Tolerance in TCR/Cognate Antigen Double-Transgenic Mice Mediated by Incomplete Thymic Deletion and Peripheral Receptor Downregulation

Influenza nucleoprotein (NP)-specific T-cell receptor transgenic mice (F5) were crossed with transgenic mice expressing the cognate antigenic protein under the control of the H- 2Kb promoter. Double-transgenic mice show negative selection of thymocytes at the CD4+8+TCR10 to CD4+8+TCRhi transition stage. A few CD8 T cells, however, escape clonal deletion, and in the peripheral lymphoid organs of these mice, they exhibit low levels of the transgenic receptor and upregulated levels of the CD44 memory marker. Such cells do not proliferate upon exposure to antigen stimulation in vivo or ex vivo, however, they can develop low but detectable levels of antigen-specific cytotoxic function after stimulation in vitro in the presence of IL-2.


system.In th
case of T-cell tolerance, this is generally accom- plished by elimination of self-reactive cells either during development in the thymus (Kappler et al.,   1987; MacDonald et al., 1988) or after encountering self-antigen in the periphery (Jones et al., 1990; Webb   et al., 1990; Kawabe and Ochi, 1991; Rocha et al.,  1992); however, some potentially autoreactive cells are not physically deleted but are rendered unre- sponsive to self-antigens (Schwartz, 1989; Blackman   et al., 1990; Ramsdell and Fowlkes, 1990).Such cells can be permanently incapacitated in their ability to respond to antigen or can be restimulated in vitro or in vivo by exposure to high levels of antigen and/or to different cytokines.The unresponsiveness main- tained in some nondeletional tolerant states has been attributed to the downregulation of the reactive TCR Corresponding author.National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK. (Sch6nrich et al., 1991) and/or the coreceptor CD4 or CD8 (Rocha et al., 1992).In addition, other fac- tors such as transcription levels of certain genes or efficiency in costimulation and signal transduction may be affected in this process (Mueller et al., 1989).

It is important to understand the mechanisms underlying the induction and maintenance o the tolerant state of T cells in order to be able to inter- vene in cases where their untimely activation causes autoimmune disease.To study the phenomenon in more controlled situations, several groups have fol- lowed a strategy that attempts to keep one or more of the participating elements constant.Such central candidates are the T-cell receptor (TCR) and the cognate antigen: Thus, T-cell-receptor (TCR) trans- genic mice have been crossed to mice that express the cognate antigen.

We have generated a TCR transgenic mouse that bears on most of its T cells a TCR (F ) that recognizes a nonamer peptide (366-374; NP peptide)   from the influenza virus (A/NT/60/68) nucleo- protein in the context of class I MHC (Db) (Townsend  et al., 1986; Mamalaki et al., 1993a).Most T cells in 300 C. MAMALAKI et al.F5 TCR transgenic mice are CD8 cytotoxic cells and can respond to the cognate antigen (peptide or viral protein) both in vivo and in vitro (Mamalaki et al.,  1992, 1993a).The utilization by the receptor of the V131 member of the -chain gene family also confers reactivity to endogenous superantigens (Mtv 8, 9,  and 11) when presented by H-2E class II MHC mol- ecules (Dyson et al., 1991).In the past, we have re- ported the creation of a tolerant state in F5 TCR transgenic mice when they are crossed with H-2E mice carrying Mtv8 and Mtv9 (Mamalaki et al.,  1993a).In that case, we demonstrated that hybrids of F5 mice bred with CBA (H-2k) or BALB/c (H-2d)  mice lack double-positive thymocytes that express high levels of TCR and as a consequence a reduc- tion in single-positive mature T-cells.CD4 cells in the periphery of these mice are more severely af- fected in that there are fewer CD4 cells expressing Vl compared to F5 H-2 bb mice.A substantial num- ber of CD8 cells, however, with low levels of V[3 transgenic chains accumulate in the peripheral lymphoid organs of F5/H2E mice.The circulating CD8 cells from E+/Mtv8+9 mice (even from those backcross animals homozygous for the H-2 k haplo- type) can be stimulated in vitro to differentiate into cytotoxic effector cells that can kill target cells in an NP-antigen-specific manner (Mamalaki et al., 1993a).This showed that T cells can be tolerant to one kind of antigen (endogenous superantigen in this case), but retain their bility to respond to nominal anti- gen (NP peptide).

To assess the development of F5 T cells and study the mechanisms of tolerance induction in transgenic mice in which the cognate antigen influenza nucleo- protein is a self-antigen, we generated transgenic mice that express the viral protein under the broadly active H-2K b promoter.Double-transgenic mice (F5TCR/H2NP) were assessed for F5 T-cell devel- opment and for their ability to respond to nucleo- protein antigen.Such mice appear tolerant mainly due to deletion of CD4+8/TCR hi thymocytes with the concomitant reduction in output of single- positive mature T cells.However, a small number of CD8+VI cells appear in the periphery.These cells express lower levels of F5 TCR in comparison with T cells from F5 mice that do not express influenza nucleoprotein, they express high levels of CD44, and they are unable to respond to peptide antigenic stimulation in vivo.In vitro, they fail to proliferate in response to peptide, but can develop effector func- tion after culture with the cytokine IL-2 for several days.

This system represents an experimental model of nondeletional tolerance that will allow the dissec- H-2K b promoter IMP1295 influenza nucleoprotein sequences  tion of biochemical mechanisms of maintenance of tolerance as well as its protential reversal, which could lead to autoimmunity.


RESULTS


Generation of Influenza Nucleoprotein Trans

In order to generate mice expressing a transgenic infl
enza nucleoprotein, we placed the expression of the transgene under the control of the widely expressed Class I MHC promoter H-2K b (H2NP) (Weiss et al., 1983).The gene for nucleoprotein in these constructs is a deletion mutant (IMP1295) of the full gene (Davey et al., 1985; Townsend et al.,   1985).The deletion removes 0c3 to 327 from the N' terminal portion of the protein and probably renders the protein nonfunctional and, thus, less likely to be harmful when expressed in the transgenic mouse cells.However, it contains the part of the protein that gives rise to the epitope (0c366-374) recognized by the F5 TCR (Townsend et al., 1986).The H-2K b promoter was joined to the deleted NP gene, and an intron and poly-A signal from the SV40 T antigen were added to generate the H2NP construct (Fig. 1).

In transgenic mice expressing this construct, the foreign antigen becomes a self-protein expressed in most cells of the body.

The construct was injected into fertilized mouse (C57B1/10) eggs and several transgenic lines were generated: four of these were used in this study (H2NP10, H2NP22, H2NP40, and H2NP47).The messenger RNA for the nucleoprotein proved to be too unstable to allow us to perform Northern analy- sis studies for expression.Polymerase chain reaction (PCR) on RNA from tissues of NP transgenic-mice assays, however, established that the transgene was expressed in these mice (data not shown).

Antigen-Presenting Cells in H2NP Transgenic Mice E

des In order to assess the level of
expression of nucleo- protein by professional antigen-presenting cells (APC) in lymphoid organs of H2NP mice, macro- phages and dendritic cells from the spleen or the thymus of these mice were isolated and used to stimulate F5 T cells.Figure 2 shows the prolifera- tion of F5 T cells after incubation with H2 bb (C57B1 / 10) APC, H2 bb APC loaded with NP peptide, or with APC from H2NP40 and H2NP47 mice.The results show that peripheral (Fig. 2A) and thymic (Fig. 2B) antigen-presenting cells from H2NP40 mice can stimulate F5 T cells with similar efficiency as H2 bb APC loaded with NP peptide.Similar results were obtained using APCs from H2NP22 mice (data not shown).This confirms expression of the NP trans- gene in APCs of these mice.Antigen-presenting cells from the spleen or thymus of H2NP47 mice, on the other hand, stimulated F5 T cells to a lesser extent but consistently above background.This indicates that expression of NP in H2NP47 transgenic mice is lower than that found in H2NP22 or H2NP40 mice.

Generation of Double (F5/NP)-Transgenic Mice To assess the effects of an antigenic molecule ex- pressed as self-protein on the development of F5 T cells, the H2NP transgenic mice were crossed with the F5 TCR transgenic mice.F5/H2NP double- transgenic mice were analyzed by FACS analysis and their T-cell development was compared with that seen in F5(H-2b) single-transgenic mice.The abso- lute numbers of thymocytes showed a tendency to be reduced in mice expressing transgenic nucleo- protein.For example, F5/H2NP10 double-transgenic mice showed approximately one-third to one-half the number of thymocytes seen in F5 control mice.Figure 3A shows thymocytes from double-transgenic mice stained for CD4 and CD8.In all double- transgenic mice, we observed that the proportion of CD4/8 and the absolute numbers of CD4 cells were not affected, whereas the proportion of thymocytes that developed into fully mature CD8/4 cells was reduced.The percentage numbers of Fig. 3A in the lower right quadrant includes cells with downregulated coreceptor levels (probably due to negative selection) and these numbers do not reflect genuine CD8 single-positive cells.

Three-color FACS analysis of F5 thymocytes stained with antibodies against CD4, CD8, and Vu normally shows two populations of cells with dif- ferent levels of TCR (Fig. 3C): one that stains dull for TCR (TCR1) and represents the majority of dou- ble-positive immature thymocytes (Mamalaki et al.,  1993a); and a brightly staining population (TCRhi) that represents mainly single-positive mature T cells and those double-positive cells that have already upregulated their TCR, including the intermediate populations CD4+8 I and CD48 3H-thymidine incorporation (cpm) FIGURE 2. In vitro proliferation of F5/RAG-/-T cells in the presence of NP macrophages and dendritic cells.T cells from F5/ RAG-/spleens were stimulated in vitro by dendritic cells (DC) and macrophages (M) isolated from (A) spleens and (B) thymuses of NP40 and NP47 mice.B10 DC and M from the same tissues, loaded with the antigenic peptide beforehand and then washed, were used as a positive control.Proliferation of the responders was measured by 3H-thymidine incorporation.FIGURE 3. Absence of TCR TM cells in the thymus of F5/H-2NP double-transgenic mice.Three color fluorometric analysis was performed on thymocytes of TCR single-transgenic mice or TCR/NP double-transgenic mice before (A) or after (B) intraperitoneal treatment for 4 days with 50 n moles of antigenic peptide.Cells were stained with anti-CD8 FITC, anti-CD4 PE, and biotinylated anti-Vu followed by Tricolor-conjugated streptavidin as described in Materials and Methods.Dot blots represent two-color ana- lysis of cells stained with CD4 and CD8.Numbers represent percent proportion of cells in the quadrant. (C) Three-parameter data files were software-gated to generate single-color staining histograms of VI expression on total thymocytes.Numbers indicate the mean fluo escence of VI staining in arbitrary units.Solid line: VI expression on total thymocytes from double-transgenic mice.Dashed line: V expression on total thymocytes from the control F5 single-transgenic mouse.FIGURE 4. Double-transgenic mice have reduced numbers of CD8 peripheral T cells with low levels of transgenic receptor.Three- color fluorometric analysis was performed on lymph node cells of TCR single-transgenic mice or TCR/NP double-transgenic mice before (A) or after (B) intraperitoneal treatment for 4 days with 50 n moles of antigenic peptide.Cells were stained with anti-CD8 FITC, anti-CD4 PE, and biotinylated anti-Vl followed by Tricolor-conjugated streptavidin as described in Materials and Methods.Dot blots represent two-color analysis of cells stained with CD4 and CD8.Numbers represent percent proportion of cells in the quadrant.(C) Three-parameter data files were software-gated to g nerate single-color staining histograms of VIlli expression on CD8/CD4 single-positive cells.Numbers indicate the mean fluorescence of V staining in arbitrary units.Solid line: V] expres- sion on total thymocytes from double-transgenic mice.Dashed line: Vu expression on total thymocytes of the control F5 single- transgenic mouse.1993a).When the Vl pro ile of total thymocytes from F5/NP double-transgenic mice was compared with that of F5 single-transgenic control mice, it be- came evident that the thymus from F5/H2NP10, F5 / H2NP22, F5/H2NP40, and F5/H2NP47 mice was almost devoid of the TCR hi population (Fig. 3C).

The findings in the thymus were reflected in the periphery of F5/H2NP double-transgenic mice.Lymph nodes of the mice described before were stained for CD4, CD8, and VI1. Figure 4A shows that the proportion of CD8 cells was drastically reduced from an average of 53-68% in F5 single- tran genic mice to 8-18% of lymph node cells in the various F5/NP double-transgenic mice.Gated CD8 cells were analyzed for expression of Vl and Fig. 4C shows such analysis.In all F5/NP double- transgenic mice, the circulating CD8 cells have re- duced levels of transgenic TCR.However, this re- duction varied from line to line, with F5/H2NP10 being most and F5/H2NP47 least affected.The majority of cells with high levels of Vl present in most double-transgenic mice probably represent cells expressing endogenous receptors as these are not as evident in mice that cannot rearrange endogenous TCR genes (see Figs 5 and 6).

Because allelic exclusion in TCR mice is not complete, particularly at the 0-chain gene locus (Mamalaki et al., 1993b), the presence of double- positive CD4/8 thymocytes and CD8 VI" periph- eral cells in F5/NP doubl.e-transgenicmice could be explained by the fact that they may express endogenous 0 and receptors, which allow them to be positive selected.

To test this possibility, we generated F5/NP dou- ble-transgenic mice unable to rearrange endogenous TCR genes.Thus, F5/H2NP mice were bred with Recombination Activating Gene-l-deficient mice (RAG-I-/-) (Spanopoulou et al., 1994).Figure 5 shows that, in the absence of endogenous TCR rearrange- ment, CD4+8 thymocytes and peripheral CD8 cells are still present in F5/H2NP22 mice.However, the CD8 T cells found in lymphoid organs are reduced in absolute numbers (not shown) and have lower levels of TCR and its coreceptor (CD8) in compari- son with CD8 cells from F5/RAG-1-/-(Fig. 5).

The absence of endogenous receptors allowed us also to compare the levels of TCR on CD8 periph- eral T cells in different double-transgenic mice.Fig-

ure 6 shows that F5/H2NP22/RAG-1-/and F5/ H2NP40/RAG-1-/mice have considerably lower levels of T-cell receptor than F5/RAG-1-/single- transgenic mice or F5/H2NP47/RAG-1-/double- transgenic mice.These results indicate that to maintain tolerance, the levels of TCR adjust to the amount of antigen present and are consistent with the F5 T-cell prolife ation studies shown in Fig. 2 that reflect variable antigen levels.Thus, mice ex- pressing high levels of NP (H2NP40 or H2NP22) downregulate the F5 TCR to lower levels than mice with reduced expression of NP (H2NP47).The levels of TCR in F5/H2NP47 mice appear to be less affected in RAG-l-/mice than in RAG-l/// mice.The reason for this is under investigation at the moment.It is possible that unresponsiveness in the different double-transgenic mice is achieved by different mechanisms.

We conclude from these results that the tolerance in the double-transgenic mice is mainly due to the reduction in output of single-positive cells by arrest and/or deletion at the transition between the CD4/8+TCR l to CD4/8/TCR hi stage.However, a small proportion of cells mature to the CD8 singlepositive stage with low levels of TCR and are re- leased in the periphery even in mice that cannot rearrange the endogenous TCR genes.No CD4-8- TCR hi T cells were detected in any of the double- transgenic mice (not shown).

CD44 Is Upregulated in CD8 from F5/H2NP Double-Transgenic Mice CD44 is a marker whose upregulation has been taken as an indication that T cells have been exposed to cognate antigen (Budd et al., 1987).F5, F5/H2NP22, and F5/H2NP47 spleen cells were stained for CD44, CD8, and Vl1.CD8 Vl cells were gated and levels of CD44 on their surface were assessed.As seen in Fig. 7, CD44 is upregulated on CD8 T cells from double-transgenic mice in compari on with CD8 T cells from F5 mice.These findings indicate that these T cells have encountered nucleoprotein.

Similar results were obtained in RAG-1-/'/F5 ex- cluding the possibility that these T cells express endogenous receptors that have responded to envi- ronmental antigens irrelevant to th F5 TCR (data not shown).These data are in agreement with stud- ies that described increased levels of CD44 on tolerant T cells in transgenic mice expressing the cognate antigen (von Boehmer et al., 1991; Alferink et al.,  1994).

F5/RAG-1 -/- F5/H2NP22/RAG-1 "/" CD4 CD8 0 Vl311 on CD8 + cells FIGURE 5. Presence of double-positive thymocytes and mature CD8 cells in F5/H2NP/RAG-l-deficient double-transgenic mice.Three-color fluorom tric analysis was performed on thymocytes or lymph node cells of TCR single-transgenic mice or TCR/NP double-transgenic mice bred onto RAG-l-deficient mice background.Cells were stained with anti-CD8 FITC, anti-CD4 PE, and biotinylated anti-Vl antibodies followed by Tricolor-conjugated streptavidin as described in Materials and Methods.Dot blots represent two-color analysis of cells stained with CD4 and CD8.Numbers represent percent proportion of cells in th

quadrant.

Three-parameters data files were software-gated
o generate signle-color staining histograms of VI on total thymocytes (thymus) or on CD8 cells (lymph node).Numbers indicate the mean fluorescence of Vu staining in arbitrary units.Solid line: V expres- sion on thymocytes or CD8 lymph node cells from F5/H2NP22/RAG-1-/-double-transgenic mice.Dashed dotted line: V ex- pression on total thymocytes of the control F5/RAG-1-/single-transgenic mouse.Vll fluorescence FIGURE 6. TCR levels on peripheral CD8 T cells in F5/H2NP/RAG-l-deficient double-transgenic mice.Three-color fluorometric analysis,was performed on lymph node cells of F5/RAG-1-/-, H2NP22/FR/RAG-1-/-, H2NP40/F5/RAG-1-/-, and H2NP47/F5/ RAG-l-/-mice.Cells were stained with anti-CD8 FITC, anti-CD4 PE, and biotynalyted anti-Vl followed by Tricolor-conjugated streptavidin as described.Three-parameter data files were software-gated to generate single-color staining histograms of Vl on CD8 cells.Peripheral CD8 cells from F5/H2NP double-transgenic mice bear upregulated levels of the CD44 memory marker.Three-color fluorometric analysis was performed on spleen cells from F5 transgenic and F5/H2NP double-transgenic mice.Cells were stained with anti-CD8 PE, anti-Vl 1FITC, and biotinylated anti-CD44 followed by Streptavidin red.Histograms represent the levels of CD44 on CD8 positive cells in F5, F5/H2NP22, and F5/H2NP47 spleens.

Response of Double-Transgenic Mice to Additional Exposure to Antigen F5 TCR-transgenic mice respond in vivo to four daily injections of 50 n moles of the antigenic peptide by proliferation of the CD8 T cells and by depletion of the double-positive CD4/8 thymocytes (Mamalaki et al., 1992).To assess to what extent these phenomena can be seen in similarly treated double- transgenic mice, we administered 4 daily inter- peritoneal injections of 50 n moles of NP peptide.

Figure 3B shows representative two-color FACS analyses of thymocytes stained with CD4 and CD8 following 4 days of peptide administration.F5 single-transgenic control mice treated with peptide for 4 days show a marked decrease (10-30-fold) of thymus cellularity.In contrast, F5/H2NP10, F5/ HPNP22, and F5/H2NP40 show a decrease in t e number of thymocytes of approximately 2-3-fold (data not shown).The exception is double-transgenic mouse F5/H2NP47, which shows extensive thymic depletion similar to that seen in the F5 singletransgenic control mouse after peptide treatment.

In the representative experiment shown in Fig. 3B, the reduction in the proportion of double-positive thymocytes in double-transgenic mice treated with peptide was from 71% to 62% in F5/H2NP10, from 73% to 56% in F5/H2NP22, from 74% to 62% in F5/ H2NP40, and from 86% to 12% in F5/H2NP47 mice.

The proportion of CD8 T cells in the lymph nodes of F5/NP double-transg6nic mice did not change upon exposure to antigenic peptide in vivo (Fig. 4B).

As expected, no change was noted in the expression of Vl on gated CD8 lymph node cells from F5/ H2NP10, F5/H2NP22, and F5/H2NP40 double- transgenic mice after, administration of antigenic peptide (Fig. 8), presumably because most of these cells express endogenous 0 and/or [ receptors.

However, when F5/H2NP22/RAG-1-/were treated with peptide, the transgenic TCR on the few circu- lating CD8 was further downmodulated (Fig. 8).We concluded from these results that tolerance in most F5/NP double-transgenic mice involves the CD8 population and is sufficient to protec the mice from self-reactivity due to the low levels of TCR and coreceptor on these cells.However, this tolerance can be enhanced by exposure to higher levels of antigenic peptide.

Functional Analysis of T Cells from Double-Transgenic Mice To test the functional capability of peripheral T cells from F5/NP double-transgenic mice or from mice capable of presenting endogenous superantigen to the F5 TCR (F5bk) (Mamalaki et al., 1993a), we as- sessed the proliferative response and the development of cytolytic activity by these cells after in vivo or in vitro stimulation with cognate peptide.Table 1 shows the results of exposing spleen cells from F5bb, F5bk, and F5/NP double-transgenic mice untreated or following peptide administration in vivo for 4 days, to H-2 b splenic APC, alone or peptide-pulsed, in the presence of low concentrations of rIL-2 (2.5 IU/ml).In each of the two experiments, spleen cells from the F5 bb control mice made good peptide- specific proliferative responses before and after 4 days in vivo peptide administration.In contrast, spleen cells from the F5 bk heterozygotes not given peptide in vivo failed to make a peptide-specific proliferative response in vitro, although they did so following exposure to peptide in vivo.None of the F5/NP double-transgenic mice made peptide- specific proliferative responses, even after exposure to peptide in vivo (Table 1).Peptide-specific cytotoxic T cells were found ex vivo in spleen cells removed from F5 bb and F5 bk mice following peptide admin- istration but never in untreated F5 bb or F5 bk mice, nor in any of the F5/NP double-transg nic mice, whether or not given peptide in vivo (data not shown).In contrast, spleen cells from all F5bb, F5bk, and F5/NP double-transgenic mice could develop peptide-specific cytotoxic effector cells after culture in vitro for 3 or 4 days in the presence of 20 IU rIL-2/ml.Table 2 shows the results of cytotoxicity developed after in vitro culture with rIL-2 and peptide-pulsed APC.Vpll FIGURE 8. Levels of TCR on peripheral CD8 T cells after treatment of double-transgenic mice with antigenic peptide.Three- parameter data fil s were software-gated to generate single-color staining histograms of Vu on CD8/CD4 single-positive cells.Numbers indicate the mean fluorescence of V[I staining in arbitrary units.Solid line: V expression on gated CD8 lymph node cells from single-or double-transgenic mice before (dashed line) or after (solid line) intraperitoneal treatment for 4 days with 50 nM of antigenic peptide.Note: TreatP: In vivo treatment of mice with NP peptide, dp: Days with NP peptide.

EL-4P: EL-4 cells loaded with NP peptide.


DISCUSSION

Experiments using normal mice have shown that mature T cells reactive to endogenous superantigens are absent in adult mice and that whereas CD4/8   double-positive thymocytes expressing the relevant TCR V[3 are present, such TCR V[3 cells are absent or reduced in single-positive T cells in the thymus and the periphery (Kappler et al., 1987; MacDonald  et al., 1988).Mice transgenic for antigen-specific T-cell r ceptors have extended these observations (Kisielow et al., 1988; Berg et al., 1989; Pircher et al.,   1989; Hhmmerling et al., 1991; Morahan et al., 1991;  Sch6nrich et al., 1991; Auphan et al., 1992; Husbands  et al., 1992; Geiger et al., 1993; Sch6nrich et al., 1993;   Oehen et al., 1994; Sponaas et al., 1994).

In this paper, we studied the forms of negative selection and tolerance induction caused in F5 TCRtransgenic mice by nominal antigen when the latter is expressed in the form of a transgene.In the thymus of F5/NP double-transgenic mice, a complete absence of F5 TCR hi thymocytes is observed.
n peripheral tissues, CD4 cells appear not to be af- fected in numbers or in the levels of Vl that they express.This is consistent with the probability that CD4 T cells in these mice are selected because they express endogenous receptors allowing their pos- itive selection (Corbella et al., 1994).The CD8 peripheral mature population on the other hand is affected both in numbers and in the levels of Vl they express.In RAG-l/// mice, the majority of these CD8 cells probably express endogenous c and/or [3 chains and this may be the reason they are posi- tively selected and released in the periphery.It is possible that these cells, due to the presence of the antigen, downregulate the transgenic receptor, caus- ing upregulation of the recombination machinery and consequently increased rearrangement of the endogenous TCR gene loci.This would be analogous to experiments describing receptor editing in self- reactive B cells (Gay et al., 1993; Tiegs et al., 1993).

In F5/RAG-I-/mice, we still found CD8 mature T cells that are tolerant due to F5 TCR and CD8 co- receptor downregulation.We found a correlation between the extent of F5 TCR downregulation and the apparent levels of NP antigen expression on antigen-presenting cells in H2NP mice.That is, the more antigen, the lower the receptor.Therefore, it appears that T cells are capable of adjusting the expression of their antigen receptor to levels not capable of responding to the self-antigen.This has implications in our understanding of autoimmunity where T cells with potential autoreactive TCRs es- cape clonal deletion in the thymus and differentiate to mature T cells with lower levels of TCR.Such cells, when encountering foreign antigen of "better fit," may be activated and following TCR upregulation could attack cells expressing self cognate antigen.An interesting observation concerns the reactiv- ity of F5 CD8 T cells present in H2 bk mice compared to those found in F5/H2NP double-transgenic mice.In both cases, CD8 T cells in peripheral lymphoid organs have low levels of TCR and are present in lower numbers.However, as we reported before (Mamalaki et al., 1993a; and this study), F5 CD8 cells from F5/H2 bk mice that are presumably tolerant to endogenous Mtv antigens still react to cognate anti- gen (Table 1).On the other hand, F5 CD8 T cells from F5/H2NP mice only respond to cognate peptide by downregulating their TCR even further.This is a similar situation reported with mice transgenic for a TCR specific for LCMV and an en- dogenous superantigen (Pircher et al., 1989; Kawai   and Ohashi, 1995).

We conclude from these findings that in F5TCR mice, negative selection of self-reactive thymocytes, whether to H-2E/Mtv (MHC Class II restricted re- a tivity) or to nucleoprotein (MHC Class I reactiv- ity), occurs at the transition from CD4+8+TCR to CD4/8+TCRhi.It is unclear at the moment whether this involves physical elimination of developing thymocytes at the point of upregulation of the receptor or arrest of these thymocytes at the TCR 1 stage due to the presence of the antigen (Takahama   et al., 1992; Swat et al., 1994).

The picture observed in F5/NP double-transgenic mice strongly resembles that developing in F5 single-transgenic mice treated with cognate peptide long term.In such long-term peptide-treated mice, the thymus contains CD4/8 cells bearing low levels of TCR and the peripheral lymphoid organs contain very few CD8 T cells with low levels of transgenic TCR (Mamalaki et al., 1993b).Thus, chronic exposure of mice to cognate antigen leads to a situation similar to tolerance toward self-antigens.

Additional exposure of tolerant T cells to high levels of antigen in other experimental models has led to further tolerization of these cells by down- modulation of the TCR and/or the coreceptor (Himmerling et al., 1991).Further antigenic exposure of T cells from F5/NP double-transgenic mice did not enhance their tolerization, judging from the levels of TCR and coreceptor: These remained un- changed after treatment with the antigenic peptide.

When we repeated this experiment using RAG-1-/mice, we saw that the few CD8 F5TCR 1 cells present in the periphery did downregulate their receptor and coreceptor even further upon exposure to higher levels of antigen given as exogenously administered peptide.This is in agreement with evidence from an alloreactive transgenic TCR model (Himmerling  et al., 1991), and confirms that whereas the tolerance induced in double-transgenic mice is sufficient for the levels of nucleoprotein transgene expressed, however, when higher levels of antigen are intro- duced, the cells downregulate their TCR even fur- ther.

Despite unresponsiveness in vivo, stimulation of T cells from double-transgenic mice was possible in vitro.Peripheral splenic T cells from all F5 mice, whether expressing a "deleting" endogenous super- antigen or a cognate peptide-generating transgene, can generate peptide-specific cytotoxicity after in vitro culture 72-96 hr in the presence of moderate doses (20 IU/ml) of rIL-2.This shows that the apparently anergic phenotype manifest in vivo can be converted to activation in the presence of IL-2.This is one of the hallmarks of anergic T cells and perhaps should not surprise us.On the other hand, the observation that the proliferative capacity of these anergic T cells is severely compromised is an inter- esting one, suggesting that whereas terminal differ- entiation into effector cells is still possible, clonal expansion is not.The F5/NP double-transgenic mice cannot be distinguished from each other in this re- spect, but the F5 bk mice are different in that their spleen cells following n vivo exposure to exogenous cognate peptide will subsequently clonally expand on in vitro stimulation with peptide-pulsed APC.The extent to which F5 T cells are restrained in vivo in the continuous presence of potentially activating antigen is a measure of the capacity of the organism to control autoreactivity.The fail-safe mechanisms could clearly involve downregulation of TCR and/ or accessory molecules, as documented in this pa- per, but the ubiquitous presence of antigen on non- professional APC cells in the periphery also could have an inhibitory effect on the activation cascade via molecular interactions at present poorly under- stood.

In a study by Oehen et al. (1994), it was shown that in double-transgenic mice for LCMV glycoprotein and a TCR specific for this antigen, when the ntigen is expressed at low levels, transgenic TCR T cells are partially deleted.However, these T cells retain their capacity to proliferate and mount a cytolytic response.In our system of double- transgenic mice with even the lowest levels of anti- gen expression, TCR T cells do not respond in vivo, do not proliferate when stimulated by antigen in vitro, and develop cytolytic activity only after cul- ture in vitro with IL-2.These differences may reflect variations in TCR/MHC-Ag affinities.

Results in this and previously published papers indicate that negative selection in the thymus can t ke place at different stages of thymocyte develop- ment with the ultimate result the absence of mature TCR hi self-reactive T cells.This can be accomplished as early as during the transition from CD4-8-TCR- to CD4/8+TCR (H-Y-, LCMV, MHC-specific TCRs)  or later during the transition from CD4/8/TCR to CD4+8/TCR hi (H-2E/Mtv+-specific TCRs, F5/H2NP).

The actual stage in development in which dele- tion of self-reactive clon s or the establishment of their unresponsiveness occurs is most likely dependent on the affinity of the TCR for the MHC/peptide ligand, on the site of expression of the deleting ligand (cortex/medulla), the type of cells that express the deleting ligand, and finally the levels of this expression.


MATERIALS AND METHODS

Mice and Gene Constructs was performed with a FACScan laser instrument and Lysis I or II program (Becton Dickinson).

Mice were generated and maintained in a conven- tional colony free of pathogens at the National Insti- tute for Medical Research in London, F5 TCR and H2NP transgenic mice were generated as described previously (Lang et al., 1988; Mamalaki et al., 1993a)   using inbred C57B1/10 mice.Influenza nucleo- protein peptide (0366-374) was dissolved in PBS and injected intraperitoneally as indicated in the figure legends.

The H2NP fragment for microinjection was con- structed by combining fragments containing the H2- K b promoter, the influenza nucleoprotein coding sequences, and the SV40 splice and polyadenylation signal in three steps: First, a BamHI-SmaI fragment from the pBG311 expression vector (Cate et al., 1986)  was cloned in the BamHI-SmaI sites of the polylinker of Bluescript (Stratagene).This fragment contains the SV40 small-t-antigen gene-splice sequence, the large-T-antigen gene-polyadenylation sequence, and part of a polylinker including SmaI-, NdeI, SstI, and BglIIrestriction sites.Second, in the BglII site of this polylin