Cytotoxic T-Cell Precursors with Low-Level CD8 in the Diabetes-Prone Biobreeding

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Proc. Natl. Acad. Sci. USA Vol. 87, pp. 313-317, January 1990 Immunology Cytotoxic T-cell precursors with low-level CD8 in the diabetes-prone Biobreeding rat: Implications for generation of an autoimmune T-cell repertoire (autoimmunity/escape from negative selection) DONALD BELLGRAU AND ANNE-CATHERINE LAGARDE The Barbara Davis Center for Childhood Diabetes and the Department of Microbiology/Immunology, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Box B140, Denver, CO 80262 Communicated by David W. Talmage, October 2, 1989

ABSTRACT Lymphocytes from diabetes-prone Biobreed- Although the BB-DP peripheral lymphocyte pool contains ing rats consistently fail to generate T-cell-mediated cytotox- a detectable percentage of CD8' cells, analysis by two-color icity under conditions where cytotoxic T lymphocyte activity is flow cytometry suggest that these cells express little, if any, readily demonstrated in normal rats. The failure is associated CD5 (5), a pan T-cell marker (10). Because natural killer (NK) with generalized T-cell lymphopenia and marked reduction in cell activity is enhanced in the BB-DP (5) and rat NK cells are the frequency ofCD8+ cells. The few remaining CD8+ cells are thought to be CD8+CD5- (11), the CD8' subset in the BB-DP widely held to be natural killer cells rather than class I major would appear to be of the NK rather than the T-cell lineage. histocompatibility complex-restricted T lymphocytes. In this This interpretation provides a plausible explanation for the report we show that a detectable percentage of CD8+ lympho- paucity of CTL activity in an abundance of NK-mediated cytes express the T-cell receptor for antigen, thus identifying lysis. However, in this report we provide evidence that the them as part of the T-cell lineage. The failure of these CD8+ BB-DP peripheral lymphocyte pool contains T cells that T-cell-receptor-positive T cells to lyse target cells that are express both CD8 and the T-cell receptor (TCR) for antigen. susceptible to T-cell mediated cytotoxicity is associated with The BB-DP peripheral lymphocyte pool can also generate markedly reduced expression of cell-surface CD8. Targets CTL-mediated lysis, but only for cells expressing high levels expressing higher than normal levels of class I major histo- of target antigen. The lysis is associated with TCR' cells compatibility complex target antigen could be lysed, suggesting expressing markedly reduced levels of CD8. How reduced that reduction in CD8 has decreased T-cell avidity for target CD8 expression might be linked to an autoimmune-prone antigen. We discuss the derivation of T cells that express low T-cell repertoire is discussed. levels of CD8 and the role they could play in generating autoimmune diabetes. MATERIALS AND METHODS Animals. DA (RT-1a haplotype) and BB-DP (RT-1u) rats The Biobreeding rat can spontaneously develop insulin- were bred and maintained at the Barbara Davis Center. dependent diabetes (1); rats from the most diabetes-prone subline (BB-DP) also express several abnormalities in their Specific pathogen-free DA breeders were originally pur- peripheral thymus-derived lymphocyte pool. These include a chased from the Trudeau Institute (Saranac Lake, NY). T-cell lymphopenia (2, 3), resulting in dramatic reduction of Specific pathogen-free BB-DP were derived in Basel, Swit- the CD4+ T-cell subset and apparent absence of the CD8+ zerland, by D.B. (6) from a breeding group supplied by A. subset, as well as functional anomalies in the proliferative (3, Naji and C. Barker (University of Pennsylvania, Philadel- 4) and cytotoxic T lymphocyte (CTL) responses (5, 6) to phia) (3). Lewis rats (RT-11) were purchased from The alloantigen in vitro. The proliferative responses of BB-DP T Charles River Breeding Laboratories. cells to alloantigen in vitro are consistently less than those Cell Staining. For indirect staining, 1-2 million thoracic from diabetes-resistant strains (3, 4, 6), whereas CTL re- duct lymphocytes (TDL) were incubated on ice for 30 min sponses of BB-DP are, for the most part, undetectable (5, 6). with supernatants from the mouse anti-rat B-cell hybridomas The association between T-cell-subset depletion and the OX-19 (anti-CD5), W3/25 (anti-CD4), and OX-8 (anti-CD8). peripheral T-cell hyporesponsiveness suggests that the lym- These hybridoma cell lines were supplied by Alan Williams phopenia causes peripheral T-cell functional anomalies. (Oxford, U.K.). After incubation, the cells were washed There are several experimental observations that cast doubt three times and incubated with fluorescein isothiocyanate on this hypothesis. (i) Enriching BB-DP peripheral lympho- (FITC)-conjugated sheep anti-mouse IgG F(ab')2 antibody cytes for T cells did not increase the proliferative responses (Cappel Laboratories). After 30 min on ice the cells were on a T-cell for T-cell basis (6), implying that the subnormal washed three times and fixed in 1% paraformaldehyde, and proliferative response cannot be explained simply as a func- the percentage of positive cells was determined using a tion of fewer T cells. (ii) The lymphopenia is associated with Coulter EPICS C flow cytometer. an absence of RT6+ lymphocytes (7, 8). The RT6 antigen, of For direct staining, ascites were purified by precipitation unknown function, is normally expressed on '50% of pe- with ammonium sulfate. For biotin conjugation, the antibody ripheral T cells. However, RT6- T cells isolated from normal was desalted in 0.1 M NaCO3 and adjusted to a concentration animals have not been reported to express the proliferative of 1 mg/ml. One hundred and twenty microliters of N- abnormalities seen in the RT6- BB-DP (7). (iii) BB-DPs can hydroxysuccinimido-d-biotin/dimethyl sulfoxide (1 mg/ml) remain lymphopenic while expressing normal T-cell prolif- was added per mg of antibody. After 4 hr at room tempera- erative responses after experimental manipulation (6, 9). Abbreviations: BB-DP, diabetes-prone Biobreeding rat; CTL, cytotoxic T lymphocyte; FITC, fluorescein isocyanate; MHC, major The publication costs of this article were defrayed in part by page charge histocompatibility complex; NK, natural killer; TCR, T-cell recep- payment. This article must therefore be hereby marked "advertisement" tor; TDL, thoracic duct lymphocytes; CD81, low-level CD8+; mAb, in accordance with 18 U.S.C. §1734 solely to indicate this fact. monoclonal antibody. 313 Downloaded by guest on September 23, 2021 314 Immunology: Bellgrau and Lagarde Proc. Natl. Acad. Sci. USA 87 (1990) 80 - described (6). Briefly, 10 million DA or BB-DP day-1 TDL, collected after surgical implantation of a thoracic duct can- * DA nula (3), were cocultured with 25 million, 2000-rad-irradiated CD 60 - * BB-DP (1 rad = 0.01 Gy) Lewis strain lymph-node lymphocytes in 30 ci) ml of Iscove's modified Dulbecco's medium (IMDM) sup- c plemented with glutamine, penicillin/streptomycin, 50 ,uM .- 40 - 2-mercaptoenthanol, and 10% selected fetal bovine serum a) u (GIBCO). The IMDM was also supplemented with 5% (vol/ w- vol) rat interleukin 2 generated from rat Con A-activated 20 - lymphocyte supernatants, as we have described (6). After 6-day culture at 370C in 5% CO2 the cells in the cultures were washed twice, counted, and tested for lytic potential against O 4- chromium-labeled targets. In some instances cells were re- 4 8 5 stimulated for an additional 6 days under the same condi- CD tions, with the exception that cells were cultured at a ratio of FIG. 1. Comparison ofcell-surface phenotypes. TDL from DA or 1 million responders to 10 million lymph node stimulators per BB-DP donors were tested with an indirect staining procedure; CD4 10 ml of culture medium. expression was identified with mAb W3/25, CD8 was identified with mAb OX-8, and CD5 was identified with mAb OX-19. RESULTS ture, the antibody was desalted on a G-25 column. For FITC Association Between BB-DP Lymphopenia and Apparent conjugation, the antibody was desalted at pH 9.6, its con- Failure to Generate CTLs. Phenotypes were analyzed to centration was adjusted to 1 mg/ml, and 1 mg of FITC on compare peripheral T cells from diabetes-resistant strain DA Celite was added for every mg. After 30-min incubation at rats with those from BB-DP. In the thoracic duct lymph of room temperature, the Celite was removed by centrifugation, normal DA rats =70% of the TDLs express the CD5 pan and the free FITC was removed by passage on a G-25 column. T-cell antigen; defined by the monoclonal antibody (mAb) Cells (2-3 x 106) were stained with 100 ul of biotin- OX-19 (Fig. 1). The CD5' T cells are divided into two conjugated antibody diluted 1:5 for 15 min at 4°C. After two subsets, class II major histocompatibility complex (MHC)- washes, 100 ,u of the FITC-conjugated antibody diluted 1:5 reactive T cells bearing CD4 and class I MHC-reactive T cells and phycoerythrin-avidin diluted 1:25 were added for 15 min bearing CD8 (10). In DA and other normal rat strains the ratio at 4°C. After two additional washes, the cells were fixed in of CD4+/CD8' is =3:1. Note also that the sum of CD4' and 0.02 M azide solution. Two-color analysis was performed by CD8+ cells correlates with the percentage ofcells expressing using the two-color-analysis EPICS C program. CD5, implying what has been formally established-that both Generation and Testing of Cytotoxic Lymphocytes. Cyto- subsets express CD5 (10). To the contrary, the BB-DP profile toxic lymphocytes were generated and tested as we have is much different. The frequency of CD5+ cells is markedly a

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o1 10 - 10 FIG. 2. Lysis of Con A blast and YAC cell targets with day-6 h Lk_tikU and -12 effectors. DA and BB-DP 1 0 0 5 0 2 5 1 2 TDLs were stimulated with Lewis 1002strain1 0 0 5 0 2 5 1 2 E/TE/TE/T populationslymphas described.node-stimulating(a) Cy- totoxic lymphocyte activity was b tested against Lewis Con A blast 60 targets (Left) or YAC targets 60 (Right) on day 6 after culture. (b) Cultures stimulated for 6 days 50 DA_A 505 DA were restimulated with Lewis an- a) B W _-*1-*BB-DP 1 tigen for an additional 6 days and co R sp CD 40 ---40 - | 1 Rat nude spleen retested on Lewis Con A and YAC CD targets as in a. Rat nude spleen, a = 30 30 - suspension of splenocytes from a ._ nude rat, was used as positive n 20 20 - control for YAC lysis. Cytotoxic- ity was specific. Stimulation with Lewis antigen generates cytotox- **ro 2- 1|1 | M M 1 0 ___10 ? icity on Con A blasts only expressing the Lewis RT-11 haplotype 0 (data not shown). SEs ofthe mean 1 0 0 5 0 2 5 1 2 1 0 0 5 0 2 5 1 2 are not >10%. E/T, effector/ EIT E/T target. Downloaded by guest on September 23, 2021 Immunology: Bellgrau and Lagarde Proc. Nati. Acad. Sci. USA 87 (1990) 315

reduced. Fig. 1 shows that the percentage of cells that are 100 _ CD5' drops from >70% to slightly <20%. In addition, the percentage of CD4' cells approximates that seen for the 80 - CD5' subset. a) CD4+TCR+ Although not formally established in this experiment, these CD8+TCR+ 0n data are consistent with the hypothesis that the CD8' subset 60 . is CD5- and therefore is not of the T-cell lineage. Functional data appear to support this view. When DA strain lympho- 0 cytes were stimulated in vitro for 6 days with MHC- 0LQf 40 . incompatible Lewis strain stimulating cells, CTLs were generated with the potential to lyse 51Cr-labeled Lewis strain 20. Con A-activated blast cell targets. BB-DP lymphocytes generated little or no lytic activity (Fig. 2a Left). Although Con A blasts were not lysed by BB-DP effector cells, the NK- 0 sensitive target YAC-1 cells showed significant chromium DA BB-DP release. The DA cultures did not generate NK cells in appreciable numbers (Fig. 2a Right). As these cultures were Population supplemented with an exogenous source of interleukin 2 (see FIG. 3. Some BB-DP lymphocytes express both CD8 and TCR. Materials and Methods) the failure to generate CTLs was DA and BB-DP TDLs were tested by using direct staining for apparently not from a deficiency in interleukin 2 production. percentage of CD4' or CD8' lymphocytes expressing TCR. Conceivably, the absence of CTLs might simply reflect lowered frequency of CD8' T cells. Therefore, day-6 BB-DP cultures were restimulated with antigen and interleukin 2 for 9L lines were derived from strains that are MHC compatible an additional 6 days. Although this procedure appeared to with the Lewis RT-11 haplotype. With a direct labeled mAb greatly reduce lysis ofthe NK-sensitive YAC-1 target (Fig. 2b specific for rat class I MHC, fluorescence-activated cell Right), it did not significantly enhance lysis of Lewis Con A sorter (FACS) analysis revealed that F-4 cells expressed blast cells (Fig. 2b Left). twice the levels of class I MHC antigen as 9L cells or Lewis Therefore, while DA strain TDLs generated significant Con A blasts, which were essentially comparable (Fig. 5). T-cell-mediated lysis and poor NK activity after culture with Neither tumor expressed class II MHC antigen. BB-DP TDL, alloantigen, BB-DP TDLs generated excellent early NK- stimulated for 6 days with Lewis antigen, lysed F-4 cells but mediated cytolysis but no significant lysis of CTL-sensitive not 9L cells, Lewis Con A targets, or any target that did not targets. BB-DP TCR Expression. The data above support the cell- 1.2 - surface phenotypic analyses suggesting that the CD8' subset in the BB-DP NK To @1 1.0- is of the rather than the T-cell lineage. c further test this hypothesis we examined the CD8' subset for 0.8- expression of the a/p TCR heterodimer, which is not present on NK cells. The R73 hybridoma produces a mAb that binds _ 0.6 - a determinant on the rat TCR het- nonpolymorphic a/8 * erodimer (12). The R73 epitope is on virtually all CD4+ T cells 0.4 - and =95% ofCD8+ cells. CD8+ cells that do not express TCR 0 are presumed to be NK cells (12). We tested whether the R73 determinant was present on CD4+ or CD8+ BB-DP lympho- 0 cytes. As expected, essentially all CD4+ cells ofboth DA and CD4 CDS BB-DP were TCR+. Somewhat surprisingly, of the 5% of BB-DP TDLs expressing CD8, -1 in 3 CD8+ lymphocytes were TCR+ (Fig. 3), suggesting that a significant portion of these cells were of the T-cell lineage. CD8 CD8 Lowered Expression of CD8 on BB-DP Lymphocytes. We examined whether the failure of CD8+TCR+ T cells to generate CTLs might be from decreased expression of TCR. Although no significant differences between DA and BB-DP lymphocytes were seen for CD4 or TCR, the level of CD8 on BB-DP CD8+TCR+ T cells was reduced relative to that seen TCR TCR on DA (Fig. 4). Therefore, reduction in CTL activity in the BB-DP lymphocytes correlated most closely with reduced FIG. 4. Reduced expression of CD8 on BB-DP lymphocytes. (Upper) Direct staining analyses were performed comparing CD4 expression of CD8 on TCR+ T cells. (mAb W3/25), CD8 (mAb OX-8), and TCR (mAb R73) antigen levels Expression of BB-DP CTL Activity on Targets Expressing on DA and BB-DP TDL. As the EPICS logarithm of fluorescence High Levels of Class I MHC Antigen. We tested whether CTL intensity is a triple logarithmic scale, mean channel values were activity could be expressed in BB-DP lymphocytes when linearized for comparison; the mean channel value was divided by target antigen levels were raised-the rationale being that the 341.3 (1024 channels divided by three logarithmic scales). For low-level expression of CD8 might prevent CTLs from bind- comparisons of mean fluorescence intensity, the loglo value of ing with sufficient avidity to lyse Con A blast targets. BB-DP BB-DP was divided by the log1o value obtained for DA. (Lower) TDL were stimulated with Lewis antigen as before. In Two-color histograms of a representative experiment comparing DA addition to testing the lytic potential on Lewis Con A blasts, (Left) and BB-DP (Right) TDL stained with phycoerythrin-labeled OX-8 (anti-CD8) and FITC-labeled R73 (anti-TCR). x axis, logarithm we also tested two additional targets, F-4 and 9L tumor lines; of green fluorescence; y axis, logarithm of red fluorescence. Quad- F-4 is an adenovirus-transformed rat embryo fibroblast line rant 1 (upper left), CD8+TCR-; quadrant 2 (upper right), and was supplied by P. Gallimore (Birmingham, U.K.) (13), CD8+TCR+; quadrant 3 (lower left), CD8-TCR-; and quadrant 4 and 9L is a methylcholanthrene-induced gliosarcoma that (lower right), CD8-TCR'. Note the shift (arrow) in BB-DP logarithm was provided by S. J. Geyer (Seattle, WA.) (14). Both F-4 and of red fluorescence in quadrant 3, indicating CD8 reduction. Downloaded by guest on September 23, 2021 316 Immunology: Bellgrau and Lagarde Proc. Nati. Acad. Sci. USA 87 (/990)

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0 a) a 30 - '0 2- a) 0 0 C a) 20 - -0- F-4 ._ 4- YAC aCU S1 1- -W Lewis ConA 0 10 - =====Ig o 4- -_ 0 9L LconA F-4 0 20 40 60 80 100 120 Target E/T

FIG. 5. Class I MHC levels on various targets. Fluorescent FIG. 7. Anti-Lewis day-12 toxicity. Cultures generated in Fig. 6 16.40.2, a mouse mAb that binds to a nonpolymorphic class I MHC were restimulated with Lewis antigen for an additional 6 days and determinant on all rat strains so far tested (D.B., unpublished tested on F-4, YAC, and Lewis Con A chromiutn-labeled targets. observation), was used to compare class I MHC antigen density on Cytotoxicity ofthe DA effectors is not shown; however, cytotoxicity Lewis Con A blasts, arbitrarily defined as 1.0, with 9L and F-4 was only seen on Lewis Con A and F-4 targets (as described in tumors. Linear conversions from the mean channel values were done Results). E/T, effector/target. as for Fig. 3. As nonspecific fluorescence, defined with a direct labeled antibody having no specificity for these tumors, differed cultured with OX-8 mAbs that bind the rat CD8 molecule among the three targets, these linear background values were sub- (data not shown). tracted from specific values before comparing populations. Only CD8 Levels on Thymocytes. One obvious explanation for Lewis Con A blasts (LconA) were positive for OX-6, a mAb that the reduction in CD8 levels is that BB-DP T-cell precursors binds to a nonpolymorphic class II rat MHC antigen. carry a genetic defect in their capacity to express cell-surface express RT-11 antigens. DA effectors lysed all RT-1'-ex- CD8. To test this hypothesis we examined BB-DP thymo- pressing targets (Fig. 6). cytes for relative levels of CD8 and found that they did not However, as shown in Fig. 2, these culture conditions also differ significantly from levels seen on normal, diabetes- resistent no generate NK cells in the BB-DP cultures. As F-4 line is an strains (Table 1). We also found obvious differ- ence in the relative frequency of NK-sensitive target, whereas 9L line and Con A blasts are CD4+/C8+ double-positive not (unpublished work), these results did not exclude the thymocytes or the absolute numbers of thymocytes (data not shown). Similar observation had been made by others possibility that the lysis of F-4 cells was NK mediated. To test (4). this further, BB-DP cultures were restimulated for an additional 6 days'with Lewis antigen and interleukin 2. These DISCUSSION day-12 culture conditions had been shown previously to Experiments were designed to determine why T cells from eliminate NK-mediated lysis (Fig. 2b). In day-12 cultures, lymphopenic BB-DPs failed to generate T-cell mediated lysis of F-4 cells remained, whereas YAC cell lysis was no cytotoxic activity after stimulation with alloantigen in vitro. longer detected (Fig. 7). Therefore, the lysis of F-4 cells by While FACS analysis demonstrated a correlation between BB-DP lymphocytes was apparently T-cell mediated. the poor CTL activity and a reduction in the absolute number Antibodies to CD8 can block target lysis by CD8+ CTLs of CD8+ cells, two-color analyses identified at least 30% of (15, 16). Weakly lytic CD8- T-cell clones can become more the CD8+ cells as expressing TCR, suggesting that a signif- lytic when transfected with the CD8 gene or, alternatively, icant number of CD8+ lymphocytes were T cells. Further presented with targets expressing increased antigen levels FACS analysis provided evidence that CD8 levels were (16). Apparently the requirement for CD8 becomes less reduced on the BB-DP CD8+ lymphocytes relative to the important if the target antigen level is optimized, suggesting levels on T cells from diabetes-resistant strains. We tested that CD8 contributes to the avidity of the TCR binding to whether BB-DP lymphocytes stimulated with alloantigen in target antigen. Consistent with this hypothesis we observed vitro could lyse targets expressing increased levels of class I no inhibition of F-4 cell lysis when BB-DP CTLs were MHC antigen. As CD8 binding to class I MHC is thought to 40 U F-4 *F-4 40 9L a) 30-9L V)m L Lewis ConA 3 Lewis ConA 5 YAC El YAC 30 - L.) 20

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FIG. 6. Anti-Lewis day-6 cytotoxicity. DA (Left) and BB-DP (Right) anti-Lewis cytotoxic effector cells were generated in day-6 cultures as described for Fig. 2 and tested on F-4, 9L, Lewis Con A and YAC chromium-labeled targets. Downloaded by guest on September 23, 2021 Immunology: Bellgrau and Lagarde Proc. Natl. Acad. Sci. USA 87 (1990) 317 Table 1. CD8 expression on thymocytes signals of negative selection that normally occur at the Population % CD8+ Mean channel % of normal CD4+CD8' stage. Thymocytes as a population may also normally express varied levels of CD8, and during normal DA TDL 16.1 161 thymocyte maturation those expressing higher levels of CD8 BB TDL 4.7 75 56.1 are preferentially selected. In this latter scenario the selection DA thymocyte 89.0 71 process could favor thymocytes expressing lowered levels of BB thymocyte 89.9 60 93.2 CD8 in BB-DPs. The reduction in CD8 might lead to a DA thymocyte 88.4 60 lowering of the TCR avidity for self-antigen and an escape BB thymocyte 91.8 53 95.3 from negative selection/self-reactivity. Thymocytes from 6-week-old DA and BB-DP donor rats were Depletion of CD8' cells in BB-DPs prevents disease (25). compared with DA and BB-DP TDL for relative density of CD8 Although the preventative effect may be entirely due to the (OX-8) antigen. Mean channel refers to the actual EPICS logarithm depletion of CD8' NK cells, CD8' T cells could also play a of green-fluorescence intensity from which linear values were cal- role in diabetic disease. Whether the depletion of CD8' T culated as described for Fig. 4. cells is ofany benefit in protecting BB-DPs from diabetes has increase the functional affinity of the TCR for antigen by yet to be tested. T cells with similar characteristics as described in BB-DPs might also be present in the diabetes- binding the a3 domain of the class I MHC molecule (17), our prone NOD mouse, where adoptive transfer of the disease rationale was that increasing target antigen levels might requires CD8' T lymphocytes (26). obviate a requirement for CD8 on BB-DP T cells. Consistent with this hypothesis, we observed lysis that was not NK We are grateful to Drs. M. McDuffie, R. Gill, and D. Gold for mediated when a target cell line was selected for increased comments and criticisms and to Dr. T. Hunig for his generosity with levels of class I MHC antigen. Therefore, we conclude that the R73 hybridoma. This work was supported by Juvenile Diabetes our previously reported failure to generate CTLs in BB-DP Foundation Grant 18891 and the Children's Diabetes Foundation at was due to more than a simple reduction in numbers of class Denver. I-reactive CD8+ T cells. Another factor is the contributing 1. Nakhooda, A. F., Like, A. A., Chapel, C. I., Murray, F. T. & reduced expression of CD8 on TCR+ T cells. Marliss, E. B. (1977) Diabetes 26, 100-112. BB-DP lymphoid cells may be inherently defective in CD8 2. Jackson, R., Rassi, N., Crump, T., Haynes, B. & Eisenbarth, G. expression, explaining the low CD8 levels observed in the (1981) Diabetes 30, 887-889. periphery. Alternatively, the CD810 (low-level CD8+) subset 3. Bellgrau, D., Naji, A., Silvers, W. K., Markmann, J. F. & Barker, C. F. (1982) Diabetologia 23, 359-364. could be preferentially selected from a normal T-cell precur- 4. Elder, M. E. & Maclaren, N. K. (1983)J. Immunol. 130,1723-1731. sor pool. We favor a selection model for the following 5. Woda, B. A., Like, A. A., Padden, C. & McFadden, M. L. (1986) reasons: (i) The expression of CD8 appears a very critical J. Immunol. 136, 856-859. event in thymocyte maturation. The CD4+CD8+ double- 6. Georgiou, H. M., Lagarde, A. C. & Bellgrau, D. (1988) J. Exp. Med. 167, 132-148. positive subset is widely held to be the precursor of single- 7. Greiner, D. L., Mordes, J. P., Handler, E. S., Angelillo, M., Na- positive, mature T cells (18-20). The double-positive subset kamura, N. & Rossini, A. A. (1987) J. Exp. Med. 166, 461-475. is thought to be derived from a CD4-CD8+ intermediate (21, 8. Angelillo, M., Greiner, D. L., Mordes, J. D., Handler, E. S., 22). It might be expected that an inherent defect in CD8 Nakamura, N., McKeever, U. & Rossini, A. (1988) J. Immunol. would lead to in 141, 4146-4151. expression dramatic alteration thymocyte 9. Scott, J., Engelhard, V. H., Curnow, R. T. & Benjamin, D. C. cell-surface phenotypes. However, no obvious abnormalities (1986) Diabetes 35, 1034-1040. appear to be present in BB-DP thymus morphology or 10. Dallman, M. J., Thomas, M. L. & Green, J. R. (1984) Eur. J. thymocyte cell-surface phenotypes thus far reported (4, 5). Immunol. 14, 260-267. (ii) Evidence from transgenic mice suggest that CD810 T cells 11. Reynolds, C. W., Sharrow, S. O., Ortaldo, J. R. & Heberman, a R. B. (1981) J. Immunol. 127, 2204-2208. can develop from pool of T-cell precursors expressing no 12. Hunig, T., Wallny, H. J., Hartley, J. K., Lawetzky, A. & obvious CD8 deficiencies (23). The transgenic animals ex- Tiefenthaler, G. (1989) J. Exp. Med. 169, 73-86. pressed functionally rearranged TCR genes isolated from a 13. Gallimore, P. J., Sharp, P. A. & Sambrook, J. (1974) J. Mol. Biol. class I-restricted T-cell clone specific for the male antigen 89, 49-57. HY in the context of class I MHC H-2Db. One in three CD8+ 14. Geyer, S. J. & Landay, A. (1983) Lab. Invest. 49, 436-444. T cells in 15. Shimonkevitz, R., Luescher, B., Cerottini, J.-C. & MacDonald, female mice expressed the transgene TCR product H. R. (1985) J. Immunol. 135, 892-899. and generated CTLs in response to HY in association with 16. Dembic, Z., Haas, W., Weiss, S., McCubrey, J., Kiefer, H., von H-2Db, demonstrating that they were preferentially or posi- Boehmer, H. & Steinmetz, M. (1986) Nature (London) 320, 232-238. tively selected. Mice that did not express H-2Db in the 17. Connolly, J. M., Potter, T. A., Wormstall, E. & Hansen, T. H. thymus did not select for the transgene TCR (24). As the male (1988) J. Exp. Med. 168, 325-336. transgenic expresses the HY antigen, it was of interest to 18. Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M. & von determine Boehmer, H. (1988) Nature (London) 333, 742-746. how these animals could survive ifthey contained, 19. MacDonald, H. R., Schneider, R., Lees, R. K., Howe, R. C., as did the female, cells with lytic potential for the HY and Acha-Orbea, H., Festenstein, H., Zinckernagel, R. M. & Hengart- MHC antigens expressed on the male cells. In the male mice ner, H. (1988) Nature (London) 332, 40-45. the vast majority of thymocytes expressing TCR for HY and 20. Fowlkes, B. J., Schwartz, R. H. & Pardoll, D. M. (1988) Nature H-2Db were eliminated-i.e., negatively selected in the thy- (London) 334, 620-623. mus. However, T cells expressing the transgenic TCR existed 21. Paterson, D. J. & Williams, A. F. (1987) J. Exp. Med. 166, 1603- 1608. in the periphery of male mice. The failure of these T cells to 22. MacDonald, H. R., Budd, R. C. & Howe, R. C. (1988) Eur. J. mediate autoreactivity was attributed to the lowered expres- Immunol. 18, 519-523. sion of CD8+. Apparently the reduction in CD8 prevented 23. Teh, H. S., Kishi, H., Scott, B. & von Boehmer, H. (1989) J. Exp. these T cells from effecting autoreactivity. Med. 169, 795-806. Virtually all CD8'° peripheral T cells in male transgenic 24. Kisielow, P., Teh, H. S., Bluthmann, H. & von Boehmer, H. (1988) mice expressed the transgenic TCR and yet they Nature (London) 335, 730-733. escaped 25. Like, A. A., Biron, C. A., Weringer, E. J., Byman, K., Sroczynski, clonal deletion/negative selection in the thymus. Conceiv- E. & Guberski, D. L. (1986) J. Exp. Med. 164, 1145-1159. ably, these thymocytes expressed TCR earlier than normal in 26. Miller, B. J., Appel, M. C., O'Neill, J. J. & Wicker, L. S. (1988) J. thymocyte maturation and were no longer sensitive to the Immunol. 140, 52-58. Downloaded by guest on September 23, 2021