Comparative Contribution of CD1 on the Development of CD4 + and CD8+ T Cell Compartments
This information is current as Bin Wang, Taehoon Chun and Chyung-Ru Wang of September 29, 2021. J Immunol 2000; 164:739-745; ; doi: 10.4049/jimmunol.164.2.739 http://www.jimmunol.org/content/164/2/739 Downloaded from
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Comparative Contribution of CD1 on the Development of CD4؉ and CD8؉ T Cell Compartments1
Bin Wang, Taehoon Chun, and Chyung-Ru Wang2
CD1 molecules are MHC class I-like glycoproteins whose expression is essential for the development of a unique subset of T cells, the NK T cells. To evaluate to what extent CD1 contributes to the development of CD4؉ and CD8؉ T cells, we generated CD1oIIo and CD1oTAPo mice and compared the generation of T cells in these double-mutant mice and IIo or TAPo mice. FACS analysis showed that the number of CD4؉ T cells in CD1oIIo mice was reduced significantly compared with the corresponding population in IIo mice. Both CD4؉ NK1.1؉ and the CD4؉ NK1.1؊ population were reduced in CD1oIIo mice, suggesting that CD1 can select not only CD4؉ NK1.1؉ T cells but also some NK1.1؊ CD4؉ T cells. Functional analysis showed that the residual CD4؉ cells in CD1oIIo can secrete large amounts of IFN-␥ and a significant amount of IL-4 during primary stimulation with anti-CD3, sug- ؉
gesting that this population may be enriched for NK T cells restricted by other class I molecules. In contrast to the CD4 Downloaded from population, no significant differences in the CD8؉ T cell compartment can be detected between TAPo and CD1oTAPo mice in all lymphoid tissues tested, including intestinal intraepithelial lymphocytes. Our data suggest that, unlike other MHC class I mole- :cules, CD1 does not contribute in a major way to the development of CD8؉ T cells. The Journal of Immunology, 2000, 164 739–745.
he CD1 molecules are cell surface glycoproteins that have (IEC) (20). This unique localization of hCD1d may allow recog- http://www.jimmunol.org/ been conserved throughout mammalian evolution (1–6). nition by intraepithelial lymphocytes (IEL). However, the expres- T The overall structure of CD1 resembles that of MHC class sion of CD1 on mouse IEC is still controversial, as anti-CD1 mAbs I molecules, with three extracellular domains (␣1, ␣2, and ␣3), a differ in detection of CD1 expression on mouse IEC (15–17, 21). transmembrane region, and a short cytoplasmic tail. The ␣3 do- Unlike MHC class I molecules, the expression of CD1 in both   3 main is noncovalently associated with 2-microglobulin ( 2m). human and mouse does not require functional TAP (22, 23). Unlike classical class I molecules, CD1 is relatively nonpolymor- Study of T cell development in mutant mice lacking MHC mol- phic and is expressed at lower levels (5, 7). Thus, CD1 molecules ecules revealed that MHC class I and class II molecules play a were classified as a member of MHC class Ib family. However, central role in the development of CD8ϩ and CD4ϩ T cells, re- unlike most of MHC class Ib genes, CD1 genes map outside of spectively (24–28). Recent studies using CD1-deficient mice have by guest on September 29, 2021 MHC both in humans and mice (8, 9), and they are significantly shown that CD1 is essential for the development of a major subset divergent from other class I genes. The sequence homology be- of NK1ϩ T cells (29–31), which use an invariant TCR ␣-chain in tween CD1 and other class I molecules is only 25–30% (5). The conjunction with a restricted set of TCR -chains (32, 33). These same degree of homology was also detected between CD1 and NK1ϩ T cells promptly produce large amounts of cytokines, in class II molecules, suggesting that CD1 may represent a third lin- particular IL-4, upon primary stimulation by TCR engagement eage of Ag-presenting molecules (10). Consistent with this idea, (34). However, the role of CD1 in the development of other T cell CD1 molecules have been shown to present lipid and glycolipid subsets was unclear. Due to the presence of other MHC class I and Ags to T cells (11–14), while MHC class I and class II molecules class II molecules in CD1o mice, no significant changes in either present peptide Ags to T cells. CD4ϩ or CD8ϩ population were detected in CD1o mice (29–31). Mouse CD1 is encoded by two closely related genes, CD1d1 Yet, several lines of evidence suggested that CD1 might be in- and CD1d2 (7). CD1d1 is widely expressed on cells of multiple volved in the development of some CD4ϩ and CD8ϩ T cells. In hemopoietic lineages (15–17), including B and T cells, macro- MHC class II-deficient mice, a small population of CD4ϩ T cells phages, and dendritic cells, while CD1d2 can be detected only on can be detected in the periphery (26–28). Many of the hybridomas thymocytes (18, 19). Human CD1d can be detected in the apical derived from the CD4ϩ T cells of class II-deficient mice have been and lateral regions of small and large intestinal epithelial cells shown to recognize CD1, implicating a role for CD1 in the devel- opment of some CD4ϩ T cells (35). Although the expression of the  o class I molecules is reduced significantly both in 2m and in Gwen Knapp Center for Lupus and Immunology Research, Committee on Immunol- o ϩ o ogy, and Department of Pathology, University of Chicago, Chicago, IL 60637 TAP mice, the residual number of CD8 T cells in TAP mice is  o Received for publication July 27, 1999. Accepted for publication November 4, 1999. slightly higher than that in 2m mice (36–38). One possible source of these residual CD8ϩ T cells may be selection by TAP- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance independent, nonclassical class I molecules, such as CD1 and TL with 18 U.S.C. Section 1734 solely to indicate this fact. molecules (22, 23, 39). 1 This work was supported by National Institutes of Health Grant R01-AI43407 (to In this report we have generated CD1oIIo and CD1oTAPo mice C.-R.W.). to directly examine the role of CD1 in the development of CD4ϩ 2 Address correspondence and reprint requests to Dr. Chyung-Ru Wang, Gwen Knapp and CD8ϩ T cells in the thymus and peripheral lymphoid organs. Center for Lupus and Immunology Research, University of Chicago, 924 East 57th ϩ Street, Chicago, IL 60637-5420. E-mail address: [email protected] In addition, the relative contributions of CD1-restricted CD4 T ϩ 3   cells and MHC class II-restricted CD4 T cells in several immune Abbreviations used in this paper: 2m, 2-microglobulin; IEL, intestinal epithelial cells; TNP, trinitrophenol. responses were analyzed.
Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 740 ROLE OF CD1 IN T CELL DEVELOPMENT
Materials and Methods Mice CD1-deficient (CD1o) mice were established by homologous recombina- tion in our laboratory as previously described (29) and were backcrossed six generations onto B6. I-A-deficient (IIo) mice, provided by Dr. Steven Reiner (University of Chicago), were backcrossed five generations onto B6. TAP1o mice were on a mixed B6 ϫ 129 background (The Jackson Laboratory, Bar Harbor, ME). CD1oIIo mice were generated by crossing CD1o mice with IIo mice in B6 background. CD1oTAPo mice with a mixed B6 ϫ 129 background were established by crossing CD1o mice with TAPo mice. Pathogen-free B6 mice were purchased from The Jackson Laboratory. Flow cytometry analysis and cell preparations The Abs used in this study include FITC-conjugated mAbs specific for CD4 (RM4-5), TCR (H57-597), CD69 (H1.2F3), V5 (MR9-4), V6 (RR4-7), V7 (TR310), V8 (MR5-2), V9 (MR10-2), V12 (MR11-1), ϩ o V14 (14-2), and V␣3 (RR3-16); PE-conjugated mAbs specific for CD8␣ FIGURE 1. Different levels of CD4 T cells in wild-type (WT), CD1 , (53-6.7), NK1.1 (PK136), CD4 (RM4-5), CD44 (IM7), V2 (B20.6), V3 IIo and CD1oIIo mice. Lymphocytes isolated from spleen, lymph nodes, (KJ25), V4 (KT4), V10 (B21.5), V11 (RR3-15), V13 (RR12-3), V␣2 and liver of the above mice were stained with mAbs against CD4 and (B20.1), V␣8 (B21.14), and V␣11 (RR8–1); biotin-conjugated mAb spe- TCR␣. The percentages of CD4ϩ T cells were analyzed by flow cytom- ϩ cific for CD62L(MEL-14); and Cy-Chrome-conjugated mAbs specific for etry and plotted as dots in the figure. The average percentages of CD4 T Downloaded from  TCR (H57-597), CD4 (RM4-5), and Cy-Chrome streptavidin (PharMin- cells are: spleen, 58.3 Ϯ 5% (WT), 57.2 Ϯ 5.1 (CD1o), 17.5 Ϯ 2.5% (IIo), gen, San Diego, CA). The lymphocytes from perfused liver were isolated and 10.7 Ϯ 1.6% (CD1oIIo); lymph node, 58.6 Ϯ 6.8% (WT), 62.1 Ϯ 3.6% according to the method described by Goossens et al. (40). The IELs were o Ϯ o Ϯ o o Ϯ prepared and purified through discontinuous 40/70% Percoll gradient cen- (CD1 ), 8.5 0.3% (II ), and 7.8 1.1% (CD1 II ); and liver, 57.2 Ϯ o Ϯ o Ϯ trifugation as described by Tagliabue et al. (41). Single-cell suspensions 8.3% (WT), 45.7 9% (CD1 ), 42.9 8.8% (II ), and 15.9 2.4% o o from thymus, spleen, and lymph node were prepared using standard pro- (CD1 II ). cedure. Cell suspensions were stained using combinations of fluorescent- conjugated Abs and were analyzed with a Becton Dickinson (Mountain http://www.jimmunol.org/ View, CA) FACS caliber flow cytometry using CellQuest software. (26–28). To address the question of whether CD1 is required for Activation of sorted cells and analysis of cytokine production the development of these residual CD4ϩ T cells, we compared the ϩ o o o CD8ϩ T cells and B cells were depleted from the splenocytes of B6, CD1o, CD4 T cell compartment in II and CD1 II mice. FACS analysis IIo, and CD1oIIo mice by incubating cells with CD8␣-FITC and B220- showed that the number of CD4ϩ T cells in liver and spleen of FITC, then cells were incubated with avidin-magnetic beads and applied to CD1oIIo mice were reduced significantly compared with the cor- magnetic separation (PerSeptive Diagnostics, Cambridge, MA). The cells o ϩ ϩ ϩ ϩ Ϫ responding population in II mice (Fig. 1). This reduction is most were then sorted for CD4 , CD4 NK1.1 , and CD4 NK1.1 by FACS, ϩ resulting in a Ͼ95% pure population. Sorted CD4ϩ T cells (2.5 ϫ 104 to prominent in liver, where NK1.1 T cells are normally prevalent ϩ 1 ϫ 105 cells/well) were stimulated in anti-CD3 (2C11)-coated 96-well (42, 43), and is moderate in spleen. CD4 T cells constitute, on the by guest on September 29, 2021 plate in a final volume of 200 l of RPMI 1640 medium (supplemented average, 42.98 Ϯ 8.8% liver lymphocytes isolated from IIo mice, with 10% FCS, 2 mM L-glutamine, 20 M 2-ME, and 100 U/ml penicillin/ while in CD1oIIo littermates, 15.88 Ϯ 2.36% of liver lymphocytes streptomycin). After 48 h, the culture supernatants were harvested, and the ϩ ϩ levels of IL-4 and IFN-␥ were quantitated by ELISA (PharMingen). are CD4 T cells. To examine whether the reduction of CD4 T cells in CD1oIIo mice is merely a result of the decrease in CD1- Isotype-specific assay for anti-trinitrophenol (anti-TNP)- restricted NK1.1ϩ T cells, we compared the distribution of CD4ϩ specific Abs NK1.1ϩ T cells and CD4ϩ NK1.1Ϫ T cells in CD1oIIo and IIo B6, CD1o,IIo, and CD1oIIo mice were immunized i.p. with 25 g of TNP- littermates. The representative CD4/NK1.1 plots (gated on TCR ϩ ϩ ϩ ϩ conjugated Ficoll (Biosearch Technologies, Novato, CA) or 50 g of TNP- ␣ cells) showed that both the CD4 NK1.1 and the CD4 LPS (provided by Dr. Guido Franzoso, University of Chicago) in 0.1% NK1.1Ϫ populations were reduced in CD1oIIo mice (Fig. 2). The alum. Animals were bled before immunization and 10 and 14 days postim- percentage of CD4ϩ NK1.1ϩ T cells was reduced by 70–75%, and munization. Anti-TNP-specific Abs in the sera were determined by isotype- ϩ Ϫ specific ELISA. Briefly, flat-bottom microtiter plates were coated over- the percentage of CD4 NK1.1 T cells was reduced by 40–50%, night at 4°C with 50 g/ml of TNP-BSA (Biosearch Technologies) in PBS respectively. These data suggested that CD1 could select not only (pH 7.4). After washing three times with PBS-Tween 20 (0.5%), serial CD4ϩ NK T cells, but also some NK1.1Ϫ CD4ϩ T cells. dilutions of murine sera in 10% FCS-HBSS were added to the plates and To determine whether CD1-dependent CD4ϩ T cells have a incubated overnight at 4°C. Plates were washed three times with PBS- ϩ restricted repertoire, we compared the TCR usage in CD4 pop- Tween 20 before adding biotinylated goat anti-mouse isotype-specific Abs o o o (1/250 in 10% FCS-HBSS; Southern Biotechnology Associates, Birming- ulations from CD1 II and II mice using a panel of mAbs specific ham, AL). After 1-h incubation at room temperature, plates were washed for various Vs and V␣s (Fig. 3A). The pattern of V segment three times with PBS-Tween. Alkaline phosphatase-conjugated streptavi- usage by CD4ϩ T cells of CD1oIIo mice was slightly different from din (1/1000; Jackson ImmunoResearch Laboratories, West Grove, PA) was that of IIo mice. In particular, the percentages of V8- and V7- then added to the plates and incubated for 25 min at room temperature. o o After five washes with PBS-Tween, the assays were developed with alka- expressing cells in CD1 II mice are significantly lower than those o ϩ line phosphatase substrate (Sigma). in II mice. The reduction of V8 T cells can be detected in both CD4ϩNK1.1ϩ and CD4ϩ NK1.1Ϫ population (Fig. 3B), suggest- Statistical analysis ing that CD1-restricted CD4ϩ T cells (both NK1.1ϩ and NK1.1Ϫ Mean values were compared using Student’s t test for independent vari- T cells) preferentially use TCR with rearranged V8 segments. ables. Statistical significance was considered to be p Ͻ 0.05. Functional characterization of CD4ϩ T cells in wild-type, CD1o, Results IIo, and CD1oIIo mice o o Analysis of T cell subsets in CD1 II mice To compare the functional potential of CD4ϩ T cells from wild- Prior studies have shown that in IIo mice, 5–15% of the wild-type type, CD1o,IIo, and CD1oIIo mice, purified CD4ϩ T cells from the numbers of CD4ϩ cells can be found in the spleen and lymph node four strains of mice were stimulated with plate-bound anti-CD3 in The Journal of Immunology 741
FIGURE 2. Reduction of CD4ϩNK1.1ϩ and CD4ϩNK1.1Ϫ T cells in the spleen and liver of CD1oIIo mice. Lymphocytes from spleen and liver of IIo and CD1oIIo littermates were stained with FITC-anti-CD4, PE-anti- NK1.1, and Cy-Chrome-anti-TCR and analyzed by flow cytometry. The Downloaded from representative dot plot depicts CD4 and NK1.1 staining in the gated TCR␣ϩ population. The numbers represent the percentages of CD4ϩNK1.1ϩ and CD4ϩNK1.1Ϫ cells relative to the total number of TCR␣ϩ cells, respectively. http://www.jimmunol.org/ vitro. Two days later, the levels of IL-2, IL-4, and IFN-␥ were measured by ELISA. Fig. 4A shows that purified CD4ϩ T cells from IIo mice produce larger amounts of IL-4 than wild-type mice FIGURE 3. Analysis of TCR V region usage of CD4ϩ T cells in IIo and in response to anti-CD3. This is presumably due to the enrichment o o o o o of CD4ϩ NK T cells in the remaining CD4ϩ population in IIo CD1 II mice. A, Splenocytes and lymph node cells from II and CD1 II littermate mice were stained and analyzed by flow cytometry for the ex- animals. Surprisingly, the residual CD4ϩ T cells from CD1oIIo pression of the indicated TCR V segments. The percentage of positive cells mice can be readily stimulated with anti-CD3 and produce large within the gated CD4ϩ populations of IIo and CD1oIIo mice are shown in ␥ amounts of IFN- and substantial amounts of IL-4. The level of the figure. Results were comparable in two experiments. B, Lymphocytes
o o by guest on September 29, 2021 IFN-␥ production in the CD1 II mice is higher than that in the from spleen and liver of II° and CD1° II° littermate mice were stained with wild-type control animals, but lower than that in the IIo mice. In FITC-anti-V8, PE-anti-NK1.1, and Cy-Chrome-anti-CD4 and analyzed contrast, the amount of IL-4 produced by CD4ϩ T cells in CD1oIIo by flow cytometry. The percentage of V8ϩ cells within the gated ϩ Ϫ ϩ ϩ is comparable to levels in wild-type mice, but much lower than that CD4 NK1.1 or CD4 NK1.1 populations of the indicated mice are in IIo mice. CD4ϩ T cells from CD1o mice do not produce sig- shown. nificant amounts of IFN-␥ and IL-4 in the same culture conditions, but produce significant amounts of IL-2 (Fig. 4A). These data sug- gest that class II-restricted, CD1-restricted, and non-class II, non- IgG1, IgG2a, and IgG2b in all four types of animals upon immu- CD1-restricted CD4ϩ T cells secrete different ratios of cytokines nization with TNP-LPS (Fig. 5A). Immunization with TNP-Ficoll upon activation. Furthermore, when CD4ϩ cells are sorted into elicited higher levels of TNP-specific IgM and IgG1 Abs in both NK1.1ϩ and NK1.1Ϫ populations, we found that NK1.1Ϫ cells are IIo and CD1oIIo mice compared with those in control and CD1o largely responsible for IFN-␥ secretion in both the CD1-restricted mice (Fig. 5B). This finding is consistent with a previous report and CD1-independent populations (Fig. 4B). However, CD1-re- that immunization with TNP-Ficoll induced higher levels of TNP- stricted CD4ϩNK1.1Ϫ T cells do secrete significant amounts of specific Abs in class II-deficient animals than in control animals IL-4, in contrast to CD1-independent CD4ϩNK1.1Ϫ T cells. The (47). However, we detected no statistically significant difference in rapid secretion of cytokines by the residual CD4ϩ cells in CD1oIIo the production of anti-TNP-specific IgM, IgG1, IgG2a, and IgG2b mice correlates with our additional finding that this population between IIo and CD1oIIo animals. Thus, CD1-restricted T cells appears to be enriched for cells that had a phenotype characteristic play little role in providing cytokines for the Ab response against of activated T cells, such as CD44highCD69high (Fig. 4C). these two T-independent Ags, contrasting with the essential role of CD1-restricted NK T cells in the IgG response to GPI-anchored o o o o Immune responses in CD1 ,II, and CD1 II mice Ag (48).
Several studies demonstrated that the response to thymic-indepen- o o dent Ags (TI Ags) could be regulated by T cells despite their in- Analysis of T cell subsets in CD1 TAP mice ability to stimulate MHC class II-dependent T cell help (44–46). To determine whether CD1 deficiency had any effect on the de- We therefore compared wild-type, CD1o,IIo, and CD1oIIo mice to velopment of CD8ϩ T cells and ␥␦ T cells, we prepared lympho- evaluate the role of CD1-restricted T cells in modulating the Ab cytes from TAPo and CD1oTAPo mice; stained them with reagents production against type I TI Ag (TNP-LPS) and type II TI Ag specific for CD4, CD8, TCR␣, and TCR ␥␦; and analyzed them (TNP-Ficoll). TNP-specific Ab responses of all isotypes could be by flow cytometry. TAPo and CD1oTAPo mice have similarly re- elicited in CD1o,IIo, CD1oIIo, and wild-type mice following im- duced numbers of CD8ϩ cells in thymus, spleen, and lymph nodes munization with TNP-LPS and TNP-Ficoll (Fig. 5). There were no (Table I). Compared with TAPo mice, the percentage of CD8ϩ T significant differences in the production of anti-TNP specific IgM, cells was increased in the liver of CD1oTAPo mice, presumably 742 ROLE OF CD1 IN T CELL DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021
FIGURE 4. Cytokine production capacity and surface phenotype of CD4ϩ T cells from CD1ϩ, CD1o,IIo, and CD1oIIo mice. A, Sorted CD4ϩ T cells from each group of mice were stimulated with plate-bound anti-CD3. After 48 h of culture, supernatants were harvested and analyzed by ELISA for IL-4, IFN-␥, and IL-2 contents. Bars represent the means and SDs of duplicate determination. Results are representative of three experiments. B, Sorted CD4ϩNK1.1ϩ and CD4ϩNK1.1Ϫ cells were stimulated with plate-bound anti-CD3 for 48 h, and culture supernatants were analyzed for the production of IL-4 and IFN-␥. C, Surface phenotype of CD4ϩ T cells from CD1ϩ, CD1o,IIo, and CD1oIIo mice. Splenocytes isolated from above mice were stained and analyzed by FACS. Histograms depict the expression of CD25, CD44, CD62L, and CD69 within gated CD4ϩ cells. The numbers represent percentages relative to CD4ϩ cells. due to the reduction of significant numbers of CD1-restricted subset of IEL. To explore the possible involvement of CD1 in the CD4ϩ T cells in the liver. No significant difference in the number development of these TAP-independent TCR␣ϩCD8ϩ IEL, we of ␥␦ T cells was detected between TAPo and CD1oTAPo mice. examined the phenotype of IEL isolated from wild-type, CD1o, TAPo, and CD1oTAPo mice by flow cytometric analysis. Surface Effect of CD1 on the development of intraepithelial lymphocytes staining for TCR␣, TCR␥␦, CD4, CD8␣, and CD8 showed no Substantial numbers of TCR␣ϩ CD8ϩ IEL are present in TAPo significant difference in the percentage of TCR␣ϩ and TCR␥␦ϩ  o mice despite their absence in 2m-deficient mice (37, 38). Most of lymphocytes between CD1 and control littermates (Fig. 6). In the the TCR␣ϩCD8ϩ IEL in TAPo mice express the CD8␣␣ ho- TCR␣ϩ population, the frequency of CD8␣␣- and CD8␣-bear-  o Ϯ modimer. This suggests that TAP-independent but 2m-dependent ing cells did not decrease substantially in CD1 animals (41.5 5 class Ib molecules may be responsible for the development of this vs 34.1 Ϯ 7.6% for CD8␣␣ϩ cells; 37.3 Ϯ 2.2 vs 31.3 Ϯ 2.4% for The Journal of Immunology 743
Table I. Frequency of lymphocyte subpopulations in TAP° and CD1° TAP° micea
Percentage of Lymphocytes
Tissue Phenotype TAP° CD1°TAP°
Thymus CD4ϩ␣ϩ 8.53 Ϯ 2.00 9.46 Ϯ 1.86 CD8ϩ␣ϩ 0.79 Ϯ 0.40 0.90 Ϯ 0.57 CD4ϪCD8Ϫ␣ϩ 2.83 Ϯ 0.84 2.51 Ϯ 1.07 ␥␦ϩ 0.51 Ϯ 0.24 0.40 Ϯ 0.08
Spleen CD4ϩ␣ϩ 95.00 Ϯ 2.50 95.70 Ϯ 1.80 CD8ϩ␣ϩ 0.65 Ϯ 0.24 0.57 Ϯ 0.10 CD4ϪCD8Ϫ␣ϩ 3.20 Ϯ 0.46 2.79 Ϯ 0.73 ␥␦ϩ 2.44 Ϯ 0.64 2.06 Ϯ 0.22
Lymph nodes CD4ϩ␣ϩ 98.70 Ϯ 0.40 98.90 Ϯ 0.25 CD8ϩ␣ϩ 0.27 Ϯ 0.15 0.23 Ϯ 0.11 CD4ϪCD8Ϫ␣ϩ 0.83 Ϯ 0.26 0.67 Ϯ 0.32 ␥␦ϩ 1.50 Ϯ 0.48 1.31 Ϯ 0.21
ϩ ϩ Liver CD4 ␣ 84.80 Ϯ 4.50 81.10 Ϯ 4.40 Downloaded from CD8ϩ␣ϩ 1.37 Ϯ 0.60 3.60 Ϯ 0.40 CD4ϪCD8Ϫ␣ϩ 14.00 Ϯ 4.40 14.60 Ϯ 4.47 ␥␦ϩ 6.30 Ϯ 2.30 9.80 Ϯ 2.30
a Values represent the mean (ϮSD) results obtained from groups of 10 mice.
probably not due to the inability of CD1 to interact with CD8, http://www.jimmunol.org/ because Teitell et al. (49) have demonstrated that mouse CD1 can bind to CD8 in redirected CTL assays. The ability of CD1 to in- teract with CD8 was further supported by the observation that constitutive expression of CD8 in transgenic mice resulted in a major depletion of CD1-restricted NK T cells that normally ex- FIGURE 5. CD1-deficient mice are able to mount a secretory Ig re- press either the CD4 coreceptor or no coreceptor at all (50). It has sponse to type I and type II TI Ags. CD1o,IIo, CD1oIIo, and control mice been suggested that NK T cells, which preferentially use an in- were immunized i.p. with 50 g of TNP-LPS (A)or25 g of TNP-Ficoll ␣ ␣ ␣
variant -chain (V 14J 281), might have high affinity for CD1. by guest on September 29, 2021 in 0.1% alum (B). Animals were bled before immunization and 10 days after being primed. Serum levels of anti-TNP-specific Abs were deter- Expression of CD8 in CD1-restricted NK T cells would lead to mined by isotype-specific ELISA. The Ab levels, expressed as OD, are their negative selection by increasing the avidity between CD1 and shown for each genotype. The levels of anti-TNP Abs were undetectable in TCR complexes. However, this hypothesis would not preclude ϩ the sera of animals without Ag challenge. Bars represent mean values and CD1 from positively selecting CD8 T cells that express TCRs SDs from four or five mice per group. Results are representative of two with lower affinity to CD1. It is worth noting that CD1-restricted experiments. CD8ϩ T cells have been isolated from mice immunized with plas- mid DNA containing chicken OVA and from mice immunized
CD8␣ϩ cells in CD1ϩ/ϩ and CD1Ϫ/Ϫ mice, respectively). In agreement with previous reports, the absolute number of TCR␣ϩ IEL decreased in mice lacking TAP (TAPo and CD1oTAPo mice), and a compensatory increase in the number of ␥␦ϩ IEL was de- tected (Fig. 6). Within the TCR ␣ϩ IEL subset, the percentage of CD8␣ IEL was reduced significantly in both TAPo and CD1oTAPo mice, and the degree of reduction is comparable be- tween TAPo and CD1oTAPo mice. In contrast to the percentage of CD8␣TCR ␣ϩ IEL, the percentage of CD8␣␣TCR ␣ϩ IEL did not change significantly among the four types of animals. Thus, our data suggest that CD1 does not play a major role in the development of either CD8␣ IELs or CD8␣␣ IELs.
Discussion In summary, analysis of the composition and functional properties of T cells in CD1o mice in IIo and TAPo backgrounds has permit- FIGURE 6. Percentages of CD8␣␣, CD8␣, and TCR␥␦ populations of ted us to examine the contribution of CD1 to the development of IEL from wild-type (WT), CD1o, TAPo, and TAPoCD1o mice. The IELs CD4 and CD8 subsets of T cells. Although CD1 was classified as were stained with a combination of FITC-anti-CD8␣, PE-anti-CD8, and  an MHC class Ib molecule due to its association with 2m, our  ␣ ␥␦ ϩ Cy-chrome-anti-TCR or FITC-anti-CD8 and PE-anti-TCR Abs. The data suggest that CD1 contributes significantly in selecting CD4 percentages of CD8␣␣ and CD8␣ cells relative to the total number of ϩ T cells but minimally in development of the CD8 subset. The TCR␣ϩ T cells and the percentage of ␥␦ T cells relative to the total ϩ limited impact of CD1 in the development of CD8 T cells was lymphocyte population were plotted. 744 ROLE OF CD1 IN T CELL DEVELOPMENT with a CD1 transfectant coated with CD1 binding peptide (51, 52). References Perhaps due to limited self-ligands, CD1-specific CD8ϩ T cells ϩ 1. Balk, S. P., P. A. Bleicher, and C. Terhorst. 1991. Isolation and expression of appear to be a minimal component of CD8 T cell subset. cDNA encoding the murine homologues of CD1. J. Immunol. 146:768. Compared with IIo mice, CD1oIIo mice have reduced numbers 2. Ichimiya, S., K. Kikuchi, and A. Matsuura. 1994. Structural analysis of the rat of both NK1.1ϩ and NK1.1Ϫ CD4ϩ T cells, suggesting that CD1 homologue of CD1: evidence for evolutionary conservation of the CD1D class ϩ and widespread transcription by rat cells. J. Immunol. 153:1112. selects both types of CD4 T cells. TCR analysis showed that both 3. Calabi, F., T. K. Belt, C.-Y. 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