Eur. J. Immunol. 1992. 22: 641-645
Lawrence R. Smith., Dwight H. Kono, Michael E. Kammullern, Robert S. Balderas and Argyrios N. Theofilopoulos Department of Immunology, The Scripps Research Institute, La Jolla
Expressed Vg repertoire in rats
641
Vp repertoire in rats and implications for endogenous superantigens* Endogenous superantigens of mice, encoded by mammary tumor virus proviral integrants, induce intrathymic deletion of entire T cell populations that express specific Vg gene products, a phenomenon proposed to be important in selftolerance and prevention of toxic responses to exogenous microbial superantigens. Evidence for the presence of Vg selecting/deleting endogenous superantigens in other species is 1acking.We report here that rats do not exhibit endogenous superantigen-induced Vp clonal deletions despite their strong response to bacterial superantigens. These findings indicate that endogenous superantigens are not obligatory in Vs repertoire shaping.
1 Introduction Superantigens are potent T cell mitogens which, unlike conventional antigens, engage specificT cell antigen receptor (TcR) Vp gene products regardless of the other TcR components (Dg/Jg,V,/J,) and bind MHC class I1 molecules outside the antigen-binding groove (reviewed in [1,21). Superantigens alter the normal distribution of the various mouse Vp-bearing T cell clonotypes either by stimulating clonal expansion if met late in life by the matureTcells, or by mediating clonal deletions if met at the neonatal stage during formation of the repertoire in the thymus (reviewed in [3]). Two classes of superantigens have been identified; endogenous superantigens expressed by certain mouse strains, previously known as minor lymphocyte-stimulating antigens (Mls), and recently shown to be encoded by mouse mammary tumor virus proviral integrants [4-61, and exogenous superantigens, products of certain bacteria [7, 81, mycoplasmas [9], or infectious retroviruses [lo]. Clonal deletion induced by endogenous superantigens is thought to represent the main mechanism by which tolerance to these self molecules is established [l-31. Moreover, the strikingly similar properties of exogenous and endogenous superantigens, including considerable overlap in specificV6 gene products engaged, have led to the hypothesis that these two types of superantigens have co-evolved [3]; if so, early-life intrathymic clonal deletion of immature T cells induced by endogenous superantigens will prevent the deleterious consequences of clonal expansion of mature T cells by cross-reacting microbial superantigens later in life [3, 111.
As noted above, recent studies have linked endogenous superantigens to several mouse type B mammary tumor proviral genes integrated on various chromosomes [4-61. Since mammary tumor viruses (MTV) are mouse viruses, the possibility exists that endogenous superantigens and the accompanying clonal deletion they elicit are unique to mice. Therefore, we addressed the question of whether endogenous superantigen expression is an integral characteristic of Tcell development by studying the mature TcR Vg repertoire in the rat, a phylogenetically close species. The recent cloning in our laboratory of rat Vg (rVg) genes having on average 89% nucleotide similarity to every mouse Vg (mV,) gene [12], facilitated this analysis and permitted direct comparison to known somatic deletions of T cells expressing mouse counterpart genes.
2 Materials and methods 2.1 Rat strains Nine strains representing several MHC haplotypes were included: Brown Norway (BN), RT1"; Buffalo, RTlb; DA, RTla; Fisher 344N and Lewis, RT1'; PVG, RTIC;Sprague Dawley (SD), outbred; Wistar Furth (WF) and diabetesprone Bio-Breeding (BB), both RTIU[13-161. All strains expressed RT1.D (35%,-60% of total spleen cells), the rat class I1 IE counterpart, as assessed by staining with OX-17 (Serotec, Indianapolis, IN). Lewis and BN strains were from the Scripps Animal Facility; PVG and DA from Bantin a n d K i n g m a n ( F r e m o n t , CA); WF, SD and Fisher from Harlan Sprague Dawley (Indianapolis, IN), and BB from the University of Massachusetts (Worcester, MA).
[I 99661 2.2 Tcell subset isolation *
This is Publication no. 6838IMM from the Department of Immunology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road/IMM3, La Jolla, CA 92037. The work reported herein was supported, in part, by grants from the National Institutes of Health. Recipient of an Irvington Institute Fellowship. A On sabbatical leave from Sandoz Pharmaceuticals.
Correspondence: Argyrios N . Theofilopoulos, Department of Immunology, The Scripps Research Institute, 10666 North Torrey Pines Road/IMM3, La Jolla, CA 92037, USA 0 VCH Verlagsgesellschaft mbH, D-6940 Weinhcim, 1992
Single-cell suspensions from thymocytes and spleens were prepared by dispersing cells through wire mesh. Seventy to 80% of total thymocytes used in this study were doublepositive (CD4+CD8+). Splenic CD4 and CD8 singlepositive cells were purified with W312.5 and OX-8 mouse monoclonal antibodies (Serotec) , respectively, and antimouse Ig-conjugated magnetic beads (Dynel, Great Neck, NY). Due to severe lymphopenia in the BB strain, total splenocytes (rather than separated single-positive populations) were analyzed. 0014-2980/92/0303-0641$3.50+ .25/0
L. R . Smith, D. H. Kono, M. E. Kammuller et al.
642
Eur. J. Immunol. 1992. 22: 641-645
2.3 RNase protection assay (RPA)
2.5 In vitro stimulation of T cells by toxins
Rat-specific RPA probes were prepared by using oligonucleotide primers derived from sequences of 20 Lewis rat Vg genes [12]. These PCR products were subsequently ligated to the riboprobe plasmid vector pGEM-7zf (Promega, Madison, WI) and sequenced to confirm product. The probes ranged in size from 90-285 base pairs. Miniprep plasmid DNA prepared for each rat Vp riboprobe was linearized and pooled into two-probe sets (Fig. 1) and used as templates for synthesizing antisense RNA with T7 RNA polymerase (Promega) as described [17, 181. Probe sets were labeled to 5000 cpmluridine residue and hybridized for 12 h at 56°C with 2 pg of RNA, prepared as described [18], from total thymocytes or 6-8 pg of RNA from magnetic bead-attached, single-positive splenic T cells. After RNase A (5 pg/ml) and T1 (10 U/ml) treatments [17, 181, “protected” probe : target mRNA duplexes were phenol/chloroform extracted, ethanol precipitated, and electrophoresed on a standard 6% polyacrylamide sequencing gel. Dried gels were autoradiographed on Kodak XAR film with intensifying screens at -70°C for 4-24 h and quantified as described below. These probes will be available for research purposes to interested parties upon request.
Spleen cells from Lewis rats (2.5 x lo6) were incubated with 1 pg/ml of specific staphylococcal enterotoxins (SE; Toxin Technologies, Sarasota, FL) in RPMI 1640 supplemented with 10% fetal calf serum, 2mM L-glutamine, 50 p~ 2-mercaptoethanol and antibiotics. After 4 days, cells were given 50 U/ml rIL2 (Genzyme, Boston, MA) for 1 day, blasts harvested by discontinuous gradient centrifugation (Histopaque 1077, Sigma, St. Louis, MO), and total RNA prepared.
2.4 Quantitation of relative rat Vg RNA levels Radioanalytic profiles were obtained by direct fi scanning of gels using an Ambis Radioanalytic System (Ambis, San Diego, CA).The net cpm of a given band corresponding to a specific protected Vg probe were calculated by the formula: (cprn Vg - cpm background)/no. uridine bases; RNA amounts were normalized between probe sets using Vp2. Vg levels were expressed as percent of the total protected Vg instead of percent of Cg since thymocytes contain large numbers of sterile transcripts (1.O-kb Cp species containing no Vf3). Cp was used to normalize between probe sets when splenic T cells were stimulated in vitro with toxins.
vp
I I 2 17
I I I I
6 16125.1
u 11
IU 13 8.1
2.6 Neonatal tolerance induction Lewis neonatal rats were injected i.p. within 12 h after birth with 5 pg SEA and every other day thereafter for 2 weeks. Total splenocyte RNA was isolated from these and uninjected control littermates, and 20 pg of RNA was subjected to RPA analysis.
3 Results and discussion A sensitive and specific rVp multiprobe set RPA was developed for quantitating Vg expression levels since a collection of rVg-specific antibodies is currently unavailable. Application of this assay with 2-probe sets encompassing 20 of the 24 known rVg genes [12] with RNA from total thymocytes is shown in Fig. 1. As revealed by the indicated autoradiographic and radioanalytic profiles, clear-cut bands or peaks, respectively, corresponding to each of the Vg analyzed are easily identifiable. As in mice [17, 181 and humans [19], the transcript levels for the various Vg in unselected thymocytes are unequal, varying from = 1% to = 12%. Eight of the assessed rVp have counterparts known to be engaged by endogenous superantigens in mice (Vg5.1, 6, 8.1, 9, 11, 12, 16 and 17) [3]. Although mVg3.1 engages Mlsc, the rVg counterpart is a pseudogene in the Lewis, DA and BB strains ([12] and unpublished data) and was not examined here. Mouse Vg clonal delections are readily
u
I
18
8.3
CP
Set 2 I I1
vp
3.3 219
I 4
I U I I 1514 10 20
I 8.2
U
9
cP
Figure I . Rat Vg RPA. Autoradiographic and radioanalytic profiles show Vp size-specific “protected” RNA bands or peaks, respectively, for rV,j genes assayed as 2-probe sets.
Expressed Vg repertoire in rats
Eur. J. Immunol. 1992. 22: 641-645
observed by RPA when Vp transcript levels in total thymocytes are compared to those in peripheral (splenic) lymphocytes of a given IE+/superantigen+ strain [17, 181. This result is based on the fact that total thymocytes contain predominantly (= 80%) immature double-positive (CD4+CD8+)T cells, while the spleen contains only singlepositive (CD4+CD8- and CD4-CD8+) T cells representing the mature TcR repertoire. Since all rat strains analyzed here are RT1.Df (the rat equivalent of mouse IE) [14], characterization of repertoire shaping was, therefore made by comparing total, mostly unselected, thymocytes vs. splcnic CD4 and CD8 populations. Fig. 2 reveals thymic and splenicVg transcript levels in nine rat strains of various backgrounds and MHC haplotypes. In contrast to endogenous superantigen-expressing mouse strains that have RNA levels for specificVp of 1%-25% of the respective levels in the thymus [17, 181, no evidence of such striking Vg clonal deletion was apparent for any Vg of the nine rat strains surveyed. However, transcript levels for
643
some Vp were either lower or higher in the single-positive mature splenic T cell subsets compared to total, mostly immature, thymocytes. Upward or downward shifts were, in most instances, of the “subset bias” type (affecting one or the other of the single-positive subsets), but, in some instances, both subsets were affected. Such somatically imposed repertoire modifications may be the result of negative selection or absence of positive selection (in the case of underexpression, i.e. Vp14) or caused by enhanced positive selection (in the case of overexpression, i. e. Vp8.1 and 9) and may be dependent on MHC, composition of self-peptide antigens and preferential pairing of certain Vg and V, gene products. Nevertheless, the results clearly indicate that these rat strains do not manifest the profound endogenous superantigen-induced somatic deletions of cells expressing specific Vp genes homologous to those known to be deleted in the mouse. We next wished to determine whether rVp are capable of engaging superantigens by taking advantage of the Vp
20
BN
I Thymus
= CD4+ eza CD8+
15
Buffalo1
DP
i
10
5
0
i
20
g
-
Fisher
PVG
I
15
0
c)
0
I-
’0
r
0
SD 15
1
VO Genes Figure 2. Relative VBexpression levels for nine rat strains. RPA were performed as in Fig. 1 using 2 bg total thymocyte RNA or 6-8 pg purified splenic CD4 or CD8 RNA. An Ambis radioanalytic imaging system was used for quantitations of protected Vp bands. The nct cpm of a given band corresponding to a specific protected Vg probe were calculated by the formula cpmVp - cpm backgroundho. uridinc bases, and RNA amounts between the two-probe sets were normalized using V,-;2.Vplevels were expressed as percent of the total protected Vg cpm.The results shown are derived from at least two independent assays using RNA from a pool of two to three 4-week-old female rats. Due to strain specific polymorphisms,V~~8.2, 13 and 19 were not quantitated for the DA or Fisher 344N strains (nor wasVg9 in these two strains due to interference by a secondary product from one of these polymorphic genes), and for the same reason.Vp4 and 16 were not quantitatedfor the BN, Buffalo and WFstrains.Vp3.,1 was also a pseudogene (2 stop codons) in the Lewis [12], DA and BB (unpublished data) rats and thus was not assayed in any of the indicated strains. Due to sevcre lymphopenia in the diabetes-prone BB rat, total splenocytes (rather than separated CD4 and CD8 cells) were asscssed.
L. R. Smith, D. H. Kono, M. E. Kammuller et al.
644
Eur. J. Immunol. 1992. 22: 641-645
Table 1. Comparison of rat and mouse Vp gene products engaged by bacterial toxinsa) Toxin
vb gene Droducts
Species
1 SEA SED
r m r
SEB
m r
+ + (+)
r
m SEC2
ExFT TSST-1
7
8.1 8.2 8.3
+ +
m SECl
3
(+)
+
+ + + + + + +
10
11
12
14
33
25
12
+ + + 64 + 13 (+) 14
+
+ 45 + +
m r m r
Probe Set 2
3.3-
4-
1020 138.1-
9 I
16
17
19
19
11
+ 19 +
8.2-
9 I
Figure 3. Induction of Vlj clonal deletions by neonatal administration of a bacterial toxin. Lewis neonate rats (within 12 h after birth) were injected i.p. with 5 pg SEA every other day for 2 weeks. Total splenocyte RNA was isolated from these and uninjected control littermates and 20 pg subjected to RPA analysis. The 2-probe set autoradiographic profiles with splenic RNA from a representative treated and control rat are shown.
9
78
(+I 68
11
7
’*
37
r 45
Probe Set 1
6-
15
+ 100 + +
55
a) Transcript levels for rVg are expressed as percent of total Vp transcripts; values > 5% are listed. rVg not assessed in these experiments areVpl, 3.1,5.2,7, 8.3 and 18. mVg reactive with toxins are derived from [3] and are indicated by f ; reactivities for mVp listed in parentheses have been reported to occur with commercial preparations, but not with recombinant sources of enterotoxins 1.31.
selecting activities of several SE [3, 7, 81. As shown in Table 1, in all instances, only certain rVp transcripts were highly represented after cells were cultured for 5 days with the indicated toxins,while the remainder represented < 5% of the total.When rat and mouseVp gene products engaging these toxins were compared, high concordance was observed for V,Jl,whileV@ and VplO were discordant.The rVpl9 and the mVp7 are also frequently selected by SE, but the engagement of the mouse and rat counterparts, respectively, has not been tested. Previous studies implicated amino acid residues 67-87 of human Vg13.2 [20] as important for binding of SEC2 and residues 22 and 70-71 of a mouse Vp8.2 variant for binding of Mls” [21]. When the amino acid sequences at these regions were compared between the homologous pairs of mouse and rat Vp genes that were concordant (Vpll, -15) or discordant (Vp8, -10) for toxin reactivities between these two species, no commonalities in primary structure distinguishing these groups were evident (data not shown). This finding suggests that other amino acid positions within spatial proximity to the previously identified binding sites may also play determining roles in toxin binding. Nonetheless, if one assumes that t h e binding sites for exogenous and endogenous superantigens are comparable [20, 211, then these data support the notion that rVB structures can interact with superantigens but rats, unlike mice, do not appear to express endogenous superantigens. To substantiate the above conclusion further, rats were also testedfor their capacity to delete clonally in vivoTcells that engage SEA to verify that neonatal tolerance to exogenous superantigens was indeed physically operative in this rodent. Lewis rat neonates (1 day old) were injected with 5 pg of SEA every other day for 2 weeks, as previously described in mice [7]. The results revealed decreases of 50%0-75%0 in transcript levels for rVgl1, -12, -17 and -19 (Fig. 3 ) , the sameVp shown to be engaged by this toxin in the in vitro testing. Transcript levels for the remaining Vp, apart from some minor compensatory changes, remained
Expressed Vg repertoire in rats
Eur. J. Immunol. 1992. 22: 641-645
essentially unaltered between these two groups. These data further strengthen the contention that despite the similar in vitvo and in v i v o behavior of rat and mouse Tcells to bacterial superantigens, the rVp repertoire does not appear to be constrained by endogenous superantigens. Despite the great similarities between mice and rats with regard to genomic TcR and MHC composition, and similarities in life-spans, generation times and environments, the absence of endogenous superantigen-mediated intrathymic Vp clonal deletions in rats suggests that different evolutionary pressures have shaped the expressed Vp repertoires of these species. Although not as thoroughly analyzed, humans, thus far, have also been found to be devoid of such phenomena [19]. The cumulative data, therefore, suggest that somatically imposed Vp clonal deletions by endogenous superantigens are unique to mice.This uniqueness is most easily explained by the restriction of MTV to this species. MTV proviral genes encode, presumably through their open reading frame (OW) gene [22, 231, the previously undefined Mls molecules as well as other Vp selecting/deleting self superantigens. The reasons why these type B retroviruses contain, uniquely of all known retroviruses [24], an ORFgene, and why the host retains MTV sequences in its genome, are unclear at present, but several speculations have been made [5-7, 10, 22, 231. The absence of clear-cut evidence for Vp clonal deletions in other species suggests that endogenous superantigenrelated Vp clonal deletions may have evolved as a unique characteristic of mice imposed by the life cycle of MTV, and are not obligatory components in Vp selection. Furthermore, although clonal deletions induced in mice by endogenous superantigens may diminish deleterious consequences by cross-reacting bacterial superantigens, the documentation that endogenous superantigens are most likely not expressed in other species (rats, humans) that nevertheless respond to bacterial superantigens makes it clear that virally encoded endogenous superantigens and bacterial superantigens need not to have co-evolved. We would like to thank Drs. E Peterson, J. Sprent, S.Webb and N. Gascoigne for critical review. Received September 20, 1991; in revised form November 14, 1991.
645
4 References 1 Blackman, M. A., Kappler, J.W. and Marrack, P., Science 1990. 248: 1335. 2 Janeway, C. A., Nature 1990. 349: 459. 3 Herman, A., Kappler, J. W., Marrack, P. and Pullen, A . M., Annu. Rev. Immunol. 1991. 9: 745. 4 Frankel,W. N., Ruby, C., Coffin, J. M. and Huber, B.T., Nature 1991. 349: 526. 5 Woodland, D., Happ, M. P., Gollob, K. J. and Palmer, E., Nature 1991. 349: 529. 6 Dyson, P. J., Knight, A. M., Fairchild, S., Simpson, E. and Tomonari, K., Nature 1991. 349: 531. 7 White, J., Cell 1989. 56: 27. 8 Marrack, F! and Kappler, J., Science 1990. 248: 705. 9 Cole, B. C., Kartchner, D. R. and Wells, D. J., J. Immunol. 1970. 144: 425. 10 Marrack, P, Kushnir, E. and Kappler, J., Nature 1991. 349: 524. 11 Marrack, F!, Blackman, M., Kushnir, E . and Kappler, J., J. Exp. Med. 1990. 171: 455. 12 Smith, L. R., Kono, D. H . and Theofilopoulos, A . N., J. Immunol. 1991. 147: 375. 13 Lindsey, J. R., in Baker, H. J., Lindsey, J. R. and Weisbroth, S. H. (Eds.), The Laboratory Rat, Academic Press, London 1979, p. 2.M 14 Gill,T. J., Smith, G. J. ,Wissler, R. W. and Kunz, H. W., Science 1989. 245: 269. 15 Chao, N. J., Timmerman, L., McDevitt, H. 0. and Jacob, C. O., Immunogenetics 1989. 29: 231. 16 Gill, T. J., Kunz, H. W., Misra, D. N. and Cortose-Hassett, A . L., Transplantation 1987. 43: 773. 17 Okada, C.Y. and Weissman, I. L., J. Exp. Med. 1989. 169: 1703. 18 Singer, P. A., Balderas, R. S. and Theofilopoulos, A . N., E M B O J. 1990. 9: 3641. 19 Baccala, R., Kono, D. H., Balderas, R. S. and Theofilopoulos, A. N., Proc. Natl. Acad. Sci. U S A 1991. 88: 2908. 20 Choi, Y , Herman, A , , DiGiusto, D., Wade, T., Marrack, P. and Kappler, J., Nature 1990. 346: 471. 21 Pullen, A. M., Wade,T., Marrack, P. and Kappler, J. W., Cell 1990. 61: 1365. 22 Choi, Y., Kappler, J. W. and Marrack, P., Nature 1991. 350: 203. 23 Acha-Orbea, H., Shakhov, A. N., Scarpellino, L., Kolb, E., Muller,V.,Vessaz-Shaw, A , , Fuchs, R.,Blochlinger, K., Rollini, F!, Billotte, J., Sarafidou, M., MacDonald, H. R. and Diggelmann, H . , Nature 1991. 350: 207. 24 Coffin, J. M., in Fields, B. N., Knipe, D. M. et al. (Eds.). Virology, 2nd Edn., Raven Press Ltd., New York 1990. p. 1437.
Lawrence R. Smith., Dwight H. Kono, Michael E. Kammullern, Robert S. Balderas and Argyrios N. Theofilopoulos Department of Immunology, The Scripps Research Institute, La Jolla
Expressed Vg repertoire in rats
641
Vp repertoire in rats and implications for endogenous superantigens* Endogenous superantigens of mice, encoded by mammary tumor virus proviral integrants, induce intrathymic deletion of entire T cell populations that express specific Vg gene products, a phenomenon proposed to be important in selftolerance and prevention of toxic responses to exogenous microbial superantigens. Evidence for the presence of Vg selecting/deleting endogenous superantigens in other species is 1acking.We report here that rats do not exhibit endogenous superantigen-induced Vp clonal deletions despite their strong response to bacterial superantigens. These findings indicate that endogenous superantigens are not obligatory in Vs repertoire shaping.
1 Introduction Superantigens are potent T cell mitogens which, unlike conventional antigens, engage specificT cell antigen receptor (TcR) Vp gene products regardless of the other TcR components (Dg/Jg,V,/J,) and bind MHC class I1 molecules outside the antigen-binding groove (reviewed in [1,21). Superantigens alter the normal distribution of the various mouse Vp-bearing T cell clonotypes either by stimulating clonal expansion if met late in life by the matureTcells, or by mediating clonal deletions if met at the neonatal stage during formation of the repertoire in the thymus (reviewed in [3]). Two classes of superantigens have been identified; endogenous superantigens expressed by certain mouse strains, previously known as minor lymphocyte-stimulating antigens (Mls), and recently shown to be encoded by mouse mammary tumor virus proviral integrants [4-61, and exogenous superantigens, products of certain bacteria [7, 81, mycoplasmas [9], or infectious retroviruses [lo]. Clonal deletion induced by endogenous superantigens is thought to represent the main mechanism by which tolerance to these self molecules is established [l-31. Moreover, the strikingly similar properties of exogenous and endogenous superantigens, including considerable overlap in specificV6 gene products engaged, have led to the hypothesis that these two types of superantigens have co-evolved [3]; if so, early-life intrathymic clonal deletion of immature T cells induced by endogenous superantigens will prevent the deleterious consequences of clonal expansion of mature T cells by cross-reacting microbial superantigens later in life [3, 111.
As noted above, recent studies have linked endogenous superantigens to several mouse type B mammary tumor proviral genes integrated on various chromosomes [4-61. Since mammary tumor viruses (MTV) are mouse viruses, the possibility exists that endogenous superantigens and the accompanying clonal deletion they elicit are unique to mice. Therefore, we addressed the question of whether endogenous superantigen expression is an integral characteristic of Tcell development by studying the mature TcR Vg repertoire in the rat, a phylogenetically close species. The recent cloning in our laboratory of rat Vg (rVg) genes having on average 89% nucleotide similarity to every mouse Vg (mV,) gene [12], facilitated this analysis and permitted direct comparison to known somatic deletions of T cells expressing mouse counterpart genes.
2 Materials and methods 2.1 Rat strains Nine strains representing several MHC haplotypes were included: Brown Norway (BN), RT1"; Buffalo, RTlb; DA, RTla; Fisher 344N and Lewis, RT1'; PVG, RTIC;Sprague Dawley (SD), outbred; Wistar Furth (WF) and diabetesprone Bio-Breeding (BB), both RTIU[13-161. All strains expressed RT1.D (35%,-60% of total spleen cells), the rat class I1 IE counterpart, as assessed by staining with OX-17 (Serotec, Indianapolis, IN). Lewis and BN strains were from the Scripps Animal Facility; PVG and DA from Bantin a n d K i n g m a n ( F r e m o n t , CA); WF, SD and Fisher from Harlan Sprague Dawley (Indianapolis, IN), and BB from the University of Massachusetts (Worcester, MA).
[I 99661 2.2 Tcell subset isolation *
This is Publication no. 6838IMM from the Department of Immunology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road/IMM3, La Jolla, CA 92037. The work reported herein was supported, in part, by grants from the National Institutes of Health. Recipient of an Irvington Institute Fellowship. A On sabbatical leave from Sandoz Pharmaceuticals.
Correspondence: Argyrios N . Theofilopoulos, Department of Immunology, The Scripps Research Institute, 10666 North Torrey Pines Road/IMM3, La Jolla, CA 92037, USA 0 VCH Verlagsgesellschaft mbH, D-6940 Weinhcim, 1992
Single-cell suspensions from thymocytes and spleens were prepared by dispersing cells through wire mesh. Seventy to 80% of total thymocytes used in this study were doublepositive (CD4+CD8+). Splenic CD4 and CD8 singlepositive cells were purified with W312.5 and OX-8 mouse monoclonal antibodies (Serotec) , respectively, and antimouse Ig-conjugated magnetic beads (Dynel, Great Neck, NY). Due to severe lymphopenia in the BB strain, total splenocytes (rather than separated single-positive populations) were analyzed. 0014-2980/92/0303-0641$3.50+ .25/0
L. R . Smith, D. H. Kono, M. E. Kammuller et al.
642
Eur. J. Immunol. 1992. 22: 641-645
2.3 RNase protection assay (RPA)
2.5 In vitro stimulation of T cells by toxins
Rat-specific RPA probes were prepared by using oligonucleotide primers derived from sequences of 20 Lewis rat Vg genes [12]. These PCR products were subsequently ligated to the riboprobe plasmid vector pGEM-7zf (Promega, Madison, WI) and sequenced to confirm product. The probes ranged in size from 90-285 base pairs. Miniprep plasmid DNA prepared for each rat Vp riboprobe was linearized and pooled into two-probe sets (Fig. 1) and used as templates for synthesizing antisense RNA with T7 RNA polymerase (Promega) as described [17, 181. Probe sets were labeled to 5000 cpmluridine residue and hybridized for 12 h at 56°C with 2 pg of RNA, prepared as described [18], from total thymocytes or 6-8 pg of RNA from magnetic bead-attached, single-positive splenic T cells. After RNase A (5 pg/ml) and T1 (10 U/ml) treatments [17, 181, “protected” probe : target mRNA duplexes were phenol/chloroform extracted, ethanol precipitated, and electrophoresed on a standard 6% polyacrylamide sequencing gel. Dried gels were autoradiographed on Kodak XAR film with intensifying screens at -70°C for 4-24 h and quantified as described below. These probes will be available for research purposes to interested parties upon request.
Spleen cells from Lewis rats (2.5 x lo6) were incubated with 1 pg/ml of specific staphylococcal enterotoxins (SE; Toxin Technologies, Sarasota, FL) in RPMI 1640 supplemented with 10% fetal calf serum, 2mM L-glutamine, 50 p~ 2-mercaptoethanol and antibiotics. After 4 days, cells were given 50 U/ml rIL2 (Genzyme, Boston, MA) for 1 day, blasts harvested by discontinuous gradient centrifugation (Histopaque 1077, Sigma, St. Louis, MO), and total RNA prepared.
2.4 Quantitation of relative rat Vg RNA levels Radioanalytic profiles were obtained by direct fi scanning of gels using an Ambis Radioanalytic System (Ambis, San Diego, CA).The net cpm of a given band corresponding to a specific protected Vg probe were calculated by the formula: (cprn Vg - cpm background)/no. uridine bases; RNA amounts were normalized between probe sets using Vp2. Vg levels were expressed as percent of the total protected Vg instead of percent of Cg since thymocytes contain large numbers of sterile transcripts (1.O-kb Cp species containing no Vf3). Cp was used to normalize between probe sets when splenic T cells were stimulated in vitro with toxins.
vp
I I 2 17
I I I I
6 16125.1
u 11
IU 13 8.1
2.6 Neonatal tolerance induction Lewis neonatal rats were injected i.p. within 12 h after birth with 5 pg SEA and every other day thereafter for 2 weeks. Total splenocyte RNA was isolated from these and uninjected control littermates, and 20 pg of RNA was subjected to RPA analysis.
3 Results and discussion A sensitive and specific rVp multiprobe set RPA was developed for quantitating Vg expression levels since a collection of rVg-specific antibodies is currently unavailable. Application of this assay with 2-probe sets encompassing 20 of the 24 known rVg genes [12] with RNA from total thymocytes is shown in Fig. 1. As revealed by the indicated autoradiographic and radioanalytic profiles, clear-cut bands or peaks, respectively, corresponding to each of the Vg analyzed are easily identifiable. As in mice [17, 181 and humans [19], the transcript levels for the various Vg in unselected thymocytes are unequal, varying from = 1% to = 12%. Eight of the assessed rVp have counterparts known to be engaged by endogenous superantigens in mice (Vg5.1, 6, 8.1, 9, 11, 12, 16 and 17) [3]. Although mVg3.1 engages Mlsc, the rVg counterpart is a pseudogene in the Lewis, DA and BB strains ([12] and unpublished data) and was not examined here. Mouse Vg clonal delections are readily
u
I
18
8.3
CP
Set 2 I I1
vp
3.3 219
I 4
I U I I 1514 10 20
I 8.2
U
9
cP
Figure I . Rat Vg RPA. Autoradiographic and radioanalytic profiles show Vp size-specific “protected” RNA bands or peaks, respectively, for rV,j genes assayed as 2-probe sets.
Expressed Vg repertoire in rats
Eur. J. Immunol. 1992. 22: 641-645
observed by RPA when Vp transcript levels in total thymocytes are compared to those in peripheral (splenic) lymphocytes of a given IE+/superantigen+ strain [17, 181. This result is based on the fact that total thymocytes contain predominantly (= 80%) immature double-positive (CD4+CD8+)T cells, while the spleen contains only singlepositive (CD4+CD8- and CD4-CD8+) T cells representing the mature TcR repertoire. Since all rat strains analyzed here are RT1.Df (the rat equivalent of mouse IE) [14], characterization of repertoire shaping was, therefore made by comparing total, mostly unselected, thymocytes vs. splcnic CD4 and CD8 populations. Fig. 2 reveals thymic and splenicVg transcript levels in nine rat strains of various backgrounds and MHC haplotypes. In contrast to endogenous superantigen-expressing mouse strains that have RNA levels for specificVp of 1%-25% of the respective levels in the thymus [17, 181, no evidence of such striking Vg clonal deletion was apparent for any Vg of the nine rat strains surveyed. However, transcript levels for
643
some Vp were either lower or higher in the single-positive mature splenic T cell subsets compared to total, mostly immature, thymocytes. Upward or downward shifts were, in most instances, of the “subset bias” type (affecting one or the other of the single-positive subsets), but, in some instances, both subsets were affected. Such somatically imposed repertoire modifications may be the result of negative selection or absence of positive selection (in the case of underexpression, i.e. Vp14) or caused by enhanced positive selection (in the case of overexpression, i. e. Vp8.1 and 9) and may be dependent on MHC, composition of self-peptide antigens and preferential pairing of certain Vg and V, gene products. Nevertheless, the results clearly indicate that these rat strains do not manifest the profound endogenous superantigen-induced somatic deletions of cells expressing specific Vp genes homologous to those known to be deleted in the mouse. We next wished to determine whether rVp are capable of engaging superantigens by taking advantage of the Vp
20
BN
I Thymus
= CD4+ eza CD8+
15
Buffalo1
DP
i
10
5
0
i
20
g
-
Fisher
PVG
I
15
0
c)
0
I-
’0
r
0
SD 15
1
VO Genes Figure 2. Relative VBexpression levels for nine rat strains. RPA were performed as in Fig. 1 using 2 bg total thymocyte RNA or 6-8 pg purified splenic CD4 or CD8 RNA. An Ambis radioanalytic imaging system was used for quantitations of protected Vp bands. The nct cpm of a given band corresponding to a specific protected Vg probe were calculated by the formula cpmVp - cpm backgroundho. uridinc bases, and RNA amounts between the two-probe sets were normalized using V,-;2.Vplevels were expressed as percent of the total protected Vg cpm.The results shown are derived from at least two independent assays using RNA from a pool of two to three 4-week-old female rats. Due to strain specific polymorphisms,V~~8.2, 13 and 19 were not quantitated for the DA or Fisher 344N strains (nor wasVg9 in these two strains due to interference by a secondary product from one of these polymorphic genes), and for the same reason.Vp4 and 16 were not quantitatedfor the BN, Buffalo and WFstrains.Vp3.,1 was also a pseudogene (2 stop codons) in the Lewis [12], DA and BB (unpublished data) rats and thus was not assayed in any of the indicated strains. Due to sevcre lymphopenia in the diabetes-prone BB rat, total splenocytes (rather than separated CD4 and CD8 cells) were asscssed.
L. R. Smith, D. H. Kono, M. E. Kammuller et al.
644
Eur. J. Immunol. 1992. 22: 641-645
Table 1. Comparison of rat and mouse Vp gene products engaged by bacterial toxinsa) Toxin
vb gene Droducts
Species
1 SEA SED
r m r
SEB
m r
+ + (+)
r
m SEC2
ExFT TSST-1
7
8.1 8.2 8.3
+ +
m SECl
3
(+)
+
+ + + + + + +
10
11
12
14
33
25
12
+ + + 64 + 13 (+) 14
+
+ 45 + +
m r m r
Probe Set 2
3.3-
4-
1020 138.1-
9 I
16
17
19
19
11
+ 19 +
8.2-
9 I
Figure 3. Induction of Vlj clonal deletions by neonatal administration of a bacterial toxin. Lewis neonate rats (within 12 h after birth) were injected i.p. with 5 pg SEA every other day for 2 weeks. Total splenocyte RNA was isolated from these and uninjected control littermates and 20 pg subjected to RPA analysis. The 2-probe set autoradiographic profiles with splenic RNA from a representative treated and control rat are shown.
9
78
(+I 68
11
7
’*
37
r 45
Probe Set 1
6-
15
+ 100 + +
55
a) Transcript levels for rVg are expressed as percent of total Vp transcripts; values > 5% are listed. rVg not assessed in these experiments areVpl, 3.1,5.2,7, 8.3 and 18. mVg reactive with toxins are derived from [3] and are indicated by f ; reactivities for mVp listed in parentheses have been reported to occur with commercial preparations, but not with recombinant sources of enterotoxins 1.31.
selecting activities of several SE [3, 7, 81. As shown in Table 1, in all instances, only certain rVp transcripts were highly represented after cells were cultured for 5 days with the indicated toxins,while the remainder represented < 5% of the total.When rat and mouseVp gene products engaging these toxins were compared, high concordance was observed for V,Jl,whileV@ and VplO were discordant.The rVpl9 and the mVp7 are also frequently selected by SE, but the engagement of the mouse and rat counterparts, respectively, has not been tested. Previous studies implicated amino acid residues 67-87 of human Vg13.2 [20] as important for binding of SEC2 and residues 22 and 70-71 of a mouse Vp8.2 variant for binding of Mls” [21]. When the amino acid sequences at these regions were compared between the homologous pairs of mouse and rat Vp genes that were concordant (Vpll, -15) or discordant (Vp8, -10) for toxin reactivities between these two species, no commonalities in primary structure distinguishing these groups were evident (data not shown). This finding suggests that other amino acid positions within spatial proximity to the previously identified binding sites may also play determining roles in toxin binding. Nonetheless, if one assumes that t h e binding sites for exogenous and endogenous superantigens are comparable [20, 211, then these data support the notion that rVB structures can interact with superantigens but rats, unlike mice, do not appear to express endogenous superantigens. To substantiate the above conclusion further, rats were also testedfor their capacity to delete clonally in vivoTcells that engage SEA to verify that neonatal tolerance to exogenous superantigens was indeed physically operative in this rodent. Lewis rat neonates (1 day old) were injected with 5 pg of SEA every other day for 2 weeks, as previously described in mice [7]. The results revealed decreases of 50%0-75%0 in transcript levels for rVgl1, -12, -17 and -19 (Fig. 3 ) , the sameVp shown to be engaged by this toxin in the in vitro testing. Transcript levels for the remaining Vp, apart from some minor compensatory changes, remained
Expressed Vg repertoire in rats
Eur. J. Immunol. 1992. 22: 641-645
essentially unaltered between these two groups. These data further strengthen the contention that despite the similar in vitvo and in v i v o behavior of rat and mouse Tcells to bacterial superantigens, the rVp repertoire does not appear to be constrained by endogenous superantigens. Despite the great similarities between mice and rats with regard to genomic TcR and MHC composition, and similarities in life-spans, generation times and environments, the absence of endogenous superantigen-mediated intrathymic Vp clonal deletions in rats suggests that different evolutionary pressures have shaped the expressed Vp repertoires of these species. Although not as thoroughly analyzed, humans, thus far, have also been found to be devoid of such phenomena [19]. The cumulative data, therefore, suggest that somatically imposed Vp clonal deletions by endogenous superantigens are unique to mice.This uniqueness is most easily explained by the restriction of MTV to this species. MTV proviral genes encode, presumably through their open reading frame (OW) gene [22, 231, the previously undefined Mls molecules as well as other Vp selecting/deleting self superantigens. The reasons why these type B retroviruses contain, uniquely of all known retroviruses [24], an ORFgene, and why the host retains MTV sequences in its genome, are unclear at present, but several speculations have been made [5-7, 10, 22, 231. The absence of clear-cut evidence for Vp clonal deletions in other species suggests that endogenous superantigenrelated Vp clonal deletions may have evolved as a unique characteristic of mice imposed by the life cycle of MTV, and are not obligatory components in Vp selection. Furthermore, although clonal deletions induced in mice by endogenous superantigens may diminish deleterious consequences by cross-reacting bacterial superantigens, the documentation that endogenous superantigens are most likely not expressed in other species (rats, humans) that nevertheless respond to bacterial superantigens makes it clear that virally encoded endogenous superantigens and bacterial superantigens need not to have co-evolved. We would like to thank Drs. E Peterson, J. Sprent, S.Webb and N. Gascoigne for critical review. Received September 20, 1991; in revised form November 14, 1991.
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4 References 1 Blackman, M. A., Kappler, J.W. and Marrack, P., Science 1990. 248: 1335. 2 Janeway, C. A., Nature 1990. 349: 459. 3 Herman, A., Kappler, J. W., Marrack, P. and Pullen, A . M., Annu. Rev. Immunol. 1991. 9: 745. 4 Frankel,W. N., Ruby, C., Coffin, J. M. and Huber, B.T., Nature 1991. 349: 526. 5 Woodland, D., Happ, M. P., Gollob, K. J. and Palmer, E., Nature 1991. 349: 529. 6 Dyson, P. J., Knight, A. M., Fairchild, S., Simpson, E. and Tomonari, K., Nature 1991. 349: 531. 7 White, J., Cell 1989. 56: 27. 8 Marrack, F! and Kappler, J., Science 1990. 248: 705. 9 Cole, B. C., Kartchner, D. R. and Wells, D. J., J. Immunol. 1970. 144: 425. 10 Marrack, P, Kushnir, E. and Kappler, J., Nature 1991. 349: 524. 11 Marrack, F!, Blackman, M., Kushnir, E . and Kappler, J., J. Exp. Med. 1990. 171: 455. 12 Smith, L. R., Kono, D. H . and Theofilopoulos, A . N., J. Immunol. 1991. 147: 375. 13 Lindsey, J. R., in Baker, H. J., Lindsey, J. R. and Weisbroth, S. H. (Eds.), The Laboratory Rat, Academic Press, London 1979, p. 2.M 14 Gill,T. J., Smith, G. J. ,Wissler, R. W. and Kunz, H. W., Science 1989. 245: 269. 15 Chao, N. J., Timmerman, L., McDevitt, H. 0. and Jacob, C. O., Immunogenetics 1989. 29: 231. 16 Gill, T. J., Kunz, H. W., Misra, D. N. and Cortose-Hassett, A . L., Transplantation 1987. 43: 773. 17 Okada, C.Y. and Weissman, I. L., J. Exp. Med. 1989. 169: 1703. 18 Singer, P. A., Balderas, R. S. and Theofilopoulos, A . N., E M B O J. 1990. 9: 3641. 19 Baccala, R., Kono, D. H., Balderas, R. S. and Theofilopoulos, A. N., Proc. Natl. Acad. Sci. U S A 1991. 88: 2908. 20 Choi, Y , Herman, A , , DiGiusto, D., Wade, T., Marrack, P. and Kappler, J., Nature 1990. 346: 471. 21 Pullen, A. M., Wade,T., Marrack, P. and Kappler, J. W., Cell 1990. 61: 1365. 22 Choi, Y., Kappler, J. W. and Marrack, P., Nature 1991. 350: 203. 23 Acha-Orbea, H., Shakhov, A. N., Scarpellino, L., Kolb, E., Muller,V.,Vessaz-Shaw, A , , Fuchs, R.,Blochlinger, K., Rollini, F!, Billotte, J., Sarafidou, M., MacDonald, H. R. and Diggelmann, H . , Nature 1991. 350: 207. 24 Coffin, J. M., in Fields, B. N., Knipe, D. M. et al. (Eds.). Virology, 2nd Edn., Raven Press Ltd., New York 1990. p. 1437.
