Positive and negative selection of T cell repertoires during differentiation in allogeneic bone marrow chimeras

Transplant Immunology 12 (2003) 79–88

Positive and negative selection of T cell repertoires during differentiation in allogeneic bone marrow chimeras Kazunori Onoe´ a,*, Toshihiko Gotohdaa, Hiroki Nishihoria, Toshimasa Aranamia, Chikako Iwabuchia, Cristina Iclozana, Taiki Morohashia, Kazumasa Ogasawarab, Robert A. Goodc, Kazuya Iwabuchia a

Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan b Faculty of Medicine, Shiga Medical School, Ohtsu, Japan c Department of Pediatrics, All Children’s Hospital, St. Petersburg, FL, USA Received 23 September 2002; accepted 24 January 2003

Abstract T cells acquire immune functions during expansion and differentiation in the thymus. Mature T cells respond to peptide antigens (Ag) derived from foreign proteins when these peptide Ag are presented on the self major histocompatibility complex (MHC) molecules but not on allo-MHC. This is termed self-MHC restriction. On the other hand, T cells do not induce aggressive responses to self Ag (self-tolerance). Self-MHC restriction and self-tolerance are not genetically determined but acquired a posteriori by positive and negative selection in the thymus in harmony with the functional maturation. Allogeneic bone marrow (BM) chimera systems have been a useful strategy to elucidate mechanisms underlying positive and negative selection. In this communication, the contribution of BM chimera systems to the investigation of the world of T-ology is discussed. 䊚 2003 Elsevier Science B.V. All rights reserved. Keywords: T cell differentiation; Positive selection; Negative selection; Self-MHC restriction; Self-tolerance; MHC class Ib-restricted T cells

1. Introduction Precursor T cells differentiate in the thymus, become immunocompetent, and emigrate to peripheral lymphoid tissues w1,2x. In the process of differentiation thymic lymphocytes undergo positive and negative selection w3–5x. The positive and negative selection of main stream T cells result from interactions between the T cell receptor (TCR) and self peptide presented in the antigen (Ag) binding groove of major histocompatibility complex (MHC) molecules w6–8x. Positive selection Abbreviations: Ab, antibody; Ag, antigen(s); APC, Ag presenting cell(s); BM, bone marrow; BMT, BM transplantation; CFA, complete Freund’s adjuvant; CTL, cytotoxic T lymphocyte; DN, double negative; DP, double positive; GVHR, graft vs. host response; MHC, major histocompatibility complex; MLR, mixed lymphocyte response; PFC, plaque forming cell; p43–58, pigeon cytochrome c peptide 43– 58; SMLR, syngeneic MLR; SRBC, sheep red blood cells; TCR, T cell Ag receptor. *Corresponding author. Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. Tel.: q81-11-706-5531; fax: q81-11-7067545. ´ E-mail address: [email protected] (K. Onoe).

generates a functional T cell repertoire restricted to selfMHC expressed on the epithelial cells of the thymic cortex w9,10x. Negative selection eliminates or anergizes T cells bearing TCR that are aggressively reactive to self-peptide Ag plus MHC. Dendritic cells or macrophages play a major role in negative selection in the thymic medulla w11–15x. Thus, the reactivity of T cells is largely determined by peptides and MHC that are expressed in the thymus, but not by the genetic background of the T cells themselves, including their own MHC. The importance of the MHC expressed on thymic epithelial cells, however, had been obscure until the specificity of the T cells from MHC incompatible chimeric mice were analyzed. The initial findings that T cells acquire MHC restriction a posteriori and are induced to be self-tolerant in the course of differentiation have been made using an irradiation bone marrow (BM) chimera system where hematopoietic cells and radio-resistant cells (e.g. epithelial cells) express different MHC molecules w16–20x. In this communication, the contribution of chimeric mice to the elucidation of the basic mechanisms under-

0966-3274/03/$ - see front matter 䊚 2003 Elsevier Science B.V. All rights reserved. PII: S 0 9 6 6 - 3 2 7 4 Ž 0 3 . 0 0 0 1 2 - 1

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Fig. 1. Time course of reconstitution of the thymus in wB6.Thy 1.1™B6x BM chimeras. Lethally irradiated (10 Gy) B6 hosts were iv transplanted with BM cells depleted of mature T cells. Eight, 16, 21 and 28 days after BMT, surface phenotype of thymocytes was analyzed by flow cytometry. The left panels (a) in each figure show proportions of donor (Thy 1.1) or host (Thy 1.2) cells. The right panels show expressions of CD4 and CD8 on Thy 1.1q or Thy 1.2q thymocytes. Four weeks after BMT almost complete donor chimerism is achieved.

lying functional differentiation and positive and negative selection of T cells is reviewed. 2. Allogeneic BM chimeras Allogeneic BM chimeras in mice are usually prepared by intravenous (i.v.) inoculation of BM cells that have been depleted of mature T cells into lethally irradiated host mice. The time course of reconstitution is influenced by various factors (e.g. the number of BM cells inoculated, mouse strains used as donors and hosts, the irradiation dose, the histocompatibility between donor and host etc.). For instance, we have shown that the population size of the thymic lymphocytes is determined by the host type w21,22x. In addition, a small number of mature T cells in the BM inoculum accelerate the reconstitution of the host immune system by donor hematopoietic cells w23,24x. The donor T cells quickly eliminate radio-resistant host T and NK cells that are involved in the rejection of donor hematopoietic cells (hybrid resistance or genetic resistance) w25,26x. Fig. 1a shows a representative time course of the thymic reconstitution of BM chimeras prepared by a combination of C57BLy6 (B6) (Thy 1.2) host and B6.Thy 1.1 congenic donor wB6.Thy 1.1™B6x. Sixteen days after BM transplantation (BMT) the majority of thymic T cells are of Thy 1.1q donor origin, and at day 28 virtually no host (Thy 1.2q) T cells are present in

the thymus. It should be noted that the remaining host cells are CD4 and CD8 double positive (DP) 16 days after BMT, whereas very few host cells in the thymus at 21 days are CD4 or CD8 single positive (SP). Similar reconstitution patterns are observed in other lymphoid organs such as spleen and lymph nodes, although the complete replacement by donor cells is achieved at slightly later stages than that in the thymus w23,24x. In these peripheral lymphoid tissues, complete replacement occurs first for B cells, a short cell cycle population, and then for T lineage cells and macrophages w24,27x. 3. Functional maturation of T lineage cells in BM chimeras When the phenotype of donor BM-derived thymocytes (Thy l.1q) from wB6.Thy 1.1™B6x chimeric mice is analyzed using CD4 and CD8 markers, most thymocytes are CD4 and CD8 double negative (DN), a very immature population, until 8 days after BMT (Fig. 1b). At 16 days approximately 90% of cells become DP, and small proportions of CD4 or CD8 SP cells are present. During the drastic phenotypic change from DN to DP, these thymocytes vigorously expand in the cortex. Almost normal proportions of CD4 SP and CD8 SP thymocytes are seen later than 21 days after BMT. When fully allogeneic wB6™AKR (H-2k, Thy 1.1)x chimeras

K. Onoe´ et al. / Transplant Immunology 12 (2003) 79–88 Table 1 Responsiveness of T cells from wB6™AKRx chimeras to alloantigens Assay

GVHR CTL MLR SMLRa a b

T cells from wB6™AKRx B6 wB6™AKRx B6 wB6™AKRx B6 wB6™AKRx B6

Target antigens B6 (H-2b) AKR (H-2k) BALByc (H-2d) y y y y y y y q

y qq y qq q qq NDb ND

qq qq qq qq qq qq ND ND

Purified dendritic cells were used as stimulators. Not done.

were analyzed, the reconstitution was slightly delayed compared to wB6.Thy 1.1™B6x chimeras w20,28x. Even within the CD4 or CD8 SP population, cells at different maturation stages are identified using several differentiation markers, i.e. TCR, heat stable Ag, etc. w29x. The responsiveness of chimera thymocytes to stimuli is dependent on the period after BMT and the nature of the stimuli w28x. Nevertheless, in most BM chimera systems, full lymphocyte functions are accomplished 4 or 5 weeks after BMT. Upon stimulation with T cell mitogens or agents such as PMA plus ionomycin, normal proliferative responses are induced in donor T cells of chimeric mice at this stage w20,28x. Thus, it appears that the individual T cells of chimeric mice possess normal immune functions when non-specific stimuli are given. Levels of reactivity comparable to those in normal mice are seen in other immune cells, B cells, NK cells and macrophages, of chimeras 5 weeks after BMT w20,27x. 4. Alloreactivity and tolerance of T cells in BM chimeras When T cells from wB6 (H-2b)™AKR (H-2k)x chimeras are intraperitoneally (ip) injected to neonatal (B6=AKR) F1 mice, no splenomegaly (graft vs. host response, GVHR) is induced (Table 1) w20x. On the other hand, these T cells induce splenomegaly in (B6=BALByc (H-2d)) F1 mice comparable to that induced by T cells of normal B6 mice. Thus, chimera T cells are specifically unresponsive to both donor and host histocompatibility Ag w20,30,31x. Similarly, no cytotoxic T cells (CTL) against donor or host targets are generated from H-2 incompatible chimeras after coculture with stimulator cells from donor or host mice w32x. Thus far, it has been demonstrated that chimera T cells are tolerant of either donor or host Ag, when the reactivity is analyzed by GVHR or CTL assays (Table 1). The tolerance of CTL to either donor or host specific Ag is largely attributed to negative selection during T cell differentiation in the host thymus w20,30x. The elucidation of mechanisms underlying negative selection

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will be discussed later. It has also been reported that a form of suppressor mechanism might be involved in the unresponsiveness of CTL to donor or host histocompatibility Ag w33–35x. When alloreactivity of T cells from wB6™AKRx chimeras is assessed by mixed lymphocyte reaction (MLR), the T cells mount a considerable proliferative response to host stimulators w36x. The responsiveness is, however, low compared to that in normal B6 mice. On the other hand, T cells from H-2 incompatible chimeras generate no proliferative responses to donor stimulators in syngeneic MLR (SMLR) (Table 1) w37,38x. Recently, we have reported that SMLR represents homeostatic proliferation of various T cell subsets w39x. We would like to postulate that SMLR reflects positive selection in the thymus, which will be discussed in the next chapter. 5. Positive selection and MHC restriction of T cells in BM chimeras To address the immune functions of H-2 incompatible wB6™AKRx chimeras, these mice were ip immunized with sheep red blood cells (SRBC), a T-dependent Ag. Primary antibody (Ab) responses were evaluated using plaque forming cell (PFC) assays after 4 days. These chimeras completely lacked Ab forming cells in the spleen w20x. By contrast, Ab responses to T-independent Ag could be seen in these chimeras w40x. Thus, it appeared that the Ab response to T-dependent Ag was specifically abrogated in wB6™AKRx chimeras. When irradiated AKR host mice were reconstituted with BM cells from B6.H-2k congenic mice, these H-2 compatible wB6.H-2k™AKRx chimeras generated normal levels of Ab response to SRBC w20x. In subsequent studies we found that histocompatibility at H-2A and H-2E loci was sufficient for chimeric mice to mount full primary PFC responses to SRBC (Fig. 2) w40,41x. The lack of a primary Ab response in H-2 incompatible chimeras prompted us to attempt to elucidate the mechanism underlying the immune deficiency. We analyzed in vitro PFC responses to SRBC using T cells from wB6™AKRx chimeras and B cells and antigen presenting cells (APC) from B10 (H-2b) or B10.BR (H-2k) mice w42x. It was clearly shown that helper T cells (Th) of chimeras could generate PFC in combination with B10.BR B cells and APC, but not with B10 cells that express H-2b products identical to those on the Th themselves (Table 2). However, when T cells from H-2 incompatible chimeras and B cells from donor or host H-2 type were adoptively transferred into irradiated (donor=host) F1 mice and then immunized with SRBC, considerable PFC responses were seen with either combination w42x. Thus, in the presence of radioresistant F1 APC, chimera T cells could cooperate with either donor or host H-2 type B cells, suggesting that

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6. Positive selection of particular T cell repertoires under the influence of thymic MHC

Fig. 2. Cooperation between helper T cells and partner cells, B cells and APC, in BM chimeras. Lethally irradiated AKR mice received BMT from B10 (Kb, Ab, E0, Db) or B10.A(2R) (Kk, Ak, Ek, Dk) mice. These wB10™AKRx and wB10.A (2R)™AKRx chimeras were immunized with SRBC and primary anti-SRBC PFC responses were analyzed. In the former wB10™AKRx chimera no PFC response was generated, whereas the wB10.A (2R)™AKRx chimera showed normal level of PFC response. Helper T cells from either chimera recognize Ak and Ek molecules as self, but only helper T cells from wB10.A (2R)™AKRx chimera encounter appropriate partner cells expressing Ak and Ek molecules.

MHC restriction operated between Th and APC but not between Th and B cells w43x. Subsequent analysis with various H-2 recombinant mice again demonstrated that both H-2A and H-2E match between Th and APC was essential for the full Ab response to T-dependent Ag w40,41x. When cell-mediated immune functions were analyzed, similar observations were made. Full anti-Listeria immunity could be achieved in H-2 compatible chimeras but not in chimeras histoincompatible at the H-2 subregions w44x. It was also demonstrated that the histocompatibility of the H-2 subregions determined the responsiveness of CTL to alloantigens in BM chimeric mice w45x. These findings reveal that positive selection by MHC expressed on the radio-resistant epithelial cells of the thymus determines the MHC restriction of the T cells. We would like to emphasize here that importance of the thymic MHC on determining the T cell restriction specificity could not be recognized until T cell functions of MHCincompatible chimera systems were analyzed.

When B6 mice are immunized with pigeon cytochrome c peptide 43–58 (AEGFSYTDANKNKGIT, p43–58) plus complete Freund’s adjuvant (CFA), T cells from regional lymph nodes mount a significant proliferative response to p43–58 in an H-2Ab restricted manner (Fig. 3) w46x. Bm12 mice that carry three amino acid substitutions on H-2Ab generate no T cell response to p43–58, although the H-2Abm12 molecule can bind p43–58 w47x. To examine whether H-2Abm12 molecules cannot positively select the T cell repertoire responding to p43–58, BMT was performed between bm12 donors and B6 hosts. These wbm12™B6x chimeric mice generated no T cell response to p43–58 because of a lack of appropriate APC bearing H-2Ab in vivo (20, 44). We then prepared wB6.Thy 1.1qbm12™B6x mixed chimeras where B6 mice simultaneously received BM cells from B6.Thy 1.1 congenic mice and bm12 mice. Eight weeks later, these chimeric mice were immunized with p43–58 and CFA, and the proliferative responses of Thy l.2q bm12 T cells were evaluated in the presence of B6 or bm12 APC. These bm12 T cells mounted a considerable proliferative response with B6 APC but not with genetically identical bm12 APC (Fig. 3). Thus, p43–58 specific T cells of bm12 origin recognized H-2Ab as self in the mixed chimeras. In addition to determining the role of thymic MHC on T cell restriction, allogeneic chimeras and flow cytometry have been used to examine positive selection of particular T cell subpopulations under the influence of specific MHC class I molecules on thymic epithelial cells. In SWR (H-2q) mice, the expression of Vb17a TCR correlates with only the CD4 SP subpopulation of thymocytes, whereas in thymocytes of SJL (H-2s) mice the expression of Vb17a is detected on both the CD4 and CD8 SP subpopulations. Analysis of allogeneic BM chimeras prepared by reciprocal combinations of SWR and SJL mice demonstrates that the size of the Vb17aq CD8 SP subpopulation is determined by the haplotype of H-2K molecules expressed on thymic Table 2 Anti-SRBC PFC response in vitro or in irradiated F1 mice T cells

B cellsqAPC Assay

Anti-SRBC PFC

wB6™AKRx

B10 B10.BR B10 B10.BR

In vitro

10yculture 248 204 182

B10 B10.BR

In (B6=AKR)F1a 122yspleen 130

(B6=AKR)F1 wB6™AKRx a

Nine Gy-irradiated (B6=AKR)F1 mice were adoptively transferred with T cells from chimeras and non-T cells from B10 or B10.BR mice.

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Amino acid substitutions at the base of the Ag binding groove in B6-H-2bm8 mice led to a significantly altered population size of Vb17aq CD8 SP T cells developing in the thymus of B6-H-2bm8 mice. The role of self peptides in the positive selection was also reported by Bevan’s group w9,49x. 7. Negative selection of self-super Ag reactive T cells in BM chimeras Mls-1a is a super Ag encoded by the open reading frame in the 39-long terminal repeat of endogenous mammary tumor virus, MTV-7 w50x. T cells bearing a Vb6 TCR respond to Mls-1a in the context of H-2E without priming. In AKR (H-2Eq, Mls-1a) mice Vb6q T cells are eliminated by negative selection during differentiation, whereas in B10.BR (H-2Eq, Mls1b) approximately 10% of T cells are Vb6q. To elucidate the mechanism of this negative selection, we prepared wB10.BR™AKRx chimeric mice and the proportion of Vb6q cells was sequentially analyzed w13x. Two weeks after BMT a considerable number of Vb6q cells were seen in the cortex and medulla of the thymus. However, at 3 weeks no Vb6q cells were detected in the medulla, although weakly positive Vb6 cells were still present in the cortex. When various B10 H-2 recombinant mice were used as BM donors, it was shown that clonal elimination was induced in chimeras where H-2Eq strains (B10.AQR) were donors but not in chimeras reconstituted by BMT from H-2Ey donor mice (B10) (Table 3). These findings demonstrate that self-reactive T cells are eliminated in the thymic medulla between 2 and 3 weeks after reconstitution and that H-2Eq donor cells of BM origin, (i.e. macrophages andyor dendritic cells), are responsi-

Fig. 3. T cell proliferative response to p43–58 peptide in B6, bml2 mice and wB6.Thy 1.1qbml2™B6x BM chimeras. Mice were immunized with 20 nmol of p43–58 plus CFA. After 10 days T cell enriched fractions (4=105) from draining lymph nodes were cultured with Ag together with 30 Gy irradiated syngeneic spleen cells (APC: 1=105) in a 96-well flat-bottomed microtiter plate. When BM chimeras were analyzed, T cell enriched fractions were depleted of Thy 1.1q cells before culture and APC were either B6 or bml2 spleen cells. After 3 days of culture 0.5 mCi of w3Hxthymidine was added and the cells were harvested 18 h later. The thymidine incorporation was measured by standard liquid scintillation counting technique.

epithelial cells (Fig. 4) w10x. Subsequent analysis with BM chimeras prepared by SWR donor and various H2K mutant host strains suggested that peptides binding to the groove of the H-2K isoform were involved in the positive selection of Vb17aq CD8 SP T cells w48x.

Fig. 4. Positive selection of Vb17aq CD8q T cells under the influence of various H-2K molecules expressed on epithelial cells in the thymic cortex. BM cells from SWR mice were transplanted into B10 (H-2Kb), B10.A (4R) (H-2Kk, DBAyI (H-2Kq ) or SJL (H-2Ks) mice and the proportion of Vb17aq CD8q T cells was determined in the thymus.

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Table 3 Elimination of Vb6q T cells in various BM chimeras Donor

Host

Vb6 cells (%)

Strain

H-2E

Mls-1

Strain

H-2E

Mls-1

B10 B10.AQR

y q

b b

AKR AKR

q q

a a

10 0

DBAy1 DBAy1

y y

a a

B10 B10.AQR

y q

b b

10 5

ble for the clonal deletion w13,51x. In a subsequent study, we found that thymic epithelial cells were also partially involved in the clonal elimination when a large amount of tolerogens produced by hematopoietic cells (DBAy1) were present (Table 3) w52x. In the presence of minor GVHR, however, clonal elimination of Vb6q cells is abrogated w53x. We demonstrated two different mechanisms underlying the abrogation of negative selection in the GVHR chimeras w54– 56x. These mechanisms have been already reported in a previous review w57x, and we will not further discuss the relationship between GVHR and negative selection in the present review. 8. Negative selection of T cell repertoires specific for p43–58 analog peptides We have determined functional sites, epitope positions and anchor positions, on the p43–58 peptide in mouse strains with various H-2 class II haplotypes w46,58–61x. The 50th and 52nd residues function as epitopes and 46th and 58th residues as anchors to H-2Ab. We then prepared various p43–58 analogs by substituting amino acid residues at these functional sites (Fig. 5). The p43– 58 analog peptides substituted at the 50th epitope posi-

Fig. 5. Amino acid sequence of p43–58 analogs. The peptides are named by substituted positions and amino acids from the original peptide, p43–58, composed of pigeon cytochrome c residues 43–58.

tion, p43–58 (50D), 50E and 50V, stimulate different T cell clones. Residues at the anchor sites determine binding affinity between the peptide and H-2Ab molecule. It has been demonstrated that the binding hierarchy is 50E54A)50E)50E54R or 50V54A)50V)50V54R w58–64x. This hierarchy is directly reflected in the proliferative response of T cells to these analogs. These analogs were then intrathymically (it) injected into wB6™B6x syngeneic BM chimeras at various stages after BMT. At 5 weeks these mice were immunized with the analogs plus CFA; T cell proliferative responses were quantitatively analyzed. When p43–58 analog peptides were it injected into wB6™B6x mice after T cell maturation had been achieved (i.e. 30 days after BMT), no influence of it peptide administration was seen on T cell responsiveness to these peptides. Mice it injected with a high affinity peptide, 50V54A, 3 weeks after BMT, however, mounted low responses to these peptides compared to wB6™B6x mice injected with phosphate buffered saline (PBS) (Fig. 6a and b). The hierarchy of the stimulator peptides, however, was maintained as 50V54A)50V)50V54R. The it administra-

Fig. 6. T cell proliferative response to p43–58 analogs in wB6™B6x BM chimeras. BM chimeras were intrathymically (it) administrated PBS (a) or 50V54A (VA) (b, c) 21 days after BMT. At 5 week post BMT these mice were immunized with VA (a, b) or 50E54A (EA) (c) plus CFA and 10 days later T cell proliferative responses to various analog peptides were determined as in legend of Fig. 3. Note it administration of VA suppresses the response to VA and 50V (b), but not that to EA and 50E (c).

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Fig. 7. T cell proliferative response to p43–58 analogs in wB6™B6x BM chimeras. BM chimeras were intrathymically (it) administrated PBS (a), 50E54A (EA) (b) or 50E54R (ER) (c) 13 days after BMT. At 5 week post BMT these mice were immunized with 50E plus CFA and 10 days later T cell proliferative responses to various analog peptides were determined as in legend of Fig. 2. Note it administration of a high affinity EA results in suppression of the response especially to EA but not to 50E (b), whereas it administration of a low affinity ER exerts no significant influence on the subsequent responses to EA, 50E and ER (c).

tion of 50V54A exerted no significant influence on the responsiveness to the 50E series of peptides that carry glutamic acid at the epitopic 50th site (Fig. 6c). The same result was obtained when the reciprocal combination of the analogs, it administration of 50E54A and immunization with 50V54A, was examined. Thus, it administration of a peptide to chimeric mice at the stage when SP thymocytes are appearing (Fig. 1) seems to lead to epitope-specific negative selection. In the next experiment, a high affinity peptide, 50E54A, was it administrated to wB6™B6x mice 13 days after reconstitution. These mice were subsequently immunized with 50E plus CFA at 5 weeks. In these mice T cell responsiveness was considerably suppressed compared to PBS-treated controls (Fig. 7a and b). In addition, 50E induced a greater response in these T cells than 50E54A in vitro, whereas 50E54A induced a greater response in T cells from mice treated with PBS or 50E54R, a low affinity peptide to H-2Ab, than 50E (Fig. 7c). In our previous study w63x, we reported two T cell clones in 50E54A-immunized B6 mice, one reactive to 50E54A alone and the other reactive to both 50E54A and 50E. Thus, it seems to us that a residue at the 54th site possesses both anchor and epitope functions. This is not truly surprising since dual functions of an amino acid have been reported by both us w60x and other laboratories w65x. It is most likely that it administration of 50E54A eliminates or inhibits the clone reactive to 50E54A alone but not the clone reactive to both 50E54A and 50E. The difference in the negative selection seen between 50E54A-reactive and 50E plus 50E54A-reactive clones may be affected by the affinity of these peptides to H-2Ab. In summary, it administration of a

peptide with high affinity to H-2Ab seems to inhibit T cell clones specific for the peptide in chimeric mice at the stage when clonal elimination of self reactive thymocytes occurs in the thymic medulla. 9. Concluding remarks T cells play a major role in determining the type of immune response generated as well as the regulation of that response. These T cells differentiate and acquire immune functions following two distinct processes, positive and negative selection, mainly in the thymus. In positive selection, MHC molecules expressed on epithelial cells of the thymic cortex interact with the TCR on thymocytes. Conversely during negative selection, MHC molecules on hematopoietic macrophages andyor dendritic cells play an essential role. Several controversial issues, however, still remain concerning the differential functions of these thymic stromal cells w66–69x. Additionally influences of the extrathymic environment on the T cell differentiation should be taken into consideration w70–72x. Indeed, we have reported that repeated or continuous stimulation of T cells with Ag generates T cell responses restricted to the MHC on donor APC in MHC incompatible chimeras w20,42,73,74x. In the past two decades, a number of T cell subsets have been identified, i.e. gdyTCRq cells w75–77x, NKT cells w78–81x, etc. in addition to the mainstream T cells. Some of these subsets undergo differentiation processes quite different from that of mainstream T cells. Indeed, we and others have reported that NK-T cells are positively selected by MHC class Ib on hematopoietic cells under the influence of epithelial cells in the thymic medulla w82–85x. We have also found that

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zeta-associated protein 70 is indispensable to TCR expression on NK-T cells but not that on main stream T cells w86,87x. Differences have been reported by several laboratories in signal pathways and growth factors involved in population expansion and differentiation between mainstream T cells and other T cells subsets w88–95x. As described for mainstream T cells, certain NK-T cells as well as gdyTCRq T cells differentiate extrathymically w88,93,96,97x. More importantly, it has been postulated that these T cell subsets appear to constitute a unique T cell population that is positively selected by non-polymorphic MHC class Ib-like molecules expressed only on hematopoietic cells w98x. Thus, further investigations are needed to completely understand the extraordinarily complex world of T-ology. Acknowledgments This study was supported by Grants-in-Aid for Scientific Research (S, B and C) and a Grant-in-Aid for Special Research Area by the Ministry of Education, Science, Sports and Culture, Japan, Research Grant for Immunology, Allergy and Organ Transplant, by the Ministry of Health and Welfare, Japan. References w1x Good RA. Experiments of nature in the development of modern immunology. Immunol Today 1991;12:283 –286. w2x Scollay R, Wilson A, D’Amico A, et al. Developmental status and reconstitution potential of subpopulations of murine thymocytes. Immunol Rev 1988;104:81 –120. w3x Sprent J, Lo D, Gao EK, Ron Y. T cell selection in the thymus. Immunol Rev 1988;101:171 –190. w4x Teh HS, Kisielow P, Scott B, et al. Thymic major histocompatibility complex antigens and the a b T-cell receptor determine the CD4yCD8 phenotype of T cells. Nature 1988;335:229 –233. w5x Sha WC, Nelson CA, Neeberry RD, Kranz DM, Russel JH, Loh DY. Positive and negative selection of an antigen receptor on T cells in transgenic mice. Nature 1988;336:73 –76. w6x Ashton-Rickardt PG, Banderia A, Delaney JR, et al. Evidence for a differential avidity model of T cell selection in the thymus. Cell 1994;76:651 –663. w7x Marrack P, Kappler J. The T cell receptor. Science 1987;238:1073 –1079. w8x Schwartz RH. T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Annu Rev Immunol 1985;3:237 –261. w9x Hogquist KA, Gavin MA, Bevan MJ. Positive selection of CD8q T cells induced by major histocompatibility complex binding peptides in fetal thymic organ culture. J Exp Med 1993;177:1469 –1473. w10x Iwabuchi K, Negishi I, Arase H, et al. Positive selection of a T-cell subpopulation in the thymus in which it develops. Proc Natl Acad Sci USA 1989;86:5089 –5093. w11x Kappler JW, Roehm N, Marrack P. T cell tolerance by clonal elimination in the thymus. Cell 1987;49:273 –280. w12x Vasquez NJ, Kaye SJ, Hedrick S. In vivo and in vitro clonal deletion of double-positive thymocytes. J Exp Med 1992;175:1307 –1316.

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