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TCR repertoire of suppressor CD8+CD28− T cell populations
TCR Repertoire of Suppressor CD81CD282 T Cell Populations Giuseppina Pennesi, Zhuoru Liu, Rodica Ciubotariu, Shiuping Jiang, Adriana Colovai, Raffaello Cortesini, Nicole Suciu-Foca, and Paul Harris ABSTRACT: The cellular basis of graft rejection and the development of strategies for specific suppression of T cell responses against allogeneic and xenogeneic transplants represents an area of active investigation. Recently, a population of MHC-class I restricted CD81CD282 T suppressor cells (Ts) which are able to inhibit specifically the proliferative response of allospecific, xenospecific and nominal-antigen specific CD41 T helper cells (Th) has been identified. We have studied the TCR Vb gene repertoire expressed by CD81CD282 Ts isolated from allospecific, xenospecific, and nominal antigen-specific T cell lines (TCL). A limited Vb repertoire has been found in all TCLs studied. The most restricted TCR Vb usage was observed within the population of Ts from xenospe-
ABBREVIATIONS MHC major histocompatibility complex Ts T suppressors Th T helpers TCR T cell receptor APC antigen presenting cell
INTRODUCTION The question of how an immune response, once initiated, is turned off is central to immunology. The original demonstration of suppression was reported by Gershon and Kondo in 1970 [1]. This work showed that antigen priming induces lymphocytes which, when adoptively transferred into immunologically naı¨ve mice, could in-
From the Department of Pathology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA (G.P., Z.L., R.C., S.J., A.C., N.S-F., P.H.), and Department of Surgery, Universita’ Degli Studi di Roma “La Sapienza,” Instituto di II Clinica Chirurgica, Servizio Trapianti d’Organo, Rome, Italy (R.C.). Address reprint requests to: Dr. Nicole Suciu-Foca, College of Physicians and Surgeons of Columbia University, Department of Pathology, 630 West 168th Street, P&S 14-401, New York, NY 10032, USA; Tel: 212-3056941; Fax: 212-305-3429; E-Mail: [email protected]. Received December 22, 1998; accepted December 29, 1998. Human Immunology 60, 291–304 (1999) © American Society for Histocompatibility and Immunogenetics, 1999 Published by Elsevier Science Inc.
cific TCLs. The TCR Vb usage within the Ts subset of TCL differs from the TCR repertoire expressed by the CD41 Th subset of the same TCL. This is consistent with the fact that Ts and Th cells recognize distinct MHC/ antigen complexes. The finding that the TCR repertoire used by Ts is limited opens new avenues for studying the mechanisms of transplant rejection. Human Immunology 60, 291–304 (1999). © American Society for Histocompatibility and Immunogenetics, 1999. Published by Elsevier Science Inc. KEYWORDS: T suppressor cells; T cell receptor; TCR Vb spectratyping
PBMCs SLA rTT rIL2 RI
peripheral blood mononuclear cells swine leukocyte antigens recombinant tetanus toxoid recombinant interleukin 2 relative intensity
hibit the immune response of the recipient animal in an antigen-specific fashion. In spite of great progress in molecular and cellular immunology, the phenotype and biological function of T suppressor cells remain a controversial subject. Ts cells were claimed to derive from both CD41 and CD81 T cell subsets [2–5]. Studies of the function of Ts cells have suggested that they can act either as inducer or effector cells and that at least in some cases their inhibitory activity is mediated by cytokine production [6 – 8]. The characterization of Ts has been hampered by the difficulty of generating Ts cell lines [6, 9 –11]. Recently, we have demonstrated that allo-, xeno- and nominal antigen-specific T suppressor cell lines can be generated by in vitro priming of human T cells with allogenic, xenogenic or autologous (antigen-loaded) 0198-8859/99/$–see front matter PII S0198-8859(98)00134-7
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TABLE 1 HLA phenotypes and genotypes of individuals used as blood donors for the generation of TCLs Class I Individual PR BN NB HM MS ES
A 29 2 3 1 26 2
Class II B
32 3 24 32 30 11
44 52 51 8 53 57
DRB1* 40 58 27 44 35 50
1101 04 0101 0101 1302 0701
0701 13 0402 0301 0701 0701
APCs [9 –11]. The Ts cells express the CD81CD282 phenotype and recognize specifically MHC class I/peptide complexes on the surface of the APCs used for priming. Ts inhibit upregulation of CD40, CD80, and CD86 on APCs, rendering them unable to elicit Th proliferation [9 –11]. The suppressor activity is triggered by the recognition of MHC classI/peptide complexes on APC via TCR a/b chain on the surface of Ts [9 –11]. TCR molecules are composed of covalently linked variable a and b chains which are non-covalently associated with the CD3 complex [12]. The genes encoding the a and b chains undergo somatic recombination in a manner similar to immunoglobulin molecules [13]. The TCR b chain is produced by the recombination of V, D, J, and C segments. This diversity is further increased by the nibbling of germline nucleotides and/or addition of nucleotides at the V-D-J junction sites [14]. The complementarily determining region 3 (CDR3), encompassing the V-D-J junction, displays the most extensive nucleotide diversity and is thought to encode a region involved in the contact of the TCR with the antigenic peptide [15]. In this report, we present results of the analysis of the TCR Vb genes expressed by CD81CD282 T suppressor lymphocytes derived from allospecific, xenospecific and antigen-specific TCLs. We have established that Ts ex-
press a limited Vb repertoire and that the most restricted repertoire was expressed by xenospecific Ts. MATERIALS AND METHODS Human Cells Blood was obtained from six blood donors (PR, BN, NB, MS, HM, and ES) who were typed for HLA class I and class II antigens by conventional serology and genomic typing by PCR-SSO. Table 1 summarizes the HLA class I phenotype and class II genotype of individuals used in these studies. Swine Cells Blood was obtained from one outbred pig and from Yucatan Miniature Swine of strain Q and Z (Sinclair Research Center, Columbia, MO) which carry different MHC class I and class II genotypes as described [16 –18]. Antigens Recombinant Tetanus Toxoid (rTT) C fragment was obtained from Boehringer Mannheim (Indianapolis, IN) and conjugated to carboxylated polystyrene microparticles (Latex beads) (Polysciences Inc., Warrington, PA), according to the manufacturer’s instructions. Generation of Allospecific and Xenospecific T Cell Lines Four allospecific TCLs (PR anti BN, NB anti BN, NB anti MS and HM anti MS) and three xenospecific TCL (HM anti outbred pig, ES anti strain Q swine, ES anti strain Z swine) were generated as previously described [9, 19] (Table 2). Briefly, human and pig PBMCs were separated from buffy coats by Ficoll-Hypaque centrifugation. Human PBMCs, used as responding cells, (1 3 106/ml) were stimulated in 24-well plates with an equal number of irradiated (1600r) stimulating cells (PBMCs) from an allogeneic (human) or xenogeneic (swine) donor. Cells were co-cultured for 7 days in complete medium (RPMI 1640 supplemented with 10% heat-inactivated
TABLE 2 CD81CD282TCLs studied and their functional characterization
TCL 1 2 3 4 5 6 7 8
Human responding cells from: ES ES HM HM NB NB PR PR
Source of stimulating cells Allogeneic
Xenogeneic
Autologous
No. of stimulation cycles
% Suppression of the reactivity
rTT*
2 2 2 2 2 2 2 3
76% 73% 50% 65% 72% 88% 50% 37%
Pig strain Q Pig strain Z Outbred pig MS MS BN BN
* T cells from individual PR were primed to rTT in the presence of autologous APCs.
TCR Repertoire of Ts Cells
FIG. 1. TCR Vb repertoire of naive CD81CD282 T lymphocytes from individuals NB (panel A), ES (panel B) and PR
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(panel C). The spectratypes show that all the Vb families are represented with relative intensity ranging from 0.001 to 0.15.
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FIG. 2. TCR Vb repertoire of CD81CD282 T lymphocytes from allospecific (panel A), xenospecific (panel B) and nominal antigen specific (panel C) TCLs. Study of the TCR Vb usage
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of Ts shows that only a few Vb families were expressed by CD81CD282 cells with relative intensity higher than 0.1. Xenospecific Ts express the highest degree of oligoclonality.
TCR Repertoire of Ts Cells
FIG. 3. Profile of the fluorescent Vb-Cb runoff products corresponding to the most represented Vb families in the spectratypes of Ts derived from allospecific TCL NB anti BN (panel A), xenospecific TCL ES anti Q (panel B) and nominal antigen specific TCL PR anti rTT (panel C) TCLs. The x-axis represents the length of the Vb-Cb segments in base pairs and the y-axis represents the fluorescence intensity in arbitrary units.
fetal calf serum, 2 mM glutamine, and 50 mg/ml gentamicin). Responding cells were restimulated at seven day intervals in medium containing 10U/ml rIL-2 (Boehringer Mannheim, Indianapolis, IN) [9, 10].
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Generation of Antigen-Specific T Cell Lines PBMCs (2 3 106/ml) from a healthy blood donor (PR) were stimulated in 24-well plates with 1 mg of rTTconjugated to latex beads in RPMI 1640 medium supplemented with 10% human serum (Sigma Chemical Co, St. Louis, MO), 2 mM L-glutamine and 50 mg/ml gentamicin (Gibco, Grand Island, NY), as described [11]. On day five, 20 U/ml of rIL-2 (Boehringer Mannheim, Indianapolis, IN) were added. Ten days after priming, T cells (2 3 106) were collected, washed and restimulated with antigen in medium containing 20 U/ml of rIL-2 and irradiated (3000r) autologous PBMCs (2 3 106).
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FIG. 4. Ts spectratypes of two allospecific TCL (NB anti BN and NB anti MS) derived from the same responder.
Antigen specific T cell lines were obtained after two or three restimulations [11] (Table 2). Cell Separation CD41 and CD81 T cells were separated from TCLs by negative selection using Dynal CD4 and CD8 magnetic beads. CD81CD282 T cell suspensions were prepared by depletion of CD281 T cells from purified CD81 T cell suspensions [9 –11]. The purity of the suspensions was monitored by cytofluorometry. Cells were rerosetted with CD28 beads when necessary, to obtain a population containing less than 2% CD281 bright cells. Proliferation Assay CD41 T cells (5 3 104/well) from TCL primed to allogeneic, xenogeneic or Tetanus Toxoid-loaded autologous APCs were stimulated with an equal number of irradiated allogeneic, xenogeneic or autologous APCs in 96-well plates. CD81CD282 Ts (5 3 104 /culture) from the same TCL were added to replicate cultures. After 48 h of incubation, the cultures were pulsed with 3 H[TdR] and harvested 18 h later. 3H[TdR] incorporation was determined by scintillation spectrometry in a LK Betaplate counter. Mean cpm of the triplicate reactions and the standard deviation to the mean were calculated [9 –11].
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The percent suppression of CD41 Th proliferation in the presence of CD81CD282 Ts was calculated as: 12
cpm in Th 1 Ts 1 APC cultures cpm in Th 1 APC cultures
TCR Spectratyping Total RNA was extracted using QIAGEN columns (Qiagen Inc., Valencia, CA) from CD81CD282 Ts and from CD41 Th. RNA was reverse transcribed into cDNA in a reaction using MMLV reverse transcriptase primed with oligo (dT)18 (Clontech Laboratories Inc., Palo Alto, CA), as recommended by the manufacturer. Aliquots of the cDNA synthesis reaction were amplified in 50 ml of the 24 Vb oligonucleotides (0.5 mM final concentration) and the Cb oligonucleotide (0.5 mM final concentration). Vb and Cb primers were used as previously described [19]. As an internal control for the cDNA amount used per reaction, a tube containing sense and antisense primers for the first exon of Cb region was included. Two microliters of the Vb-Cb PCR products were subjected to elongation with a fluorophore-labeled Cb specific primer (0.5 mM final concentration). The size and the fluorescence intensity of labeled run off products were determined on a 377 DNA sequencer (Perkin Elmer Applied Biosystem Division, Foster City, CA) and analyzed using the ABI PRISM 377 GENESCAN analysis program (Perkin Elmer Applied Biosystem Division, Foster City, CA).
TCR Repertoire of Ts Cells
FIG. 5. Ts spectratypes of two xenospecific TCL (ES anti strain Q and ES anti strain Z) derived from the same responder.
The relative intensity of each Vb family was calculated as the peak areas corresponding to each Vb family divided by the sum of all the peak areas of all the Vb families [14]. The sharing rate of Vb families in two different spectratypes was calculated as number of shared families divided by number of families present in the two spectratypes. The statistical significance of differences among sharing rates was calculated by the Fisher test. Vb-Jb Gene Segment Analysis Two microliters of the Vb-Cb PCR products, corresponding to the Vb families with the highest relative intensity in each spectratype, were subjected to elongation with 13 fluorophore-labeled Jb specific primers [19]. The size and the fluorescence intensity of the labeled run off products were determined on a 377 DNA sequencer as previously described. The relative intensity of each Vb-Jb gene segment was calculated. RESULTS Functional Characterization of Ts We have previously shown that CD81CD282 Ts recognize MHC class I/peptide complexes and inhibit the ability of CD41 Th to react against MHC class II/
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peptide complexes expressed by the APCs used for priming [9 –11]. CD81CD282 T cells from each of the TCL included in this study were tested for their ability to inhibit the proliferative response of CD41 Th from the same TCL in 3-day blastogenesis assay. Table 2 summarizes the results. Xenospecific Ts (TCL 1, 2, 3) inhibited Th reactivity to swine MHC-class II antigens by 50 to 76% (Table 2). Inhibition was specific since Ts primed to strain Q did not suppress the reactivity of Th primed to strain Z or vice versa [10]. Similarly, allospecific Ts (TCL 4, 5, 6, 7) suppressed Th reactivity to the specific stimulator by 50 – 88% (Table 2). Ts from TCL 8 recognize Tetanus Toxoid peptides presented by MHC-class I antigens expressed by autologous APCs [11]. Spectratyping Study of the TCR Vb gene repertoire expressed by allospecific, xenospecific and nominal antigen-specific Ts shows an oligoclonal expansion of Vb families. As illustrated in Fig. 1, before priming, CD81CD282 T lymphocytes express all the 24 Vb families with relative intensity ranging from 0.001 to 0.15. After multiple stimulations with either allogeneic, xenogeneic or rTTloaded-self APCs, the expression pattern of Vb gene segments changes dramatically. The Ts population of each TCL expresses only a few Vb families (3–5 per spectratype) at a relative intensity level greater than 0.1 (Fig. 2). Other families found within the naive population of CD81CD282 T cells from the same blood donor are either absent or expressed with a relative intensity
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FIG. 6. Comparison between the TCR Vb repertoire of allospecific and xenospecific TCL from the same responder.
lower than 0.05 following multiple priming. In each TCL a unique TCR Vb family seems to prevail in terms of relative intensity. For example, Vb19 has the highest relative intensity (RI 5 0.38) in the alloreactive TCL NB anti BN (TCL 6), while Vb23 and Vb5 have the highest RI in TCL ES anti-Q (TCL 1) and PR anti-rTT (TCL 8) respectively (Fig. 2, panel A, panel B, and panel C). The TCR repertoire used by xenospecific CD81CD282 T lymphocytes (Fig. 2, panel B) is more restricted than the repertoire of allospecific TCLs (Fig. 2, panel A) To study the clonal expansion of particular TCR Vb families within a spectratype, we analyzed the profile and peak distribution of Vb-Cb fragments of each TCR Vb family. Prior to antigenic stimulation, human PBMCs exhibit 6-8 peaks spaced by 3 nucleotides corresponding to in-frame transcripts of the TCR b chain CDR3 region. The area under each peak is proportional to the amount of transcripts of the corresponding CDR3 size in the sample. Each peak corresponding to the length of a given CDR3 usually contains multiple distinct sequences. An increase in the height and area of a peak that modifies the bell-shaped CDR3 size distribution indicates oligoclonal or monoclonal expansion occurring after antigenic stimulation [14]. In the allospecific Ts population, the distribution of peaks within each Vb family was prevalently
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gaussian-like, suggesting that certain Vb-Cb combinations are broadly used by different TCRs (Fig. 3, panel A). In the xenospecific CD81CD282 population, the shape of the TCR Vb families was mono- or bi-modal (with the exception of Vb23) suggesting that only a few subfamilies of Vb genes are used (Fig. 3, panel B). Similarly, only few TCR Vb subfamilies seem to recognize epitopes of the rTT molecule are indicated by the presence of mono- or bi-modal peaks within the spectratype of Tetanus Toxoid specific Ts (Fig. 3, panel C) To determine whether the Vb repertoire of Ts is influenced by the responder’s or stimulator’s HLA-DR antigens, we compared the spectratypes of: a) TCLs generated by stimulating the T cells from the same responder with APCs from different stimulators and b) TCLs generated by stimulating TCLs from different responders with APCs from the same stimulator. When T cells from the same responder (NB) were stimulated with APCs from two HLA-DR different individuals (BN and MS), 36% of the Vb families appeared to be shared (Fig. 4). Similarly, when T cells from a single responder (ES) were stimulated with APCs from two SLA disparate swine (strain Q and Z), 37.5% of the Vb families seen in the spectratype were shared (Fig. 5). Furthermore, 28.5% of Vb families were shared by allospecific and xenospecific Ts generated by stimulating T cells from the same responder (HM) with human and pig APC respectively (Figs. 6 and 7), and 43% were shared
TCR Repertoire of Ts Cells
FIG. 7. TCR Vb gene usage in allospecific and Tetanus Toxoid specific Ts from a single individual (PR).
among allospecific and rTT-specific Ts obtained from the same individual (PR). However, comparison of the spectratype of TCLs obtained from different responders (NB and HM) stimulated with the same allogeneic APCs (MS) also revealed a sharing rate of 27% (Fig. 8). Hence, the rate of TCR-Vb gene sharing among Ts from different individuals primed to the same antigen is not different from the FIG. 8. TCR-Vb repertoire of allospecific Ts obtained by priming T cells from two different responders with APCs from the same stimulator.
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sharing rate seen among Ts from the same individual primed to different antigens. The extent to which the recipient’s MHC genotype and the source of antigenic stimulation influence TCR Vb usage Ts population, remains unclear. Because Th recognize peptide/MHC-class II complexes while Ts recognize peptide/MHC class I complexes on the same APC, the TCR Vb repertoire of CD41 Th and CD81CD282 Ts from the same TCL is expected to be different. Although Th and Ts from the same line shared less than 50% of TCR-Vb families, certain Vb genes were used only by Ts or by Th consistent with the notion that they recognize distinct antigenic determinants. Comparison of the TCR repertoire used by CD81CD282 Ts with that used by
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FIG. 9. Comparison between TCR Vb repertoires of allospecific (panel A), xenospecific (panel B), and nominal antigen specific (panel C) Th and Ts from the same TCL subpopulations showed a not coincident TCR Vb usage.
CD81CD281 T cells from the same TCL showed although certain TCR families are shared, Ts lack certain families which are present in the population of allospecific CD81CD281 T cells.
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Vb-Jb Gene Segment Analysis To further analyze the Vb repertoire of Ts, we studied the Vb-Jb gene segment distribution within prevailing TCR Vb families. The distribution of Vb-Jb gene segments within each TCR Vb family has a gaussian-like profile in naive PBMCs while in Ts cell lines it is unior bi-modal (data not shown). In general, modifications of the bell-shaped pattern mirror oligoclonal expansion
TCR Repertoire of Ts Cells
FIG. 10. Comparison of the TCR Vb repertoire expressed by allospecific CD81CD281 and CD81CD82 T cells from the same TCL.
of particular subfamilies or preferential usage of a Jb gene variant. The relative intensity of each peak in the Vb-Jb spectratype is the measure of the frequency of each Vb-Jb recombination fragment [14]. In Ts cell lines only a few Vb-Jb combinations are represented (Figs. 9 –11). The most restricted Vb-Jb repertoire is displayed by TCR Vb families from xenospecific Ts lines. The Vb-Jb repertoires found in different Ts cell lines are not superimposable, indicating that no Jb1 or Jb2 gene variants are preferentially selected by Ts (Fig. 11). DISCUSSION The molecular mechanism of T cell suppression is still unknown. It has been recently shown that xenospecific, allospecific and nominal antigen-specific human Ts can be generated in vitro by multiple T cell stimulations [9 –11]. Ts generated in this manner carry the CD81 CD282 phenotype, recognize specifically MHC-class I antigens expressed by the APCs used for priming, and are able to suppress T helper cells reactivity to MHCclass II/peptide complexes on the same APCs. The suppressive effect was shown to result from downregulation of CD40, CD80, and CD86 costimulatory molecules on the surface of APCs exposed to Ts [9 –11]. In this report we have analyzed and compared the TCR Vb usage in CD81CD282 Ts from allospecific, xenospecific and nominal-antigen specific TCLs.
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An expansion of a limited number of families was observed in all TCL regardless of the antigen used for Ts priming. This observation is reminiscent of previous reports showing that the CD81CD282 population of lymphocytes present in human peripheral blood is oligoclonal [20, 21] These cells were shown to have relatively shorter telomeres, compared to other lymphocyte subsets, and to be unable to proliferate in culture, suggesting that they are aging, terminally differentiated cells, marked for apoptosis [20, 21]. In contrast to these studies our data indicate that the CD81CD282 subset of T lymphocytes comprises an oligoclonal population of Ts which can be readily primed and expanded in vitro [9 –11]. Since Ts inhibit Th reactivity in vitro in an antigen-specific manner it is likely that they play an important role as regulators of the immune response to both nominal and self antigens in vivo [11]. We found that CD81CD282 and CD81CD281 T cells from the same TCL display a superimposable oligoclonal repertoire, suggesting that they derive from the same precursor [24]. However, the Ts population lacks certain TCR Vb families which are present among the population of CD81CD281 T cells, known to display cytotoxic function [25, 26]. Hence, suppressor and cytotoxic T lymphocytes differ not only functionally but also with respect to the TCR Vb genes which they use to recognize MHC-class I/peptide complexes. The comparison between the TCR repertoire expressed by CD81CD282 T cells with suppressor activity and CD41 T helper lymphocytes indicated that most of the Vb families present in the two subsets were different. This finding suggests that the antigenic determinants recognized by Ts and Th are not the same. Comparison
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FIG. 11. Jb usage of the most frequent Vb families in allospecific HM anti MS (panel A), xenospecific ES anti Q (panel B) and nominal antigen specific PR anti rTT (panel C) T suppressor TCLs. Only few Jb-Vb recombination fragments were present in these cell lines.
of allospecific, xenospecific, and nominal antigen specific Ts cell lines showed that xeno-specific Ts display the highest degree of oligoclonality. It is possible that, only few TCRs are able to recognize the Ag/SLA complexes on the surface of the pig APCs due to the extent of the antigenic differences between these species [27–30]. TCRs engage in xenoreactivity should have a favorable
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TCR-Ag/SLA complex association/dissociation rate in order to activate the suppressor activity of the cells [31, 32]. An alternative explanation for the skewed TCR repertoire found in xenospecific Ts lines stems from the molecular incompatibility between certain human and swine co-receptor and accessory molecules which are involved in the immune response [33–36]. Beside the signal through the TCR, activation of T cells generally requires a costimulatory signal provided by the ligation of one of the various accessory molecules expressed by T cells. It is possible that if the co-receptors on the surface
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of human T cells are not able to fully interact with their counter-parts on pig APCs, the signal that leads to the T cell activation will be provided by only few TCRs, with high avidity for Ag/SLA complexes. Thus, the self-MHC driven selection of T cells may lead to a T cell repertoire, which recognizes only closely related MHC structures. MHC class I restricted Ts may play a physiological role in regulating the immune response of Th cells against self or non-self peptide MHC class II complexes. The finding that there is a cross-talk between the MHC class I and class II pathway of peptide processing supports the notion that the same APCs present both helper and suppressor inducing peptides. It is possible that recognition by Ts of MHC class I bound peptides is play a role in controlling the activation of antigen-specific Th cells. Identification of suppressor-inducing peptides may be useful for induction of unresponsiveness to auto-, alloor xenoantigens. Furthermore, the ability to generate Ts cells after exposure to antigen in vitro may be an advantage for further functional analysis and identification of new potential therapeutic strategies for autoimmune diseases and graft rejection. REFERENCES 1. Gershon RK, Kondo K: Cell interactions in the induction of tolerance: the role of thymic lymphocytes. Immunol 18:723, 1970. 2. Groux H, O’Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, Roncarolo MG: A CD41 T-cell subset inhibits antigen-specific T-cell responses and prevent colitis. Nature 389:737, 1997. 3. Han HS, Jun HS, Utsugi T, Yoon JW: A new type of CD41 Suppressor T cell completely prevents spontaneous autoimmune diabetes in syngenic islet-transplanted NOD mice. J Autoimmunity 9:331, 1996. 4. Noble A, Pestano GA, Cantor H: Suppression of immune responses by CD8 cells. I. Superantigen-activated CD8 cells induce unidirectional Fas-mediated apoptosis of antigen-activated CD4 cells. J Immunol 159:539, 1998. 5. Noble A, Zhao ZS, Cantor H: Suppression of immune responses by CD8 cells. II. Qa-1 on activated B cells stimulates CD8 cell suppression of T helper 2 responses. J Immunol 160:566, 1998. 6. O’Hara RM: Antigen-specific suppressor factor: missing pieces in the puzzle. Immunol Res 14:252, 1995. 7. Horwitz DA, Dixon Gray J, Ohtsuka K, Hirokawa M, Takahashi T: The immunoregulatory effects of NK cells: the role of TGF-b and implications for autoimmunity. Immunol. Today 18:538, 1997. 8. Groux H., Bigler M., de Vries JE, Roncarolo MG: Inhibitory and stimulatory effects of Il-10 on human CD81 T cells. J Immunol 160:3188, 1998.
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tween allogenic and xenogenic lymphocyte reactivity. Transpl Proc 28:2915, 1996. Wu GD, Cramer DV, Shirvan H, Borie D, Chapman FA: Genetic evidence that antibody response to xenografts is directed at a restricted number of target antigens. Transpl Proc 29:954, 1997. Garcia KC, Degano M, Pease LR, Huang M, Peterson PA, Teyton L, Wilson IA: Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science 279:1166, 1998. Valitutti S, Lanzavecchia A: Serial triggering of TCRs: a basis for the sensitivity and specificity of antigen recognition. Immunol Today 18:299, 1997. Hudrisier D, Kessler B, Valitutti S, Horvath C, Cerottini C, Luescher IF: The efficiency of antigen recognition by CD81 CTL clones is determined by the frequency of serial TCR engagement. J Immunol 161:553, 1998. Maher SE, Karmann K, Min W, Hughes CC, Pober JS, Bothwell ALM: Porcine endothelial CD86 is a major costimulator of xenogeneic human T cells. J Immunol 157:3838, 1996. Murray AG, Khodadoust MM, Pober JS, Bothwell AL: Porcine aortic endothelial cells activate human T cells: direct presentation of MHC antigens and costimulation by ligands for human CD2 and CD28. Immunity 1:57, 1994. Rollins SA, Kennedy SP, Chodera AJ, Elliott EA, Zavoico GB, Matis LA: Evidence that activation of human T cells by porcine endothelium involves direct recognition of porcine SLA and costimulation by porcine ligands for LFA-1 and CD2. Transplantation 57:1709, 1994
ABBREVIATIONS MHC major histocompatibility complex Ts T suppressors Th T helpers TCR T cell receptor APC antigen presenting cell
INTRODUCTION The question of how an immune response, once initiated, is turned off is central to immunology. The original demonstration of suppression was reported by Gershon and Kondo in 1970 [1]. This work showed that antigen priming induces lymphocytes which, when adoptively transferred into immunologically naı¨ve mice, could in-
From the Department of Pathology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA (G.P., Z.L., R.C., S.J., A.C., N.S-F., P.H.), and Department of Surgery, Universita’ Degli Studi di Roma “La Sapienza,” Instituto di II Clinica Chirurgica, Servizio Trapianti d’Organo, Rome, Italy (R.C.). Address reprint requests to: Dr. Nicole Suciu-Foca, College of Physicians and Surgeons of Columbia University, Department of Pathology, 630 West 168th Street, P&S 14-401, New York, NY 10032, USA; Tel: 212-3056941; Fax: 212-305-3429; E-Mail: [email protected]. Received December 22, 1998; accepted December 29, 1998. Human Immunology 60, 291–304 (1999) © American Society for Histocompatibility and Immunogenetics, 1999 Published by Elsevier Science Inc.
cific TCLs. The TCR Vb usage within the Ts subset of TCL differs from the TCR repertoire expressed by the CD41 Th subset of the same TCL. This is consistent with the fact that Ts and Th cells recognize distinct MHC/ antigen complexes. The finding that the TCR repertoire used by Ts is limited opens new avenues for studying the mechanisms of transplant rejection. Human Immunology 60, 291–304 (1999). © American Society for Histocompatibility and Immunogenetics, 1999. Published by Elsevier Science Inc. KEYWORDS: T suppressor cells; T cell receptor; TCR Vb spectratyping
PBMCs SLA rTT rIL2 RI
peripheral blood mononuclear cells swine leukocyte antigens recombinant tetanus toxoid recombinant interleukin 2 relative intensity
hibit the immune response of the recipient animal in an antigen-specific fashion. In spite of great progress in molecular and cellular immunology, the phenotype and biological function of T suppressor cells remain a controversial subject. Ts cells were claimed to derive from both CD41 and CD81 T cell subsets [2–5]. Studies of the function of Ts cells have suggested that they can act either as inducer or effector cells and that at least in some cases their inhibitory activity is mediated by cytokine production [6 – 8]. The characterization of Ts has been hampered by the difficulty of generating Ts cell lines [6, 9 –11]. Recently, we have demonstrated that allo-, xeno- and nominal antigen-specific T suppressor cell lines can be generated by in vitro priming of human T cells with allogenic, xenogenic or autologous (antigen-loaded) 0198-8859/99/$–see front matter PII S0198-8859(98)00134-7
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TABLE 1 HLA phenotypes and genotypes of individuals used as blood donors for the generation of TCLs Class I Individual PR BN NB HM MS ES
A 29 2 3 1 26 2
Class II B
32 3 24 32 30 11
44 52 51 8 53 57
DRB1* 40 58 27 44 35 50
1101 04 0101 0101 1302 0701
0701 13 0402 0301 0701 0701
APCs [9 –11]. The Ts cells express the CD81CD282 phenotype and recognize specifically MHC class I/peptide complexes on the surface of the APCs used for priming. Ts inhibit upregulation of CD40, CD80, and CD86 on APCs, rendering them unable to elicit Th proliferation [9 –11]. The suppressor activity is triggered by the recognition of MHC classI/peptide complexes on APC via TCR a/b chain on the surface of Ts [9 –11]. TCR molecules are composed of covalently linked variable a and b chains which are non-covalently associated with the CD3 complex [12]. The genes encoding the a and b chains undergo somatic recombination in a manner similar to immunoglobulin molecules [13]. The TCR b chain is produced by the recombination of V, D, J, and C segments. This diversity is further increased by the nibbling of germline nucleotides and/or addition of nucleotides at the V-D-J junction sites [14]. The complementarily determining region 3 (CDR3), encompassing the V-D-J junction, displays the most extensive nucleotide diversity and is thought to encode a region involved in the contact of the TCR with the antigenic peptide [15]. In this report, we present results of the analysis of the TCR Vb genes expressed by CD81CD282 T suppressor lymphocytes derived from allospecific, xenospecific and antigen-specific TCLs. We have established that Ts ex-
press a limited Vb repertoire and that the most restricted repertoire was expressed by xenospecific Ts. MATERIALS AND METHODS Human Cells Blood was obtained from six blood donors (PR, BN, NB, MS, HM, and ES) who were typed for HLA class I and class II antigens by conventional serology and genomic typing by PCR-SSO. Table 1 summarizes the HLA class I phenotype and class II genotype of individuals used in these studies. Swine Cells Blood was obtained from one outbred pig and from Yucatan Miniature Swine of strain Q and Z (Sinclair Research Center, Columbia, MO) which carry different MHC class I and class II genotypes as described [16 –18]. Antigens Recombinant Tetanus Toxoid (rTT) C fragment was obtained from Boehringer Mannheim (Indianapolis, IN) and conjugated to carboxylated polystyrene microparticles (Latex beads) (Polysciences Inc., Warrington, PA), according to the manufacturer’s instructions. Generation of Allospecific and Xenospecific T Cell Lines Four allospecific TCLs (PR anti BN, NB anti BN, NB anti MS and HM anti MS) and three xenospecific TCL (HM anti outbred pig, ES anti strain Q swine, ES anti strain Z swine) were generated as previously described [9, 19] (Table 2). Briefly, human and pig PBMCs were separated from buffy coats by Ficoll-Hypaque centrifugation. Human PBMCs, used as responding cells, (1 3 106/ml) were stimulated in 24-well plates with an equal number of irradiated (1600r) stimulating cells (PBMCs) from an allogeneic (human) or xenogeneic (swine) donor. Cells were co-cultured for 7 days in complete medium (RPMI 1640 supplemented with 10% heat-inactivated
TABLE 2 CD81CD282TCLs studied and their functional characterization
TCL 1 2 3 4 5 6 7 8
Human responding cells from: ES ES HM HM NB NB PR PR
Source of stimulating cells Allogeneic
Xenogeneic
Autologous
No. of stimulation cycles
% Suppression of the reactivity
rTT*
2 2 2 2 2 2 2 3
76% 73% 50% 65% 72% 88% 50% 37%
Pig strain Q Pig strain Z Outbred pig MS MS BN BN
* T cells from individual PR were primed to rTT in the presence of autologous APCs.
TCR Repertoire of Ts Cells
FIG. 1. TCR Vb repertoire of naive CD81CD282 T lymphocytes from individuals NB (panel A), ES (panel B) and PR
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(panel C). The spectratypes show that all the Vb families are represented with relative intensity ranging from 0.001 to 0.15.
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FIG. 2. TCR Vb repertoire of CD81CD282 T lymphocytes from allospecific (panel A), xenospecific (panel B) and nominal antigen specific (panel C) TCLs. Study of the TCR Vb usage
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of Ts shows that only a few Vb families were expressed by CD81CD282 cells with relative intensity higher than 0.1. Xenospecific Ts express the highest degree of oligoclonality.
TCR Repertoire of Ts Cells
FIG. 3. Profile of the fluorescent Vb-Cb runoff products corresponding to the most represented Vb families in the spectratypes of Ts derived from allospecific TCL NB anti BN (panel A), xenospecific TCL ES anti Q (panel B) and nominal antigen specific TCL PR anti rTT (panel C) TCLs. The x-axis represents the length of the Vb-Cb segments in base pairs and the y-axis represents the fluorescence intensity in arbitrary units.
fetal calf serum, 2 mM glutamine, and 50 mg/ml gentamicin). Responding cells were restimulated at seven day intervals in medium containing 10U/ml rIL-2 (Boehringer Mannheim, Indianapolis, IN) [9, 10].
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Generation of Antigen-Specific T Cell Lines PBMCs (2 3 106/ml) from a healthy blood donor (PR) were stimulated in 24-well plates with 1 mg of rTTconjugated to latex beads in RPMI 1640 medium supplemented with 10% human serum (Sigma Chemical Co, St. Louis, MO), 2 mM L-glutamine and 50 mg/ml gentamicin (Gibco, Grand Island, NY), as described [11]. On day five, 20 U/ml of rIL-2 (Boehringer Mannheim, Indianapolis, IN) were added. Ten days after priming, T cells (2 3 106) were collected, washed and restimulated with antigen in medium containing 20 U/ml of rIL-2 and irradiated (3000r) autologous PBMCs (2 3 106).
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FIG. 4. Ts spectratypes of two allospecific TCL (NB anti BN and NB anti MS) derived from the same responder.
Antigen specific T cell lines were obtained after two or three restimulations [11] (Table 2). Cell Separation CD41 and CD81 T cells were separated from TCLs by negative selection using Dynal CD4 and CD8 magnetic beads. CD81CD282 T cell suspensions were prepared by depletion of CD281 T cells from purified CD81 T cell suspensions [9 –11]. The purity of the suspensions was monitored by cytofluorometry. Cells were rerosetted with CD28 beads when necessary, to obtain a population containing less than 2% CD281 bright cells. Proliferation Assay CD41 T cells (5 3 104/well) from TCL primed to allogeneic, xenogeneic or Tetanus Toxoid-loaded autologous APCs were stimulated with an equal number of irradiated allogeneic, xenogeneic or autologous APCs in 96-well plates. CD81CD282 Ts (5 3 104 /culture) from the same TCL were added to replicate cultures. After 48 h of incubation, the cultures were pulsed with 3 H[TdR] and harvested 18 h later. 3H[TdR] incorporation was determined by scintillation spectrometry in a LK Betaplate counter. Mean cpm of the triplicate reactions and the standard deviation to the mean were calculated [9 –11].
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The percent suppression of CD41 Th proliferation in the presence of CD81CD282 Ts was calculated as: 12
cpm in Th 1 Ts 1 APC cultures cpm in Th 1 APC cultures
TCR Spectratyping Total RNA was extracted using QIAGEN columns (Qiagen Inc., Valencia, CA) from CD81CD282 Ts and from CD41 Th. RNA was reverse transcribed into cDNA in a reaction using MMLV reverse transcriptase primed with oligo (dT)18 (Clontech Laboratories Inc., Palo Alto, CA), as recommended by the manufacturer. Aliquots of the cDNA synthesis reaction were amplified in 50 ml of the 24 Vb oligonucleotides (0.5 mM final concentration) and the Cb oligonucleotide (0.5 mM final concentration). Vb and Cb primers were used as previously described [19]. As an internal control for the cDNA amount used per reaction, a tube containing sense and antisense primers for the first exon of Cb region was included. Two microliters of the Vb-Cb PCR products were subjected to elongation with a fluorophore-labeled Cb specific primer (0.5 mM final concentration). The size and the fluorescence intensity of labeled run off products were determined on a 377 DNA sequencer (Perkin Elmer Applied Biosystem Division, Foster City, CA) and analyzed using the ABI PRISM 377 GENESCAN analysis program (Perkin Elmer Applied Biosystem Division, Foster City, CA).
TCR Repertoire of Ts Cells
FIG. 5. Ts spectratypes of two xenospecific TCL (ES anti strain Q and ES anti strain Z) derived from the same responder.
The relative intensity of each Vb family was calculated as the peak areas corresponding to each Vb family divided by the sum of all the peak areas of all the Vb families [14]. The sharing rate of Vb families in two different spectratypes was calculated as number of shared families divided by number of families present in the two spectratypes. The statistical significance of differences among sharing rates was calculated by the Fisher test. Vb-Jb Gene Segment Analysis Two microliters of the Vb-Cb PCR products, corresponding to the Vb families with the highest relative intensity in each spectratype, were subjected to elongation with 13 fluorophore-labeled Jb specific primers [19]. The size and the fluorescence intensity of the labeled run off products were determined on a 377 DNA sequencer as previously described. The relative intensity of each Vb-Jb gene segment was calculated. RESULTS Functional Characterization of Ts We have previously shown that CD81CD282 Ts recognize MHC class I/peptide complexes and inhibit the ability of CD41 Th to react against MHC class II/
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peptide complexes expressed by the APCs used for priming [9 –11]. CD81CD282 T cells from each of the TCL included in this study were tested for their ability to inhibit the proliferative response of CD41 Th from the same TCL in 3-day blastogenesis assay. Table 2 summarizes the results. Xenospecific Ts (TCL 1, 2, 3) inhibited Th reactivity to swine MHC-class II antigens by 50 to 76% (Table 2). Inhibition was specific since Ts primed to strain Q did not suppress the reactivity of Th primed to strain Z or vice versa [10]. Similarly, allospecific Ts (TCL 4, 5, 6, 7) suppressed Th reactivity to the specific stimulator by 50 – 88% (Table 2). Ts from TCL 8 recognize Tetanus Toxoid peptides presented by MHC-class I antigens expressed by autologous APCs [11]. Spectratyping Study of the TCR Vb gene repertoire expressed by allospecific, xenospecific and nominal antigen-specific Ts shows an oligoclonal expansion of Vb families. As illustrated in Fig. 1, before priming, CD81CD282 T lymphocytes express all the 24 Vb families with relative intensity ranging from 0.001 to 0.15. After multiple stimulations with either allogeneic, xenogeneic or rTTloaded-self APCs, the expression pattern of Vb gene segments changes dramatically. The Ts population of each TCL expresses only a few Vb families (3–5 per spectratype) at a relative intensity level greater than 0.1 (Fig. 2). Other families found within the naive population of CD81CD282 T cells from the same blood donor are either absent or expressed with a relative intensity
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FIG. 6. Comparison between the TCR Vb repertoire of allospecific and xenospecific TCL from the same responder.
lower than 0.05 following multiple priming. In each TCL a unique TCR Vb family seems to prevail in terms of relative intensity. For example, Vb19 has the highest relative intensity (RI 5 0.38) in the alloreactive TCL NB anti BN (TCL 6), while Vb23 and Vb5 have the highest RI in TCL ES anti-Q (TCL 1) and PR anti-rTT (TCL 8) respectively (Fig. 2, panel A, panel B, and panel C). The TCR repertoire used by xenospecific CD81CD282 T lymphocytes (Fig. 2, panel B) is more restricted than the repertoire of allospecific TCLs (Fig. 2, panel A) To study the clonal expansion of particular TCR Vb families within a spectratype, we analyzed the profile and peak distribution of Vb-Cb fragments of each TCR Vb family. Prior to antigenic stimulation, human PBMCs exhibit 6-8 peaks spaced by 3 nucleotides corresponding to in-frame transcripts of the TCR b chain CDR3 region. The area under each peak is proportional to the amount of transcripts of the corresponding CDR3 size in the sample. Each peak corresponding to the length of a given CDR3 usually contains multiple distinct sequences. An increase in the height and area of a peak that modifies the bell-shaped CDR3 size distribution indicates oligoclonal or monoclonal expansion occurring after antigenic stimulation [14]. In the allospecific Ts population, the distribution of peaks within each Vb family was prevalently
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gaussian-like, suggesting that certain Vb-Cb combinations are broadly used by different TCRs (Fig. 3, panel A). In the xenospecific CD81CD282 population, the shape of the TCR Vb families was mono- or bi-modal (with the exception of Vb23) suggesting that only a few subfamilies of Vb genes are used (Fig. 3, panel B). Similarly, only few TCR Vb subfamilies seem to recognize epitopes of the rTT molecule are indicated by the presence of mono- or bi-modal peaks within the spectratype of Tetanus Toxoid specific Ts (Fig. 3, panel C) To determine whether the Vb repertoire of Ts is influenced by the responder’s or stimulator’s HLA-DR antigens, we compared the spectratypes of: a) TCLs generated by stimulating the T cells from the same responder with APCs from different stimulators and b) TCLs generated by stimulating TCLs from different responders with APCs from the same stimulator. When T cells from the same responder (NB) were stimulated with APCs from two HLA-DR different individuals (BN and MS), 36% of the Vb families appeared to be shared (Fig. 4). Similarly, when T cells from a single responder (ES) were stimulated with APCs from two SLA disparate swine (strain Q and Z), 37.5% of the Vb families seen in the spectratype were shared (Fig. 5). Furthermore, 28.5% of Vb families were shared by allospecific and xenospecific Ts generated by stimulating T cells from the same responder (HM) with human and pig APC respectively (Figs. 6 and 7), and 43% were shared
TCR Repertoire of Ts Cells
FIG. 7. TCR Vb gene usage in allospecific and Tetanus Toxoid specific Ts from a single individual (PR).
among allospecific and rTT-specific Ts obtained from the same individual (PR). However, comparison of the spectratype of TCLs obtained from different responders (NB and HM) stimulated with the same allogeneic APCs (MS) also revealed a sharing rate of 27% (Fig. 8). Hence, the rate of TCR-Vb gene sharing among Ts from different individuals primed to the same antigen is not different from the FIG. 8. TCR-Vb repertoire of allospecific Ts obtained by priming T cells from two different responders with APCs from the same stimulator.
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sharing rate seen among Ts from the same individual primed to different antigens. The extent to which the recipient’s MHC genotype and the source of antigenic stimulation influence TCR Vb usage Ts population, remains unclear. Because Th recognize peptide/MHC-class II complexes while Ts recognize peptide/MHC class I complexes on the same APC, the TCR Vb repertoire of CD41 Th and CD81CD282 Ts from the same TCL is expected to be different. Although Th and Ts from the same line shared less than 50% of TCR-Vb families, certain Vb genes were used only by Ts or by Th consistent with the notion that they recognize distinct antigenic determinants. Comparison of the TCR repertoire used by CD81CD282 Ts with that used by
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FIG. 9. Comparison between TCR Vb repertoires of allospecific (panel A), xenospecific (panel B), and nominal antigen specific (panel C) Th and Ts from the same TCL subpopulations showed a not coincident TCR Vb usage.
CD81CD281 T cells from the same TCL showed although certain TCR families are shared, Ts lack certain families which are present in the population of allospecific CD81CD281 T cells.
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Vb-Jb Gene Segment Analysis To further analyze the Vb repertoire of Ts, we studied the Vb-Jb gene segment distribution within prevailing TCR Vb families. The distribution of Vb-Jb gene segments within each TCR Vb family has a gaussian-like profile in naive PBMCs while in Ts cell lines it is unior bi-modal (data not shown). In general, modifications of the bell-shaped pattern mirror oligoclonal expansion
TCR Repertoire of Ts Cells
FIG. 10. Comparison of the TCR Vb repertoire expressed by allospecific CD81CD281 and CD81CD82 T cells from the same TCL.
of particular subfamilies or preferential usage of a Jb gene variant. The relative intensity of each peak in the Vb-Jb spectratype is the measure of the frequency of each Vb-Jb recombination fragment [14]. In Ts cell lines only a few Vb-Jb combinations are represented (Figs. 9 –11). The most restricted Vb-Jb repertoire is displayed by TCR Vb families from xenospecific Ts lines. The Vb-Jb repertoires found in different Ts cell lines are not superimposable, indicating that no Jb1 or Jb2 gene variants are preferentially selected by Ts (Fig. 11). DISCUSSION The molecular mechanism of T cell suppression is still unknown. It has been recently shown that xenospecific, allospecific and nominal antigen-specific human Ts can be generated in vitro by multiple T cell stimulations [9 –11]. Ts generated in this manner carry the CD81 CD282 phenotype, recognize specifically MHC-class I antigens expressed by the APCs used for priming, and are able to suppress T helper cells reactivity to MHCclass II/peptide complexes on the same APCs. The suppressive effect was shown to result from downregulation of CD40, CD80, and CD86 costimulatory molecules on the surface of APCs exposed to Ts [9 –11]. In this report we have analyzed and compared the TCR Vb usage in CD81CD282 Ts from allospecific, xenospecific and nominal-antigen specific TCLs.
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An expansion of a limited number of families was observed in all TCL regardless of the antigen used for Ts priming. This observation is reminiscent of previous reports showing that the CD81CD282 population of lymphocytes present in human peripheral blood is oligoclonal [20, 21] These cells were shown to have relatively shorter telomeres, compared to other lymphocyte subsets, and to be unable to proliferate in culture, suggesting that they are aging, terminally differentiated cells, marked for apoptosis [20, 21]. In contrast to these studies our data indicate that the CD81CD282 subset of T lymphocytes comprises an oligoclonal population of Ts which can be readily primed and expanded in vitro [9 –11]. Since Ts inhibit Th reactivity in vitro in an antigen-specific manner it is likely that they play an important role as regulators of the immune response to both nominal and self antigens in vivo [11]. We found that CD81CD282 and CD81CD281 T cells from the same TCL display a superimposable oligoclonal repertoire, suggesting that they derive from the same precursor [24]. However, the Ts population lacks certain TCR Vb families which are present among the population of CD81CD281 T cells, known to display cytotoxic function [25, 26]. Hence, suppressor and cytotoxic T lymphocytes differ not only functionally but also with respect to the TCR Vb genes which they use to recognize MHC-class I/peptide complexes. The comparison between the TCR repertoire expressed by CD81CD282 T cells with suppressor activity and CD41 T helper lymphocytes indicated that most of the Vb families present in the two subsets were different. This finding suggests that the antigenic determinants recognized by Ts and Th are not the same. Comparison
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FIG. 11. Jb usage of the most frequent Vb families in allospecific HM anti MS (panel A), xenospecific ES anti Q (panel B) and nominal antigen specific PR anti rTT (panel C) T suppressor TCLs. Only few Jb-Vb recombination fragments were present in these cell lines.
of allospecific, xenospecific, and nominal antigen specific Ts cell lines showed that xeno-specific Ts display the highest degree of oligoclonality. It is possible that, only few TCRs are able to recognize the Ag/SLA complexes on the surface of the pig APCs due to the extent of the antigenic differences between these species [27–30]. TCRs engage in xenoreactivity should have a favorable
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TCR-Ag/SLA complex association/dissociation rate in order to activate the suppressor activity of the cells [31, 32]. An alternative explanation for the skewed TCR repertoire found in xenospecific Ts lines stems from the molecular incompatibility between certain human and swine co-receptor and accessory molecules which are involved in the immune response [33–36]. Beside the signal through the TCR, activation of T cells generally requires a costimulatory signal provided by the ligation of one of the various accessory molecules expressed by T cells. It is possible that if the co-receptors on the surface
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of human T cells are not able to fully interact with their counter-parts on pig APCs, the signal that leads to the T cell activation will be provided by only few TCRs, with high avidity for Ag/SLA complexes. Thus, the self-MHC driven selection of T cells may lead to a T cell repertoire, which recognizes only closely related MHC structures. MHC class I restricted Ts may play a physiological role in regulating the immune response of Th cells against self or non-self peptide MHC class II complexes. The finding that there is a cross-talk between the MHC class I and class II pathway of peptide processing supports the notion that the same APCs present both helper and suppressor inducing peptides. It is possible that recognition by Ts of MHC class I bound peptides is play a role in controlling the activation of antigen-specific Th cells. Identification of suppressor-inducing peptides may be useful for induction of unresponsiveness to auto-, alloor xenoantigens. Furthermore, the ability to generate Ts cells after exposure to antigen in vitro may be an advantage for further functional analysis and identification of new potential therapeutic strategies for autoimmune diseases and graft rejection. REFERENCES 1. Gershon RK, Kondo K: Cell interactions in the induction of tolerance: the role of thymic lymphocytes. Immunol 18:723, 1970. 2. Groux H, O’Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, Roncarolo MG: A CD41 T-cell subset inhibits antigen-specific T-cell responses and prevent colitis. Nature 389:737, 1997. 3. Han HS, Jun HS, Utsugi T, Yoon JW: A new type of CD41 Suppressor T cell completely prevents spontaneous autoimmune diabetes in syngenic islet-transplanted NOD mice. J Autoimmunity 9:331, 1996. 4. Noble A, Pestano GA, Cantor H: Suppression of immune responses by CD8 cells. I. Superantigen-activated CD8 cells induce unidirectional Fas-mediated apoptosis of antigen-activated CD4 cells. J Immunol 159:539, 1998. 5. Noble A, Zhao ZS, Cantor H: Suppression of immune responses by CD8 cells. II. Qa-1 on activated B cells stimulates CD8 cell suppression of T helper 2 responses. J Immunol 160:566, 1998. 6. O’Hara RM: Antigen-specific suppressor factor: missing pieces in the puzzle. Immunol Res 14:252, 1995. 7. Horwitz DA, Dixon Gray J, Ohtsuka K, Hirokawa M, Takahashi T: The immunoregulatory effects of NK cells: the role of TGF-b and implications for autoimmunity. Immunol. Today 18:538, 1997. 8. Groux H., Bigler M., de Vries JE, Roncarolo MG: Inhibitory and stimulatory effects of Il-10 on human CD81 T cells. J Immunol 160:3188, 1998.
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