Proc. Natl. Acad. Sci. USAVol. 87, pp. 313-317, January 1990Immunology

Cytotoxic T-cell precursors with low-level CD8 in thediabetes-prone Biobreeding rat: Implications forgeneration of an autoimmune T-cell repertoire

(autoimmunity/escape from negative selection)

DONALD BELLGRAU AND ANNE-CATHERINE LAGARDEThe Barbara Davis Center for Childhood Diabetes and the Department of Microbiology/Immunology, University of Colorado Health Sciences Center, 4200East 9th Avenue, Box B140, Denver, CO 80262

Communicated by David W. Talmage, October 2, 1989

ABSTRACT Lymphocytes from diabetes-prone Biobreed-ing rats consistently fail to generate T-cell-mediated cytotox-icity under conditions where cytotoxic T lymphocyte activity isreadily demonstrated in normal rats. The failure is associatedwith generalized T-cell lymphopenia and marked reduction inthe frequency ofCD8+ cells. The few remaining CD8+ cells arewidely held to be natural killer cells rather than class I majorhistocompatibility complex-restricted T lymphocytes. In thisreport we show that a detectable percentage of CD8+ lympho-cytes express the T-cell receptor for antigen, thus identifyingthem as part of the T-cell lineage. The failure of these CD8+T-cell-receptor-positive T cells to lyse target cells that aresusceptible to T-cell mediated cytotoxicity is associated withmarkedly reduced expression of cell-surface CD8. Targetsexpressing higher than normal levels of class I major histo-compatibility complex target antigen could be lysed, suggestingthat reduction in CD8 has decreased T-cell avidity for targetantigen. We discuss the derivation of T cells that express lowlevels of CD8 and the role they could play in generatingautoimmune diabetes.

The Biobreeding rat can spontaneously develop insulin-dependent diabetes (1); rats from the most diabetes-pronesubline (BB-DP) also express several abnormalities in theirperipheral thymus-derived lymphocyte pool. These include aT-cell lymphopenia (2, 3), resulting in dramatic reduction ofthe CD4+ T-cell subset and apparent absence of the CD8+subset, as well as functional anomalies in the proliferative (3,4) and cytotoxic T lymphocyte (CTL) responses (5, 6) toalloantigen in vitro. The proliferative responses of BB-DP Tcells to alloantigen in vitro are consistently less than thosefrom diabetes-resistant strains (3, 4, 6), whereas CTL re-sponses of BB-DP are, for the most part, undetectable (5, 6).The association between T-cell-subset depletion and the

peripheral T-cell hyporesponsiveness suggests that the lym-phopenia causes peripheral T-cell functional anomalies.There are several experimental observations that cast doubton this hypothesis. (i) Enriching BB-DP peripheral lympho-cytes for T cells did not increase the proliferative responseson a T-cell for T-cell basis (6), implying that the subnormalproliferative response cannot be explained simply as a func-tion of fewer T cells. (ii) The lymphopenia is associated withan absence of RT6+ lymphocytes (7, 8). The RT6 antigen, ofunknown function, is normally expressed on '50% of pe-ripheral T cells. However, RT6- T cells isolated from normalanimals have not been reported to express the proliferativeabnormalities seen in the RT6- BB-DP (7). (iii) BB-DPs canremain lymphopenic while expressing normal T-cell prolif-erative responses after experimental manipulation (6, 9).

Although the BB-DP peripheral lymphocyte pool containsa detectable percentage ofCD8' cells, analysis by two-colorflow cytometry suggest that these cells express little, if any,CD5 (5), a pan T-cell marker (10). Because natural killer (NK)cell activity is enhanced in the BB-DP (5) and rat NK cells arethought to be CD8+CD5- (11), the CD8' subset in the BB-DPwould appear to be of the NK rather than the T-cell lineage.This interpretation provides a plausible explanation for thepaucity of CTL activity in an abundance of NK-mediatedlysis. However, in this report we provide evidence that theBB-DP peripheral lymphocyte pool contains T cells thatexpress both CD8 and the T-cell receptor (TCR) for antigen.The BB-DP peripheral lymphocyte pool can also generateCTL-mediated lysis, but only for cells expressing high levelsof target antigen. The lysis is associated with TCR' cellsexpressing markedly reduced levels of CD8. How reducedCD8 expression might be linked to an autoimmune-proneT-cell repertoire is discussed.

MATERIALS AND METHODSAnimals. DA (RT-1a haplotype) and BB-DP (RT-1u) rats

were bred and maintained at the Barbara Davis Center.Specific pathogen-free DA breeders were originally pur-chased from the Trudeau Institute (Saranac Lake, NY).Specific pathogen-free BB-DP were derived in Basel, Swit-zerland, by D.B. (6) from a breeding group supplied by A.Naji and C. Barker (University of Pennsylvania, Philadel-phia) (3). Lewis rats (RT-11) were purchased from TheCharles River Breeding Laboratories.

Cell Staining. For indirect staining, 1-2 million thoracicduct lymphocytes (TDL) were incubated on ice for 30 minwith supernatants from the mouse anti-rat B-cell hybridomasOX-19 (anti-CD5), W3/25 (anti-CD4), and OX-8 (anti-CD8).These hybridoma cell lines were supplied by Alan Williams(Oxford, U.K.). After incubation, the cells were washedthree times and incubated with fluorescein isothiocyanate(FITC)-conjugated sheep anti-mouse IgG F(ab')2 antibody(Cappel Laboratories). After 30 min on ice the cells werewashed three times and fixed in 1% paraformaldehyde, andthe percentage of positive cells was determined using aCoulter EPICS C flow cytometer.For direct staining, ascites were purified by precipitation

with ammonium sulfate. For biotin conjugation, the antibodywas desalted in 0.1 M NaCO3 and adjusted to a concentrationof 1 mg/ml. One hundred and twenty microliters of N-hydroxysuccinimido-d-biotin/dimethyl sulfoxide (1 mg/ml)was added per mg of antibody. After 4 hr at room tempera-

Abbreviations: BB-DP, diabetes-prone Biobreeding rat; CTL, cyto-toxic T lymphocyte; FITC, fluorescein isocyanate; MHC, majorhistocompatibility complex; NK, natural killer; TCR, T-cell recep-tor; TDL, thoracic duct lymphocytes; CD81, low-level CD8+; mAb,monoclonal antibody.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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CDci)

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FIG. 1. Comparison of cell-surface phenotypes. TDL from DA orBB-DP donors were tested with an indirect staining procedure; CD4expression was identified with mAb W3/25, CD8 was identified withmAb OX-8, and CD5 was identified with mAb OX-19.

ture, the antibody was desalted on a G-25 column. For FITCconjugation, the antibody was desalted at pH 9.6, its con-centration was adjusted to 1 mg/ml, and 1 mg of FITC onCelite was added for every mg. After 30-min incubation atroom temperature, the Celite was removed by centrifugation,and the free FITC was removed by passage on a G-25 column.Cells (2-3 x 106) were stained with 100 ul of biotin-conjugated antibody diluted 1:5 for 15 min at 4°C. After twowashes, 100 ,u of the FITC-conjugated antibody diluted 1:5and phycoerythrin-avidin diluted 1:25 were added for 15 minat 4°C. After two additional washes, the cells were fixed in0.02 M azide solution. Two-color analysis was performed byusing the two-color-analysis EPICS C program.

Generation and Testing of Cytotoxic Lymphocytes. Cyto-toxic lymphocytes were generated and tested as we have

described (6). Briefly, 10 million DA or BB-DP day-1 TDL,collected after surgical implantation of a thoracic duct can-nula (3), were cocultured with 25 million, 2000-rad-irradiated(1 rad = 0.01 Gy) Lewis strain lymph-node lymphocytes in 30ml of Iscove's modified Dulbecco's medium (IMDM) sup-plemented with glutamine, penicillin/streptomycin, 50 ,uM2-mercaptoenthanol, and 10% selected fetal bovine serum(GIBCO). The IMDM was also supplemented with 5% (vol/vol) rat interleukin 2 generated from rat Con A-activatedlymphocyte supernatants, as we have described (6). After6-day culture at 370C in 5% CO2 the cells in the cultures werewashed twice, counted, and tested for lytic potential againstchromium-labeled targets. In some instances cells were re-stimulated for an additional 6 days under the same condi-tions, with the exception that cells were cultured at a ratio of1 million responders to 10 million lymph node stimulators per10 ml of culture medium.

RESULTSAssociation Between BB-DP Lymphopenia and Apparent

Failure to Generate CTLs. Phenotypes were analyzed tocompare peripheral T cells from diabetes-resistant strain DArats with those from BB-DP. In the thoracic duct lymph ofnormal DA rats =70% of the TDLs express the CD5 panT-cell antigen; defined by the monoclonal antibody (mAb)OX-19 (Fig. 1). The CD5' T cells are divided into twosubsets, class II major histocompatibility complex (MHC)-reactive T cells bearing CD4 and class I MHC-reactive T cellsbearing CD8 (10). In DA and other normal rat strains the ratioof CD4+/CD8' is =3:1. Note also that the sum of CD4' andCD8+ cells correlates with the percentage of cells expressingCD5, implying what has been formally established-that bothsubsets express CD5 (10). To the contrary, the BB-DP profileis much different. The frequency of CD5+ cells is markedly

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10 FIG. 2. Lysis of Con A blasth Lk_tikU and YAC cell targets with day-6

and -12 effectors. DA and BB-DP1 0 0 5 0 2 5 1 2 1 0 0 5 0 2 5 1 2 TDLs were stimulated with Lewis

E/T1002strain lymph node-stimulatingE/TE/T populations as described. (a) Cy-totoxic lymphocyte activity wastested against Lewis Con A blast

60 targets (Left) or YAC targets(Right) on day 6 after culture. (b)

_A 50 DACultures stimulated for 6 days

-*DA 5B

DA were restimulated with Lewis an-*_-1 BB-DP

R

1 sp tigen for an additional 6 days and---40 - | 1 Rat nude spleen retested on Lewis Con A and YAC

targets as in a. Rat nude spleen, a30 - suspension of splenocytes from a

nude rat, was used as positive20 - control for YAC lysis. Cytotoxic-

ity was specific. Stimulation with

10 -**ro 2 1|1 | M M Lewis antigen generates cytotox-10___ ? icity on Con A blasts only express-

ing the Lewis RT-11 haplotype(data not shown). SEs of the 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.

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reduced. Fig. 1 shows that the percentage of cells that areCD5' drops from >70% to slightly <20%. In addition, thepercentage of CD4' cells approximates that seen for theCD5' subset.Although not formally established in this experiment, these

data are consistent with the hypothesis that the CD8' subsetis CD5- and therefore is not of the T-cell lineage. Functionaldata appear to support this view. When DA strain lympho-cytes were stimulated in vitro for 6 days with MHC-incompatible Lewis strain stimulating cells, CTLs were gen-erated with the potential to lyse 51Cr-labeled Lewis strainCon A-activated blast cell targets. BB-DP lymphocytes gen-erated little or no lytic activity (Fig. 2a Left). Although ConA blasts were not lysed by BB-DP effector cells, the NK-sensitive target YAC-1 cells showed significant chromiumrelease. The DA cultures did not generate NK cells inappreciable numbers (Fig. 2a Right). As these cultures weresupplemented with an exogenous source of interleukin 2 (seeMaterials and Methods) the failure to generate CTLs wasapparently not from a deficiency in interleukin 2 production.Conceivably, the absence of CTLs might simply reflectlowered frequency of CD8' T cells. Therefore, day-6 BB-DPcultures were restimulated with antigen and interleukin 2 foran additional 6 days. Although this procedure appeared togreatly reduce lysis ofthe NK-sensitive YAC-1 target (Fig. 2bRight), it did not significantly enhance lysis of Lewis Con Ablast cells (Fig. 2b Left).

Therefore, while DA strain TDLs generated significantT-cell-mediated lysis and poor NK activity after culture withalloantigen, BB-DP TDLs generated excellent early NK-mediated cytolysis but no significant lysis of CTL-sensitivetargets.BB-DP TCR Expression. The data above support the cell-

surface phenotypic analyses suggesting that the CD8' subsetin the BB-DP is of the NK rather than the T-cell lineage. Tofurther test this hypothesis we examined the CD8' subset forexpression of the a/p TCR heterodimer, which is not presenton NK cells. The R73 hybridoma produces a mAb that bindsa nonpolymorphic determinant on the rat a/8 TCR het-erodimer (12). The R73 epitope is on virtually all CD4+ T cellsand =95% ofCD8+ cells. CD8+ cells that do not express TCRare presumed to be NK cells (12). We tested whether the R73determinant was present on CD4+ or CD8+ BB-DP lympho-cytes. As expected, essentially all CD4+ cells of both DA andBB-DP were TCR+. Somewhat surprisingly, of the 5% ofBB-DP TDLs expressing CD8, -1 in 3 CD8+ lymphocyteswere TCR+ (Fig. 3), suggesting that a significant portion ofthese cells were of the T-cell lineage.Lowered Expression of CD8 on BB-DP Lymphocytes. We

examined whether the failure of CD8+TCR+ T cells togenerate CTLs might be from decreased expression of TCR.Although no significant differences between DA and BB-DPlymphocytes were seen for CD4 or TCR, the level of CD8 onBB-DP CD8+TCR+ T cells was reduced relative to that seenon DA (Fig. 4). Therefore, reduction in CTL activity in theBB-DP lymphocytes correlated most closely with reducedexpression of CD8 on TCR+ T cells.

Expression of BB-DP CTL Activity on Targets ExpressingHigh Levels of Class I MHC Antigen. We tested whether CTLactivity could be expressed in BB-DP lymphocytes whentarget antigen levels were raised-the rationale being that thelow-level expression of CD8 might prevent CTLs from bind-ing with sufficient avidity to lyse Con A blast targets. BB-DPTDL were stimulated with Lewis antigen as before. Inaddition to testing the lytic potential on Lewis Con A blasts,we also tested two additional targets, F-4 and 9L tumor lines;F-4 is an adenovirus-transformed rat embryo fibroblast lineand was supplied by P. Gallimore (Birmingham, U.K.) (13),and 9L is a methylcholanthrene-induced gliosarcoma thatwas provided by S. J. Geyer (Seattle, WA.) (14). Both F-4 and

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FIG. 3. Some BB-DP lymphocytes express both CD8 and TCR.DA and BB-DP TDLs were tested by using direct staining forpercentage of CD4' or CD8' lymphocytes expressing TCR.

9L lines were derived from strains that are MHC compatiblewith the Lewis RT-11 haplotype. With a direct labeled mAbspecific for rat class I MHC, fluorescence-activated cellsorter (FACS) analysis revealed that F-4 cells expressedtwice the levels of class I MHC antigen as 9L cells or LewisCon A blasts, which were essentially comparable (Fig. 5).Neither tumor expressed class II MHC antigen. BB-DP TDL,stimulated for 6 days with Lewis antigen, lysed F-4 cells butnot 9L cells, Lewis Con A targets, or any target that did not

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FIG. 4. Reduced expression of CD8 on BB-DP lymphocytes.(Upper) Direct staining analyses were performed comparing CD4(mAb W3/25), CD8 (mAb OX-8), and TCR (mAb R73) antigen levelson DA and BB-DP TDL. As the EPICS logarithm of fluorescenceintensity is a triple logarithmic scale, mean channel values werelinearized for comparison; the mean channel value was divided by341.3 (1024 channels divided by three logarithmic scales). Forcomparisons of mean fluorescence intensity, the loglo value ofBB-DP was divided by the log1o value obtained for DA. (Lower)Two-color histograms of a representative experiment comparing DA(Left) and BB-DP (Right) TDL stained with phycoerythrin-labeledOX-8 (anti-CD8) and FITC-labeled R73 (anti-TCR). x axis, logarithmof green fluorescence; y axis, logarithm of red fluorescence. Quad-rant 1 (upper left), CD8+TCR-; quadrant 2 (upper right),CD8+TCR+; quadrant 3 (lower left), CD8-TCR-; and quadrant 4(lower right), CD8-TCR'. Note the shift (arrow) in BB-DP logarithmof red fluorescence in quadrant 3, indicating CD8 reduction.

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40 -

a)

0a)

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30 -

20 -

10 -

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-0- F-44- YAC-W Lewis ConA

0 20 40 60E/T

FIG. 5. Class I MHC levels on various targets. Fluorescent16.40.2, a mouse mAb that binds to a nonpolymorphic class I MHCdeterminant on all rat strains so far tested (D.B., unpublishedobservation), was used to compare class I MHC antigen density onLewis Con A blasts, arbitrarily defined as 1.0, with 9L and F-4tumors. Linear conversions from the mean channel values were doneas for Fig. 3. As nonspecific fluorescence, defined with a directlabeled antibody having no specificity for these tumors, differedamong the three targets, these linear background values were sub-tracted from specific values before comparing populations. OnlyLewis Con A blasts (LconA) were positive for OX-6, a mAb thatbinds to a nonpolymorphic class II rat MHC antigen.

express RT-11 antigens. DA effectors lysed all RT-1'-ex-pressing targets (Fig. 6).However, as shown in Fig. 2, these culture conditions also

generate NK cells in the BB-DP cultures. As F-4 line is anNK-sensitive target, whereas 9L line and Con A blasts arenot (unpublished work), these results did not exclude thepossibility that the lysis of F-4 cells was NK mediated. To testthis further, BB-DP cultures were restimulated for an addi-tional 6 days'with Lewis antigen and interleukin 2. Theseday-12 culture conditions had been shown previously toeliminate NK-mediated lysis (Fig. 2b). In day-12 cultures,lysis of F-4 cells remained, whereas YAC cell lysis was nolonger detected (Fig. 7). Therefore, the lysis of F-4 cells byBB-DP lymphocytes was apparently T-cell mediated.

Antibodies to CD8 can block target lysis by CD8+ CTLs(15, 16). Weakly lytic CD8- T-cell clones can become morelytic when transfected with the CD8 gene or, alternatively,presented with targets expressing increased antigen levels(16). Apparently the requirement for CD8 becomes lessimportant if the target antigen level is optimized, suggestingthat CD8 contributes to the avidity of the TCR binding totarget antigen. Consistent with this hypothesis we observedno inhibition of F-4 cell lysis when BB-DP CTLs were

a)

V)m

L.)

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CO

40

FIG. 7. Anti-Lewis day-12 toxicity. Cultures generated in Fig. 6were restimulated with Lewis antigen for an additional 6 days andtested on F-4, YAC, and Lewis Con A chromiutn-labeled targets.Cytotoxicity of the DA effectors is not shown; however, cytotoxicitywas only seen on Lewis Con A and F-4 targets (as described inResults). E/T, effector/target.

cultured with OX-8 mAbs that bind the rat CD8 molecule(data not shown).CD8 Levels on Thymocytes. One obvious explanation for

the reduction in CD8 levels is that BB-DP T-cell precursorscarry a genetic defect in their capacity to express cell-surfaceCD8. To test this hypothesis we examined BB-DP thymo-cytes for relative levels of CD8 and found that they did notdiffer significantly from levels seen on normal, diabetes-resistent strains (Table 1). We also found no obvious differ-ence in the relative frequency of CD4+/C8+ double-positivethymocytes or the absolute numbers of thymocytes (data notshown). Similar observation had been made by others (4).

DISCUSSIONExperiments were designed to determine why T cells fromlymphopenic BB-DPs failed to generate T-cell mediatedcytotoxic activity after stimulation with alloantigen in vitro.While FACS analysis demonstrated a correlation betweenthe poor CTL activity and a reduction in the absolute numberof CD8+ cells, two-color analyses identified at least 30% ofthe CD8+ cells as expressing TCR, suggesting that a signif-icant number of CD8+ lymphocytes were T cells. FurtherFACS analysis provided evidence that CD8 levels werereduced on the BB-DP CD8+ lymphocytes relative to thelevels on T cells from diabetes-resistant strains. We testedwhether BB-DP lymphocytes stimulated with alloantigen invitro could lyse targets expressing increased levels of class IMHC antigen. As CD8 binding to class I MHC is thought to

40

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L Lewis ConA 3 Lewis ConA5 YAC El YAC

20

30 -

20 -

10 -

5 0 2 5ET

1 2 5 0 2 5ET

1 2

FIG. 6. Anti-Lewis day-6 cytotoxicity. DA (Left) and BB-DP (Right) anti-Lewis cytotoxic effector cells were generated in day-6 culturesas described for Fig. 2 and tested on F-4, 9L, Lewis Con A and YAC chromium-labeled targets.

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0

a

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o 4-9L LconA F-4

Target80 100 120

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Table 1. CD8 expression on thymocytes

Population % CD8+ Mean channel % of normalDA TDL 16.1 161BB TDL 4.7 75 56.1DA thymocyte 89.0 71BB thymocyte 89.9 60 93.2DA thymocyte 88.4 60BB thymocyte 91.8 53 95.3

Thymocytes from 6-week-old DA and BB-DP donor rats werecompared with DA and BB-DP TDL for relative density of CD8(OX-8) antigen. Mean channel refers to the actual EPICS logarithmof green-fluorescence intensity from which linear values were cal-culated as described for Fig. 4.

increase the functional affinity of the TCR for antigen bybinding the a3 domain of the class I MHC molecule (17), ourrationale was that increasing target antigen levels mightobviate a requirement for CD8 on BB-DP T cells. Consistentwith this hypothesis, we observed lysis that was not NKmediated when a target cell line was selected for increasedlevels of class I MHC antigen. Therefore, we conclude thatour previously reported failure to generate CTLs in BB-DPwas due to more than a simple reduction in numbers of classI-reactive CD8+ T cells. Another contributing factor is thereduced expression of CD8 on TCR+ T cells.BB-DP lymphoid cells may be inherently defective in CD8

expression, explaining the low CD8 levels observed in theperiphery. Alternatively, the CD810 (low-level CD8+) subsetcould be preferentially selected from a normal T-cell precur-sor pool. We favor a selection model for the followingreasons: (i) The expression of CD8 appears a very criticalevent in thymocyte maturation. The CD4+CD8+ double-positive subset is widely held to be the precursor of single-positive, mature T cells (18-20). The double-positive subsetis thought to be derived from a CD4-CD8+ intermediate (21,22). It might be expected that an inherent defect in CD8expression would lead to dramatic alteration in thymocytecell-surface phenotypes. However, no obvious abnormalitiesappear to be present in BB-DP thymus morphology orthymocyte cell-surface phenotypes thus far reported (4, 5).(ii) Evidence from transgenic mice suggest that CD810 T cellscan develop from a pool of T-cell precursors expressing noobvious CD8 deficiencies (23). The transgenic animals ex-pressed functionally rearranged TCR genes isolated from aclass I-restricted T-cell clone specific for the male antigenHY in the context of class I MHC H-2Db. One in three CD8+T cells in female mice expressed the transgene TCR productand generated CTLs in response to HY in association withH-2Db, demonstrating that they were preferentially or posi-tively selected. Mice that did not express H-2Db in thethymus did not select for the transgene TCR (24). As the maletransgenic expresses the HY antigen, it was of interest todetermine how these animals could survive if they contained,as did the female, cells with lytic potential for the HY andMHC antigens expressed on the male cells. In the male micethe vast majority of thymocytes expressing TCR for HY andH-2Db were eliminated-i.e., negatively selected in the thy-mus. However, T cells expressing the transgenic TCR existedin the periphery of male mice. The failure of these T cells tomediate autoreactivity was attributed to the lowered expres-sion of CD8+. Apparently the reduction in CD8 preventedthese T cells from effecting autoreactivity.

Virtually all CD8'° peripheral T cells in male transgenicmice expressed the transgenic TCR and yet they escapedclonal deletion/negative selection in the thymus. Conceiv-ably, these thymocytes expressed TCR earlier than normal inthymocyte maturation and were no longer sensitive to the

signals of negative selection that normally occur at theCD4+CD8' stage. Thymocytes as a population may alsonormally express varied levels of CD8, and during normalthymocyte maturation those expressing higher levels of CD8are preferentially selected. In this latter scenario the selectionprocess could favor thymocytes expressing lowered levels ofCD8 in BB-DPs. The reduction in CD8 might lead to alowering of the TCR avidity for self-antigen and an escapefrom negative selection/self-reactivity.

Depletion of CD8' cells in BB-DPs prevents disease (25).Although the preventative effect may be entirely due to thedepletion of CD8' NK cells, CD8' T cells could also play arole in diabetic disease. Whether the depletion of CD8' Tcells is of any benefit in protecting BB-DPs from diabetes hasyet to be tested. T cells with similar characteristics asdescribed in BB-DPs might also be present in the diabetes-prone NOD mouse, where adoptive transfer of the diseaserequires CD8' T lymphocytes (26).

We are grateful to Drs. M. McDuffie, R. Gill, and D. Gold forcomments and criticisms and to Dr. T. Hunig for his generosity withthe R73 hybridoma. This work was supported by Juvenile DiabetesFoundation Grant 18891 and the Children's Diabetes Foundation atDenver.

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