Eur. J. Immunol. 1991. 21: 1411-1417
Elizabeth K. Bikoff Department of Obstetrics, Gynecology, and Reproductive Sciences, Mount S i n a i School of Medicine, New York
Presentation of a signal-minus IgG2ab antigen
1411
Class 11-restricted IgG2ab-specific T cells recognize a signal-minus form of the V-CH3b antigen* To study the question when and where self peptides become associated with major histocompatibility complex class I1 molecules for tolerance induction, we recently developed a system in which the intracellular site(s) of antigen expression could be manipulated using gene cloning techniques. We previously constructed a truncated IgG2a gene comprising a variable (V) domain and the C H domain ~ (not including the membrane exons) from the IgG2ab heavy (H) chain.The secreted form of theV-CH3bprotein was expressed at high levels under control of the Ig H chain enhancer in Ia+ B lymphoma cells and was efficiently recognized by class 11-restrictedIgG2ab-specificTcel1hybrids. Here we describe a modified V-CH3b gene construct in which the sequences encoding the signal peptide were deleted. A strong argument can be made that the ~ignal-lessV-CH3~ protein is predominantly expressed in the cytosol.We show that transfected L cell lines expressing the signal-less form of the V-CH3b protein can stimulate class 11-restricted IgG2ab-specificT cells. Cell mixing experiments indicate that this response cannot be due to passive uptake of soluble antigenic peptides released into culture supernatants. These experiments demonstrate that a cytoplasmic protein having no obvious means of reaching the cell surface can be presented to class 11-restricted T cells.
1 Introduction
known constraints concerning intracellular site(s) of expression of antigenic peptides that can interact with According to recent X-ray crystal studies, the shapes of the MHC-encoded class I molecules. Class I-restricted CTL sites responsible for binding antigenic peptides and for that recognize fragments of the influenza virus nucleoprobeing recognized by MHC class I- and class 11-restricted T tein [lo, 111, a cytoplasmic form of the influenza virus cells are remarkably similar [l]. Consistent with this, hemagglutinin (HA) lacking the amino terminal signal several investigators have described peptides that are sequence [12], and antigenic peptides synthesized in transrecognized by both class I- and class 11-restricted T cells fected cell lines from minigene constructs [4] have all been [2-4].Thus, there are no obvious structural differences that described. Based on these data, it seems clear that peptides distinguish antigenic peptides seen in association with derived from endogenously synthesized self proteins can MHC-encoded class I molecules and those recognized in readily be presented to class I-restricted Tcells for tolerance the context of class I1 molecules. Recent experiments have induction. Similar experiments attempting to demonstrate suggested that divergent pathways may exist for class I and class 11-restricted T cell recognition of endogenously synclass I1 gene products with respect to the availability of thesized intracellular antigens have not met with success. binding site (s) during biosynthesis and intracellular trans- Thus, Morrison et al. [5] found that the same transfected port [ M I . The ability of any particular antigen to become target cells expressing influenza HA antigenic peptides associated with class I as opposed to class I1 molecules recognized by class I-restricted CTL failed to stimulate might be dictated by its movement inside cells and thus its responses of class 11-restrictedTcells. The inability of these likelihood of contact with class I or class I1 molecules. As a target cells to be recognized by class 11-restricted Tcells is general rule, exogenous protein antigens are recognized by consistent with the suggestion that class I1 molecules may class 11-restrictedTcells. Recent biochemical and morpho- recycle inside cells in such a way as to preclude an logical studies indicate that the pathway involved in the association with endogenously cytoplasmic proteins [6]. transport of class I1 molecules to the cell surface intersects Although several recent studies have described class IIthe endocytic route followed by internalized antigens [7-91. restricted T cell recognition of antigenic peptides derived Data presented by Neefjes et al. suggest that interactions from endogenously synthesized antigens [13-16], the between antigenic peptides and newly synthesized class I1 site(s) of antigen expression was not precisely localized. molecules may involve joint residence within a specialized Thus, these observations do not exclude the possibility that intracellular compartment [9]. By contrast, there are no the conventional pathway involving uptake of soluble antigen, internalization, and proteolytic degradation in endocytic vesicles may be essential for presentation of self peptides to class 11-restricted T cells.
*
This work was supported by National Institutes of Health grant
AI19047. Correspondence: Elizabeth K.Bikoff, Department of Obstetrics, Gynecology, and Reproductive Sciences, Annenberg 20-84, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
To learn more about intracellular pathway(s) involved in presentation of self proteins to class 11-restricted T cells, this laboratory has been studying T cell recognition of endogenously synthesized IgG2a antigens expressed in Ia+ B lymphoma cells. So that the intracellular site@) of antigen expression could be readily manipulated using gene cloning techniques, it was important to develop IgG2abOO14-2980/91/0606-1411$3S O + .25/0
1412
Eur. J. Immunol. 1991. 21: 1411-1417
E. K. Bikoff
specificTcell hybridomas and expression vectors encoding IgG2ab antigenic determinants recognized by these Tcells. The previous report describes the construction of a truncated IgG2a gene comprising a V domain and the cH3 domain (not including the membrane exons) from the IgG2ab H chain [171.The V-CH3bprotein was expressed at high levels under control of the Ig H chain enhancer in Ia+ B lymphoma cells and was efficiently recognized by BALB/cT cells specific for IgG2ab in association with I-Ad.This report describes a modified V-CH3b gene construct in which the sequences encoding the signal peptide were deleted. We show that transfected L cell lines expressing the signal-less form of the V-CH3bprotein can stimulate class 11-restricted IgG2ab-specific T cells. Cell mixing experiments indicate that this response cannot be due to passive uptake of soluble IgG2ab CH3 peptides that were released into culture SN. These findings strongly suggest that binding site(s) present on MHC-encoded class I1 molecules are available to antigenic peptides expressed intracellularly.
2 Materials and methods 2.1 Gene constructions and DNA-mediated gene transfer A pSV2gpt vector encoding t h e secreted form of the C57BL/6 IgG2ab CH3 was described previously [ 171. Briefly, three fragments - the Igh enhancer + VMpcI1+ the C57BL/6 IgG2ab cH3 were inserted into pSV2gpt as shown in Fig. 1A. The 0.9-kb fragment from the MPCll VH contained the promoter region, transcriptional start site, the leader,V region exon and 3' flanking sequence. Our strategy for the construction of a V-CH3b gene encoding a signal-less form of the V-CH3bprotein is shown in Fig. 1B. We replaced most of the exon encoding the signal peptide and the intervening sequences between the leader and theV region exon with a synthetic oligonucleotide. A unique Bcl I site was located between the transcriptional start site and the ATG initiation codon. The 5' 162-bp Eco RI/Bcl I fragment contained the promoter region [181.The MPCll VHgene contained in addition, a unique Pvu I1 site located near the 5' end of the V region exon 15 nucleotides downstream of the 5' splice acceptor site. A 60-bp oligonucleotide containing the Bcl I recognition site, all 40 nucleotides of 5' flanking sequence, the ATG initiation codon, and CAG GTC CAG encoding the amino terminal portion of the V region, was inserted into the vMpC11 subclone as shown. Plasmids containing the 786-bp Eco RI/Hind I11 signal-lessVMPClIwere identified by restriction enzyme digestion. The structure of the resultant plasmid was confirmed by sequencing through the junction of the introduced oligonucleotide. The 786-bp Eco RI/Hind I11 signal-less Vlllpcll fragment recovered from this subclone and the 666-bp Hind IIIEcoRI B6CH3 fragment were then ligated to Eco RI-digested, calf intestinal phosphatase-treated pSV-J11, a plasmid containing the 2.1-kb Bam HIEco RI Igh enhancer fragment inserted into Bam HIEco RI-digested pSV2gpt.The resultant plasmid including the Igh enhancer, the signal-lessVMpcll and B6CH3 was designated PSV-S-V-CH~~. An expression vector in which the signal-less vMpCl1 was inserted under control of the human p actin promoter is shown in Fig. 1C. The parental LK444 plasmid was obtained from Dr. Larry Kedes, Stanford University School of Medicine, Palo Alto, CA
[19]. The G4MR gene from pSV2neo was also present on this 10-kb vector. The 624-bp BcI I/Hind I11 signal-less V~pc11fragment and the 656-bp Hind III/Bam HI B6CH3 fragment were ligated to Bam HI-digested, calf intestinal phosphatase-treated LK444 (Fig. 1C). The resultant plasmid containing the signal-less vMvIpCI1 + B6CH3 in the correct orientation was designated H u A c ~ s S - V - C H ~ ~ . J558L and A20/2J cells were transfected using the protoplast fusion technique.The H u A c ~ S - V - C Hconstruct ~~ was introduced into I-Aa-expressing L cells by calcium phosphate precipitation. In the case of the PSV-S-V-CH~~ vector, transformants were selected in medium containing mycophenolic acid and xanthine. Cells carrying the H u A c ~ S - V - C Hconstruct ~~ were selected in the presence of G418.
2.2 Radiolabeling and analysis of V-CH3 proteins Exponentially growing cells were harvested, washed with warm HBSS containing 2% FCS and antibiotics, and resuspended (5 x lo6 cells/ml) in warm methionine-free DMEM supplemented with 4 mM L-glutamine and 5% dialyzed FCS. Following a 40-min incubation at 37 "C.,
h a t Glu GLNVALGLN GATCAGTG-----CCACCATG GAA CAG GTC CAG O m a r ,TCAC GGTGGTAC CTTGTC CAGGTC 5 6 ~ 1
4
Bcl 1 R
Pvull &It
hull
H
Signol-lerr VMpcII
L L
CARPUP
Dipest with Bcll+ Hind Ill
?
/
s
Digeat with BamHltHindm
CAL Bc!l/B
HuActS- V-CH3'
Figure 1. Gene constructs for expression of a signal-less V-CH3" protein. (A) Structure of the pSV436CH3 vector encoding the secreted form of the C57BL/6 IgG2ab CH3. This plasmid was comprised of three fragments: the Igh enhancer + VhlPcll+ the C57BL/6 IgG2ah CH3 inserted into pSV2gpt. (B) Construction of the signal-lessVMPCl gene in which sequences encoding the leader peptide were deleted. (C) Structure of the H~ActS-V-CH3~vector in which fragments encoding the signal-less VMPCII + the B6CH3 were inserted under control of the human p-actin promoter. These maps are not drawn to scale.
Eur. J. Immunol. 1991. 21: 1411-1417
[”S]methionine was added (100 pCi/ml = 3.7 MBq/ml) for 30 min. After washing twice with ice-cold PBS containing 2 mM methionine, lysates were prepared by resuspending the cell pellet in lysis buffer [1% (v/v) NP40, 20 mM Tris-HC1, pH 7.5, 150 M NaCl, 5 M EDTA] containing 1 mM PMSF and 10 pg/ml aprotinin as protease inhibitors. After incubation on ice for 15 min, and centrifugation at loo00 rpm for 10 min, lysates were stored frozen at - 70 “C. Upon thawing, lysates were centrifuged for 30 min at 4 “C, and precleared twice with protein A-agarose (BRL, Bethesda, MD), before the addition of affinity-purified rabbit anti-mouse IgG (Fc fragment specific) antibodies (Zymed, San Francisco, CA). After 30 min on ice, protein A-agarose beads were added, and antigen-antibody complexes isolated by a 30-min incubation at 4 “C. Immunoprecipitates were washed three times with buffer containing 0.05 M Tris-HC1, pH 8, 0.45 M NaCI, 0.5% NP40, 0.05% sodium azide, and 1 pg/ml aprotinin, solubilized in Laemmli buffer containing SDS and 2 - h E , and analyzed by SDS-PAGE [20].
Presentation of a signal-minus IgG2ah antigen
1413
previously shown to direct high levels of expression of a heterologous protein (i.e.,the bacterial enzyme gpt), it was possible that deleted sequences encoding the leader peptide and/or the intervening sequences that had been removed might contain additional regulatory elements required for efficient transcription of the Ig H chain gene. If so, the signal-less vMpC11 coding sequences might be expressed at high levels under the control of a heterologous promoter. To test this, the fragments encoding the signalless vMpCI1 plus the B6CH3 were inserted into a mammalian expression vector containing the human p actin gene promoter. Significant levels of CH3” mRNA were detected in the majority of HuActS-V-CH3” x J558L transfected cell lines (data not shown).
The next priority was to test whether the H u A c ~ S - V - C H ~ ~ x J558L transformants synthesized serologically detectable S-V-CH3h protein. Because we anticipated that signal-less molecules probably turnover rapidly, the cells were labeled for a relatively short period of time (30 min). As shown in Fig. 2, we identified a protein migrating at the correct position predicted for the signal-less V-CH3” that was expressed in HuActS-V-CH3” x J558L transformants but 2.3 T cell antigen recognition assays not in parental J558L myeloma cells. The signal-less BALB/cTcell hybrids specific for IgG2a of the b allotype in V-CH3b appeared to be slightly larger than the secreted association with I-Ad were described previously [17]. The form of the protein, consistent with the prediction that this 7B7.3 T cell hybridoma was derived from BALBk mice molecule contains two additional amino acid residues immunized with the bacteriophage repressor cI protein, present at its amino terminus.To confirm that the product and can be stimulated with the peptide P12-26 in the encoded by the signal-less V-CH3” gene was not secreted context of I-Ad [21]. The T cell hybridoma 3D054.8 into the culture medium, cells were labeled for 3 h to allow recognizes chicken OVA in association with I-Ad [22]. sufficient time for transport. Under these conditions, the These Tcells and the P12-26 cI peptide were the gift of Dr. signal-less V-CH3b protein was barely detectable in cell Tom Briner, Immunologic, Cambridge, MA. IL 2 produc- lysates implying that this molecule is relatively short-lived. ~ tion by Tcell hybridomas was assessed by incubating Tcell As expected, the H u A c ~ S - V - C Hx~ J558L transformants hybrids (5 x lo5) with APC (5 x 105) in 1 ml complete did not export any serologically detectable V-CH3bprotein medium. SN were collected after 20 h and assayed for IL 2 into the culture SN (data not shown). Thus, we conclude content in a secondary culture with CTLL cells; 5 x 103 that V-CH3” protein lacking the signal peptide is rapidly CTLL cells were incubated in the presence of 50% primary degraded and does not accumulate in culture SN. culture SN in 0.2 ml of complete medium. The degree of stimulation was measured after 24 h by a 16-18-h exposure to 1 pCi of [3H]dThd. All results shown are mean cpm of J HuAct S V C H 3 b triplicate cultures. $ xJ558LClones 0 In
TLNrr) 0 (glcgr
-
2!2,5
3 Results 3.1 Gene constructions and characterization of the signal-lessV-CH3bprotein
99 66
-
The pSV-S-V-CH3” construct was initially tested in J558L because these myeloma cells are efficient recipients for 4 5 DNA-mediated gene transfer and in comparison with A20/2J lymphoma cells produce higher levels of Ig. To be 31certain before undertaking immunoprecipitation experiments that the signal-less V M P C I I - B ~ Cconstruct H~ was 2 Itranscriptionally active, mRNA expression was evaluated 12345678910 using an RNAse protection assay. Although 15 lines were analyzed in 3 independent experiments, we found only one Figure2. Expression of a signal-less V-CH3” protein in ~ transfected cell lines. Parental JSS8L clone that weakly expressed CH3b transcripts (data not H u A c ~ S - V - C HX~ J558L myelomacells, the pSV-B6CH3 x J558L transfected cell line 1A10. shown). or H U A C ~ S - V - C Hx~ J558L ~ transformants as indicated were biosynthetically labeled with [35S]methionine for 30 min. IgG2a” These observations suggested that the signal-less Vmc11 CH3 proteins were immunoprecipitated using rabbit anti-mouse gene was for some reason defective in its ability to initiate IgG antibodies and analyzed by SDS-PAGE under reducing mRNA transcription. Although the Igh enhancer and 5‘ conditions.The positions of the secreted and the ~ignal-lessV-CH3~ promoter region included in this construct had been proteins are indicated.
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E. K. Bikoff
3.2 Transfected AU)/21 lymphoma cells expressing low levels of the signal-less IgG2ab CH3 cannot stimulate IgG2ab-specificT cells
Table 1. Stably transfected A20/2J lymphoma cells expressing a signal-less form of the IgG2ah C”3 protein lack the ability to stimulate Igh-lb-specific T cell hybrids”)
As a prerequisite for antigen presentation studies, H U A C ~ S - V C Hx~ ~A20/2J transfected cell lines were characterized with respect to expression of signal-less V-CH3b protein and Ia surface antigens. We found that clone #6A most strongly expressed the signal-less V-CH3b protein (Fig. 3A). Surface staining experiments using antiI-Ad antibodies confirmed that this line was strongly Ia+. Clone #6A was therefore selected as the most promising candidate stimulator expressing the highest level of signalless V-CH3“ protein and I-Ad surface antigens.
1L 2 production (cpm) Exp. 1
Exp. 2
Stimulator cells
Medium
+BEPC7Y
A20/U 1B4 8C3 HuActS-V x A20121 #6A #8A # 14A
1657 68634 107499
116887 94 454 108447
3 047 13% 3 683
117494 127044 96798
I540
88 123
A20D.l
The ability of H u A c ~ S - V - C Hx~ A20/2J ~ transformants to 1B4 46002 57 787 present the signal-lessV-CH3bprotein to class 11-restricted 2c12 50 118 70 450 Tcells was tested in the presence of IgG2ab-specificTcell HuActS-V x A20/21 hybrids.To control for clonal differences in the level of I-Ad #6A 060 1085 19 #YA 659 -W672 expression or other accessory molecules required for antigen presentation and/or Tcell stimulation, we analyzed responses in the presence of exogenous soluble IgG2ab. As a positive control, 1B4 and two other pSV-B6CH3 x A20/2J a) 0 - 1 0 Igh-lb-specificTcells (5 x los) were incubated with APC (5 x lo5) in 1 ml complete medium alone, or in the presence of transformants expressing high levels of the secreted form of BEPC69 myeloma protein, 500 @ml. SN were collected after theV-CH3b protein, gave strong responses in the absence of 20 h and assayed for IL 2 content in a secondary culture with exogenous antigen (Table 1). By contrast, IL 2 production CTLL cells; 5 x 1@ CTLL cells were incubated in the presence by IgG2ab-specificTcell hybrids was not stimulated in the of 50% primary culture SN in 0.2 ml of complete medium.The presence of clone #6A or other H U A C ~ S - V - C XHA20/2J ~~ degree of stimulation was measured after 24 h, by a 16-18 h transformants.These cells functioned perfectly well as APC exposure to 1 pCi of [“HIdThd. Results are expressed as mean cpm of triplicate cultures. C5-10 is one of several BALB/c in the presence of exogenous soluble IgG2ab.These results Igh-lb-specific T cell hybrids previously shown to recognize are consistent with previous findings suggesting that cytodeterminants in the c H 3 domain of the IgG2ab H chain. 1B4, plasmic antigens cannot be presented to class 11-restrictedT 8C3, and 2C12 are pSV-B6-CH3 X A20/2J transformants that cells. An important point, however, with respect to this interpretation was that HuActS-V-CH3” x A20/2J transformants synthesized much less signal-less V-CH3h protein in comparison with the amount of secreted V-CH3” protein produced by 1B4 and other pSV-B6CH3 x A2012J transfected cell lines recognized by IgG2ab-specificT cells (see Fig. 3B). It was possible that the inability of HuActS-VCH3b x A2012J transfected cell lines t o stimulate class 11-restrictedTcells might simply be due to the relatively low concentration of antigen. Consistent with this, it should be
97-
97-
66-
(#
express the secreted form of IgG2ah c H 3 protein. HuActS-V X A20/2J-transfected cell lines expressing the signal-less form of the IgG2ah c H 3 protein are described in the text.
emphasized that our previous studies indicated that pSVB6CH3 x A20/2J transformants expressing low levels of the secreted form of the V-CH3b protein lack the ability to stimulate IgG2ab-specific T cell hybrids. To address this issue, it was necessary to achieve higher levels of expression of the signal-less V-CH3b protein.
9766-
45-
45-I
-s-v-cns
31-
31-
-V-CH3
45d*
qS-V-CH3 V-CH3
2l-
I23456789
21-
I 2 3 4 5 6 7 8 9 1 0
/ I
+-v-cw VCH3
3’-
-
21
I 2 3 4 5678
Figure 3. Expression of signal-le~sV-CH3~ protein in H u A c ~ S - V - C Hx~ A20/2J ~ transfected cell lines. Control and transfected cell lines were labeled with [3SS]methioninefor 30 min as follows: parental A20/21; the pSVB6CH3 X A20/2J transformants 184, 13A8.8C3, or 2C12, expressing high levels of the secreted V-CH3h protein; J558L; H U A C ~ S - V - C Hx~ ~J558L clone #12; or the indicated H U A C ~ S - V - C XH A20/2J ~~ transfected cell lines.The positions of the x light chain, the secreted and the signal-le~sV-CH3~ proteins are indicated.
Eur. J. Immunol. 1991. 21: 1411-1417
Presentation of a signal-minus IgG2ah antigen
3.3 Responses of IgG2ab-specificT cells in the presence of transfected L cells strongly expressing the signal-less IgG2ab CH3 protein
1415
Table 2. Responsiveness of IgG2ab-specificTcells in the presence of transfected L cells as stimulatorsa)
IL 2 production (cpm) It was our impression that the H u A c ~ S - V - C Hx~ ~ A20/2J Medium +BEPC7Y Stimulator cells transformants generally synthesized less signal-lessV-CH3b protein in comparison with that used by H u A c ~ S - V - C H ~ ~ Exp. 1 A20D.I 1427 81 980 x J558L-transfected cell lines (see Fig. 3C). For this 1B4 42 SO3 46 161 reason, we believed that the efficiency of expression of the 2c12 21 226 50419 signal-less V-CH3b protein might be dependent on the RT2.3.3H-D6 1386 89 653 recipient cell line. Additionally, the ability of transformants HuActS-V x RT X26 1398 91 615 to function as APC required that potential recipients 9 113 97 79 1 X31 strongly express I-Ad surface antigens. I-Ad-expressing L X32 1 308 96 445 cells were previously co-transfected with the herpes thyX39 2595 94 401 midine kinase gene [23]. To test whether the signal-less Exp. 2 A20lJ 861 62 816 V-CH3b protein might be more efficiently expressed in 1B4 40411 48 799 fibroblasts, this I-Ad-expressing L cell line (RT2.3.3H) was 2c12 -36551 21 495 transfected with the H u A c ~ S - V - C H construct ~~ using RT2.3.3H-D6 1120 79 197 G418R as the selectable marker. A panel of randomly HuActS-V x RT selected H u A c ~ S - V - C Hx~ ~RT2.3.3H transfected cell X26 912 57 225 lines was analyzed. As shown in Fig. 4, we initially X3 1 7300 92416 identified one clone, #31, which strongly expressed the X32 1430 80 332 signal-less V-CH3b protein. Moreover, the amount of 1914 77 973 X39 immunoprecipitable signal-less V-CH3b protein produced Exp. 3 A20121 2 005 96 726 by clone #31 during this short-term labeling experiment 1B4 51 940 73 889 was similar to the amount of secreted V-CH3b synthesized HuActS-V x RT by 1B4 (Fig. 4B, compare lanes 2 and 4).These HuActS-VX105 1647 76 587 CH3b x RT2.3.3H transformants were tested for their 10933 7011~ 4109 13526 76 261 X137 ability to stimulate IgG2ab-specificTcel1hybrids. As shown 11798 70431 X144 in Table 2, we consistently observed a significant positive response in the presence of clone #31 (Exp. 1and 2).These findings were subsequently confirmed by analyzing a a) C5-10 (Exps. 1 and 2) or C5-46 (Exp. 3) Igh-lb-specificTcells second independently selected panel of H u A ~ t s - v - C H 3 ~ - (5 x 105) were incubated with APC (5 x 105) in 1 ml complete transfected L cell lines. We again found that only clones medium alone, or in the presence of BEPC 79 myeloma protein. strongly expressing the signal-less V-CH3b protein were 500 pg/ml. SN were collected after 20 h and assayed for IL 2 capable of stimulating IL 2 production by IgG2ab-specificT content in a secondary culture with CTLL cells; 5 x 103 CTLL cells (Table 2, Exp. 3 and data not shown). Thus, we cells were incubated in the presence of 50% primary culture SN in 0.2 ml of complete medium. The degree of stimulation was conclude that the ~ignal-lessV-CH3~ protein was presented measured after 24 h by a 16-18 h exposure to 1pCi of ['HldThd. to class 11-restricted T cells.
To rule out the possibility that this response might be due to passive uptake of antigenic peptides released into culture
Results are expressed as mean cpm of triplicate cultures. C5-10 and C5-46 are BALBk Igh-lb-specificTcell hybrids previously shown to recognize determinants in the C H domain ~ of the IgG2ah H chain. 1B4 and 2C12 are pSV-B6-CH3 X A20/2J transformants expressing the secreted form of IgG2ab CH3 protein. The RT2.3.3H-D6 I-Ad expressing L cells were generously provided by Dr. Ronald Germain, National Institutes of Health Bethesda, MD. The RT2.3.3H.D6 cell line was transfected with the HuActS-V construct encoding the signal-less form of the IgG2ab C"3 protein and clones were selected as described in the text.
91-
9'7-
66-
66-
45-
45-
-
4-v-cn3 v-cn3
31-
-
21
21-
I 2 3 4 5 6 7 8 9
I 2 3 4 5 6
Figure 4. Expression of the signal-less V-CH3b protein in x ~RT2.3.3H transfected cell lines. Control and HUAC~S-V-CH ~ transfected cell lines were analyzed as follows: the parental RT2.3.3H cell line; A2012J; the pSV-B6CH3 x A20I2J transformants, 1B4 and 2C12, expressing secreted V-CH3b protein or the indicated H U A C ~ S - V - C x H ~RT2.3.3H ~ transfected cell lines.
SN,we carried out cell mixingexperiments. IL 2 production by IgG2ab-specific T cell hybrids was analyzed in the presence of A20/2J lymphoma cells and as the only potential source of antigen, we added the pSV-B6CH3 x J558L transformant lAlO which expresses high levels of the secreted form of theV-CH3b protein (see Fig. 2). As shown in Table 3, the relatively high concentration of secreted V-CH3bprotein produced by these cells failed to stimulate a response despite the presence of optimal numbers of A20/2J APC. These results support the view that responses directed against the signal-less V-CH3b protein cannot be due to the release of soluble antigenic peptides.
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E. K . Bikoff
Table3. Lack of responsiveness in the presence of A20/2J lymphoma cells and IgG2ah CH3-transfccted myeloma cell linesa) IL 2 production (cpm) Mcdium t BEPC79
S t im u lat o r ccl I\
Exp. I
X45 50 x43
A20/2J I B4 1c12
3.5 083
A20/2J + JSSXL. + IAlO + HuActS V x J I2 Exp. 2
x27 71X 717
S1960 54 1 I2
1147 30 I16
9x216 64 120 67 120
141s I272 1201
Yo 193 85 55s 85 409
3-1743 AZO/2J +J5M. t IAI(I
+ i luAr1S v
x .II2
57 232 5x 7x5 4s 275
S4 107
a) C5-10 Igh-lb-specific T cells ( 5 X 10s) were incubated with control, or pSV-B6-CH3-transfected A20/2J lymphoma cells ( 5 x lo5)in 1 ml complete medium alone, or in the presence of SO0 pg/ml BEPC7Y myeloma protein.Where indicated, cultures also contained 5 x 105 myeloma cells. SN were collected after 20 h and assayed f o r IL 2 content in a secondary culture with CTLL cells; 5 x 103CTLL cells were incubated in the presence of 50% primary culture SN in 0.2 ml of complete medium.The degree of stimulation was measured after 24 h by a 16-18-h exposure to I pCi of [3H]dThd. Results are expressed as mean cpm of triplicate cultures. (25-10 is one of several BALB/c Igh-l b-specific T cell hybrids previously shown to recognize determinants in the CtJ domain of the IgG2ah H chain. 1B4 and 2C12 are independently cloned pSV-B6-CH3 X A20/2J transformants that express the secreted form o f IgG2abCH3protein. lAl0 is a pSV-Bh-CH3 x J5S8L transformant that produces significant amounts o f soluble IgG2ah CI13protein in culture SN. HuActS-V x JS58L #12. one of several JS58L transformants expressing the signal-less form of the IgG2ah CH3 protein. is described in the text.
4 Discussion The present study addresses the question whether self peptides can become associated with MHC class I1 molecules intracellularly.The application of recombinant DNA techniques to this problem offers the opportunity to study events involved in antigen presentation in healthy viable cells, avoiding the use of drugs such as chloroquine and brefeldin that dramatically affect membrane recycling pathways. In the system described previously, secreted V-CH3” protein expressed at high levels in transfected A20/2J lymphoma cells was recognized by IgG2ah-specificT cell hybrids [17]. These findings were entirely consistent with current thinking since the secreted V-CH3b protein was probably accessible to MHC class I1 gene products during transport to the cell surface. Moreover, soluble V-CH3”released from cells was potentially internalized and co-localized with class I1 molecules inside endocytic vesicles. Here we describe a modified V-CH3” gene construct lacking sequences that encode the leader peptide. A strong argument can be made that the signal-IessV-CH3”protein is
predominantly expressed in the cytosol. Experiments described in this report demonstrate that a cytoplasmic protein having no obvious means of reaching the cell surface can be presented to class 11-restricted T cells. The strategy used for the construction of a signal-less V-CH3” gene was to utilize a shortened V region gene in which the sequences encoding the signal peptide were deleted. This initially caused some difficulty because the construct made originally with the signal-less VMPCII gene under control of the Igh enhancer was poorly transcribed. Although we were aware that a requirement for RNA splicing for efficient transcription of Ig H chain genes had been described previously, the sequences involved were not specifically localized to the first intron [24]. Our RNase protection experiments showed that pre-mRNA were correct in the context of the pSV-B6CH3 construct. Thus, we conclude that the original pSV-S-V-CH3” contruct did contain a functional 3‘ intron. These observations suggest that regulatory elements specificallyrequired for Ig H chain transcription are located in the first intron. The signal-less V-CH3” gene was strongly expressed under the control of the human p actin promoter. Additionally, the use of this strong ubiquitous promoter opened up the possibility for expression in a wide variety of cell types. Consistent with this, the HuActS-V-CH3” construct directed high levels of CH3” mRNA expression in mouse L cells. It should also be mentioned that previous experiments showed that the secreted V-CH3” protein does not interact with light chains [17], so a requirement for light chain expression probably does not influence expression of the V-CH3” protein in non-B cells. The response stimulated in the presence of signal-less V-CH3h protein was relatively low in comparison with that stimulated by transfected A20/2J lymphoma cells expressing the secreted form of theV-CH3”protein.To explain this, we considered the possibility that IgG2a”-specific T cells were responding to a small amount of signal-less V-CH3” antigen that somehow got released into culture SN, internalized, and presented to class 11-restrictedTcells through the conventional pathway. However, we found no evidence for serologically detectable signal-less V-CH3h protein released into the medium in immunoprecipitation experiments. Additionally, results of cell mixing experiments demonstrate that the response stimulated in the presence of transfected L cells cannot be directed against soluble antigenic peptides. Thus, we conclude that an intracellular form of the V-CH3“ protein was presented to IgG2a”specific T cells. High rates of degradation of the signal-lessV-CH3”protein in the cytoplasm did not seem to result in enhanced abilities of these stimulator cells to be recognized by class IIrestricted T cells. One possibility is that proteolytic enzymes required for production of appropriate antigenic peptides for presentation fo IgG2ah-specificTcells may be predominantly expressed in a specialized intracellular compartment, and only present at low levels in the cytosol. Alternatively, cytoplasmic V-CH3h protein may be degraded to yield the appropriate peptide(s), but these may have limited access to class I1 molecules. According to this way of thinking, t h e response stimulated in the presence of transfected L cells may be directed towards small amounts
Eur. J. Immunol. 1991. 21: 1411-1417
of peptide that inefficiently gain entry into the endoplasmic reticulum and/or possibly early endosomes. With respect to the intracellular transport of peptides once formed, relatively little is known. Although results of immunoprecipitation experiments suggested that 1B4 and clone #31 synthesize comparable amounts of antigen, the amount of serologically dete~tableV-CH3~ protein may not accurately reflect intracellular concentrations of antigenic peptide. Thus, another possible interpretation is that the signal-less V-CH3b antigen is simply present at a lower concentration in comparison with the amount of secreted V-CH3b available for presentation to class 11-restricted T cells. Finally, although it seems likely that the ability of transfected L cells to function as stimulators simply reflects the increased amount of signal-less V-CH3b antigen, these responses may also be influenced by differences in intracellular transport pathways in different types of cells. There is evidence that fibroblast cell lines are relatively inefficient APC with respect to their ability to present exogenous soluble protein antigens, and that this defect is associated with the absence of expression of the invariant (Ii) chain [25].Since the I-Ad-expressingL cells used here were found to efficiently present exogenous soluble IgG2abprotein and to express significant levels of Ii chain mRNA (Dr. Ronald N. Germain, personal communication), the question whether expression of the Ii chain influences presentation of endogenous IgG2ab antigens has not yet been examined in this system. The V-CH3bantigens described here can be expressed under control of the human p actin promoter in a wide variety of cell types. This will allow us to study presentation of self peptides by functionally diverse APC having different characteristics with respect to membrane fluidity, proteolytic enzymes, endocytosis, lymphokine production and expression of the Ii chain. I thank Enuma Okoye and Yvonne Duzant for excellent technical assistanceand Laurel Eckhardt for critical review of the manuscript. Regretfully a number of relevant previous experiments by others could not be discussed because of space contraints. Received November 8, 1990; in final revised form February 8, 1991.
5 References
Presentation of a signal-minus IgG2ah antigen
2 Perkins, D. L., Lai, M. Z., Smith, J. A. and Gefter. M. L.. J. Exp. Med. 1989. 170: 279. 3 Takahashi, H., Germain, R. N., Moss, B. and Berzofsky, J. A , , J. Exp. Med. 1990. 171: 571. 4 Sweetser, M.T., Morrison. L. A., Braciale,V. L. and Bracia1e.T. J., Nature 1989. 342: 180. 5 Morrison, L. A., Lukacher, A. E., Braciale,V. L., Fan, D. F? and Braciale, T. J., J. Exp. Med. 1986. 163: 903. 6 Germain, R. N., Nature 1986. 322: 687. 7 Cresswell, F?, Proc. Natl. Acad. Sci. USA 1985. 82: 8188. 8 Guargliardi, L. E., Koppelman, B., Blum, J. S., Marks, M. S., Cresswell, P. and Brodsky, F. M., Nature 1990. 343: 133. 9 Neefjes, J. J., Stollorz,V., Peters, F? J., Geuze, H. J. and Ploegh, H. L., Cell 1990. 61: 171. 10 Townsend, A. R. M., Gotch, F. M. and Davey, J.. Cell 1985.42: 457. 11 Townsend, A. R. M., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. and McMichael, A. J., Cell 1986. 44: 959. 12 Townsend, A. R. M., Bastin, J., Gould, K. and Brownlee, G. G . , Nature 1986. 324: 575. 13 Sekaly, R. I?, Jacobson, S., Richert, J. R., Tonnelle, C., McFarland, H. F. and Long, E. O., Proc. Natl. Acad. Sci. USA 1988. 85: 1209. 14 Bikoff, E. K.,Yu, H. and Eckhardt, L. A., Eur. J. Immunol. 1988. 18: 341. 15 Weiss, S. and Bogen, B., Proc. Natl. Acad. Sci. USA 1989.86: 282. 16 Nuchtern, J. G., Biddison, W. E. and Klausner, R. D., Nature 1990. 343: 74. 17 Bikoff, E. K. and Eckhardt, L. A., Eur. J. Immunol. 1989.19: 1903. 18 Zaller, D. M. and Eckhardt, L. A., Mol. Cell. Biol. 1988. 8: 1923. 19 Gunning, F?, Leavitt, J., Muscat, G., Ng, S.Y. and Kedes, L.. Proc. Natl. Acad. Sci. USA 1987. 84: 4831. 20 Laemmli, U. K., Nature 1970. 227: 680. 21 Guillet, J. G., Lai, M. Z., Briner,T. J., Smith, J. A. and Gefter, M. L., Nature 1986. 324: 260. 22 Shimonkevitz, R., Kappler, J., Marrack, P. and Grey, H.. J. Exp. Med. 1983. 158: 303. 23 Germain, R. N., Ashwell, J. D., Lechler, R. I., Margulies, D. H., Nickerson, 1. M., Suzuki, G. and Tou, J.Y. L., Proc. Natl. Acad. Sci. USA 1985. 82: 2940. 24 Neuberger, M. S. and Williams, G.T., Nucleic Acids Res. 1988. 16: 6713. 25 Stockinger, B., Pessara, U.,Lin, R. H., Habicht, J., Grez, M. and Koch, N., Cell 1989. 56: 683. Note added in proof: Recent experiments of Weiss and Bogen (Weiss, S. and Bogen, B., MHC class 11-restricted presentation of intercellular antigen, Cell 1991. 64: 767) similarly show class IIrestricted T cells can recognize intracellular antigens.
1 Brown, J. H., Jardetzky, T., Saper, M. A., Samraoui, B.,
Bjorkmnn. F? J. and Wiley. D. C.. Narurc. 1987. A??: 845.
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Received April 4, 1991.
Elizabeth K. Bikoff Department of Obstetrics, Gynecology, and Reproductive Sciences, Mount S i n a i School of Medicine, New York
Presentation of a signal-minus IgG2ab antigen
1411
Class 11-restricted IgG2ab-specific T cells recognize a signal-minus form of the V-CH3b antigen* To study the question when and where self peptides become associated with major histocompatibility complex class I1 molecules for tolerance induction, we recently developed a system in which the intracellular site(s) of antigen expression could be manipulated using gene cloning techniques. We previously constructed a truncated IgG2a gene comprising a variable (V) domain and the C H domain ~ (not including the membrane exons) from the IgG2ab heavy (H) chain.The secreted form of theV-CH3bprotein was expressed at high levels under control of the Ig H chain enhancer in Ia+ B lymphoma cells and was efficiently recognized by class 11-restrictedIgG2ab-specificTcel1hybrids. Here we describe a modified V-CH3b gene construct in which the sequences encoding the signal peptide were deleted. A strong argument can be made that the ~ignal-lessV-CH3~ protein is predominantly expressed in the cytosol.We show that transfected L cell lines expressing the signal-less form of the V-CH3b protein can stimulate class 11-restricted IgG2ab-specificT cells. Cell mixing experiments indicate that this response cannot be due to passive uptake of soluble antigenic peptides released into culture supernatants. These experiments demonstrate that a cytoplasmic protein having no obvious means of reaching the cell surface can be presented to class 11-restricted T cells.
1 Introduction
known constraints concerning intracellular site(s) of expression of antigenic peptides that can interact with According to recent X-ray crystal studies, the shapes of the MHC-encoded class I molecules. Class I-restricted CTL sites responsible for binding antigenic peptides and for that recognize fragments of the influenza virus nucleoprobeing recognized by MHC class I- and class 11-restricted T tein [lo, 111, a cytoplasmic form of the influenza virus cells are remarkably similar [l]. Consistent with this, hemagglutinin (HA) lacking the amino terminal signal several investigators have described peptides that are sequence [12], and antigenic peptides synthesized in transrecognized by both class I- and class 11-restricted T cells fected cell lines from minigene constructs [4] have all been [2-4].Thus, there are no obvious structural differences that described. Based on these data, it seems clear that peptides distinguish antigenic peptides seen in association with derived from endogenously synthesized self proteins can MHC-encoded class I molecules and those recognized in readily be presented to class I-restricted Tcells for tolerance the context of class I1 molecules. Recent experiments have induction. Similar experiments attempting to demonstrate suggested that divergent pathways may exist for class I and class 11-restricted T cell recognition of endogenously synclass I1 gene products with respect to the availability of thesized intracellular antigens have not met with success. binding site (s) during biosynthesis and intracellular trans- Thus, Morrison et al. [5] found that the same transfected port [ M I . The ability of any particular antigen to become target cells expressing influenza HA antigenic peptides associated with class I as opposed to class I1 molecules recognized by class I-restricted CTL failed to stimulate might be dictated by its movement inside cells and thus its responses of class 11-restrictedTcells. The inability of these likelihood of contact with class I or class I1 molecules. As a target cells to be recognized by class 11-restricted Tcells is general rule, exogenous protein antigens are recognized by consistent with the suggestion that class I1 molecules may class 11-restrictedTcells. Recent biochemical and morpho- recycle inside cells in such a way as to preclude an logical studies indicate that the pathway involved in the association with endogenously cytoplasmic proteins [6]. transport of class I1 molecules to the cell surface intersects Although several recent studies have described class IIthe endocytic route followed by internalized antigens [7-91. restricted T cell recognition of antigenic peptides derived Data presented by Neefjes et al. suggest that interactions from endogenously synthesized antigens [13-16], the between antigenic peptides and newly synthesized class I1 site(s) of antigen expression was not precisely localized. molecules may involve joint residence within a specialized Thus, these observations do not exclude the possibility that intracellular compartment [9]. By contrast, there are no the conventional pathway involving uptake of soluble antigen, internalization, and proteolytic degradation in endocytic vesicles may be essential for presentation of self peptides to class 11-restricted T cells.
*
This work was supported by National Institutes of Health grant
AI19047. Correspondence: Elizabeth K.Bikoff, Department of Obstetrics, Gynecology, and Reproductive Sciences, Annenberg 20-84, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
To learn more about intracellular pathway(s) involved in presentation of self proteins to class 11-restricted T cells, this laboratory has been studying T cell recognition of endogenously synthesized IgG2a antigens expressed in Ia+ B lymphoma cells. So that the intracellular site@) of antigen expression could be readily manipulated using gene cloning techniques, it was important to develop IgG2abOO14-2980/91/0606-1411$3S O + .25/0
1412
Eur. J. Immunol. 1991. 21: 1411-1417
E. K. Bikoff
specificTcell hybridomas and expression vectors encoding IgG2ab antigenic determinants recognized by these Tcells. The previous report describes the construction of a truncated IgG2a gene comprising a V domain and the cH3 domain (not including the membrane exons) from the IgG2ab H chain [171.The V-CH3bprotein was expressed at high levels under control of the Ig H chain enhancer in Ia+ B lymphoma cells and was efficiently recognized by BALB/cT cells specific for IgG2ab in association with I-Ad.This report describes a modified V-CH3b gene construct in which the sequences encoding the signal peptide were deleted. We show that transfected L cell lines expressing the signal-less form of the V-CH3bprotein can stimulate class 11-restricted IgG2ab-specific T cells. Cell mixing experiments indicate that this response cannot be due to passive uptake of soluble IgG2ab CH3 peptides that were released into culture SN. These findings strongly suggest that binding site(s) present on MHC-encoded class I1 molecules are available to antigenic peptides expressed intracellularly.
2 Materials and methods 2.1 Gene constructions and DNA-mediated gene transfer A pSV2gpt vector encoding t h e secreted form of the C57BL/6 IgG2ab CH3 was described previously [ 171. Briefly, three fragments - the Igh enhancer + VMpcI1+ the C57BL/6 IgG2ab cH3 were inserted into pSV2gpt as shown in Fig. 1A. The 0.9-kb fragment from the MPCll VH contained the promoter region, transcriptional start site, the leader,V region exon and 3' flanking sequence. Our strategy for the construction of a V-CH3b gene encoding a signal-less form of the V-CH3bprotein is shown in Fig. 1B. We replaced most of the exon encoding the signal peptide and the intervening sequences between the leader and theV region exon with a synthetic oligonucleotide. A unique Bcl I site was located between the transcriptional start site and the ATG initiation codon. The 5' 162-bp Eco RI/Bcl I fragment contained the promoter region [181.The MPCll VHgene contained in addition, a unique Pvu I1 site located near the 5' end of the V region exon 15 nucleotides downstream of the 5' splice acceptor site. A 60-bp oligonucleotide containing the Bcl I recognition site, all 40 nucleotides of 5' flanking sequence, the ATG initiation codon, and CAG GTC CAG encoding the amino terminal portion of the V region, was inserted into the vMpC11 subclone as shown. Plasmids containing the 786-bp Eco RI/Hind I11 signal-lessVMPClIwere identified by restriction enzyme digestion. The structure of the resultant plasmid was confirmed by sequencing through the junction of the introduced oligonucleotide. The 786-bp Eco RI/Hind I11 signal-less Vlllpcll fragment recovered from this subclone and the 666-bp Hind IIIEcoRI B6CH3 fragment were then ligated to Eco RI-digested, calf intestinal phosphatase-treated pSV-J11, a plasmid containing the 2.1-kb Bam HIEco RI Igh enhancer fragment inserted into Bam HIEco RI-digested pSV2gpt.The resultant plasmid including the Igh enhancer, the signal-lessVMpcll and B6CH3 was designated PSV-S-V-CH~~. An expression vector in which the signal-less vMpCl1 was inserted under control of the human p actin promoter is shown in Fig. 1C. The parental LK444 plasmid was obtained from Dr. Larry Kedes, Stanford University School of Medicine, Palo Alto, CA
[19]. The G4MR gene from pSV2neo was also present on this 10-kb vector. The 624-bp BcI I/Hind I11 signal-less V~pc11fragment and the 656-bp Hind III/Bam HI B6CH3 fragment were ligated to Bam HI-digested, calf intestinal phosphatase-treated LK444 (Fig. 1C). The resultant plasmid containing the signal-less vMvIpCI1 + B6CH3 in the correct orientation was designated H u A c ~ s S - V - C H ~ ~ . J558L and A20/2J cells were transfected using the protoplast fusion technique.The H u A c ~ S - V - C Hconstruct ~~ was introduced into I-Aa-expressing L cells by calcium phosphate precipitation. In the case of the PSV-S-V-CH~~ vector, transformants were selected in medium containing mycophenolic acid and xanthine. Cells carrying the H u A c ~ S - V - C Hconstruct ~~ were selected in the presence of G418.
2.2 Radiolabeling and analysis of V-CH3 proteins Exponentially growing cells were harvested, washed with warm HBSS containing 2% FCS and antibiotics, and resuspended (5 x lo6 cells/ml) in warm methionine-free DMEM supplemented with 4 mM L-glutamine and 5% dialyzed FCS. Following a 40-min incubation at 37 "C.,
h a t Glu GLNVALGLN GATCAGTG-----CCACCATG GAA CAG GTC CAG O m a r ,TCAC GGTGGTAC CTTGTC CAGGTC 5 6 ~ 1
4
Bcl 1 R
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H
Signol-lerr VMpcII
L L
CARPUP
Dipest with Bcll+ Hind Ill
?
/
s
Digeat with BamHltHindm
CAL Bc!l/B
HuActS- V-CH3'
Figure 1. Gene constructs for expression of a signal-less V-CH3" protein. (A) Structure of the pSV436CH3 vector encoding the secreted form of the C57BL/6 IgG2ab CH3. This plasmid was comprised of three fragments: the Igh enhancer + VhlPcll+ the C57BL/6 IgG2ah CH3 inserted into pSV2gpt. (B) Construction of the signal-lessVMPCl gene in which sequences encoding the leader peptide were deleted. (C) Structure of the H~ActS-V-CH3~vector in which fragments encoding the signal-less VMPCII + the B6CH3 were inserted under control of the human p-actin promoter. These maps are not drawn to scale.
Eur. J. Immunol. 1991. 21: 1411-1417
[”S]methionine was added (100 pCi/ml = 3.7 MBq/ml) for 30 min. After washing twice with ice-cold PBS containing 2 mM methionine, lysates were prepared by resuspending the cell pellet in lysis buffer [1% (v/v) NP40, 20 mM Tris-HC1, pH 7.5, 150 M NaCl, 5 M EDTA] containing 1 mM PMSF and 10 pg/ml aprotinin as protease inhibitors. After incubation on ice for 15 min, and centrifugation at loo00 rpm for 10 min, lysates were stored frozen at - 70 “C. Upon thawing, lysates were centrifuged for 30 min at 4 “C, and precleared twice with protein A-agarose (BRL, Bethesda, MD), before the addition of affinity-purified rabbit anti-mouse IgG (Fc fragment specific) antibodies (Zymed, San Francisco, CA). After 30 min on ice, protein A-agarose beads were added, and antigen-antibody complexes isolated by a 30-min incubation at 4 “C. Immunoprecipitates were washed three times with buffer containing 0.05 M Tris-HC1, pH 8, 0.45 M NaCI, 0.5% NP40, 0.05% sodium azide, and 1 pg/ml aprotinin, solubilized in Laemmli buffer containing SDS and 2 - h E , and analyzed by SDS-PAGE [20].
Presentation of a signal-minus IgG2ah antigen
1413
previously shown to direct high levels of expression of a heterologous protein (i.e.,the bacterial enzyme gpt), it was possible that deleted sequences encoding the leader peptide and/or the intervening sequences that had been removed might contain additional regulatory elements required for efficient transcription of the Ig H chain gene. If so, the signal-less vMpC11 coding sequences might be expressed at high levels under the control of a heterologous promoter. To test this, the fragments encoding the signalless vMpCI1 plus the B6CH3 were inserted into a mammalian expression vector containing the human p actin gene promoter. Significant levels of CH3” mRNA were detected in the majority of HuActS-V-CH3” x J558L transfected cell lines (data not shown).
The next priority was to test whether the H u A c ~ S - V - C H ~ ~ x J558L transformants synthesized serologically detectable S-V-CH3h protein. Because we anticipated that signal-less molecules probably turnover rapidly, the cells were labeled for a relatively short period of time (30 min). As shown in Fig. 2, we identified a protein migrating at the correct position predicted for the signal-less V-CH3” that was expressed in HuActS-V-CH3” x J558L transformants but 2.3 T cell antigen recognition assays not in parental J558L myeloma cells. The signal-less BALB/cTcell hybrids specific for IgG2a of the b allotype in V-CH3b appeared to be slightly larger than the secreted association with I-Ad were described previously [17]. The form of the protein, consistent with the prediction that this 7B7.3 T cell hybridoma was derived from BALBk mice molecule contains two additional amino acid residues immunized with the bacteriophage repressor cI protein, present at its amino terminus.To confirm that the product and can be stimulated with the peptide P12-26 in the encoded by the signal-less V-CH3” gene was not secreted context of I-Ad [21]. The T cell hybridoma 3D054.8 into the culture medium, cells were labeled for 3 h to allow recognizes chicken OVA in association with I-Ad [22]. sufficient time for transport. Under these conditions, the These Tcells and the P12-26 cI peptide were the gift of Dr. signal-less V-CH3b protein was barely detectable in cell Tom Briner, Immunologic, Cambridge, MA. IL 2 produc- lysates implying that this molecule is relatively short-lived. ~ tion by Tcell hybridomas was assessed by incubating Tcell As expected, the H u A c ~ S - V - C Hx~ J558L transformants hybrids (5 x lo5) with APC (5 x 105) in 1 ml complete did not export any serologically detectable V-CH3bprotein medium. SN were collected after 20 h and assayed for IL 2 into the culture SN (data not shown). Thus, we conclude content in a secondary culture with CTLL cells; 5 x 103 that V-CH3” protein lacking the signal peptide is rapidly CTLL cells were incubated in the presence of 50% primary degraded and does not accumulate in culture SN. culture SN in 0.2 ml of complete medium. The degree of stimulation was measured after 24 h by a 16-18-h exposure to 1 pCi of [3H]dThd. All results shown are mean cpm of J HuAct S V C H 3 b triplicate cultures. $ xJ558LClones 0 In
TLNrr) 0 (glcgr
-
2!2,5
3 Results 3.1 Gene constructions and characterization of the signal-lessV-CH3bprotein
99 66
-
The pSV-S-V-CH3” construct was initially tested in J558L because these myeloma cells are efficient recipients for 4 5 DNA-mediated gene transfer and in comparison with A20/2J lymphoma cells produce higher levels of Ig. To be 31certain before undertaking immunoprecipitation experiments that the signal-less V M P C I I - B ~ Cconstruct H~ was 2 Itranscriptionally active, mRNA expression was evaluated 12345678910 using an RNAse protection assay. Although 15 lines were analyzed in 3 independent experiments, we found only one Figure2. Expression of a signal-less V-CH3” protein in ~ transfected cell lines. Parental JSS8L clone that weakly expressed CH3b transcripts (data not H u A c ~ S - V - C HX~ J558L myelomacells, the pSV-B6CH3 x J558L transfected cell line 1A10. shown). or H U A C ~ S - V - C Hx~ J558L ~ transformants as indicated were biosynthetically labeled with [35S]methionine for 30 min. IgG2a” These observations suggested that the signal-less Vmc11 CH3 proteins were immunoprecipitated using rabbit anti-mouse gene was for some reason defective in its ability to initiate IgG antibodies and analyzed by SDS-PAGE under reducing mRNA transcription. Although the Igh enhancer and 5‘ conditions.The positions of the secreted and the ~ignal-lessV-CH3~ promoter region included in this construct had been proteins are indicated.
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E. K. Bikoff
3.2 Transfected AU)/21 lymphoma cells expressing low levels of the signal-less IgG2ab CH3 cannot stimulate IgG2ab-specificT cells
Table 1. Stably transfected A20/2J lymphoma cells expressing a signal-less form of the IgG2ah C”3 protein lack the ability to stimulate Igh-lb-specific T cell hybrids”)
As a prerequisite for antigen presentation studies, H U A C ~ S - V C Hx~ ~A20/2J transfected cell lines were characterized with respect to expression of signal-less V-CH3b protein and Ia surface antigens. We found that clone #6A most strongly expressed the signal-less V-CH3b protein (Fig. 3A). Surface staining experiments using antiI-Ad antibodies confirmed that this line was strongly Ia+. Clone #6A was therefore selected as the most promising candidate stimulator expressing the highest level of signalless V-CH3“ protein and I-Ad surface antigens.
1L 2 production (cpm) Exp. 1
Exp. 2
Stimulator cells
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+BEPC7Y
A20/U 1B4 8C3 HuActS-V x A20121 #6A #8A # 14A
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The ability of H u A c ~ S - V - C Hx~ A20/2J ~ transformants to 1B4 46002 57 787 present the signal-lessV-CH3bprotein to class 11-restricted 2c12 50 118 70 450 Tcells was tested in the presence of IgG2ab-specificTcell HuActS-V x A20/21 hybrids.To control for clonal differences in the level of I-Ad #6A 060 1085 19 #YA 659 -W672 expression or other accessory molecules required for antigen presentation and/or Tcell stimulation, we analyzed responses in the presence of exogenous soluble IgG2ab. As a positive control, 1B4 and two other pSV-B6CH3 x A20/2J a) 0 - 1 0 Igh-lb-specificTcells (5 x los) were incubated with APC (5 x lo5) in 1 ml complete medium alone, or in the presence of transformants expressing high levels of the secreted form of BEPC69 myeloma protein, 500 @ml. SN were collected after theV-CH3b protein, gave strong responses in the absence of 20 h and assayed for IL 2 content in a secondary culture with exogenous antigen (Table 1). By contrast, IL 2 production CTLL cells; 5 x 1@ CTLL cells were incubated in the presence by IgG2ab-specificTcell hybrids was not stimulated in the of 50% primary culture SN in 0.2 ml of complete medium.The presence of clone #6A or other H U A C ~ S - V - C XHA20/2J ~~ degree of stimulation was measured after 24 h, by a 16-18 h transformants.These cells functioned perfectly well as APC exposure to 1 pCi of [“HIdThd. Results are expressed as mean cpm of triplicate cultures. C5-10 is one of several BALB/c in the presence of exogenous soluble IgG2ab.These results Igh-lb-specific T cell hybrids previously shown to recognize are consistent with previous findings suggesting that cytodeterminants in the c H 3 domain of the IgG2ab H chain. 1B4, plasmic antigens cannot be presented to class 11-restrictedT 8C3, and 2C12 are pSV-B6-CH3 X A20/2J transformants that cells. An important point, however, with respect to this interpretation was that HuActS-V-CH3” x A20/2J transformants synthesized much less signal-less V-CH3h protein in comparison with the amount of secreted V-CH3” protein produced by 1B4 and other pSV-B6CH3 x A2012J transfected cell lines recognized by IgG2ab-specificT cells (see Fig. 3B). It was possible that the inability of HuActS-VCH3b x A2012J transfected cell lines t o stimulate class 11-restrictedTcells might simply be due to the relatively low concentration of antigen. Consistent with this, it should be
97-
97-
66-
(#
express the secreted form of IgG2ah c H 3 protein. HuActS-V X A20/2J-transfected cell lines expressing the signal-less form of the IgG2ah c H 3 protein are described in the text.
emphasized that our previous studies indicated that pSVB6CH3 x A20/2J transformants expressing low levels of the secreted form of the V-CH3b protein lack the ability to stimulate IgG2ab-specific T cell hybrids. To address this issue, it was necessary to achieve higher levels of expression of the signal-less V-CH3b protein.
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21
I 2 3 4 5678
Figure 3. Expression of signal-le~sV-CH3~ protein in H u A c ~ S - V - C Hx~ A20/2J ~ transfected cell lines. Control and transfected cell lines were labeled with [3SS]methioninefor 30 min as follows: parental A20/21; the pSVB6CH3 X A20/2J transformants 184, 13A8.8C3, or 2C12, expressing high levels of the secreted V-CH3h protein; J558L; H U A C ~ S - V - C Hx~ ~J558L clone #12; or the indicated H U A C ~ S - V - C XH A20/2J ~~ transfected cell lines.The positions of the x light chain, the secreted and the signal-le~sV-CH3~ proteins are indicated.
Eur. J. Immunol. 1991. 21: 1411-1417
Presentation of a signal-minus IgG2ah antigen
3.3 Responses of IgG2ab-specificT cells in the presence of transfected L cells strongly expressing the signal-less IgG2ab CH3 protein
1415
Table 2. Responsiveness of IgG2ab-specificTcells in the presence of transfected L cells as stimulatorsa)
IL 2 production (cpm) It was our impression that the H u A c ~ S - V - C Hx~ ~ A20/2J Medium +BEPC7Y Stimulator cells transformants generally synthesized less signal-lessV-CH3b protein in comparison with that used by H u A c ~ S - V - C H ~ ~ Exp. 1 A20D.I 1427 81 980 x J558L-transfected cell lines (see Fig. 3C). For this 1B4 42 SO3 46 161 reason, we believed that the efficiency of expression of the 2c12 21 226 50419 signal-less V-CH3b protein might be dependent on the RT2.3.3H-D6 1386 89 653 recipient cell line. Additionally, the ability of transformants HuActS-V x RT X26 1398 91 615 to function as APC required that potential recipients 9 113 97 79 1 X31 strongly express I-Ad surface antigens. I-Ad-expressing L X32 1 308 96 445 cells were previously co-transfected with the herpes thyX39 2595 94 401 midine kinase gene [23]. To test whether the signal-less Exp. 2 A20lJ 861 62 816 V-CH3b protein might be more efficiently expressed in 1B4 40411 48 799 fibroblasts, this I-Ad-expressing L cell line (RT2.3.3H) was 2c12 -36551 21 495 transfected with the H u A c ~ S - V - C H construct ~~ using RT2.3.3H-D6 1120 79 197 G418R as the selectable marker. A panel of randomly HuActS-V x RT selected H u A c ~ S - V - C Hx~ ~RT2.3.3H transfected cell X26 912 57 225 lines was analyzed. As shown in Fig. 4, we initially X3 1 7300 92416 identified one clone, #31, which strongly expressed the X32 1430 80 332 signal-less V-CH3b protein. Moreover, the amount of 1914 77 973 X39 immunoprecipitable signal-less V-CH3b protein produced Exp. 3 A20121 2 005 96 726 by clone #31 during this short-term labeling experiment 1B4 51 940 73 889 was similar to the amount of secreted V-CH3b synthesized HuActS-V x RT by 1B4 (Fig. 4B, compare lanes 2 and 4).These HuActS-VX105 1647 76 587 CH3b x RT2.3.3H transformants were tested for their 10933 7011~ 4109 13526 76 261 X137 ability to stimulate IgG2ab-specificTcel1hybrids. As shown 11798 70431 X144 in Table 2, we consistently observed a significant positive response in the presence of clone #31 (Exp. 1and 2).These findings were subsequently confirmed by analyzing a a) C5-10 (Exps. 1 and 2) or C5-46 (Exp. 3) Igh-lb-specificTcells second independently selected panel of H u A ~ t s - v - C H 3 ~ - (5 x 105) were incubated with APC (5 x 105) in 1 ml complete transfected L cell lines. We again found that only clones medium alone, or in the presence of BEPC 79 myeloma protein. strongly expressing the signal-less V-CH3b protein were 500 pg/ml. SN were collected after 20 h and assayed for IL 2 capable of stimulating IL 2 production by IgG2ab-specificT content in a secondary culture with CTLL cells; 5 x 103 CTLL cells (Table 2, Exp. 3 and data not shown). Thus, we cells were incubated in the presence of 50% primary culture SN in 0.2 ml of complete medium. The degree of stimulation was conclude that the ~ignal-lessV-CH3~ protein was presented measured after 24 h by a 16-18 h exposure to 1pCi of ['HldThd. to class 11-restricted T cells.
To rule out the possibility that this response might be due to passive uptake of antigenic peptides released into culture
Results are expressed as mean cpm of triplicate cultures. C5-10 and C5-46 are BALBk Igh-lb-specificTcell hybrids previously shown to recognize determinants in the C H domain ~ of the IgG2ah H chain. 1B4 and 2C12 are pSV-B6-CH3 X A20/2J transformants expressing the secreted form of IgG2ab CH3 protein. The RT2.3.3H-D6 I-Ad expressing L cells were generously provided by Dr. Ronald Germain, National Institutes of Health Bethesda, MD. The RT2.3.3H.D6 cell line was transfected with the HuActS-V construct encoding the signal-less form of the IgG2ab C"3 protein and clones were selected as described in the text.
91-
9'7-
66-
66-
45-
45-
-
4-v-cn3 v-cn3
31-
-
21
21-
I 2 3 4 5 6 7 8 9
I 2 3 4 5 6
Figure 4. Expression of the signal-less V-CH3b protein in x ~RT2.3.3H transfected cell lines. Control and HUAC~S-V-CH ~ transfected cell lines were analyzed as follows: the parental RT2.3.3H cell line; A2012J; the pSV-B6CH3 x A20I2J transformants, 1B4 and 2C12, expressing secreted V-CH3b protein or the indicated H U A C ~ S - V - C x H ~RT2.3.3H ~ transfected cell lines.
SN,we carried out cell mixingexperiments. IL 2 production by IgG2ab-specific T cell hybrids was analyzed in the presence of A20/2J lymphoma cells and as the only potential source of antigen, we added the pSV-B6CH3 x J558L transformant lAlO which expresses high levels of the secreted form of theV-CH3b protein (see Fig. 2). As shown in Table 3, the relatively high concentration of secreted V-CH3bprotein produced by these cells failed to stimulate a response despite the presence of optimal numbers of A20/2J APC. These results support the view that responses directed against the signal-less V-CH3b protein cannot be due to the release of soluble antigenic peptides.
1416
Eur. J. lmmunol. 1991. 21: 1411-1417
E. K . Bikoff
Table3. Lack of responsiveness in the presence of A20/2J lymphoma cells and IgG2ah CH3-transfccted myeloma cell linesa) IL 2 production (cpm) Mcdium t BEPC79
S t im u lat o r ccl I\
Exp. I
X45 50 x43
A20/2J I B4 1c12
3.5 083
A20/2J + JSSXL. + IAlO + HuActS V x J I2 Exp. 2
x27 71X 717
S1960 54 1 I2
1147 30 I16
9x216 64 120 67 120
141s I272 1201
Yo 193 85 55s 85 409
3-1743 AZO/2J +J5M. t IAI(I
+ i luAr1S v
x .II2
57 232 5x 7x5 4s 275
S4 107
a) C5-10 Igh-lb-specific T cells ( 5 X 10s) were incubated with control, or pSV-B6-CH3-transfected A20/2J lymphoma cells ( 5 x lo5)in 1 ml complete medium alone, or in the presence of SO0 pg/ml BEPC7Y myeloma protein.Where indicated, cultures also contained 5 x 105 myeloma cells. SN were collected after 20 h and assayed f o r IL 2 content in a secondary culture with CTLL cells; 5 x 103CTLL cells were incubated in the presence of 50% primary culture SN in 0.2 ml of complete medium.The degree of stimulation was measured after 24 h by a 16-18-h exposure to I pCi of [3H]dThd. Results are expressed as mean cpm of triplicate cultures. (25-10 is one of several BALB/c Igh-l b-specific T cell hybrids previously shown to recognize determinants in the CtJ domain of the IgG2ah H chain. 1B4 and 2C12 are independently cloned pSV-B6-CH3 X A20/2J transformants that express the secreted form o f IgG2abCH3protein. lAl0 is a pSV-Bh-CH3 x J5S8L transformant that produces significant amounts o f soluble IgG2ah CI13protein in culture SN. HuActS-V x JS58L #12. one of several JS58L transformants expressing the signal-less form of the IgG2ah CH3 protein. is described in the text.
4 Discussion The present study addresses the question whether self peptides can become associated with MHC class I1 molecules intracellularly.The application of recombinant DNA techniques to this problem offers the opportunity to study events involved in antigen presentation in healthy viable cells, avoiding the use of drugs such as chloroquine and brefeldin that dramatically affect membrane recycling pathways. In the system described previously, secreted V-CH3” protein expressed at high levels in transfected A20/2J lymphoma cells was recognized by IgG2ah-specificT cell hybrids [17]. These findings were entirely consistent with current thinking since the secreted V-CH3b protein was probably accessible to MHC class I1 gene products during transport to the cell surface. Moreover, soluble V-CH3”released from cells was potentially internalized and co-localized with class I1 molecules inside endocytic vesicles. Here we describe a modified V-CH3” gene construct lacking sequences that encode the leader peptide. A strong argument can be made that the signal-IessV-CH3”protein is
predominantly expressed in the cytosol. Experiments described in this report demonstrate that a cytoplasmic protein having no obvious means of reaching the cell surface can be presented to class 11-restricted T cells. The strategy used for the construction of a signal-less V-CH3” gene was to utilize a shortened V region gene in which the sequences encoding the signal peptide were deleted. This initially caused some difficulty because the construct made originally with the signal-less VMPCII gene under control of the Igh enhancer was poorly transcribed. Although we were aware that a requirement for RNA splicing for efficient transcription of Ig H chain genes had been described previously, the sequences involved were not specifically localized to the first intron [24]. Our RNase protection experiments showed that pre-mRNA were correct in the context of the pSV-B6CH3 construct. Thus, we conclude that the original pSV-S-V-CH3” contruct did contain a functional 3‘ intron. These observations suggest that regulatory elements specificallyrequired for Ig H chain transcription are located in the first intron. The signal-less V-CH3” gene was strongly expressed under the control of the human p actin promoter. Additionally, the use of this strong ubiquitous promoter opened up the possibility for expression in a wide variety of cell types. Consistent with this, the HuActS-V-CH3” construct directed high levels of CH3” mRNA expression in mouse L cells. It should also be mentioned that previous experiments showed that the secreted V-CH3” protein does not interact with light chains [17], so a requirement for light chain expression probably does not influence expression of the V-CH3” protein in non-B cells. The response stimulated in the presence of signal-less V-CH3h protein was relatively low in comparison with that stimulated by transfected A20/2J lymphoma cells expressing the secreted form of theV-CH3”protein.To explain this, we considered the possibility that IgG2a”-specific T cells were responding to a small amount of signal-less V-CH3” antigen that somehow got released into culture SN, internalized, and presented to class 11-restrictedTcells through the conventional pathway. However, we found no evidence for serologically detectable signal-less V-CH3h protein released into the medium in immunoprecipitation experiments. Additionally, results of cell mixing experiments demonstrate that the response stimulated in the presence of transfected L cells cannot be directed against soluble antigenic peptides. Thus, we conclude that an intracellular form of the V-CH3“ protein was presented to IgG2a”specific T cells. High rates of degradation of the signal-lessV-CH3”protein in the cytoplasm did not seem to result in enhanced abilities of these stimulator cells to be recognized by class IIrestricted T cells. One possibility is that proteolytic enzymes required for production of appropriate antigenic peptides for presentation fo IgG2ah-specificTcells may be predominantly expressed in a specialized intracellular compartment, and only present at low levels in the cytosol. Alternatively, cytoplasmic V-CH3h protein may be degraded to yield the appropriate peptide(s), but these may have limited access to class I1 molecules. According to this way of thinking, t h e response stimulated in the presence of transfected L cells may be directed towards small amounts
Eur. J. Immunol. 1991. 21: 1411-1417
of peptide that inefficiently gain entry into the endoplasmic reticulum and/or possibly early endosomes. With respect to the intracellular transport of peptides once formed, relatively little is known. Although results of immunoprecipitation experiments suggested that 1B4 and clone #31 synthesize comparable amounts of antigen, the amount of serologically dete~tableV-CH3~ protein may not accurately reflect intracellular concentrations of antigenic peptide. Thus, another possible interpretation is that the signal-less V-CH3b antigen is simply present at a lower concentration in comparison with the amount of secreted V-CH3b available for presentation to class 11-restricted T cells. Finally, although it seems likely that the ability of transfected L cells to function as stimulators simply reflects the increased amount of signal-less V-CH3b antigen, these responses may also be influenced by differences in intracellular transport pathways in different types of cells. There is evidence that fibroblast cell lines are relatively inefficient APC with respect to their ability to present exogenous soluble protein antigens, and that this defect is associated with the absence of expression of the invariant (Ii) chain [25].Since the I-Ad-expressingL cells used here were found to efficiently present exogenous soluble IgG2abprotein and to express significant levels of Ii chain mRNA (Dr. Ronald N. Germain, personal communication), the question whether expression of the Ii chain influences presentation of endogenous IgG2ab antigens has not yet been examined in this system. The V-CH3bantigens described here can be expressed under control of the human p actin promoter in a wide variety of cell types. This will allow us to study presentation of self peptides by functionally diverse APC having different characteristics with respect to membrane fluidity, proteolytic enzymes, endocytosis, lymphokine production and expression of the Ii chain. I thank Enuma Okoye and Yvonne Duzant for excellent technical assistanceand Laurel Eckhardt for critical review of the manuscript. Regretfully a number of relevant previous experiments by others could not be discussed because of space contraints. Received November 8, 1990; in final revised form February 8, 1991.
5 References
Presentation of a signal-minus IgG2ah antigen
2 Perkins, D. L., Lai, M. Z., Smith, J. A. and Gefter. M. L.. J. Exp. Med. 1989. 170: 279. 3 Takahashi, H., Germain, R. N., Moss, B. and Berzofsky, J. A , , J. Exp. Med. 1990. 171: 571. 4 Sweetser, M.T., Morrison. L. A., Braciale,V. L. and Bracia1e.T. J., Nature 1989. 342: 180. 5 Morrison, L. A., Lukacher, A. E., Braciale,V. L., Fan, D. F? and Braciale, T. J., J. Exp. Med. 1986. 163: 903. 6 Germain, R. N., Nature 1986. 322: 687. 7 Cresswell, F?, Proc. Natl. Acad. Sci. USA 1985. 82: 8188. 8 Guargliardi, L. E., Koppelman, B., Blum, J. S., Marks, M. S., Cresswell, P. and Brodsky, F. M., Nature 1990. 343: 133. 9 Neefjes, J. J., Stollorz,V., Peters, F? J., Geuze, H. J. and Ploegh, H. L., Cell 1990. 61: 171. 10 Townsend, A. R. M., Gotch, F. M. and Davey, J.. Cell 1985.42: 457. 11 Townsend, A. R. M., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith, D. and McMichael, A. J., Cell 1986. 44: 959. 12 Townsend, A. R. M., Bastin, J., Gould, K. and Brownlee, G. G . , Nature 1986. 324: 575. 13 Sekaly, R. I?, Jacobson, S., Richert, J. R., Tonnelle, C., McFarland, H. F. and Long, E. O., Proc. Natl. Acad. Sci. USA 1988. 85: 1209. 14 Bikoff, E. K.,Yu, H. and Eckhardt, L. A., Eur. J. Immunol. 1988. 18: 341. 15 Weiss, S. and Bogen, B., Proc. Natl. Acad. Sci. USA 1989.86: 282. 16 Nuchtern, J. G., Biddison, W. E. and Klausner, R. D., Nature 1990. 343: 74. 17 Bikoff, E. K. and Eckhardt, L. A., Eur. J. Immunol. 1989.19: 1903. 18 Zaller, D. M. and Eckhardt, L. A., Mol. Cell. Biol. 1988. 8: 1923. 19 Gunning, F?, Leavitt, J., Muscat, G., Ng, S.Y. and Kedes, L.. Proc. Natl. Acad. Sci. USA 1987. 84: 4831. 20 Laemmli, U. K., Nature 1970. 227: 680. 21 Guillet, J. G., Lai, M. Z., Briner,T. J., Smith, J. A. and Gefter, M. L., Nature 1986. 324: 260. 22 Shimonkevitz, R., Kappler, J., Marrack, P. and Grey, H.. J. Exp. Med. 1983. 158: 303. 23 Germain, R. N., Ashwell, J. D., Lechler, R. I., Margulies, D. H., Nickerson, 1. M., Suzuki, G. and Tou, J.Y. L., Proc. Natl. Acad. Sci. USA 1985. 82: 2940. 24 Neuberger, M. S. and Williams, G.T., Nucleic Acids Res. 1988. 16: 6713. 25 Stockinger, B., Pessara, U.,Lin, R. H., Habicht, J., Grez, M. and Koch, N., Cell 1989. 56: 683. Note added in proof: Recent experiments of Weiss and Bogen (Weiss, S. and Bogen, B., MHC class 11-restricted presentation of intercellular antigen, Cell 1991. 64: 767) similarly show class IIrestricted T cells can recognize intracellular antigens.
1 Brown, J. H., Jardetzky, T., Saper, M. A., Samraoui, B.,
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Received April 4, 1991.
