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The Journal of Immunology, 2001, 166: 3090-3097.
Copyright © 2001 by The American Association of Immunologists

Involvement of CD1 in Peripheral Deletion of T Lymphocytes Is Independent of NK T Cells1

Tao Dao*, Mark Exley{dagger}, Wajahat Z. Mehal*, Syed Muhammad Ali Tahir{dagger}, Scott Snapper{ddagger}, Masaru Taniguchi§, Steven P. Balk{dagger} and I. Nicholas Crispe2,*

* Immunobiology Section, Yale University School of Medicine, New Haven, CT 06510; {dagger} Cancer Biology Program, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215; {ddagger} Gastrointestinal Unit (Medical Services), Massachusetts General Hospital, Boston, MA 02116; and § Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chuoku Chiba, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During peripheral T cell deletion, lymphocytes accumulate in nonlymphoid organs including the liver, a tissue that expresses the nonclassical, MHC-like molecule, CD1. Injection of anti-CD3 Ab results in T cell activation, which in normal mice is followed by peripheral T cell deletion. However, in CD1-deficient mice, the deletion of the activated T cells from the lymph nodes was impaired. This defect in peripheral T cell deletion was accompanied by attenuated accumulation of CD8+ T cells in the liver. In tetra-parental bone marrow chimeras, expression of CD1 on the T cells themselves was not required for T cell deletion, suggesting a role for CD1 on other cells with which the T cells interact. We tested whether this role was dependent on the Ag receptor-invariant, CD1-reactive subset of NK T cells using two other mutant mouse lines that lack most NK T cells, due to deletion of the genes encoding either {beta}2-microglobulin or the TCR element J{alpha}281. However, these mice had no abnormality of peripheral T cell deletion. These findings indicate a novel role for CD1 in T cell deletion, and show that CD1 functions in this process through mechanisms that does not involve the major, TCR-invariant set of NK T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The activation of T cells by nonphysiological stimuli, such as superantigens and Abs against the TCR, results in activation-induced cell death and deletion of the T cells from the periphery (1, 2, 3, 4, 5). Similarly, injection of specific antigenic peptide into TCR-transgenic mice leads to a truncated, abortive immune response in which T cell activation is rapidly followed by peripheral deletion (6, 7). In some experimental models, peripheral deletion is due to apoptosis of the activated T cells in lymphoid tissues (8). In other models, apoptosis is difficult to detect in the lymphoid tissue, and activated T cells recirculate to specific destruction sites (9, 10, 11).

Our previous research has identified the liver as a site for the accumulation and destruction of CD8+ T cells during peripheral deletion, induced in a TCR-transgenic mouse by the injection of antigenic peptide (10). Intrahepatic accumulation of apoptotic T cells has been reported in several other models of peripheral T cell deletion, raising the possibility that this is a general site of T cell death (11, 12, 13). The liver is an unusual immunologic environment, in which a population of T cells similar to the circulating CD4+ and CD8+ T cells coexists with a subset of CD4-, CD8-,B220+,TCR{alpha}{beta}+ cells that contains a high frequency of apoptotic cells, and with an abundant subset of NK-1.1+ T cells (reviewed in Refs. 14 and 15)). This latter population contains a subset of cells that express a distinctive TCR{alpha}{beta}, in which the TCR{beta}-chain is of very limited diversity and the TCR{alpha}-chain is essentially monomorphic. These cells depend for their development and function on CD1 molecules, which are {beta}2-microglobulin-associated, MHC class I-like molecules encoded outside the gene complex on chromosome 17 (reviewed in Refs. 16, 17, 18)).

Mice with induced mutations in key recognition molecules make it possible to determine precisely which subsets of CD1-reactive or NK-like T cells are involved in any biological process. Thus, mice lacking the invariant TCR J region, J{alpha}281, specifically lack CD1-reactive T cells with this invariant Ag receptor (19, 20, 21). Mice deficient in CD1 lack these invariant T cells, and also other CD1-reactive T cells. Mice deficient in {beta}2-microglobulin display a similar defect to CD1-deficient mice (22, 23), except that they contain a small population of {beta}2-microglobulin-independent T cells with NK markers, which increases with age (24). All of these cell populations include NK-like T cells, and in this report we use the generic term NK T cells to embrace all of them.

The intrahepatic population of NK T cells is cytotoxic though both Fas ligand-mediated and perforin-mediated mechanisms. The cytokine IL-18, which is produced in large amounts by the resident liver macrophages (termed Kupffer cells), can augment the perforin-dependent cytotoxicity of liver NK T cells (25). We postulated that these cells might be involved in the intrahepatic killing of activated T cells, perhaps through recognition of CD1-associated ligands (for example, specific glycolipids) on their surface. Significantly, both mouse and rat liver express CD1 (26, 27). The present study was conducted to test the hypothesis that CD1 is involved in T cell deletion, which we have done by inducing T cell activation and peripheral deletion in mice lacking CD1, and, therefore, devoid of the CD1-reactive, largely TCR-invariant subset of NK T cells. The results show that peripheral T cell deletion was indeed impaired in CD1-deficient mice; however, this impairment was not due to the lack of NK T cells. Furthermore, CD1 on the T cells could not be acting as a target for a T cell deletion mechanism, because the requirement for CD1 expression was non-T cell autonomous. Therefore, we conclude that an interaction between the activated T cells and another cell expressing CD1 promotes the deletion of activated peripheral T cells.


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

C57BL/6 (B6),3 (B6 x 129)F1 or F2, and {beta}2-microglobulin-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). CD1d-deficient mice (on the B6 x 129 background) were created at Beth Israel Deaconess Hospital (Harvard University, Boston, MA). The J{alpha}281-deficient mice were created at Chiba University (Chuoku Chiba, Japan). The mice were kept in the Immunobiology Mouse Unit at Yale Medical School (New Haven, CT), or at Beth Israel Deaconess Hospital. All mice were used between 6–9 wk of age.

Reagents

PE-coupled anti-NK1.1 Ab, PE-anti-mouse CD1 Ab, CyChrome-anti-mouse TCR{alpha}{beta} Ab, biotinylated anti-hamster IgG, and strepavidin-PE were obtained from PharMingen (San Diego, CA). PE-anti-mouse CD4 Ab and FITC-coupled anti-mouse CD8 Ab were purchased from Life Technologies (Gaithersburg, MD). Hybridomas producing anti-mouse CD3 Ab (145-2C11) and anti-murine NK (PK-136) Ab were obtained from American Type Culture Collection (Manassass, VA). These Abs were purified using staphylococcal protein A-Sepharose (Pharmacia, Piscataway, NJ). An ELISA kit to measure murine TNF-{alpha} was purchased from Endogen (Woburn, MA).

Induction of T cell deletion

Mice were i.p. injected with either 100 µg anti-murine CD3 Ab in 0.2 ml PBS, or with 0.2 ml PBS as control. Two to 8 days after injection, four LNs (two axillary and two inguinal) were removed by dissection, homogenized, and then suspended lymph node cells rinsed in Bruff’s culture medium. Cells were counted and kept on ice until staining.

FACS analysis

Cells suspended (0.5–1 x 106) in 0.1 ml staining buffer (PBS with 1% BSA and 0.02% sodium azide) were incubated with the following directly conjugated Abs: anti-murine TCR{alpha}{beta}-CyChrome, anti-murine CD4-PE and anti-murine CD8-FITC, or TCR{alpha}{beta}-CyChrome vs PE-anti-NK1.1 Ab or vs PE-anti-mouse CD1 Ab for 30 min, and were analyzed using a FACScan (Becton Dickinson, Mountain View, CA). The percentage of TCR{alpha}{beta} vs CD4- or CD8-positive cells was determined using CellQuest software on an Apple Macintosh computer (Apple Computer, Cupertino, CA) .

Cell proliferation

To determine the proliferation potential of wild-type vs CD1-deficient T cells in vitro, LN or spleen cells (1 x 106 cells/ml) were incubated in RPMI 1640 medium containing 10% FCS and supplemented with L-glutamine, antibiotics, and 50 µM 2-ME, with or without 1 µg/ml immobilized anti-CD3 Ab for 2 days. [3H]Thymidine (25 µCi/ml) was added to the culture for the last 4 h of the incubation period. To determine the proliferative response of T cells in vivo, mice were injected i.p. with anti-CD3 Ab on day 0, and on day 2 were injected i.p. with 5-bromo-2'-deoxyuridine (BrdU; 1 mg/mouse) four times at 4-h intervals. Twelve hours after the last injection, mice were killed and LN and spleen cells were isolated. The cells were stained with TCR{alpha}{beta}-CyChrome vs CD4-PE or CD8-PE, then were fixed with 70% ethanol followed by 1% paraformaldehyde, and then were stained to detect incorporated BrdU using anti-BrdU-FITC Ab (Becton Dickinson).

Construction of bone marrow chimeras

Eight- to 10-wk-old (B6 x 129) F1 mice were given a single dose of 9 Gy (900 rad) of whole body irradiation from a {gamma} source. Bone marrow cells from B6- or CD1-deficient mice were mixed at a 50/50 ratio, and injected i.v. 5 h after the irradiation. Four weeks later, chimeras were injected with either anti-CD3 as previously described, or with PBS as a control.

Isolation of liver lymphocytes

Intrahepatic lymphocytes (IHL) were isolated by a standard method (28). Briefly, in an anesthetized mouse, the portal vein was perfused with 5 ml of "digestion buffer" (i.e., medium containing 0.2 µg/ml collagenase, 0.02 µg/ml DNase, and 5% FCS). After perfusion, the livers were homogenized by forcing through a metal strainer, and were then digested with 10 ml of digestion buffer at 37°C for 45 min. The hepatocytes were removed by centrifugation at 30 x g for 3 min. The supernatant was centrifuged at 650 x g for 10 min to obtain a pellet of nonparenchymal cells. The pellet from each liver was suspended with Bruff’s medium to a final volume of 1 ml, before mixing with 4 ml of 30% (w/v) metrizamide in PBS. The cell suspension in metrizamide was overlaid with serum-free Bruff’s medium, and centrifuged at 1500 x g for 20 min. The cells at the interface were collected, washed with PBS, and counted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Defective peripheral T cell deletion

The down-regulation of the TCR is a recognized feature of T cell activation leading to deletion, and has been previously reported in peptide- and superantigen-induced peripheral T cell deletion (2, 10). Similarly, the injection of 100 µg of anti-CD3 resulted in the down-regulation of the TCR. Fig. 1GoA shows the effect of anti-CD3 injection of CD4+ and CD8+ T cells of wild-type and CD1-deficient mice at 2 days after injection of the Ab. Lymph node cells of individual, representative wild-type mice are shown in the upper panels of Fig 1GoA. In PBS-injected controls, 50% of the lymph node cells were CD4+, TCR{alpha}{beta}+ T cells and only 1% were CD4+, TCR{alpha}{beta}- T cells. In an anti-CD3-injected mouse, there were 23% of CD4+, TCR{alpha}{beta}+ T cells and 18% of CD4+, TCR{alpha}{beta}- T cells, making 41% of CD4+ T cells overall. Thus, at this early time point, the main effect of anti-CD3 treatment was the loss of TCR{alpha}{beta} expression rather than T cell deletion, yet there was also a small loss of CD4+ T cells. Similarly, the TCR{alpha}{beta} was lost from many of the CD8+ cells. The lower panels of Fig. 1GoA show the effects of anti-CD3 injection on the T cells of individual, representative CD1-deficient mice. The basic pattern was similar, with down-regulation of the TCR on both CD4+ and CD8+ cells. However, the total of CD4+, TCR{alpha}{beta}+ T cells and CD4+, TCR{alpha}{beta}- T cells (25 and 26%, respectively) was 51%, against 54% in the PBS-injected controls. Both of these numbers are within the normal range for PBS-injected CD1-deficient mice, in contrast to the loss of 10% of T cells from the anti-CD3-injected normal mice.



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  		FIGURE 1. Anti-CD3-induced peripheral T cell deletion is delayed in CD1-deficient mice. A, Down-regulation of the TCR on CD4+ and CD8+ T cells at 2 days after anti-CD3 injection. At this stage, there is no difference between wild-type mice (upper panels) and CD1-deficient mice (lower panels). B, Deletion of CD4+ and CD8+ T cells at 4 days after anti-CD4 injection. The percentage of CD4+ cells is reduced to half in the wild-type mice (upper panels) but not in the CD1-deficient mice (lower panels).

 
This difference was accentuated 2 days later. Fig. 1GoB shows that at day 4, the TCR-low cells had disappeared from both the CD4+ and the CD8+ subsets of the anti-CD3-injected wild-type mice. The total number of CD4+ cells was reduced from 58% in a representative PBS-injected control to 27% in a representative anti-CD3-injected mouse (Fig. 1GoB, upper panels). Similar changes of lesser magnitude occurred in the CD8+ cells. In CD1-deficient mice the TCR-low cells had also disappeared, but, in contrast, the total CD4+ cell frequencies in anti-CD3-injected mice were within the range of the controls. Among the CD8+ cells, the modest deletion observed in wild-type mice was not evident in the CD1-deficient mice.

The results from groups of four mice each are summarized in Fig. 2Go, which shows the means and SD estimates of the total numbers of CD4+ and CD8+ T cells in the lymph node, for each experimental group, at 2, 4, 6, and 8 days after a single injection of anti-CD3. We chose to represent absolute CD4+ and CD8+ cell numbers to show that the effects of CD1 deficiency on the relative depletion of CD4+ T cells were not due to the fluctuations in the absolute numbers of other cell types. To obtain these numbers, we multiplied the lymph node cell yield from the pooled superficial (axillary and inguinal) lymph nodes of each individual animal by the percentage of CD4+ and CD8+ cells from that animal. In wild-type mice, the absolute number of CD4+ T cells was already depleted (from 4.4 x 106 to 1.8 x 106) by day 2, while there was less depletion in the CD1-deficient cells (from 4.6 x 106 to 3.0 x 106). By day 4, there was deletion of CD4+ T cells from both wild-type and CD1-deficient mice, but much less deletion in the CD1-deficient mice. This difference persisted throughout the time-course of the experiment, until the latest time-point evaluated on day 8. We conclude first that, even at early time-points, peripheral T cell deletion was impaired in CD1-deficient mice. Second, the calculation of absolute cell numbers revealed that the deletion of CD8+ cells, as well as CD4+ cells, was impaired in CD1-/- mice, which was not obvious from the study of percentages alone. Thus, at days 4, 6, and 8, respectively, in wild-type mice the number of CD8+ cells was reduced from mean values of 2.2, 2.4, and 2.6 million in PBS-injected control groups to 0.5, 0.2, and 0.1 million in anti-CD3-injected groups of mice. In the CD1-deficient mice, anti-CD3 reduced the mean numbers of CD8+ cells from similar control group values of 2.0, 1.5, and 2.5 million to 0.8, 1.0, and 0.3 million in anti-CD3-injected groups of mice. Thus the deletion of CD8+ T cells was more profound in wild-type mice.



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  		FIGURE 2. Kinetics of peripheral CD4+ and CD8+ T cell deletion in wild-type and CD1-deficient mice. Solid bars ({blacksquare}) represent PBS-injected controls, and hatched bars () represent anti-CD3-injected mice. The CD1-deficient mice showed defective deletion of both CD4+ and CD8+ T cells. The data are mean ± SD of numbers (in millions) of CD4+ or CD8+ cells in pooled superficial (inguinal, axillary) lymph nodes of groups of four mice.

 
The limited availability of CD1-deficient mice backcrossed to B6 limited our ability to perform a full kinetic analysis on such a uniform background. However, to confirm the difference in T cell deletion, we analyzed the effect of anti-CD3 treatment in groups of B6 vs fifth-generation B6.CD1-/- backcross mice at 4 days. In this direct comparison, the inguinal plus axillary lymph nodes of PBS-injected wild-type mice contained 5.3 ± 0.9 x 106 CD4+ cells. Injection of four such mice with anti-CD3 caused very substantial deletion of the cells, such that 0.6 ± 0.2 x 106 CD4+ cells remained. In the four congenic CD1-/- mice, the corresponding numbers were 4.0 x 106 and 1.03 ± 0.3 x 106. Because the numbers of CD4+ T cells were not identical in the PBS-injected controls, the numbers of cells in each anti-CD3-injected animal was normalized to "the percent of mean PBS control", and these numbers subjected to a t test. The deletion in the CD1-/- mice was significantly less (p = 0.0026).

An identical analysis was applied to the CD8+ T cells in the B6 control vs congenic CD1-/- mice. Lymph nodes of wild-type mice contained 4.4 ± 0.7 x 106 CD8+ cells, depleted to 0.4 ± 0.1 x 106 by anti-CD3. Lymph nodes of CD1-/- mice contained 3.8 ± 0.2 x 106 CD8+ cells, depleted to 1.0 ± 0.4 x 106 with anti-CD3. The difference in T cell deletion was significant (p = 0.0025).

Normal activation of CD1-/- T cells

One possible explanation for these findings could be that CD1-deficient T cells simply failed to become activated. Therefore, we verified that the lack of CD1 expression did not compromise T cell activation. In a 48-h in vitro proliferative response to anti-CD3 Ab, in which the T cell response was measured using a [3H]thymidine incorporation assay, the activation of CD1-/- T cells was identical with the activation of wild-type T cells (the data were: means of 260, 534 cpm in wild-type T cells and 264, 279 cpm in CD1-/- cells, with backgrounds of less than 2,000 cpm in both cases). In vivo injection of 100 µg of purified 145-2C11 anti-CD3 Ab resulted in a burst of T cell proliferation, which was very similar between CD1-deficient and wild-type mice based on the percentage of T cells that incorporated 2-bromo-deoxyuridine (20.4% in wild type, and 24.2% in CD1-/-).

Defective accumulation of T cells in liver in CD1-deficient mice

The activation and deletion of T cells is associated with their accumulation in the liver. Our previous studies have shown that the accumulation of T cells in the liver during peripheral T cell deletion is independent of Fas function (29). To test whether such accumulation was dependent on CD1, we isolated IHL from the livers of wild-type control mice and CD1-deficient mice during anti-CD3-induced peripheral T cell deletion. In normal mice, anti-CD3 induced a transient increase in the IHL count, with a peak at 5 x 106 at day 4 after anti-CD3 injection (Fig. 3Go, left, dark shading). Control mice injected with PBS showed an IHL count of around 1 x 106 throughout the experiment, which is in the normal range for unmanipulated mice. Mice lacking CD1 had a normal number of IHL in the PBS-injected controls. However, in anti-CD3-injected CD1-deficient mice, the increase in IHL numbers was smaller, with a peak of 3 x 106, and the peak was delayed until day 6. Therefore, the normal process of liver accumulation of T cells during peripheral T cell deletion was defective in CD1-deficient mice.



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  		FIGURE 3. Delayed kinetics of liver accumulation during anti-CD3 induced peripheral T cell deletion in CD1-deficient mice. In anti-CD3-injected wild-type mice, the liver lymphocyte count increased to four times control values, with a maximum on day 4. The percentage of CD8+ cells peaked on day 2, while there was no effect on the percentage of either CD4+ cells of NK-1.1+ cells. In CD1-deficient mice, the liver lymphocytosis was reduced and delayed. Similarly, there was a smaller increase in the percentage of CD8+ cells.

 
To validate the conclusions, we normalized the mean number of liver lymphocytes in PBS-injected control mice on day 4 to 100%. Day 4 was chosen because it was the peak of the liver lymphocytosis in normal mice. In wild-type mice, the IHL count in anti-CD3-injected mice on day 4 was 4.6 ± 0.6 x 106, compared with 1.0 x 106 in the PBS-injected controls. In CD1-/- mice, the IHL count in anti-CD3-injected mice on day 4 was 2.7 ± 0.4 x 106, compared with 0.91 ± 0.3 x 106 in the PBS controls. The difference between wild-type and CD1-/- mice was significant, using a t test (p = 0.0008).

The isolated IHL were stained for CD4 and CD8 expression (Fig. 3Go, center left and center right panels). As expected, the frequency of CD4+ cells was unchanged among the IHL, and fluctuations were within the limits of normal. However, a dramatic effect was evident in the CD8+ cells. In wild-type mice, anti-CD3 injection caused a 4-fold increase in the percentage of CD8+ IHL at day 2, followed by a return to normal on day 4. In contrast, in CD1-deficient mice, the percentage of CD8+ IHL showed a much smaller increase on day 2, but remained elevated by 2- to 3-fold on days 4 and 6 (Fig. 3Go, inner left and right panels). Thus in terms both of cell accumulation and phenotypic change, the liver phase of peripheral T cell deletion was attenuated and delayed in CD1-deficient mice. The most striking difference between IHL subsets of wild-type and CD1-/- mice was in the percentage of CD8+ cells, observed in anti-CD3-injected mice on day 2. This difference was statistically significant (p = 0.002).

It is noteworthy that in anti-CD3-injected normal mice, the peak in the percentage of CD8+ cells occurred at 2 days, while the peak in total IHL numbers occurred at 4 days. This could have been because CD8+ T cells, trapped in the liver, undergo loss of recognition molecules resulting in CD4-, CD8- T cells, as we have reported before (10, 30). Alternatively, it could have been because of expansion of the liver NK T cells, induced by the anti-CD3. The right panels of Fig. 3Go show that anti-CD3 treatment did not change the percentage of NK-1.1+ cells in the liver, either in wild-type of in CD1-/- mice.

Fig. 4Go shows examples of the changes in TCR{alpha}{beta}, CD4, and CD8 expression in liver lymphocytes during anti-CD3 induced T cell deletion. All data are taken from day 4. In wild-type mice, anti-CD3 treatment caused down-regulation of the TCR{alpha}{beta}, both on T cells and on NK T cells (Fig. 4Go, upper left panels). In CD1-/- mice, there was less down-modulation of the TCR{alpha}{beta}, and very few NK T cells, as expected (Fig. 4Go, lower left panels). Within the population of TCR{alpha}{beta}+ cells, anti-CD3 treatment caused a decrease in the percentages of CD4+ cells, with a compensatory increase in the percentage of both DN and CD8+ cells (Fig. 4Go, upper right panels). Similar, but less dramatic, effects were observed in CD1-/- mice (Fig. 4Go, lower left panels). We conclude that the increase in IHL numbers is not fully accounted for by CD4+ and CD8+ cells, but also includes some TCR{alpha}{beta}+ DN cells, which we have elsewhere proposed to be the end-stage cells of the intrahepatic deletion pathway.



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  		FIGURE 4. Changes in subsets of liver lymphocytes on day 4. Injection of anti-CD3 into wild-type mice caused reduced expression of the TCR{alpha}{beta} on both T cells and NK T cells (two upper left panels). In CD1-deficient mice, there was no such anti-CD3-induced loss of TCR staining, and NK T cells were very rare, as expected (two lower left panels). Among TCR{alpha}{beta}+ liver lymphocytes of wild-type mice, anti-CD3 caused relative loss of CD4+ cells, with compensating increases in both CD8+ and DN cells (two upper right panels). Among CD1-deficient liver lymphocytes, DN T cells were rare, due in part to the lack of NK T cells. Anti-CD3 injection caused some loss of CD4+ cells, but the magnitude of the loss was smaller than in wild-type mice.

 
These data suggested that CD1 is an active player in the trapping of CD8+ T cells in liver during T cell deletion, but the nature of its involvement is not yet clear. Cell interactions that involve CD1 may change the adhesion or recirculation properties of activated CD8+ T cells in the circulating pool, increasing the chance that they home to liver. Alternatively, CD1 expressed in the liver may have a direct role in the trapping of CD8+ T cells.

The role of CD1 is non-T cell autonomous

CD1 molecules are expressed on lymphoid cells, as well as on nonlymphoid bone marrow-derived cells and on extrahepatic tissues, particularly the liver and the intestine. To determine whether the CD1 on the activated T cells themselves was a target of the T cell deletion mechanism, tetraparental bone marrow chimeras were constructed in which 50% of the bone marrow was CD1-intact, and 50% was CD1-deficient. The radioresistant host tissues in these chimeras were also CD1-intact. Four weeks after irradiation and reconstitution we obtained stable, balanced chimeras.

These chimeras were injected with anti-CD3 Ab, and the effect on the CD1-intact and the CD1-deficient T cells in each chimera was determined. Fig. 5GoA shows two chimeras. One has been injected with PBS as a control; this chimera contained 47% of CD1+ lymphocytes, most of which were T cells. The other chimeras were injected with anti-CD3. Although the expression of the TCR was reduced, the overall frequency of CD1+ lymphocytes was the same, at 46%. Fig. 5GoB shows the CD4 expression on the CD1+/+ and the CD1-/- cells within two chimeras. The upper panels of Fig. 5Go illustrate a PBS-injected control chimera, in which the CD1-/- lymph node cells are 43% CD4+ cells, while the CD1+/+ lymph node cells are 53% CD4+ cells. In anti-CD3-injected chimeras, the frequency of CD4+ cells was reduced by >50% in both the CD1-deficient and the CD1-intact lymphocytes. Thus, the lower panels of Fig. 5GoB show the CD1-/- and the CD1+/+ lymphocytes in an anti-CD3-injected chimera. The CD1-/- cells are only 11% CD4+ cells, while the CD1+/+ cells are similarly depleted and contain only 18% CD4+ cells. This shows that both CD1-/- and CD1+/+ CD4+ cells are susceptible to deletion. Fig. 5GoC shows the means and SDs of the percentage of CD4+ cells from the CD1-/- and the CD1+/+ cell populations in four PBS-injected control chimeras, and four anti-CD3-injected chimeras. These data confirm that the presence or absence of CD1 on the T cells made no difference to their deletion. Therefore, this experiment shows that the role of CD1 in peripheral T cell deletion is non-T cell autonomous; instead, the presence of CD1 on other cells in the chimera permitted the effective deletion of CD1-/- T cells.



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  		FIGURE 5. The effect of CD1 deficiency on peripheral T cell deletion is non-T cell autonomous. Mixed bone marrow chimeras containing 50% wild-type and 50% CD1-deficient marrow were stable with approximately half of their lymphocytes expressing CD1. A, Lymph node cells from two such chimeras, with the gates used to define the CD1- and CD1+ subsets of cells. The center panels (B) show representative data from two chimeras. Upper panels show the expression of CD4 in the CD1- and CD1+ lymphocytes in a PBS-injected control chimera, while the lower panels show the CD4 expression in the two cell populations from a chimera injected with anti-CD3. Peripheral T cell deletion occurred in both CD1- and CD1+ cells. C, The data from four control and four anti-CD3-injected chimeras. There was no difference between the CD1- and the CD1+ cells in their potential for deletion. We conclude that the CD1 that is important in peripheral T cell deletion is not expressed on the T cells.

 
Abnormal T cell deletion was not due to lack of V{alpha}14, J{alpha}281 NK T cells

The impairment of peripheral T cell deletion in CD1-deficient mice might be accounted for by various mechanisms. These mice lack TCR-invariant NK T cells, and a mechanism could be envisaged in which such NK T cells engage CD1 ligands on activated T cells, and deliver a death signal. In support of this concept, we have shown that liver NK T cells are cytotoxic by both Fas ligand-dependent and perforin-dependent mechanisms (25). Therefore, we tested this hypothesis by repeating the anti-CD3 deletion experiment in young {beta}2-microglobulin-deficient mice. These mice contain very few NK T cells, although the defect is not absolute and the cells increase in number as the mice age (24). They also lack CD8+ T cells, but we were able to evaluate the peripheral deletion of CD4+ T cells. Fig. 6GoA shows that anti-CD3-induced peripheral CD4+ T cell deletion was normal in lymph nodes of {beta}2-microglobulin-deficient mice, with the disappearance of around half of all the CD4+ T cells at 4 days (compare with Fig. 1GoB). However, {beta}2-microglobulin-deficient mice could potentially be abnormal in ways that would confound the interpretation of the experiment, for example due to the lack of CD8+ T cells. To overcome this problem, we addressed the same issue using an alternative mutant mouse line that lacks the same population of NK T cells, but contains CD8+ cells. Because most NK T cells use a highly specific TCR{alpha}-chain in their Ag receptors, namely V{alpha}14 J{alpha}281, it is possible to test the involvement of these NK T cells in mice unable to make this specific TCR due to lack of the J region. In J{alpha}281-deficient mice, the deletion of peripheral lymph node CD4+ T cells by anti-CD3 was intact (Fig. 6GoB). These two experiments, taken together, argue strongly against a role for most, if not all NK T cells in the CD1-dependent component of peripheral T cell deletion.



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  		FIGURE 6. Deficiency of NK T cells does not result in a similar phenotype to CD1-deficiency. A, Young (6-wk-old) mice lacking {beta}2-microglobulin, and hence deficient in NK T cells, responded normally to anti-CD3 injection and showed peripheral lymph node CD4+ T cell deletion identical with wild-type. B, Mice deficient in TCR J{alpha}281, and hence in NK T cells, also showed normal peripheral deletion of lymph node CD4+ cells in response to anti-CD3. All experimental data were at day 4; bar graphs show mean ± SD of groups of three to four mice.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The highly polymorphic, peptide-binding "classical" molecules of the MHC are clustered on chromosome 17 in the mouse, and on chromosome 6 in the human. In addition to this gene complex, both humans and mice express structurally related but less polymorphic "nonclassical" MHC like molecules encoded elsewhere. In the human, these are termed CD1a, b, c, d, and e. In mice, there is only a tandem duplication of a homologue of CD1d (31). The CD1 molecules, like their classical MHC counterparts, contain a pocket on the apical surface. However, this cavity is deeper and its inner recesses are more hydrophobic that is the case for classical MHC molecules (32), and recent evidence suggests that this is because the pocket of all CD1 molecules binds and presents glycolipids instead of peptides (33). These complexes are recognized not by classical CD4+ and CD8+ T cells, but by a distinct set of TCR{alpha}{beta}+ lymphocytes, many of which express a monomorphic TCR{alpha}-chain paired with a limited set of V{beta}-chains. In addition to the TCR{alpha}{beta}, these cells express surface markers characteristic of NK cells (16).

The tissue distribution of CD1 molecules is more restricted than the distribution of classical MHC molecules, and differs between species. In the mouse, CD1 expression has been reported on thymocytes and peripheral T lymphocytes, B cells, dendritic cells, and macrophages (34), on hepatocytes in the liver, and on intestinal epithelial cells where it appears to act as a T cell ligand (26, 35). The reported CD1 expression in the livers of mice, and also of rats (27), is provocative because this tissue site has been implicated in the trapping of activated T cells (10, 11, 13). Furthermore, the liver is a site in which NK T cells are unusually abundant (36, 37), raising the possibility that the NK T cells are involved in the liver component of peripheral T cell deletion. The availability of mice deficient in CD1 allowed us to examine these issues. In the present study, we have induced peripheral T cell activation leading to T cell deletion in normal vs CD1-deficient mice.

We tested peripheral T cell deletion using a single injection of anti-CD3, which was chosen in preference to a peptide Ag because it allowed us to induce the activation and deletion of both CD4+ and CD8+ T cells in the same experimental animals. This technique suffers from the disadvantage that the TCR is ligated at an unusual point (i.e., on the extracellular domain of the CD3{epsilon}-chain), but offers the compensating advantage that general conclusions may be drawn without the risk of artifacts due to the vagaries of an individual transgenic TCR. After the injection of anti-CD3, lymph node cells and IHL were isolated at 2, 4, 6, and 8 days. As previously reported by others (4, 5, 38), this treatment caused peripheral T cell activation followed by deletion in wild-type mice.

We used this model to show that CD1d was required for the early phase of peripheral T cell deletion in mice. This role was independent of TCR-invariant, CD1-reactive NK T cells, because neither {beta}2-microglobulin-deficient mice nor J{alpha}281-deficient mice reproduced the abnormality found in CD1-deficient mice. The defect appears to control the homing of activated T cells to the liver. Chimera experiments have shown that the expression of CD1d on the T cells is not important for its role in T cell deletion. To explain these data, we propose that activated T cells interact with CD1d expressed on non-T cells, and that this interaction predisposes them to undergo deletion from the periphery. At present, we do not know whether the relevant CD1d is expressed on specialized, bone marrow-derived APCs such as macrophages, B cells, or dendritic cells (39, 40), or whether it is on tissue cells such as intestinal epithelium or hepatocytes (41, 42).

Liver shows a variety of unusual immunological properties, apart from the expression of CD1 and the presence of populations of lymphocytes distinct from those found in secondary lymphoid organs or in the general circulation. It has been proposed as a site of extrathymic T cell development (43, 44, 45), and is a site at which systemic tolerance may be induced. This tolerance is manifest as failure to reject liver allografts (46), as failure to reject pancreatic islet allografts when they are introduced into the liver via the portal vein (47), and as the induction of tolerance in T cells specific for allogeneic MHC Ags expressed on hepatocytes through transgenesis (48, 49). Liver is also a site at which an important pathogenic virus, Hepatitis C virus, establishes a persistent infection (50). The present study suggests an important role for CD1 in T cell accumulation in the liver, and thus raises the possibility that its presence at that site may be linked to liver tolerance.

This study documents the importance of CD1-based mechanisms in the peripheral deletion of T cells induced by anti-CD3 Ab. Presumably these mechanisms also have a role in the immune response to Ag. It is clear from our data that CD1 is important in the earliest phase of the deletion process, because when it is missing, abnormalities are evident as early as 2 days after anti-CD3 injection. In a normal immune response to specific peptide Ag, this is the time of the clonal expansion phase, while T cell deletion does not start until day 5 or later (51, 52, 53). On this basis, we speculate that the CD1-based mechanism is involved in removing cells that did not receive a full activation signal, i.e., in the induction of tolerance rather than in the termination of a full-blown immune response.


   Acknowledgments
 
We acknowledge the critical input of our colleagues in the Yale Immunobiology Section. S.S. was a postdoctoral fellow in the laboratory of Dr. F. Alt during the creation of the CD1-deficient mice; he thanks Dr. Alt for his support. T.D. thanks Dr. Derek Sant’Angelo for support and the freedom to complete these studies.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI37554 (to I.N.C.) and AI42955 (to S.P.B.), and by an Howard Hughes Medical Institute Postdoctoral Fellowship (to W.Z.M.). Back

2 Address correspondence and reprint requests to Dr. I. Nicholas Crispe, The Center for Vaccine Biology and Immunology, The Aab Institute for Biomedical Research, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14620. Back

3 Abbreviations used in this paper: B6, C57BL/6; BrdU, 5-bromo-2'-deoxyuridine; IHL, intrahepatic lymphocytes. Back

Received for publication March 24, 2000. Accepted for publication December 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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