Cells T Lymphocytes Is Independent of NK T Involvement of CD1 in Peripheral Deletion Of

Involvement of CD1 in Peripheral Deletion of T Lymphocytes Is Independent of NK T Cells Tao Dao, Mark Exley, Wajahat Z. Mehal, Syed Muhammad Ali Tahir, Scott Snapper, Masaru Taniguchi, Steven P. Balk This information is current as and I. Nicholas Crispe of September 28, 2021. J Immunol 2001; 166:3090-3097; ; doi: 10.4049/jimmunol.166.5.3090 http://www.jimmunol.org/content/166/5/3090 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Involvement of CD1 in Peripheral Deletion of T Lymphocytes Is Independent of NK T Cells1

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

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 Downloaded from ␤ ␣ encoding either 2-microglobulin or the TCR element J 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. The Journal of Immunology, 2001, 166: 3090–3097.

he activation of T cells by nonphysiological stimuli, such (reviewed in Refs. 14 and 15)). This latter population contains a

as superantigens and Abs against the TCR, results in ac- subset of cells that express a distinctive TCR␣␤, in which the http://www.jimmunol.org/ T tivation-induced cell death and deletion of the T cells TCR␤-chain is of very limited diversity and the TCR␣-chain is from the periphery (1–5). Similarly, injection of specific antigenic essentially monomorphic. These cells depend for their develop- ␤ peptide into TCR-transgenic mice leads to a truncated, abortive ment and function on CD1 molecules, which are 2-microglobu- immune response in which T cell activation is rapidly followed by lin-associated, MHC class I-like molecules encoded outside the peripheral deletion (6, 7). In some experimental models, peripheral gene complex on chromosome 17 (reviewed in Refs. 16–18)). deletion is due to apoptosis of the activated T cells in lymphoid Mice with induced mutations in key recognition molecules tissues (8). In other models, apoptosis is difficult to detect in the make it possible to determine precisely which subsets of CD1- lymphoid tissue, and activated T cells recirculate to specific de- reactive or NK-like T cells are involved in any biological process. by guest on September 28, 2021 struction sites (9–11). Thus, mice lacking the invariant TCR J region, J␣281, specifically Our previous research has identified the liver as a site for the ϩ lack CD1-reactive T cells with this invariant Ag receptor (19–21). accumulation and destruction of CD8 T cells during peripheral Mice deficient in CD1 lack these invariant T cells, and also other deletion, induced in a TCR-transgenic mouse by the injection of ␤ CD1-reactive T cells. Mice deficient in 2-microglobulin display a antigenic peptide (10). Intrahepatic accumulation of apoptotic T similar defect to CD1-deficient mice (22, 23), except that they cells has been reported in several other models of peripheral T cell ␤ contain a small population of 2-microglobulin-independent T deletion, raising the possibility that this is a general site of T cell cells with NK markers, which increases with age (24). All of these death (11–13). The liver is an unusual immunologic environment, cell populations include NK-like T cells, and in this report we use in which a population of T cells similar to the circulating CD4ϩ the generic term NK T cells to embrace all of them. and CD8ϩ T cells coexists with a subset of CD4Ϫ, The intrahepatic population of NK T cells is cytotoxic though CD8Ϫ,B220ϩ,TCR␣␤ϩ cells that contains a high frequency of ap- ϩ both Fas ligand-mediated and perforin-mediated mechanisms. The optotic cells, and with an abundant subset of NK-1.1 T cells 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 postu- *Immunobiology Section, Yale University School of Medicine, New Haven, CT 06510; †Cancer Biology Program, Division of Hematology/Oncology, Beth Israel lated that these cells might be involved in the intrahepatic killing Deaconess Medical Center, Boston, MA 02215; ‡Gastrointestinal Unit (Medical Ser- of activated T cells, perhaps through recognition of CD1-associ- vices), Massachusetts General Hospital, Boston, MA 02116; and §Department of Mo- lecular Immunology, Graduate School of Medicine, Chiba University, Chuoku Chiba, ated ligands (for example, specific glycolipids) on their surface. Japan Significantly, both mouse and rat liver express CD1 (26, 27). The Received for publication March 24, 2000. Accepted for publication December present study was conducted to test the hypothesis that CD1 is 22, 2000. involved in T cell deletion, which we have done by inducing T cell The costs of publication of this article were defrayed in part by the payment of page activation and peripheral deletion in mice lacking CD1, and, there- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. fore, devoid of the CD1-reactive, largely TCR-invariant subset of NK T cells. The results show that peripheral T cell deletion was 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 Post- indeed impaired in CD1-deficient mice; however, this impairment doctoral Fellowship (to W.Z.M.). was not due to the lack of NK T cells. Furthermore, CD1 on the T 2 Address correspondence and reprint requests to Dr. I. Nicholas Crispe, The Center cells could not be acting as a target for a T cell deletion mecha- for Vaccine Biology and Immunology, The Aab Institute for Biomedical Research, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14620. E-mail ad- nism, because the requirement for CD1 expression was non-T cell dress: [email protected] autonomous. Therefore, we conclude that an interaction between

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 3091 the activated T cells and another cell expressing CD1 promotes the ␮g/ml DNase, and 5% FCS). After perfusion, the livers were homogenized deletion of activated peripheral T cells. 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 ϫ g for 3 min. The supernatant was centrifuged at Materials and Methods 650 ϫ g for 10 min to obtain a pellet of nonparenchymal cells. The pellet Mice 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 C57BL/6 (B6),3 (B6 ϫ 129)F or F , and ␤ -microglobulin-deficient mice 1 2 2 suspension in metrizamide was overlaid with serum-free Bruff’s medium, were purchased from The Jackson Laboratory (Bar Harbor, ME). CD1d- and centrifuged at 1500 ϫ g for 20 min. The cells at the interface were deficient mice (on the B6 ϫ 129 background) were created at Beth Israel collected, washed with PBS, and counted. Deaconess Hospital (Harvard University, Boston, MA). The J␣281-deficient mice were created at Chiba University (Chuoku Chiba, Japan). The mice were kept in the Immunobiology Mouse Unit at Yale Medical School Results (New Haven, CT), or at Beth Israel Deaconess Hospital. All mice were Defective peripheral T cell deletion used between 6–9 wk of age. The down-regulation of the TCR is a recognized feature of T cell Reagents activation leading to deletion, and has been previously reported in PE-coupled anti-NK1.1 Ab, PE-anti-mouse CD1 Ab, CyChrome-anti- peptide- and superantigen-induced peripheral T cell deletion (2, mouse TCR␣␤ Ab, biotinylated anti-hamster IgG, and strepavidin-PE were 10). Similarly, the injection of 100 ␮g of anti-CD3 resulted in the obtained from PharMingen (San Diego, CA). PE-anti-mouse CD4 Ab and down-regulation of the TCR. Fig. 1A shows the effect of anti-CD3 FITC-coupled anti-mouse CD8 Ab were purchased from Life Technologies ϩ ϩ (Gaithersburg, MD). Hybridomas producing anti-mouse CD3 Ab (145- injection of CD4 and CD8 T cells of wild-type and CD1-defi-

2C11) and anti-murine NK (PK-136) Ab were obtained from American cient mice at 2 days after injection of the Ab. Lymph node cells of Downloaded from Type Culture Collection (Manassass, VA). These Abs were puriﬁed using individual, representative wild-type mice are shown in the upper staphylococcal protein A-Sepharose (Pharmacia, Piscataway, NJ). An ␣ panels of Fig 1A. In PBS-injected controls, 50% of the lymph node ELISA kit to measure murine TNF- was purchased from Endogen ϩ ␣␤ϩ ϩ (Woburn, MA). cells were CD4 , TCR T cells and only 1% were CD4 , TCR␣␤Ϫ T cells. In an anti-CD3-injected mouse, there were 23% Induction of T cell deletion of CD4ϩ, TCR␣␤ϩ T cells and 18% of CD4ϩ, TCR␣␤Ϫ T cells, ϩ

Mice were i.p. injected with either 100 ␮g anti-murine CD3 Ab in 0.2 ml making 41% of CD4 T cells overall. Thus, at this early time http://www.jimmunol.org/ PBS, or with 0.2 ml PBS as control. Two to 8 days after injection, four LNs point, the main effect of anti-CD3 treatment was the loss of (two axillary and two inguinal) were removed by dissection, homogenized, TCR␣␤ expression rather than T cell deletion, yet there was also and then suspended lymph node cells rinsed in Bruff’s culture medium. a small loss of CD4ϩ T cells. Similarly, the TCR␣␤ was lost from Cells were counted and kept on ice until staining. many of the CD8ϩ cells. The lower panels of Fig. 1A show the FACS analysis effects of anti-CD3 injection on the T cells of individual, repre- Cells suspended (0.5–1 ϫ 106) in 0.1 ml staining buffer (PBS with 1% BSA sentative CD1-deﬁcient mice. The basic pattern was similar, with ϩ ϩ and 0.02% sodium azide) were incubated with the following directly con- down-regulation of the TCR on both CD4 and CD8 cells. How- jugated Abs: anti-murine TCR␣␤-CyChrome, anti-murine CD4-PE and anti- ever, the total of CD4ϩ, TCR␣␤ϩ T cells and CD4ϩ, TCR␣␤Ϫ T

␣␤ by guest on September 28, 2021 murine CD8-FITC, or TCR -CyChrome vs PE-anti-NK1.1 Ab or vs PE- cells (25 and 26%, respectively) was 51%, against 54% in the anti-mouse CD1 Ab for 30 min, and were analyzed using a FACScan (Becton Dickinson, Mountain View, CA). The percentage of TCR␣␤ vs PBS-injected controls. Both of these numbers are within the nor- CD4- or CD8-positive cells was determined using CellQuest software on mal range for PBS-injected CD1-deficient mice, in contrast to the an Apple Macintosh computer (Apple Computer, Cupertino, CA) . loss of 10% of T cells from the anti-CD3-injected normal mice. This difference was accentuated 2 days later. Fig. 1B shows that Cell proliferation at day 4, the TCR-low cells had disappeared from both the CD4ϩ To determine the proliferation potential of wild-type vs CD1-deficient T and the CD8ϩ subsets of the anti-CD3-injected wild-type mice. ϫ 6 cells in vitro, LN or spleen cells (1 10 cells/ml) were incubated in RPMI The total number of CD4ϩ cells was reduced from 58% in a rep- 1640 medium containing 10% FCS and supplemented with L-glutamine, antibiotics, and 50 ␮M 2-ME, with or without 1 ␮g/ml immobilized anti- resentative PBS-injected control to 27% in a representative anti- CD3 Ab for 2 days. [3H]Thymidine (25 ␮Ci/ml) was added to the culture CD3-injected mouse (Fig. 1B, upper panels). Similar changes of forthelast4hoftheincubation period. To determine the proliferative lesser magnitude occurred in the CD8ϩ cells. In CD1-deficient response of T cells in vivo, mice were injected i.p. with anti-CD3 Ab on mice the TCR-low cells had also disappeared, but, in contrast, the day 0, and on day 2 were injected i.p. with 5-bromo-2Ј-deoxyuridine ϩ total CD4 cell frequencies in anti-CD3-injected mice were within (BrdU; 1 mg/mouse) four times at 4-h intervals. Twelve hours after the last ϩ injection, mice were killed and LN and spleen cells were isolated. The cells the range of the controls. Among the CD8 cells, the modest de- were stained with TCR␣␤-CyChrome vs CD4-PE or CD8-PE, then were letion observed in wild-type mice was not evident in the fixed with 70% ethanol followed by 1% paraformaldehyde, and then were CD1-deficient mice. stained to detect incorporated BrdU using anti-BrdU-FITC Ab (Becton The results from groups of four mice each are summarized in Dickinson). Fig. 2, which shows the means and SD estimates of the total num- Construction of bone marrow chimeras bers of CD4ϩ and CD8ϩ T cells in the lymph node, for each ϫ experimental group, at 2, 4, 6, and 8 days after a single injection Eight- to 10-wk-old (B6 129) F1 mice were given a single dose of 9 Gy ϩ ϩ (900 rad) of whole body irradiation from a ␥ source. Bone marrow cells of anti-CD3. We chose to represent absolute CD4 and CD8 cell from B6- or CD1-deficient mice were mixed at a 50/50 ratio, and injected numbers to show that the effects of CD1 deficiency on the relative i.v. 5 h after the irradiation. Four weeks later, chimeras were injected with depletion of CD4ϩ T cells were not due to the fluctuations in the either anti-CD3 as previously described, or with PBS as a control. absolute numbers of other cell types. To obtain these numbers, we Isolation of liver lymphocytes multiplied the lymph node cell yield from the pooled superficial (axillary and inguinal) lymph nodes of each individual animal by Intrahepatic lymphocytes (IHL) were isolated by a standard method (28). ϩ ϩ Briefly, in an anesthetized mouse, the portal vein was perfused with 5 ml the percentage of CD4 and CD8 cells from that animal. In wild- ϩ of “digestion buffer” (i.e., medium containing 0.2 ␮g/ml collagenase, 0.02 type mice, the absolute number of CD4 T cells was already depleted (from 4.4 ϫ 106 to 1.8 ϫ 106) by day 2, while there was less 6 6 3 Ј depletion in the CD1-deficient cells (from 4.6 ϫ 10 to 3.0 ϫ 10 ). Abbreviations used in this paper: B6, C57BL/6; BrdU, 5-bromo-2 -deoxyuridine; ϩ IHL, intrahepatic lymphocytes. By day 4, there was deletion of CD4 T cells from both wild-type 3092 CD1 FUNCTION INDEPENDENT OF NK T CELLS

FIGURE 1. Anti-CD3-induced peripheral T cell deletion is delayed in CD1-deﬁcient mice. A, Down- regulation of the TCR on CD4ϩ and CD8ϩ 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 CD4ϩ and CD8ϩ T cells at 4 days after anti-CD4 ϩ

injection. The percentage of CD4 cells is reduced to Downloaded from half in the wild-type mice (upper panels) but not in the CD1-deficient mice (lower panels). http://www.jimmunol.org/ by guest on September 28, 2021 and CD1-deficient mice, but much less deletion in the CD1-defi- numbers subjected to a t test. The deletion in the CD1Ϫ/Ϫ mice was cient mice. This difference persisted throughout the time-course of significantly less ( p ϭ 0.0026). the experiment, until the latest time-point evaluated on day 8. We An identical analysis was applied to the CD8ϩ T cells in the B6 conclude first that, even at early time-points, peripheral T cell de- control vs congenic CD1Ϫ/Ϫ mice. Lymph nodes of wild-type letion was impaired in CD1-deficient mice. Second, the calculation mice contained 4.4 Ϯ 0.7 ϫ 106 CD8ϩ cells, depleted to 0.4 Ϯ 0.1 ϫ of absolute cell numbers revealed that the deletion of CD8ϩ cells, 106 by anti-CD3. Lymph nodes of CD1Ϫ/Ϫ mice contained 3.8 Ϯ as well as CD4ϩ cells, was impaired in CD1Ϫ/Ϫ mice, which was 0.2 ϫ 106 CD8ϩ cells, depleted to 1.0 Ϯ 0.4 ϫ 106 with anti-CD3. not obvious from the study of percentages alone. Thus, at days 4, The difference in T cell deletion was significant ( p ϭ 0.0025). ϩ 6, and 8, respectively, in wild-type mice the number of CD8 cells Ϫ/Ϫ was reduced from mean values of 2.2, 2.4, and 2.6 million in Normal activation of CD1 T cells PBS-injected control groups to 0.5, 0.2, and 0.1 million in anti- One possible explanation for these findings could be that CD1- CD3-injected groups of mice. In the CD1-deficient mice, anti-CD3 deficient T cells simply failed to become activated. Therefore, we reduced the mean numbers of CD8ϩ cells from similar control verified that the lack of CD1 expression did not compromise T cell group values of 2.0, 1.5, and 2.5 million to 0.8, 1.0, and 0.3 million activation. In a 48-h in vitro proliferative response to anti-CD3 Ab, in anti-CD3-injected groups of mice. Thus the deletion of CD8ϩ T in which the T cell response was measured using a [3H]thymidine cells was more profound in wild-type mice. incorporation assay, the activation of CD1Ϫ/Ϫ T cells was identical The limited availability of CD1-deficient mice backcrossed to with the activation of wild-type T cells (the data were: means of B6 limited our ability to perform a full kinetic analysis on such a 260, 534 cpm in wild-type T cells and 264, 279 cpm in CD1Ϫ/Ϫ uniform background. However, to confirm the difference in T cell cells, with backgrounds of less than 2,000 cpm in both cases). In deletion, we analyzed the effect of anti-CD3 treatment in groups of vivo injection of 100 ␮g of purified 145-2C11 anti-CD3 Ab re- B6 vs fifth-generation B6.CD1Ϫ/Ϫ backcross mice at 4 days. In sulted in a burst of T cell proliferation, which was very similar this direct comparison, the inguinal plus axillary lymph nodes of between CD1-deficient and wild-type mice based on the percent- PBS-injected wild-type mice contained 5.3 Ϯ 0.9 ϫ 106 CD4ϩ age of T cells that incorporated 2-bromo-deoxyuridine (20.4% in cells. Injection of four such mice with anti-CD3 caused very sub- wild type, and 24.2% in CD1Ϫ/Ϫ). stantial deletion of the cells, such that 0.6 Ϯ 0.2 ϫ 106 CD4ϩ cells remained. In the four congenic CD1Ϫ/Ϫ mice, the corresponding Defective accumulation of T cells in liver in CD1-deficient mice numbers were 4.0 ϫ 106 and 1.03 Ϯ 0.3 ϫ 106. Because the The activation and deletion of T cells is associated with their ac- numbers of CD4ϩ T cells were not identical in the PBS-injected cumulation in the liver. Our previous studies have shown that the controls, the numbers of cells in each anti-CD3-injected animal accumulation of T cells in the liver during peripheral T cell dele- was normalized to “the percent of mean PBS control”, and these tion is independent of Fas function (29). To test whether such The Journal of Immunology 3093

tosis in normal mice. In wild-type mice, the IHL count in anti- CD3-injected mice on day 4 was 4.6 Ϯ 0.6 ϫ 106, compared with 1.0 ϫ 106 in the PBS-injected controls. In CD1Ϫ/Ϫ mice, the IHL count in anti-CD3-injected mice on day 4 was 2.7 Ϯ 0.4 ϫ 106, compared with 0.91 Ϯ 0.3 ϫ 106 in the PBS controls. The difference between wild-type and CD1Ϫ/Ϫ mice was significant, using a t test ( p ϭ 0.0008). The isolated IHL were stained for CD4 and CD8 expression (Fig. 3, center left and center right panels). As expected, the frequency of CD4ϩ cells was unchanged among the IHL, and fluctuations were within the limits of normal. However, a dramatic effect was evident in the CD8ϩ cells. In wild-type mice, anti-CD3 injection caused a 4-fold increase in the percentage of CD8ϩ IHL at day 2, followed by a return to normal on day 4. In contrast, in CD1-deficient mice, the percentage of CD8ϩ IHL showed a much smaller increase on day 2, but remained elevated by 2- to 3-fold on days 4 and 6 (Fig. 3, inner left and right panels). Thus in terms

both of cell accumulation and phenotypic change, the liver phase Downloaded from of peripheral T cell deletion was attenuated and delayed in CD1- ϩ ϩ deficient mice. The most striking difference between IHL subsets FIGURE 2. Kinetics of peripheral CD4 and CD8 T cell deletion in Ϫ/Ϫ ϩ wild-type and CD1-deficient mice. Solid bars (f) represent PBS-injected of wild-type and CD1 mice was in the percentage of CD8 controls, and hatched bars (o) represent anti-CD3-injected mice. The CD1- cells, observed in anti-CD3-injected mice on day 2. This difference deficient mice showed defective deletion of both CD4ϩ and CD8ϩ T cells. The was statistically significant ( p ϭ 0.002). data are mean Ϯ SD of numbers (in millions) of CD4ϩ or CD8ϩ cells in It is noteworthy that in anti-CD3-injected normal mice, the peak http://www.jimmunol.org/ pooled superficial (inguinal, axillary) lymph nodes of groups of four mice. in the percentage of CD8ϩ cells occurred at 2 days, while the peak in total IHL numbers occurred at 4 days. This could have been because CD8ϩ T cells, trapped in the liver, undergo loss of rec- accumulation was dependent on CD1, we isolated IHL from the Ϫ Ϫ ognition molecules resulting in CD4 , CD8 T cells, as we have livers of wild-type control mice and CD1-deficient mice during reported before (10, 30). Alternatively, it could have been because anti-CD3-induced peripheral T cell deletion. In normal mice, anti- of expansion of the liver NK T cells, induced by the anti-CD3. The CD3 induced a transient increase in the IHL count, with a peak at right panels of Fig. 3 show that anti-CD3 treatment did not change 5 ϫ 106 at day 4 after anti-CD3 injection (Fig. 3, left, dark shad- the percentage of NK-1.1ϩ cells in the liver, either in wild-type of ing). Control mice injected with PBS showed an IHL count of Ϫ/Ϫ by guest on September 28, 2021 around 1 ϫ 106 throughout the experiment, which is in the normal in CD1 mice. ␣␤ range for unmanipulated mice. Mice lacking CD1 had a normal Fig. 4 shows examples of the changes in TCR , CD4, and number of IHL in the PBS-injected controls. However, in anti- CD8 expression in liver lymphocytes during anti-CD3 induced T CD3-injected CD1-deficient mice, the increase in IHL numbers cell deletion. All data are taken from day 4. In wild-type mice, was smaller, with a peak of 3 ϫ 106, and the peak was delayed anti-CD3 treatment caused down-regulation of the TCR␣␤, both Ϫ/Ϫ until day 6. Therefore, the normal process of liver accumulation of on T cells and on NK T cells (Fig. 4, upper left panels). In CD1 T cells during peripheral T cell deletion was defective in mice, there was less down-modulation of the TCR␣␤, and very CD1-deficient mice. few NK T cells, as expected (Fig. 4, lower left panels). Within the ϩ To validate the conclusions, we normalized the mean number of population of TCR␣␤ cells, anti-CD3 treatment caused a deliver lymphocytes in PBS-injected control mice on day 4 to 100%. crease in the percentages of CD4ϩ cells, with a compensatory Day 4 was chosen because it was the peak of the liver lymphocy- increase in the percentage of both DN and CD8ϩ cells (Fig. 4,

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 lympho- cyte count increased to four times control values, with a maximum on day 4. The percentage of CD8ϩ cells peaked on day 2, while there was no effect on the percentage of either CD4ϩ cells of NK-1.1ϩ cells. In CD1-deﬁcient mice, the liver lymphocytosis was reduced and delayed. Simi- larly, there was a smaller increase in the percentage of CD8ϩ cells. 3094 CD1 FUNCTION INDEPENDENT OF NK T CELLS

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␣␤ 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␣␤ϩ liver lymphocytes of wild-type mice, anti-CD3 caused relative loss of CD4ϩ cells, with compensating increases in both CD8ϩ 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 CD4ϩ cells, but the magnitude of the loss was smaller than in wild-type mice. Downloaded from upper right panels). Similar, but less dramatic, effects were ob- injected control chimeras, and four anti-CD3-injected chimeras. served in CD1Ϫ/Ϫ mice (Fig. 4, lower left panels). We conclude These data confirm that the presence or absence of CD1 on the T that the increase in IHL numbers is not fully accounted for by cells made no difference to their deletion. Therefore, this experi- CD4ϩ and CD8ϩ cells, but also includes some TCR␣␤ϩ DN cells, ment shows that the role of CD1 in peripheral T cell deletion is which we have elsewhere proposed to be the end-stage cells of the non-T cell autonomous; instead, the presence of CD1 on other cells Ϫ/Ϫ

intrahepatic deletion pathway. in the chimera permitted the effective deletion of CD1 T cells. http://www.jimmunol.org/ These data suggested that CD1 is an active player in the trapping ϩ of CD8 T cells in liver during T cell deletion, but the nature of Abnormal T cell deletion was not due to lack of V␣14, J␣281 its involvement is not yet clear. Cell interactions that involve CD1 NK T cells may change the adhesion or recirculation properties of activated CD8ϩ T cells in the circulating pool, increasing the chance that The impairment of peripheral T cell deletion in CD1-deﬁcient they home to liver. Alternatively, CD1 expressed in the liver may mice might be accounted for by various mechanisms. These mice have a direct role in the trapping of CD8ϩ T cells. lack TCR-invariant NK T cells, and a mechanism could be envis- aged in which such NK T cells engage CD1 ligands on activated

The role of CD1 is non-T cell autonomous T cells, and deliver a death signal. In support of this concept, we by guest on September 28, 2021 have shown that liver NK T cells are cytotoxic by both Fas ligand- CD1 molecules are expressed on lymphoid cells, as well as on dependent and perforin-dependent mechanisms (25). Therefore, nonlymphoid bone marrow-derived cells and on extrahepatic tis- we tested this hypothesis by repeating the anti-CD3 deletion ex- sues, particularly the liver and the intestine. To determine whether periment in young ␤ -microglobulin-deficient mice. These mice the CD1 on the activated T cells themselves was a target of the T 2 contain very few NK T cells, although the defect is not absolute cell deletion mechanism, tetraparental bone marrow chimeras were and the cells increase in number as the mice age (24). They also constructed in which 50% of the bone marrow was CD1-intact, and lack CD8ϩ T cells, but we were able to evaluate the peripheral 50% was CD1-deficient. The radioresistant host tissues in these deletion of CD4ϩ T cells. Fig. 6A shows that anti-CD3-induced chimeras were also CD1-intact. Four weeks after irradiation and peripheral CD4ϩ T cell deletion was normal in lymph nodes of reconstitution we obtained stable, balanced chimeras. ␤ -microglobulin-deficient mice, with the disappearance of around These chimeras were injected with anti-CD3 Ab, and the effect 2 half of all the CD4ϩ T cells at 4 days (compare with Fig. 1B). on the CD1-intact and the CD1-deficient T cells in each chimera However, ␤ -microglobulin-deficient mice could potentially be ab- was determined. Fig. 5A shows two chimeras. One has been in- 2 ϩ normal in ways that would confound the interpretation of the ex- jected with PBS as a control; this chimera contained 47% of CD1 periment, for example due to the lack of CD8ϩ T cells. To over- lymphocytes, most of which were T cells. The other chimeras were come this problem, we addressed the same issue using an injected with anti-CD3. Although the expression of the TCR was ϩ alternative mutant mouse line that lacks the same population of reduced, the overall frequency of CD1 lymphocytes was the ϩ ϩ ϩ NK T cells, but contains CD8 cells. Because most NK T cells use same, at 46%. Fig. 5B shows the CD4 expression on the CD1 / Ϫ Ϫ a highly specific TCR␣-chain in their Ag receptors, namely V␣14 and the CD1 / cells within two chimeras. The upper panels of J␣281, it is possible to test the involvement of these NK T cells in Fig. 5 illustrate a PBS-injected control chimera, in which the Ϫ Ϫ ϩ ϩ ϩ mice unable to make this specific TCR due to lack of the J region. CD1 / lymph node cells are 43% CD4 cells, while the CD1 / ϩ In J␣281-deficient mice, the deletion of peripheral lymph node lymph node cells are 53% CD4 cells. In anti-CD3-injected chi- ϩ ϩ CD4 T cells by anti-CD3 was intact (Fig. 6B). These two exper- meras, the frequency of CD4 cells was reduced by Ͼ50% in both iments, taken together, argue strongly against a role for most, if not the CD1-deficient and the CD1-intact lymphocytes. Thus, the Ϫ Ϫ ϩ ϩ all NK T cells in the CD1-dependent component of peripheral T lower panels of Fig. 5B show the CD1 / and the CD1 / lym- Ϫ Ϫ cell deletion. phocytes in an anti-CD3-injected chimera. The CD1 / cells are only 11% CD4ϩ cells, while the CD1ϩ/ϩ cells are similarly depleted and contain only 18% CD4ϩ cells. This shows that both Discussion CD1Ϫ/Ϫ and CD1ϩ/ϩ CD4ϩ cells are susceptible to deletion. Fig. The highly polymorphic, peptide-binding “classical” molecules of 5C shows the means and SDs of the percentage of CD4ϩ cells the MHC are clustered on chromosome 17 in the mouse, and on from the CD1Ϫ/Ϫ and the CD1ϩ/ϩ cell populations in four PBS- chromosome 6 in the human. In addition to this gene complex, The Journal of Immunology 3095

FIGURE 6. Deficiency of NK T cells does not result in a similar phe- ␤ notype to CD1-deficiency. A, Young (6-wk-old) mice lacking 2-microglobulin, and hence deficient in NK T cells, responded normally to anti- CD3 injection and showed peripheral lymph node CD4ϩ T cell deletion Downloaded from identical with wild-type. B, Mice deficient in TCR J␣281, and hence in NK T cells, also showed normal peripheral deletion of lymph node CD4ϩ cells in response to anti-CD3. All experimental data were at day 4; bar graphs show mean Ϯ SD of groups of three to four mice. http://www.jimmunol.org/

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 FIGURE 5. The effect of CD1 deficiency on peripheral T cell deletion the trapping of activated T cells (10, 11, 13). Furthermore, the liver is non-T cell autonomous. Mixed bone marrow chimeras containing 50% wild-type and 50% CD1-deficient marrow were stable with approximately is a site in which NK T cells are unusually abundant (36, 37), half of their lymphocytes expressing CD1. A, Lymph node cells from two raising the possibility that the NK T cells are involved in the liver such chimeras, with the gates used to define the CD1Ϫ and CD1ϩ subsets component of peripheral T cell deletion. The availability of mice by guest on September 28, 2021 of cells. The center panels (B) show representative data from two chimeras. deficient in CD1 allowed us to examine these issues. In the present Upper panels show the expression of CD4 in the CD1Ϫ and CD1ϩ lym- study, we have induced peripheral T cell activation leading to T phocytes in a PBS-injected control chimera, while the lower panels show cell deletion in normal vs CD1-deficient mice. the CD4 expression in the two cell populations from a chimera injected We tested peripheral T cell deletion using a single injection of ϩ with anti-CD3. Peripheral T cell deletion occurred in both CD1- and CD1 anti-CD3, which was chosen in preference to a peptide Ag because cells. C, The data from four control and four anti-CD3-injected chimeras. ϩ Ϫ ϩ it allowed us to induce the activation and deletion of both CD4 There was no difference between the CD1 and the CD1 cells in their ϩ and CD8 T cells in the same experimental animals. This tech- potential for deletion. We conclude that the CD1 that is important in peripheral T cell deletion is not expressed on the T cells. nique suffers from the disadvantage that the TCR is ligated at an unusual point (i.e., on the extracellular domain of the CD3⑀-chain), but offers the compensating advantage that general conclusions may be drawn without the risk of artifacts due to the vagaries of an both humans and mice express structurally related but less poly- individual transgenic TCR. After the injection of anti-CD3, lymph morphic “nonclassical” MHC like molecules encoded elsewhere. node cells and IHL were isolated at 2, 4, 6, and 8 days. As pre- In the human, these are termed CD1a, b, c, d, and e. In mice, there viously reported by others (4, 5, 38), this treatment caused periph- is only a tandem duplication of a homologue of CD1d (31). The eral T cell activation followed by deletion in wild-type mice. CD1 molecules, like their classical MHC counterparts, contain a We used this model to show that CD1d was required for the pocket on the apical surface. However, this cavity is deeper and its early phase of peripheral T cell deletion in mice. This role was inner recesses are more hydrophobic that is the case for classical independent of TCR-invariant, CD1-reactive NK T cells, because ␤ ␣ MHC molecules (32), and recent evidence suggests that this is neither 2-microglobulin-deficient mice nor J 281-deficient mice because the pocket of all CD1 molecules binds and presents gly- reproduced the abnormality found in CD1-deficient mice. The de- colipids instead of peptides (33). These complexes are recognized fect appears to control the homing of activated T cells to the liver. not by classical CD4ϩ and CD8ϩ T cells, but by a distinct set of Chimera experiments have shown that the expression of CD1d on TCR␣␤ϩ lymphocytes, many of which express a monomorphic the T cells is not important for its role in T cell deletion. To explain TCR␣-chain paired with a limited set of V␤-chains. In addition to these data, we propose that activated T cells interact with CD1d the TCR␣␤, these cells express surface markers characteristic of expressed on non-T cells, and that this interaction predisposes NK cells (16). them to undergo deletion from the periphery. At present, we do not The tissue distribution of CD1 molecules is more restricted than know whether the relevant CD1d is expressed on specialized, bone the distribution of classical MHC molecules, and differs between marrow-derived APCs such as macrophages, B cells, or dendritic species. In the mouse, CD1 expression has been reported on thy- cells (39, 40), or whether it is on tissue cells such as intestinal mocytes and peripheral T lymphocytes, B cells, dendritic cells, and epithelium or hepatocytes (41, 42). 3096 CD1 FUNCTION INDEPENDENT OF NK T CELLS

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