Brief Definitive Report Qa-2–dependent Selection of CD8␣ր␣ T Cell Receptor ␣ր␤ϩ Cells in Murine Intestinal Intraepithelial Lymphocytes By Gobardhan Das,* Dina S. Gould,‡ Mathew M. Augustine,* Gladis Fragoso,¶ Edda Scitto,¶ Iwona Stroynowski,ʈ Luc Van Kaer,§ Danny J. Schust,‡ Hidde Ploegh,‡ and Charles A. Janeway, Jr.* From the *Section of Immunobiology, Yale University School of Medicine, and the Howard Hughes Medical Institute, New Haven, Connecticut 06520-8011; the ‡Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115; the §Department of Microbiology and Immunology and The Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; the ʈCenter for Immunology and the Department of Microbiology and the Department of Internal Medicine, Southwestern Medical Center, Dallas, Texas 75235-9093; and the ¶Department of Immunology, Instituto de Investigaciones Biomedicas, Universidad Nacional Avtonoma de Mexico, Mexico D.F. 04510 Abstract Murine intestinal intraepithelial lymphocytes (iIELs) are made up of a heterogeneous mix of T cells with unique phenotypes. Whereas CD8ϩ T cells in peripheral lymphoid organs use CD8␣ր␤ and are selected on MHC class Ia molecules, a majority of iIELs use CD8␣ր␣. Here, we report that the presence of CD8␣ր␣ TCR-␣ր␤ cells in iIELs is independent of classical MHC class I molecules Kb and Db, as illustrated by their presence in Kb/Db double-knockout mice and in mice lacking a nonclassical MHC class I molecule, CD1d. Most strikingly, their in mice lacking transporter associated with antigen processing %70ف presence is decreased by (TAP). The TAP-dependent nonclassical MHC class I molecule Qa-2 is strongly implicated in the presence of these cells, as inferred from the low numbers of CD8␣ր␣ TCR-␣ր␤ T cells in mice deficient in Qa-2 genes. Second, a Qa-2–transgenic mouse made in a Qa-2Ϫ strain showed an increase in the numbers of CD8␣ր␣ cells among its iIELs. Thus, the presence of CD8␣ր␣ TCR-␣ր␤ cells in iIELs is mainly dependent on the nonclassical MHC class I mole- cule Qa-2. Key words: CD8␣ր␣ TCR-␣ր␤ cells • MHC class I–deficient mice • Qa-2–transgenic mice • Qa-2–deficient mice • intestinal intraepithelial lymphocyte Introduction In contrast to conventional thymus-derived ␣ր␤ TCR TCR-␣ր␤ subset of iIELs is still lacking information about cells, intestinal intraepithelial lymphocytes (iIELs) are a dis- the MHC molecules that they recognize and that present tinct population having several unique features. The vast antigen to them. They appear to be a resident subset, do majority of such cells are CD8ϩ, the frequency of ␣ր␤ and not participate in antigen-specific immune responses, and ␥ր␦ T cells is roughly equal, and most ␥ր␦ T cells express a may play a role(s) in localized immune regulation (15, 16). CD8␣ր␣ homodimer and may develop extrathymically (1– Mice aged 6–8 wk and raised in specific pathogen–free 8). TCR-␣ր␤ cells in the iIEL compartment are generally conditions contain an equal number of ␣ր␤ and ␥ր␦ T cells heterogeneous in terms of their expression of CD8␣ր␣ and among their iIELs, which are primarily, but not solely, CD8␣ր␤. CD8␣ր␤ cells are reported to govern conven- CD8␣ր␣ (17). Previously, it had been shown that the clas- tional immune responses against pathogens and environ- sical MHC class I molecules, Kb and Db, are required for mental antigens (9–14). Information regarding the CD8␣ր␣ the presence of CD8␣ր␤ TCR-␣ր␤ cells in peripheral lym- phoid organs as well as in iIELs. On the other hand, CD8␣ր␣ TCR-␣ր␤ cells were found only in intestinal epi- thelium and do not require the classical MHC class I mole- Address correspondence to Charles A. Janeway, Jr., Section of Im- b b munobiology, P.O. Box 208011, LH416, 310 Cedar St., New Haven, cules K and D (17–19). However, the presence of such CT 06520-8011. Phone: 203-785-2793; Fax: 203-737-1765; E-mail: cells is dependent on ␤2-microglobulin (␤2m), suggesting a [email protected] requirement for an MHC-related protein (20). 1521 J. Exp. Med. The Rockefeller University Press • 0022-1007/2000/11/1521/07 $5.00 Volume 192, Number 10, November 20, 2000 1521–1527 http://www.jem.org/cgi/content/full/192/10/1521 Here, we report that while the maintenance of CD8ϩ CD1Ϫ/ϪTAPϪ/Ϫ mice were generated by an F1 brother and sister Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ TCR-␣ր␤ iIELs is dependent on ␤2m and transporter asso- mating of CD1 and TAP mice. CD1d mice were de- ciated with antigen processing (TAP) function, the absence scribed previously (28), as were Qa-2–transgenic mice (29). In of the Kb and Db molecules does not alter the development brief, these mice were produced in (C57BL/6 ϫ BALB/AnN) or maintenance of such cells. This observation allowed us F1 and backcrossed to BALB/cAnN for 10 generations. 6–8-wk- old mice, irrespective of their sex, were used throughout the to study the involvement of TAP-dependent nonclassical study. Mice were maintained in a pathogen-free colony and fed MHC molecules on the maintenance of TCR-␣ր␤ cells sterile food prepared at the Yale Animal Research Center. Anti– among the iIELs. All of the MHC class I molecules and the TCR-␣ր␤ (H-57), anti–TCR-␥ր␦ (GL-3), anti-CD8␤ (Ly-3.2), well studied, nonclassical MHC class Ib molecules Qa-2 and anti–Qa-2 (1-1-2), anti-V␤2 (B20), -V␤3 (KJ25), -V␤4 proteins are known to be expressed in a TAP-dependent (KT4), -V␤5.1,5.2 (MR9-4), -V␤6 (RR4-7), -V␤7 (TR310), fashion (21). These proteins are encoded by four genes -V␤8.1,8.1 (MR5-2), -V␤9 (MR10-2), -V␤10b (B21.5), -V␤11 (Q6, Q7, Q8, and Q9) in C57BL/6 mice (22), contribut- (RR3-15), -V␤12 (MR12-3), -V␤14 (14-2), and anti-CD1D ing to a Qa-2high phenotype (23). BALB/cJ mice carry only (1B1) antibodies were purchased from PharMingen. Anti-CD8␣ two functional Qa-2 genes (Q6 and Q7; reference 24) and (53-6.7) was purchased from Sigma-Aldrich. Anti-MHC class I b b express Qa-2 at an intermediate level (Qa-2dull; reference antibody (K and D ; HB-51) was purified from the culture su- 25). We find that BALB/cJ mice have a corresponding de- pernatant. Preparation of iIELs. iIELs were prepared as described earlier crease in the percentage of CD8␣ր␣ TCR-␣ր␤ cells in (30) with a minor modification. In brief, small intestines were their iIEL compartments. BALB/cByJ and C3H/HeJ mice, harvested and washed by swirling in PBS. Mesentery and Peyer’s which lack functional Qa-2 genes altogether and are there- patches were carefully removed. The intestines were cut longitu- -cm pieces. Intestinal pieces were agi-0.5ف fore deficient in Qa-2 expression (23, 25, 26), have a severe dinally and then into deficit in the number of CD8␣ր␣ TCR-␣ր␤ϩ iIELs. To tated in 25 ml of extraction buffer (PBS, 3% FCS, 1 mM dithio- further confirm the apparent importance of Qa-2 on the threitol, 1 mM EDTA) for 30 min at 37ЊC. This slurry was passed presence of CD8␣ր␣ TCR-␣ր␤ T cells in iIELs, we exam- through a loosely packed nylon wool column to remove the ag- ined their abundance in Qa-2–transgenic mice. Indeed, the gregates. The follow-through was layered on a discontinuous Qa-2–transgenic mice produce higher numbers of CD8␣ր␣ Percoll gradient (Amersham Pharmacia Biotech). This gradient TCR-␣ր␤ iIELs. Thus, Qa-2 by itself can account for most was then centrifuged at 900 g for 20 min. Cells at the interface of the 40/70% layer were collected and washed in staining buffer. ␣ր␣ ␣ր␤ of the TAP-dependent presence of CD8 TCR- FACS® Staining and Analysis. Cells were suspended in stain- cells among iIELs. ing buffer at a concentration of 107 cells/ml. 100 ␮l of suspension was incubated either with directly conjugated antibodies or biotin- ylated antibodies for 30 min on ice. For the latter, streptavidin– Materials and Methods PE was used as the secondary reagent for an additional incubation Mice and Antibodies. C57BL/6, BALB/c, BALB/cByJ, C3H/ of 30 min on ice. Cells were washed twice with staining buffer Ϫ/Ϫ HeJ, B6C3F1/J, CByB6F1/J, and B6-␤2m mice were pur- and fixed with 1% paraformaldehyde. Fluorescence intensities chased from The Jackson Laboratory. TAPϪ/Ϫ mice, which had were measured with FACScan™ (Becton Dickinson). Cells used been backcrossed with C57BL/6 mice for 12 generations, were a for sorting were stained in Click’s Earls Hanks amino acids, 5% gift from Dr. K.A. Hogquist (University of Minnesota, Minneap- FCS media. Cells were sorted by using a FACStarPLUS™ (Becton olis, MN). The KbDbϪ/Ϫ mice were described previously (27). Dickinson). Figure 1. CD8␣ր␣ TCR-␣ր␤ iIELs are present in Kb/Db double-knock- out mice but absent in TAP- and/or Ϫ/Ϫ ␤2m mice. Comparison of iIELs from C57BL/6, KbDbϪ/Ϫ, and KbDbϪ/Ϫ CD1Ϫ/Ϫ mice showed almost equal numbers of CD8␣ր␣ TCR-␣ր␤ cells, whereas TAPϪ/Ϫ, TAPϪ/ϪCD1Ϫ/Ϫ, and Ϫ/Ϫ ␤2m mice had only a few of these cells. TCR-␥ր␦ cell numbers are not af- fected in any of these mice. The data shown represents six independent ex- periments. 1522 TAP-dependent CD8␣/␣ TCR-␣/␤ Cells in iIELs Are Qa-2 Dependent Results and Discussion Mice deficient in MHC class I expression lack peripheral Table I.