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The Journal of Immunology, 2000, 164: 739-745.
Copyright © 2000 by The American Association of Immunologists

Comparative Contribution of CD1 on the Development of CD4+ and CD8+ T Cell Compartments1

Bin Wang, Taehoon Chun and Chyung-Ru Wang2

Gwen Knapp Center for Lupus and Immunology Research, Committee on Immunology, and Department of Pathology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-{gamma} and a significant amount of IL-4 during primary stimulation with anti-CD3, suggesting that this population may be enriched for NK T cells restricted by other class I molecules. In contrast to the CD4+ 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 molecules, CD1 does not contribute in a major way to the development of CD8+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD1 molecules are cell surface glycoproteins that have been conserved throughout mammalian evolution (1, 2, 3, 4, 5, 6). The overall structure of CD1 resembles that of MHC class I molecules, with three extracellular domains ({alpha}1, {alpha}2, and {alpha}3), a transmembrane region, and a short cytoplasmic tail. The {alpha}3 domain is noncovalently associated with {beta}2-microglobulin ({beta}2m).3 Unlike classical class I molecules, CD1 is relatively nonpolymorphic and is expressed at lower levels (5, 7). Thus, CD1 molecules were classified as a member of MHC class Ib family. However, unlike most of MHC class Ib genes, CD1 genes map outside of MHC both in humans and mice (8, 9), and they are significantly divergent from other class I genes. The sequence homology between CD1 and other class I molecules is only 25–30% (5). The same degree of homology was also detected between CD1 and class II molecules, suggesting that CD1 may represent a third lineage of Ag-presenting molecules (10). Consistent with this idea, CD1 molecules have been shown to present lipid and glycolipid Ags to T cells (11, 12, 13, 14), while MHC class I and class II molecules present peptide Ags to T cells.

Mouse CD1 is encoded by two closely related genes, CD1d1 and CD1d2 (7). CD1d1 is widely expressed on cells of multiple hemopoietic lineages (15, 16, 17), including B and T cells, macrophages, and dendritic cells, while CD1d2 can be detected only on thymocytes (18, 19). Human CD1d can be detected in the apical and lateral regions of small and large intestinal epithelial cells (IEC) (20). This unique localization of hCD1d may allow recognition by intraepithelial lymphocytes (IEL). However, the expression of CD1 on mouse IEC is still controversial, as anti-CD1 mAbs differ in detection of CD1 expression on mouse IEC (15, 16, 17, 21). Unlike MHC class I molecules, the expression of CD1 in both human and mouse does not require functional TAP (22, 23).

Study of T cell development in mutant mice lacking MHC molecules revealed that MHC class I and class II molecules play a central role in the development of CD8+ and CD4+ T cells, respectively (24, 25, 26, 27, 28). Recent studies using CD1-deficient mice have shown that CD1 is essential for the development of a major subset of NK1+ T cells (29, 30, 31), which use an invariant TCR {alpha}-chain in conjunction with a restricted set of TCR {beta}-chains (32, 33). These NK1+ T cells promptly produce large amounts of cytokines, in particular IL-4, upon primary stimulation by TCR engagement (34). However, the role of CD1 in the development of other T cell subsets was unclear. Due to the presence of other MHC class I and class II molecules in CD1o mice, no significant changes in either CD4+ or CD8+ population were detected in CD1o mice (29, 30, 31). Yet, several lines of evidence suggested that CD1 might be involved in the development of some CD4+ and CD8+ T cells. In MHC class II-deficient mice, a small population of CD4+ T cells can be detected in the periphery (26, 27, 28). Many of the hybridomas derived from the CD4+ T cells of class II-deficient mice have been shown to recognize CD1, implicating a role for CD1 in the development of some CD4+ T cells (35). Although the expression of the class I molecules is reduced significantly both in {beta}2mo and in TAPo mice, the residual number of CD8+ T cells in TAPo mice is slightly higher than that in {beta}2mo mice (36, 37, 38). One possible source of these residual CD8+ T cells may be selection by TAP-independent, nonclassical class I molecules, such as CD1 and TL molecules (22, 23, 39).

In this report we have generated CD1oIIo and CD1oTAPo mice to directly examine the role of CD1 in the development of CD4+ and CD8+ T cells in the thymus and peripheral lymphoid organs. In addition, the relative contributions of CD1-restricted CD4+ T cells and MHC class II-restricted CD4+ T cells in several immune responses were analyzed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

CD1-deficient (CD1o) mice were established by homologous recombination in our laboratory as previously described (29) and were backcrossed six generations onto B6. I-A{beta}-deficient (IIo) mice, provided by Dr. Steven Reiner (University of Chicago), were backcrossed five generations onto B6. TAP1o mice were on a mixed B6 x 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 x 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{beta} (H57-597), CD69 (H1.2F3), V{beta}5 (MR9-4), V{beta}6 (RR4-7), V{beta}7 (TR310), V{beta}8 (MR5-2), V{beta}9 (MR10-2), V{beta}12 (MR11-1), V{beta}14 (14-2), and V{alpha}3 (RR3-16); PE-conjugated mAbs specific for CD8{alpha} (53-6.7), NK1.1 (PK136), CD4 (RM4-5), CD44 (IM7), V{beta}2 (B20.6), V{beta}3 (KJ25), V{beta}4 (KT4), V{beta}10 (B21.5), V{beta}11 (RR3-15), V{beta}13 (RR12-3), V{alpha}2 (B20.1), V{alpha}8 (B21.14), and V{alpha}11 (RR8–1); biotin-conjugated mAb specific for CD62L(MEL-14); and Cy-Chrome-conjugated mAbs specific for TCR{beta} (H57-597), CD4 (RM4-5), and Cy-Chrome streptavidin (PharMingen, San Diego, CA). The lymphocytes from perfused liver were isolated according to the method described by Goossens et al. (40). The IELs were prepared and purified through discontinuous 40/70% Percoll gradient centrifugation as described by Tagliabue et al. (41). Single-cell suspensions from thymus, spleen, and lymph node were prepared using standard procedure. Cell suspensions were stained using combinations of fluorescent-conjugated Abs and were analyzed with a Becton Dickinson (Mountain View, CA) FACS caliber flow cytometry using CellQuest software.

Activation of sorted cells and analysis of cytokine production

CD8+ T cells and B cells were depleted from the splenocytes of B6, CD1o, IIo, and CD1oIIo mice by incubating cells with CD8{alpha}-FITC and B220-FITC, then cells were incubated with avidin-magnetic beads and applied to magnetic separation (PerSeptive Diagnostics, Cambridge, MA). The cells were then sorted for CD4+, CD4+NK1.1+, and CD4+ NK1.1- by FACS, resulting in a >95% pure population. Sorted CD4+ T cells (2.5 x 104 to 1 x 105 cells/well) were stimulated in anti-CD3 (2C11)-coated 96-well plate in a final volume of 200 µl of RPMI 1640 medium (supplemented with 10% FCS, 2 mM L-glutamine, 20 µM 2-ME, and 100 U/ml penicillin/streptomycin). After 48 h, the culture supernatants were harvested, and the levels of IL-4 and IFN-{gamma} were quantitated by ELISA (PharMingen).

Isotype-specific assay for anti-trinitrophenol (anti-TNP)- specific Abs

B6, CD1o, IIo, and CD1oIIo mice were immunized i.p. with 25 µg of TNP-conjugated Ficoll (Biosearch Technologies, Novato, CA) or 50 µg of TNP-LPS (provided by Dr. Guido Franzoso, University of Chicago) in 0.1% alum. Animals were bled before immunization and 10 and 14 days postimmunization. Anti-TNP-specific Abs in the sera were determined by isotype-specific ELISA. Briefly, flat-bottom microtiter plates were coated overnight at 4°C with 50 µg/ml of TNP-BSA (Biosearch Technologies) in PBS (pH 7.4). After washing three times with PBS-Tween 20 (0.5%), serial dilutions of murine sera in 10% FCS-HBSS were added to the plates and incubated overnight at 4°C. Plates were washed three times with PBS-Tween 20 before adding biotinylated goat anti-mouse isotype-specific Abs (1/250 in 10% FCS-HBSS; Southern Biotechnology Associates, Birmingham, AL). After 1-h incubation at room temperature, plates were washed three times with PBS-Tween. Alkaline phosphatase-conjugated streptavidin (1/1000; Jackson ImmunoResearch Laboratories, West Grove, PA) was then added to the plates and incubated for 25 min at room temperature. After five washes with PBS-Tween, the assays were developed with alkaline phosphatase substrate (Sigma).

Statistical analysis

Mean values were compared using Student’s t test for independent variables. Statistical significance was considered to be p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of T cell subsets in CD1oIIo mice

Prior studies have shown that in IIo mice, 5–15% of the wild-type numbers of CD4+ cells can be found in the spleen and lymph node (26, 27, 28). To address the question of whether CD1 is required for the development of these residual CD4+ T cells, we compared the CD4+ T cell compartment in IIo and CD1oIIo mice. FACS analysis showed that the number of CD4+ T cells in liver and spleen of CD1oIIo mice were reduced significantly compared with the corresponding population in IIo mice (Fig. 1Go). This reduction is most prominent in liver, where NK1.1+ T cells are normally prevalent (42, 43), and is moderate in spleen. CD4+ T cells constitute, on the average, 42.98 ± 8.8% liver lymphocytes isolated from IIo mice, while in CD1oIIo littermates, 15.88 ± 2.36% of liver lymphocytes 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-restricted NK1.1+ T cells, we compared the distribution of CD4+ NK1.1+ T cells and CD4+ NK1.1- T cells in CD1oIIo and IIo littermates. The representative CD4/NK1.1 plots (gated on TCR {alpha}{beta}+ cells) showed that both the CD4+ NK1.1+ and the CD4+ NK1.1- populations were reduced in CD1oIIo mice (Fig. 2Go). The percentage of CD4+ NK1.1+ T cells was reduced by 70–75%, and the percentage of CD4+ NK1.1- T cells was reduced by 40–50%, respectively. These data suggested that CD1 could select not only CD4+ NK T cells, but also some NK1.1- CD4+ T cells.



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  		FIGURE 1. Different levels of CD4+ T cells in wild-type (WT), CD1o, IIo and CD1oIIo mice. Lymphocytes isolated from spleen, lymph nodes, and liver of the above mice were stained with mAbs against CD4 and TCR{alpha}{beta}. The percentages of CD4+ T cells were analyzed by flow cytometry and plotted as dots in the figure. The average percentages of CD4+ T cells are: spleen, 58.3 ± 5% (WT), 57.2 ± 5.1 (CD1o), 17.5 ± 2.5% (IIo), and 10.7 ± 1.6% (CD1oIIo); lymph node, 58.6 ± 6.8% (WT), 62.1 ± 3.6% (CD1o), 8.5 ± 0.3% (IIo), and 7.8 ± 1.1% (CD1oIIo); and liver, 57.2 ± 8.3% (WT), 45.7 ± 9% (CD1o), 42.9 ± 8.8% (IIo), and 15.9 ± 2.4% (CD1oIIo).

 


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  		FIGURE 2. Reduction of CD4+NK1.1+ and CD4+NK1.1- 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{beta} and analyzed by flow cytometry. The representative dot plot depicts CD4 and NK1.1 staining in the gated TCR{alpha}{beta}+ population. The numbers represent the percentages of CD4+NK1.1+ and CD4+NK1.1- cells relative to the total number of TCR{alpha}{beta}+ cells, respectively.

 
To determine whether CD1-dependent CD4+ T cells have a restricted repertoire, we compared the TCR usage in CD4+ populations from CD1oIIo and IIo mice using a panel of mAbs specific for various V{beta}s and V{alpha}s (Fig. 3GoA). The pattern of V{beta} segment usage by CD4+ T cells of CD1oIIo mice was slightly different from that of IIo mice. In particular, the percentages of V{beta}8- and V{beta}7-expressing cells in CD1oIIo mice are significantly lower than those in IIo mice. The reduction of V{beta}8+ T cells can be detected in both CD4+NK1.1+ and CD4+ NK1.1- population (Fig. 3GoB), suggesting that CD1-restricted CD4+ T cells (both NK1.1+ and NK1.1- T cells) preferentially use TCR with rearranged V{beta}8 segments.



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  		FIGURE 3. Analysis of TCR V region usage of CD4+ T cells in IIo and CD1oIIo mice. A, Splenocytes and lymph node cells from IIo and CD1oIIo littermate mice were stained and analyzed by flow cytometry for the expression of the indicated TCR V segments. The percentage of positive cells within the gated CD4+ populations of IIo and CD1oIIo mice are shown in the figure. Results were comparable in two experiments. B, Lymphocytes from spleen and liver of II° and CD1° II° littermate mice were stained with FITC-anti-V{beta}8, PE-anti-NK1.1, and Cy-Chrome-anti-CD4 and analyzed by flow cytometry. The percentage of V{beta}8+ cells within the gated CD4+NK1.1- or CD4+NK1.1+ populations of the indicated mice are shown.

 
Functional characterization of CD4+ T cells in wild-type, CD1o, IIo, and CD1oIIo mice

To compare the functional potential of CD4+ T cells from wild-type, CD1o, IIo, and CD1oIIo mice, purified CD4+ T cells from the four strains of mice were stimulated with plate-bound anti-CD3 in vitro. Two days later, the levels of IL-2, IL-4, and IFN-{gamma} were measured by ELISA. Fig. 4GoA shows that purified CD4+ T cells from IIo mice produce larger amounts of IL-4 than wild-type mice in response to anti-CD3. This is presumably due to the enrichment of CD4+ NK T cells in the remaining CD4+ population in IIo animals. Surprisingly, the residual CD4+ T cells from CD1oIIo mice can be readily stimulated with anti-CD3 and produce large amounts of IFN-{gamma} and substantial amounts of IL-4. The level of IFN-{gamma} production in the CD1oIIo mice is higher than that in the wild-type control animals, but lower than that in the IIo mice. In contrast, the amount of IL-4 produced by CD4+ T cells in CD1oIIo is comparable to levels in wild-type mice, but much lower than that in IIo mice. CD4+ T cells from CD1o mice do not produce significant amounts of IFN-{gamma} and IL-4 in the same culture conditions, but produce significant amounts of IL-2 (Fig. 4GoA). These data suggest that class II-restricted, CD1-restricted, and non-class II, non-CD1-restricted CD4+ T cells secrete different ratios of cytokines upon activation. Furthermore, when CD4+ cells are sorted into NK1.1+ and NK1.1- populations, we found that NK1.1- cells are largely responsible for IFN-{gamma} secretion in both the CD1-restricted and CD1-independent populations (Fig. 4GoB). However, CD1-restricted CD4+NK1.1- T cells do secrete significant amounts of IL-4, in contrast to CD1-independent CD4+NK1.1- T cells. The rapid secretion of cytokines by the residual CD4+ cells in CD1oIIo mice correlates with our additional finding that this population appears to be enriched for cells that had a phenotype characteristic of activated T cells, such as CD44highCD69high (Fig. 4GoC).



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  		FIGURE 4. Cytokine production capacity and surface phenotype of CD4+ T cells from CD1+, CD1o, IIo, and CD1oIIo mice. A, Sorted CD4+ 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-{gamma}, and IL-2 contents. Bars represent the means and SDs of duplicate determination. Results are representative of three experiments. B, Sorted CD4+NK1.1+ and CD4+NK1.1- cells were stimulated with plate-bound anti-CD3 for 48 h, and culture supernatants were analyzed for the production of IL-4 and IFN-{gamma}. C, Surface phenotype of CD4+ T cells from CD1+, 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 CD4+ cells. The numbers represent percentages relative to CD4+ cells.

 
Immune responses in CD1o, IIo, and CD1oIIo mice

Several studies demonstrated that the response to thymic-independent Ags (TI Ags) could be regulated by T cells despite their inability to stimulate MHC class II-dependent T cell help (44, 45, 46). We therefore compared wild-type, CD1o, IIo, and CD1oIIo mice to evaluate the role of CD1-restricted T cells in modulating the Ab production against type I TI Ag (TNP-LPS) and type II TI Ag (TNP-Ficoll). TNP-specific Ab responses of all isotypes could be elicited in CD1o, IIo, CD1oIIo, and wild-type mice following immunization with TNP-LPS and TNP-Ficoll (Fig. 5Go). There were no significant differences in the production of anti-TNP specific IgM, IgG1, IgG2a, and IgG2b in all four types of animals upon immunization with TNP-LPS (Fig. 5GoA). Immunization with TNP-Ficoll elicited higher levels of TNP-specific IgM and IgG1 Abs in both IIo and CD1oIIo mice compared with those in control and CD1o mice (Fig. 5GoB). This finding is consistent with a previous report that immunization with TNP-Ficoll induced higher levels of TNP-specific Abs in class II-deficient animals than in control animals (47). However, we detected no statistically significant difference in the production of anti-TNP-specific IgM, IgG1, IgG2a, and IgG2b between IIo and CD1oIIo animals. Thus, CD1-restricted T cells play little role in providing cytokines for the Ab response against these two T-independent Ags, contrasting with the essential role of CD1-restricted NK T cells in the IgG response to GPI-anchored Ag (48).



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  		FIGURE 5. CD1-deficient mice are able to mount a secretory Ig response to type I and type II TI Ags. CD1o, IIo, CD1oIIo, and control mice were immunized i.p. with 50 µg of TNP-LPS (A) or 25 µg of TNP-Ficoll in 0.1% alum (B). Animals were bled before immunization and 10 days after being primed. Serum levels of anti-TNP-specific Abs were determined by isotype-specific ELISA. The Ab levels, expressed as OD, are shown for each genotype. The levels of anti-TNP Abs were undetectable in the sera of animals without Ag challenge. Bars represent mean values and SDs from four or five mice per group. Results are representative of two experiments.

 
Analysis of T cell subsets in CD1oTAPo mice

To determine whether CD1 deficiency had any effect on the development of CD8+ T cells and {gamma}{delta} T cells, we prepared lymphocytes from TAPo and CD1oTAPo mice; stained them with reagents specific for CD4, CD8, TCR{alpha}{beta}, and TCR {gamma}{delta}; and analyzed them by flow cytometry. TAPo and CD1oTAPo mice have similarly reduced numbers of CD8+ cells in thymus, spleen, and lymph nodes (Table IGo). Compared with TAPo mice, the percentage of CD8+ T cells was increased in the liver of CD1oTAPo mice, presumably due to the reduction of significant numbers of CD1-restricted CD4+ T cells in the liver. No significant difference in the number of {gamma}{delta} T cells was detected between TAPo and CD1oTAPo mice.


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  		Table I. Frequency of lymphocyte subpopulations in TAP° and CD1° TAP° mice1

 
Effect of CD1 on the development of intraepithelial lymphocytes

Substantial numbers of TCR{alpha}{beta}+ CD8+ IEL are present in TAPo mice despite their absence in {beta}2m-deficient mice (37, 38). Most of the TCR{alpha}{beta}+CD8+ IEL in TAPo mice express the CD8{alpha}{alpha} homodimer. This suggests that TAP-independent but {beta}2m-dependent class Ib molecules may be responsible for the development of this subset of IEL. To explore the possible involvement of CD1 in the development of these TAP-independent TCR{alpha}{beta}+CD8+ IEL, we examined the phenotype of IEL isolated from wild-type, CD1o, TAPo, and CD1oTAPo mice by flow cytometric analysis. Surface staining for TCR{alpha}{beta}, TCR{gamma}{delta}, CD4, CD8{alpha}, and CD8{beta} showed no significant difference in the percentage of TCR{alpha}{beta}+ and TCR{gamma}{delta}+ lymphocytes between CD1o and control littermates (Fig. 6Go). In the TCR{alpha}{beta}+ population, the frequency of CD8{alpha}{alpha}- and CD8{alpha}{beta}-bearing cells did not decrease substantially in CD1o animals (41.5 ± 5 vs 34.1 ± 7.6% for CD8{alpha}{alpha}+ cells; 37.3 ± 2.2 vs 31.3 ± 2.4% for CD8{alpha}{beta}+ cells in CD1+/+ and CD1-/- mice, respectively). In agreement with previous reports, the absolute number of TCR{alpha}{beta}+ IEL decreased in mice lacking TAP (TAPo and CD1oTAPo mice), and a compensatory increase in the number of {gamma}{delta}+ IEL was detected (Fig. 6Go). Within the TCR {alpha}{beta}+ IEL subset, the percentage of CD8{alpha}{beta} IEL was reduced significantly in both TAPo and CD1oTAPo mice, and the degree of reduction is comparable between TAPo and CD1oTAPo mice. In contrast to the percentage of CD8{alpha}{beta}TCR {alpha}{beta}+ IEL, the percentage of CD8{alpha}{alpha}TCR {alpha}{beta}+ 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{alpha}{beta} IELs or CD8{alpha}{alpha} IELs.



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  		FIGURE 6. Percentages of CD8{alpha}{alpha}, CD8{alpha}{beta}, and TCR{gamma}{delta} populations of IEL from wild-type (WT), CD1o, TAPo, and TAPoCD1o mice. The IELs were stained with a combination of FITC-anti-CD8{alpha}, PE-anti-CD8{beta}, and Cy-chrome-anti-TCR{beta} or FITC-anti-CD8{alpha} and PE-anti-TCR{gamma}{delta} Abs. The percentages of CD8{alpha}{alpha} and CD8{alpha}{beta} cells relative to the total number of TCR{alpha}{beta}+ T cells and the percentage of {gamma}{delta} T cells relative to the total lymphocyte population were plotted.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In summary, analysis of the composition and functional properties of T cells in CD1o mice in IIo and TAPo backgrounds has permitted us to examine the contribution of CD1 to the development of CD4 and CD8 subsets of T cells. Although CD1 was classified as an MHC class Ib molecule due to its association with {beta}2m, our data suggest that CD1 contributes significantly in selecting CD4+ T cells but minimally in development of the CD8+ subset. The limited impact of CD1 in the development of CD8+ T cells was probably not due to the inability of CD1 to interact with CD8, because Teitell et al. (49) have demonstrated that mouse CD1 can bind to CD8 in redirected CTL assays. The ability of CD1 to interact 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 express either the CD4 coreceptor or no coreceptor at all (50). It has been suggested that NK T cells, which preferentially use an invariant {alpha}-chain (V{alpha}14J{alpha}281), might have high affinity for CD1. Expression of CD8 in CD1-restricted NK T cells would lead to their negative selection by increasing the avidity between CD1 and TCR complexes. However, this hypothesis would not preclude CD1 from positively selecting CD8+ T cells that express TCRs with lower affinity to CD1. It is worth noting that CD1-restricted CD8+ T cells have been isolated from mice immunized with plasmid DNA containing chicken OVA and from mice immunized with a CD1 transfectant coated with CD1 binding peptide (51, 52). Perhaps due to limited self-ligands, CD1-specific CD8+ T cells appear to be a minimal component of CD8+ T cell subset.

Compared with IIo mice, CD1oIIo mice have reduced numbers of both NK1.1+ and NK1.1- CD4+ T cells, suggesting that CD1 selects both types of CD4+ T cells. TCR analysis showed that both NK1.1+ and NK1.1- CD1-restricted CD4+ T cells preferentially express TCR with V{beta}8 and V{beta}7. Our data are consistent with a recent report that CD4+ hybridomas derived from both NK1.1+CD4+ and NK1.1- CD4+ T cells of class IIo mice recognize CD1, although recognition may require ligands derived from different cellular compartments (53). It has been shown that most of the NK1.1+CD4+ T cells express an invariant V{alpha}-chain with V{alpha}14J{alpha}281 rearrangement, while most of the NK1.1- CD4+ T cells express different V{alpha}-chains (53). Therefore, NK1.1+CD4+ and NK1.1- CD4+ T cells might be selected by different sets of CD1-bound ligands, while structural features of CD1 might be responsible for preferential selection of T cells expressing V{beta}8 and, to a lesser extent, for T cells expressing V{beta}7. Preferential selection of V{beta}5+ T cells by some MHC class I molecules has also been demonstrated (54).

A detectable pool of CD4+ T cells still exists in CD1oIIo mice. This T cell population, presumably selected by other class I molecules, shows diverse TCR usage, suggesting recognition of heterogeneous ligands. Compared with CD4+ T cells in IIo mice, the residual CD4+ T cells in CD1oIIo mice secrete comparable amounts of IFN-{gamma} but significantly lower amounts of IL-4 and IL-2 in response to anti-CD3 stimulation. These data indicate that CD1-restricted CD4+ T cells are the major source of IL-4 production, while non-CD1-restricted CD4+ T cells play a substantial role in IFN-{gamma} production in the anti-CD3 induction model. It is important to note that the amount of IFN-{gamma} secreted by the CD4+ cells from CD1oIIo mice is significantly higher than the amount produced by conventional class II-restricted CD4+ T cells. Thus, this subset of the CD4+ T cell population might play an important role in modulating the immune response in vivo.

In contrast to classical class I molecules, the surface expression of CD1 and TL does not depend on TAP (22, 23, 39). The expression of TL on IEC has been clearly demonstrated (55), whereas the expression of CD1 on IEC is controversial. Our FACS analysis showed that the IEL in CD1oTAPo mice are phenotypically similar to the IEL in TAPo mice, suggesting that CD1 may not be responsible for intestinal selection of TAP-independent CD8+ IEL. Intrathymic selection of TCR{alpha}{beta}+CD8+ IEL by TL Ag seems unlikely, because thymocytes from all mice used in this study were TL- (data not shown). However, it is possible that TCR{alpha}{beta}+ CD8{alpha}{alpha} T cells may be selected by TL extrathymically. Alternatively, low levels of MHC class I molecules expressed in the absence of TAP might be responsible for the selection of CD8+ T cells in TAPo mice.

Recent studies indicated that NK T cells are heterogeneous (29, 56, 57). Although a major population of NK T cells are CD1 restricted, some NK T cells are restricted by other MHC molecules. Our study of CDIoIIo mice clearly demonstrates the existence of a functionally distinct NK T cell subset. Further analyses of CDIoIIo mice might provide information on the immunological functions and ligand specificities of these remnant CD4+NK T cells.


   Acknowledgments
 
We thank Nancy Chiu and Cherita White-Morris for their critical reading of the manuscript, and Lillian Zhao and Candice Mulder for technical assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R01-AI43407 (to C.-R.W.). Back

2 Address correspondence and reprint requests to Dr. Chyung-Ru Wang, Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, 924 East 57th Street, Chicago, IL 60637-5420. E-mail address: Back

3 Abbreviations used in this paper: {beta}2m, {beta}2-microglobulin; IEL, intestinal epithelial cells; TNP, trinitrophenol. Back

Received for publication July 27, 1999. Accepted for publication November 4, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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