 fluorescence 410͞490 nm (410, Ca2ϩ-bound; 490, Ca2ϩ-free) after stimulation in a representative pair of clones is pictured. Control experiments showed no differences in calcium flux in response to thapsigargin treatment (data not shown). was hybridized overnight to Genechips (Affymetrix, San Jose, Because the defect in IL-4 secretion was likely the result of CA). After staining with phycoerythrin-streptavidin, the flu- multiple differences, a representative clone pair was chosen for orescence of bound RNA was quantitated by using a Genechip intensive analysis with DNA microarrays that monitor the expres- Reader (a modified confocal microscope; Affymetrix). sion of Ϸ6,800 genes (Unigene collection; National Center for Biotechnology Information, Bethesda, MD). The DNA microar- Results and Discussion rays provide a practical and reproducible approach for large-scale To identify which of the known signaling cascades initiated by T study of complex differences in gene expression (11–14). Expres- cell antigen receptor ligation played a dominant role in IL-4 sion profiles were determined after4hofstimulationwithanti-CD3 secretion, a series of inhibitor studies was performed. Inhibitors or control IgG. This time point was selected because it was used in of specific kinase cascades were used in conjunction with anti- a previous analysis of cytokine secretion in clones derived from CD3 stimulation. Both phosphoinositide-3-OH kinase (PI3- monozygotic twin pairs discordant for type 1 diabetes (8). The kinase) inhibitors wortmannin and LY294002 blocked anti-CD3- number of genes with detectable expression either before or after induced IL-4 secretion from the IL-4ϩ clone, but had no effect stimulation was nearly identical for the IL-4 null and IL-4-secreting on the secretion of IFN-␥ from either the IL-4ϩ or IL-4-null clones (1,523 and 1,558, respectively). As expected, the frequency clones (Fig. 1). In contrast, inhibition of the mitogen-activated of the majority of transcripts was unchanged. Interestingly, only protein kinase kinase by PD98059 or the JNK and p38 cascades ͞ with SB203580 (9, 10) had no effect. After inhibition of PI3- about 2 3 of this set (988) were shared between the two clones. The kinase, IL-4 secretion could be rescued in the IL-4ϩ clone by the number of genes whose expression after anti-CD3 stimulation was found to increase or decrease by at least 2-fold relative to unstimu- inclusion of the phorbol ester PMA or the calcium ionophore ϩ ionomycin.