Autoreactive responses to an environmental factor. 2. Phthalate-induced anti-DNA specificity is downregulated by autoreactive cytotoxic T cells

Abstract

An environmental factor (phthalate) was shown, in our previous study, to induce serum anti-DNA responses in BALB/c, NZB and lupus-prone NZB/W F1 mice. Out of such anti-phthalate responses, cross-reactive populations were identified that strongly bind phthalate, DNA, or both. A phthalate-specific BALB/c monoclonal antibody, 2C3-Ig (γ1,κ), showed considerable affinity for DNA and had extensive sequence homology with the heavy and light chain variable regions of a known anti-DNA immunoglobulin, BV04-01, from lupus-prone NZB/W F1 mice. This study was initiated to address how BALB/c mice, but not NZB/W F1 mice, are protected from these adverse autoreactive B cells. Using 2C3 hybridoma cells as the prototype autoreactive BALB/c B cell, we determined whether its DNA-binding monoclonal antibody would induce any regulatory cell-mediated immune responses. Synthetic idiopeptides corresponding to the heavy and light chain variable regions of 2C3-Ig were found to be effective at inducing specific effector cells in BALB/c mice, but not in lupus-prone F1 mice. The splenocytes from BALB/c mice incubated in vitro with the idiopeptides, particularly the complementarity-determining region 1 (VL1) of the 2C3-Ig light chain, showed significant proliferative and cytolytic responses. A CD8⁺ cytotoxic T-lymphocyte (CTL) response was elicited that recognized the VL1 peptide presented by the K^d allele, and affected the growth of 2C3 cells. In vivo depletion of CD8⁺ T cells in BALB/c mice significantly decreased this CTL activity but increased the anti-DNA humoral response. These results suggest that autoreactive CTLs are induced in non-autoimmune prone mice as a mechanism to downregulate self-reactive B cells.

Introduction

Although immune reactivity to self-antigens is contrary to immunological paradigms, such responses are not uncommon and often engender persistent autoimmune disorders. Autoreactive cells are not completely deleted and escape to the periphery. The breakdown of self-tolerance by various factors can lead to their activation in susceptible individuals.^1,2 What factors contribute to induction of autoimmune responses is largely unknown. Possibly, the aetiology lies in multifactorial events triggered by environmental, genetic and hormonal factors.^3–6

The ability to produce anti-DNA immunoglobulins is not restricted to mice that develop systemic lupus erythematosus (SLE).^7,8 Even normal mice can develop such antibodies with ageing or upon antigen-specific stimulation of their B cells. However, they rarely develop pathological autoimmune disorders.^9,10 Such reactions are probably under the control of regulatory mechanisms in bone marrow and thymus in unaffected individuals.^11,12 There is also an increasing body of evidence suggesting that self-reactive autoimmune cells are controlled by idiotype and an anti-idiotypic network of specific antibodies or T cells.^13,14 However, the functional and structural properties of anti-idiotypic T cells and antibodies remain unclear. We previously showed that anti-phthalate immunoglobulins induced in autoimmune-prone NZB/W F1 and non-susceptible BALB/c mice exhibit considerable affinity for DNA and, in particular, for an oligonucleotide, d(pT)4. However, only susceptible mice are affected with progressive signs of lupus-like syndromes.¹⁵ These observations raise several questions. Are the antibodies from lupus mice qualitatively different and more pathogenic than their counterparts in normal BALB/c mice? Do normal and autoimmune-prone mice possess identical anti-DNA antibody-producing clones whose regulation is impaired in the lupus strains? In this report, we have addressed some of these issues by examining the role of anti-idiotypic immune regulation by autoreactive B cells that produce anti-DNA immunoglobulins in non-susceptible BALB/c mice.

The splenocytes of autoimmune-prone and resistant mice have been stimulated in vitro with synthetic peptides corresponding to the heavy- and light-chain variable regions (idiotype) of a monoclonal antibody (mAb), 2C3-Ig. This mAb, secreted by an anti-phthalate hybridoma clone, has extensive homology with BV04-01, an anti-DNA immunoglobulin identified in autoimmune-susceptible, lupus-prone NZB/W F1 mice.¹⁶ Despite such homology and self-reactivity, BALB/c mice, but not NZB/W F1 mice, are protected from the adverse effects of such anti-DNA B cells. We report here that peptides corresponding to the heavy- and light-chain variable regions (idiotype) of 2C3-Ig stimulate splenic T cells from BALB/c mice, but not from susceptible NZB/W F1 mice, and generate idiotype-specific CD8⁺ T cells that are highly cytotoxic for DNA-binding 2C3 hybridoma clones. Our study shows that autoreactive cytotoxic T lymphocytes (CTLs) are induced in BALB/c mice as a mechanism to delete or render autoreactive B cells inactive.

Materials and methods

Mice

BALB/c female mice were bred and housed in the animal facility of Indiana State University. NZB/W F1 mice were purchased from the Harlan Sprague Dawley (Indianapolis, IN). Mice were used at age 8–12 weeks. All animals were housed in the animal facility of Indiana State University according to principles of laboratory animal care (NIH publication 85–23) followed under a specific protocol approved by the Animal Care and Use Committee (ACUC) of Indiana State University.

Cell lines

The hybridoma 2C3 clone secreting anti-phthalate 2C3-Ig (γ1,κ) was generated from fusion of phthalate–keyhole limpet haemocyanin (KLH)-primed BALB/c splenocytes with a non-secreting myeloma, X63-Ag8.653.¹⁷ They were propagated in Dulbecco's modified Eagle's minimal essential medium (DMEM) containing 10% horse serum. 2C3 cells have been extensively studied and maintained for more than 18 years without any phenotypic changes.^18–21 Hybridomas secreting anti-CD4 (TIB 207), anti-CD8 (TIB 105), anti-K^d (HB 159), anti-D^d (HB102), and anti-L^d (HB31) were obtained from the American Type Culture Collection (Rockville, MD) and used in inhibition assays.

Peptide prediction analysis

Sequences of heavy- and light-chain variable regions were examined for peptides of 9–10 amino acids in length that might bind to H-2 K^d, D^d, or L^d major histocompatibility complex (MHC) alleles, using computer prediction analysis (http://www.uni-tuebingen.de/uni/kxi, http://bimas.dcrt.nih.gov/molbio/hla/bind).^22,23 Primary amino acid sequences of 2C3-Ig were scanned for prediction of antigenic characteristics by using protean software²⁴ and several of these sequences were also included in immune reactivity of both non-autoimmune-prone and autoimmune-prone mice studies.

Synthetic peptides

Synthetic peptides corresponding to variable regions of 2C3-Ig, other anti-DNA immunoglobulins, and an unrelated control peptide, CEMSDMSFSKDWSFYI, used as detailed in Table 1, were purchased from Alpha Diagnostic International, Inc. (San Antonio, TX). Throughout the text, unless stated otherwise, the 9-mer peptides corresponding to CDR1, CDR2 and CDR3 regions of immunoglobulin heavy and light chains are designated as VH1-9, VH2-9, VH3-9, and VL1-9, VL2-9, and VL3-9, respectively. Extended peptides containing MHC class II epitopes are designated as VL1-15, etc.

Table 1.

Peptides corresponding to the variable regions used in the study

Synthetic peptide	Origin	Region	Sequences
9-mer
VH1-9	2C3	CDR1 of VH	TFSDYYIYW
VH2-9	2C3	CDR2 of VH	TISDGGSYT
VH3-9	2C3	CDR3 of VH	GYYGSSYGFA
VL1-9	2C3	CDR1 of VL	VHSNGNTYL
VL2-9	2C3	CDR2 of VL	IYKVSNRFS
VL3-9	2C3	CDR3 of VL	YFCSQSTHV
FR1-9	2C3	FR1 of VH	GFTFSDYYI
VK2-9	19–19b133	CDR1 of VL	LESNGKTYL
VK9-9	2–5c1233	CDR1 of VL	ASQDIHGYL
VK41-9	18–26c733	FR3 of VL	DYSLTISSL
VK41-9	18–26c733	CDR1 of VL	ASQDIGSSL
9-mer+MHC class II-binding motif
VH1-12	2C3	CDR1 of VH	SGFTFSDYYIYW
VH2-17	2C3	CDR2 of VH	TISDGGSYTYYPDSVKG
VH3-21	2C3	CDR3 of VH	EDTAMYYCARGGYYGSSYGFA
VL1-15	2C3	CDR1 of VL	RSSQSLVHSNGNTYL
VL2-13	2C3	CDR2 of VL	IYKVSNRFSGVPD
VL3-20	2C3	CDR3 of VL	EDLGVYFCSQSTHVPWTFGG
Control
β2m	Mouse β2m		EMSDMSFSKDWSFYI

β2m, β₂-microglobulin peptide; CDR, complementarity-determining region; FR, framework region; MHC, major histocompatibility complex; V_H, heavy chain variable region; V_L, light chain variable region.

Immunizations

Experimental mice (five or more) were grouped and primed intraperitoneally (i.p.) with phthalate–KLH conjugates emulsified in complete Freund's adjuvant. Control groups of mice were immunized with either adjuvant or KLH. Boosters in phosphate-buffered saline (PBS) were given at 10-day intervals. The mice were bled through retro-orbital veins 5 days after each immunization.

Preparation of different T2 cell lysates

Adopting a procedure reported by Zeh et al.,²⁵ these lysates were used to identify idiopeptides that preferentially bound to MHC class I alleles. Dr William Storkus of the University of Pittsburgh School of Medicine kindly provided different T2 cell lines. About 2 × 10⁷ cells of T2 K^d or T2 D^d cells were harvested and washed in PBS. The cells were then lysed in 1 ml of lysis buffer that contained 0·5% Triton-X-100, 0·5% Mega-9 and 5 mm EDTA, in 0·01 m Tris, 0·15 m NaCl (pH 7·4), 2 mm phenylmethylsulphonyl fluoride (PMSF), 5 mm iodoacetic acid and 20 µl of protease inhibitor cocktail. Nuclei and other debris were removed from the lysates by centrifugation (13 000 g, 5 min, 4°). Aliquots were precleared with protein A–agarose beads for 14 hr, with or without the addition of β₂-microglobulin (β2m) (Sigma Chemical Co., St Louis, MO) and/or peptides. After removing protein A–agarose beads by centrifugation at 250 g, anti-K^d or anti-D^d mAbs (prepared, respectively, from HB159, and the HB102 cell line from the ATCC) were added and incubated for 1 hr followed by treatment with protein G–sepharose for 30 min at 4°. Beads were then separated, washed thoroughly and used for sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE).

SDS–PAGE and Western blot

Proteins bound to the beads above were eluted using SDS–PAGE reducing sample buffer (Bio-Rad Laboratories, Hercules, CA) run on 16·5% Tris–tricine gels (Bio-Rad Laboratories) and then transferred to nitrocellulose membranes. The incorporation of β2m in the lysate complex was determined using rabbit anti-β2m immunoglobulins (Sigma Chemical Co.). Goat anti-rabbit immunoglobulin conjugated to horseradish peroxidase (HRP) was added followed by colour development with Supersignal® chemiluminescence (Pierce, Rockford, IL).

Production of effector cells

Splenic effector cells were generated as described by Lim et al.²¹ Briefly, splenocytes from naïve mice (BALB/c, NZB and NZB/W F1) were harvested and seeded into six-well tissue culture flasks at 6 × 10⁶ cells/well in 2 ml of RPMI/10% fetal bovine serum (FBS), and stimulated with either killed 2C3 cells (1·2 × 10⁶ cells/well) or synthetic peptides (20 µg/ml) corresponding to the heavy- and light-chain variable regions of 2C3-Ig, for 5 days in the presence of 10% CO₂ at 37°. Splenocytes were obtained from both BALB/c and NZB/W F1 mice immunized with phthalate–KLH, stimulated as described above and then used in cytotoxicity assays as such or as a CD8⁺ T-cell-enriched population.

Cytotoxicity assay

Cytotoxicity assays were carried out using 2C3 hybridoma and P815 cells as targets for effector CTLs generated as described above. The target cells were labelled at 37° with 150 µCi of sodium ⁵¹Cr for 1 hr in the presence or absence of idiopeptides or control peptides, washed three times in PBS and then resuspended in RPMI/10% FBS. The labelled target cells, at 5 × 10³ cells/well, were then dispensed into 96-well plates. Effector CTLs were added at various effector/target (E : T) ratios to the appropriate target cells seeded in 96-well plates. The total volume of the reaction was 200 µl/well. The plates were incubated at 37° for 6 hr, after which they were centrifuged, and 30 µl of supernatant, removed from each well, was added to 96-well lumina plates to assess ⁵¹Cr release in a Top Count-NXT plate reader (Packard Instruments, Meriden, CT). The percentage specific lysis was determined by the following formula:

All cytolytic analyses described in this study were performed in triplicate and repeated at least three times in separate experiments.

Blocking of T-cell responses by inhibitory antibodies

To characterize the specific T-cell responses, we incubated rat mAbs (10 µg/ml) against CD4 and CD8 with effector cells and mAbs against BALB/c H-2 alleles (K^d, D^d and L^d), separately with the target cells, for 30 min prior to addition to a 96-well plate in the cytotoxicity assay described above. As a control, a non-relevant IgG1 mAb, 1BF7, was used. Results were expressed as percentage inhibition of cytotoxicity in the presence of blocking mAbs compared to the control run without these antibodies.

In vivo CD8⁺T-cell depletion

In vivo CD8⁺ T-cell depletion was accomplished, as previously described,²⁶ by daily administration of 500 µg of affinity-purified mouse anti-CD8 mAb i.p. to a group of five or more mice. This procedure has been shown to significantly deplete CD8⁺ T cells. Three days following this treatment, the mice were immunized with phthalate–KLH conjugates, as described above. Sera were collected and assessed for anti-DNA levels by enzyme-linked immunosorbent assay (ELISA) on plates coated with calf thymus DNA. The effector CTL activity was determined by a cytotoxicity assay using BALB/c splenocytes or CD8⁺ T-enriched fractions, as described above.

Proliferation assay (DNA synthesis)

Splenocytes (1 × 10⁶ cells/well) from naïve BALB/c mice were stimulated with different concentrations of six idiopeptides (1–30 µg/ml) corresponding to the hypervariable regions of 2C3-Ig, and cultured for ≈3 days. After 48 hr, [³H]thymidine ([³H]TdR) (1 µCi) was added to each well and incubated for 16 hr. To assess whether CD8⁺ CTL induction would be facilitated in the presence of helper T cells (CD4⁺),^27,28 MHC class II binding motifs, as determined using the protean software,²⁴ were incorporated into 9-mer idiopeptides, whereby the latter become 15–21 mer peptides (Table 1). These peptides were then used in the activation of splenocytes, as described above. To test the involvement of T helper cells in the induction of CTLs, CD4⁺ T cells were removed by panning²⁹ on anti-CD4 antibody-coated bacterial dishes, and CD8⁺ T-cell-enriched splenocytes were incubated in the presence of the peptides. The incorporated radioactivity was calculated for each sample in triplicate wells by subtracting the mean radioactivity taken up by unstimulated cells.

Role of CTLs in the inhibition of 2C3 cell proliferation and anti-DNA immunoglobulin secretion

To investigate the regulatory functions of the CD8⁺ effector cells, we established a suppression assay, adopted from Najafian et al., using a [³H]TdR proliferation assay.³⁰ Splenocytes from naïve or immunized mice (BALB/c and NZB/W F1, respectively) were stimulated in vitro with a VL1-15 mer peptide that has been shown to have a significant proliferative effect on splenocytes, as described above, and then were incubated for 48 hr, at different ratios, with 2C3 hybridoma cells in 96-well plates. The cultures were then treated with [³H]TdR (1 µCi/well) for 18 hr. Appropriate control cultures were set up using only BALB/c splenocytes or 2C3 hybridoma cells. Incorporation of [³H]TdR was measured as described above. To quantify the 2C3-Ig secretion, supernatants harvested at various time-points from 2C3 hybridoma-splenic effector cell culture were assayed on DNA-coated ELISA plates, as described above.

Results

Prediction of MHC-binding idiopeptide in immunoglobulin

2C3-Ig exhibits considerable binding affinity for DNA, and has extensive sequence homology with a well-characterized anti-DNA immunoglobulin, BV04-01.¹⁶ In spite of this, the anti-DNA response is downregulated in phthalate-hyperimmunized BALB/c mice, but not in NZB/W F1 mice. To determine if this is because of regulatory Id-specific CTLs, we searched for the presence of CTL epitopes on the heavy- and light-chain variable regions of 2C3-Ig. These epitopes would ideally be peptides containing 9 and 10 amino acids, with the potential ability to bind MHC class I alleles, as reported by others.^22,23 Six idiopeptides corresponding to the complementarity-determining regions of the heavy and light chain of 2C3-Ig were chosen, which are usually the most immunogenic domains of immunoglobulin proteins. One peptide, corresponding to the framework region 1 of the heavy chain, was also selected for investigation, as it has the highest MHC class I-binding score. The scores summarized in Table 2 were obtained using two programs, syfpeithi and bimas. The I^A-binding regions in 2C3-Ig sequences were identified using the protean software (shown in Fig. 1). All information was utilized to identify immunogenic peptides that we have reported here.

Table 2.

2C3–Ig sequence analysis by prediction software (syfpeithi and bimas programs) to identify cytotoxic T-lymphocyte (CTL) epitopes from associated complementarity-determining regions (CDRs) and framework regions (FRs)

			MHC binding score
			syfpeithi22			bimas23
Origin	Position	Sequence	Kd	Ld	Kd	Dd	Ld
IgH	CDR3	YYGSSYGFA	20	13	72	2	13
IgH	FR1 + CDR1	GFTFSDYYI	18	9	960	2·88	10
IgH	FR1	RLVESGGGL	17	11	115·2	1·2	5
IgH	FR1	GLVKPGGSL	16	5	80	1	5
IgH	CDR1	IYWVRQTPE	16	3	100	<1	<1
IgH	CDR3	GYYGSSYGF	13	12	60	<1	10
IgH	CDR2	SDGGSYTYY	12	3	<1	<1	<1
IgH	CDR1	TFSDYYIYW	10	8	24	<1	<1
IgKappa	FR1	TPLSLPVSL	20	21	115·2	<1	150
IgKappa	CDR3	YFCSQSTHV	19	<1	288·0	<1	<1
IgKappa	CDR3 + FR4	WTFGGGTKL	18	13	57·6	<1	6·5
IgKappa	FR1	VSLGDQASI	17	14	96	<1	<1
IgKappa	FR1	SCRSSQSLV	17	<1	57·6	<1	<1
IgKappa	FR3	SGSGTDFTL	16	12	48	12	<1
IgKappa	FR1	ISCRSSQSL	15	20	48	<1	25
IgKappa	CDR1	VHSNGNTYL	15	15	<1	<1	10
IgKappa	CDR2	IYKVSNRFS	15	<1	60	<1	10
IgKappa	CDR3 + FR4	TFGGGTKLE	13	3	<1	<1	6·5

MHC, major histocompatibility complex.

Figure 1.

Analysis of the 2C3-Ig sequence for I^A-binding regions (major histocompatibility complex class II epitopes) using protean²⁴ prediction software.

Induction of idiotype-specific CTLs by 2C3-Ig idiopeptides

The peptides listed in Table 1, which correspond to hypervariable regions of different 2C3-Ig heavy and light chains, were tested in vitro to determine if any would be cytolytic effector cell epitopes. BALB/c splenocytes were stimulated with killed 2C3 cells. After a 5-day exposure to stimulator at 37° in 10% CO₂, these splenocytes were used as effector cells in cytotoxicity assays against ⁵¹Cr-labelled and peptide-pulsed P815. The results in Fig. 2 clearly show that only VL1-9 (corresponding to CDR1 of the light chain) was most significantly recognized by cytolytic effector cells in BALB/c splenocytes.

Figure 2.

Cytolysis of P815 target cells pulsed with 2C3 idiopeptides (9-mer). Splenocytes from unprimed BALB/c mice were stimulated in vitro with mitomycin C-treated 2C3 cells and assayed on day 5 for the presence of cytotoxic effector cells, by using the ⁵¹Cr-release assay. E : T ratio, effector : target cell ratio.

Cytolytic effector cells are induced in BALB/c mice, but not in NZB/W F1 mice

The results in Fig. 3(a) show that splenocytes from BALB/c mice stimulated with mitomycin C-treated 2C3 cells could evoke effector cells that recognize both 2C3- and VL1-9-pulsed P815 target cells. Similarly, an in vitro stimulation of BALB/c splenocytes with VL1-15 mer peptide containing MHC class II epitope also induced significant levels of cytolytic activities in BALB/c splenocytes. A K^d-binding peptide (p53, LLGRNSFEV)²⁵ was used as a control to determine whether this cytolytic response was specific to VL1. The CTLs from BALB/c mice recognized VL1-9, but not p53 peptide-loaded P815 cells. However, no such response was elicited by similarly stimulated NZB/W F1 splenocytes (Fig. 3b). When control 2C3-unrelated peptides, corresponding to the variable regions of anti-DNA immunoglobulin found in both normal and non-autoimmune prone mice (listed in Table 1) were tested likewise, no significant cytotoxic effector cells were generated from either BALB/c or NZB/W F1 mice (Table 3).

Figure 3.

VL1-9-recognizing cytolytic effector cells are induced in BALB/c mice, but not in NZB/W F1 mice. Splenocytes from both unprimed BALB/c and NZB/W F1 mice were stimulated in vitro with either mitomycin C-treated 2C3 cells or VL1-15 and assayed on day 5 for the cytolytic effect on 2C3 and VL1-9-loaded P815 as target cells. The assay was performed using stimulated splenocytes from (a) BALB/c mice or (b) NZB/W F1 mice.

Table 3.

Comparison of idiopeptide recognition by splenic effector cells from BALB/c and NZB/W F1 mice using peptide-pulsed and ⁵¹Cr-labelled P815 target cells

	% Target lysis (E:T =100:1)
Peptide used to pulse P815 target cells	BALB/c splenocyte effectors*	NZB/W F1 splenocyte effectors*
VK2-9	10·00 ± 2·64	ND†
VK8-9	16·33 ± 1·15	9·00 ± 7·0
VK9-9	13·00 ± 3·60	17·67 ± 4·61
VK41-9 (FR3)	6·33 ± 4·04	14·33 ± 1·15
VK41-9 (CDR1)	9·00 ± 7·0	18·33 ± 3·05

^*Naïve splenocytes were stimulated in vitro with each peptide for 5 days as described in the Materials and methods.

^†ND, not detected.

CDR, complementarity-determining region; E : T, effector : target; FR, framework region.

Hyperimmunization with phthalate–KLH induces active CD8⁺ T cells that regulate the anti-DNA response in BALB/c mice

We previously showed that the anti-DNA response evoked by immunization with phthalate–KLH was gradually downregulated in BALB/c mice.¹⁵ To understand the basis for this muted response against autoreactivity in BALB/c mice, splenocytes from phthalate–KLH-immunized animals were tested for the induction of any specific cytolytic effectors. As shown in Fig. 4(a), 2C3 target cells were killed by splenocytes from phthalate–KLH-immunized BALB/c mice that were stimulated with VL1-15 mer peptide in vitro. This induction was evident only after the third immunization of BALB/c with phthalate–KLH. No such cytolytic activity was observed with splenocytes from NZB/W F1 mice immunized with phthalate (Fig. 4b).

Figure 4.

Induction of cytotoxic effector cells by immunization with phthalate–keyhole limpet haemocyanin (phthalate–KLH). Splenocytes from immunized mice were stimulated in vitro with VL1-15 idiopeptide and assayed using ⁵¹Cr-labelled 2C3 target cells. (a) Cytotoxicity caused by BALB/c splenocytes. (b) Cytotoxicity caused by NZB/W F1 splenocytes. E/T ratio, effector : target cell ratio.

The nature and specificity of cytolytic effector cells

To further elucidate the nature of these effector cells, cytotoxicity assays were performed in the presence of blocking mAbs such as anti-CD4, anti-CD8, anti-β2m, anti-K^d, anti-D^d and anti-L^d. The results shown in Fig. 5(a) indicate that these were mostly CD8⁺ CTLs that recognized a 2C3-Ig- or P815-associated VL1-9 mer peptide in the context of an MHC class I molecule, particularly K^d. A similar inhibition assay (Fig. 5b) using anti-Id, anti-Fc and a control isotype-matched 1BF7 mAb, indicated that the CTL response was mediated through the idiotypic determinants of 2C3. The inhibition of cytolytic effector cells from BALB/c mice by anti-Id immunoglobulin parallels our previous finding with 2C3-idiotype-specific CTLs.^19–21 Similar observations were also reported by others.^31,32

Figure 5.

Inhibition of splenic effector cell cytotoxicity towards ⁵¹Cr-labelled 2C3 cells by different blocking antibodies. (a) The inhibitory monoclonal antibodies (mAbs) used were directed to CD4, CD8 and BALB/c H-2 alleles. (b) The inhibitory antibodies used were purified anti-idiotypic, anti-isotypic and isotype-matched control antibodies.

Effects of in vivo depletion of CD8⁺ CTLs on the anti-DNA response

To address this issue, we depleted CD8⁺ T cells in BALB/c mice, prior to phthalate immunization, by administration of high doses of anti-CD8 mAb for three consecutive days. This significantly increased the anti-DNA immunoglobulin levels in these mice compared to non-CD8-depleted controls (Fig. 6a). A cytotoxicity assay carried out in parallel also revealed a significant decrease in the cytolytic activities of BALB/c splenocytes following the in vivo depletion of CD8⁺ T cells (Fig. 6b).

Figure 6.

Effects of CD8⁺ T-cell depletion on the phthalate-induced anti-DNA immunoglobulin response in BALB/c mice. Mice were treated, intraperitoneally (i.p.) with 500 µg of anti-mouse CD8 monoclonal antibody (mAb), 3 days, 2 days and 1 day before immunization with phthalate–keyhole limpet haemocyanin (phthalate–KLH). (a) The anti-DNA immunoglobulin response induced with phthalate was measured using enzyme-linked immunosorbent assay (ELISA; A = absorbance). (b) Cytolytic effects of cytotoxic T lymphocytes (CTLs) from those BALB/c mice were compared. E/T ratio, effector : target cell ratio.

Association of VL1-9 with the MHC class I molecule was confirmed using the T2 K^d cell-stabilization assay

The validity of the above finding, that VL1-9 could be a CTL epitope, came also from a β2m-dependent peptide-binding assay that utilized mutant T2 cell lines.²⁵ Different T2 cell lysates were incubated overnight with VL1-9 and human β2m, and the stabilized heavy chains of MHC class I captured by H-2 allele-specific mAb were visualized by Western blot. Only the K^d allele involvement was discernible. Band intensities of VL1-9 and the positive-control p53 peptide were strong, indicating K^d allele specificity of the VL1 peptide (Fig. 7).

Figure 7.

Stabilization assay²⁵ of the K^d allele of the T2 K^d mutant cell line with the VL1-9 mer peptide, Lane 1, 2C3 cell lysate (>2000 molecular weight cut-off) as a tumour control; lane 2, lysates of T2 K^d-VL1-9 peptide (20 µg); lane 3, lysates of T2 K^d-VL1-9 peptide (2 µg); lane 4, lysates of T2 K^d only (no peptide); lane 5, lysates of T2 K^d-p53 (positive K^d-binding peptide, 20 µg);²⁵ lane 6, lysates of T2 K^d-p53 (positive K^d-binding peptide, 2 µg); lane 7, lysates of T2 K^d-VL1-9 peptide in the absence of β₂-microglobulin.

A larger VL1-15, but not VL1-9, helps to recruit T helper cells during the activation of cytolytic effector cells

The 2C3 light chain sequence analysis in Fig. 1 showed that there is a segment of a naturally occurring MHC class II-binding motif, RSSQSL, in the CDR1 region. Inclusion of this sequence in VL1-9 resulted in VL1-15, which was compared with VL1-9 for the ability to activate the CD8⁺ CTL. Results of the [³H]TdR-uptake assay, in which six idiopeptides containing MHC class II epitopes were co-incubated with naïve BALB/c splenocytes in separate experiments, showed that only VL1-15 was effective in significantly augmenting [³H]TdR uptake (data not shown). This response was not detected when CD4⁺ helper T cells were removed by panning (Fig. 8a). However, VL1-9 seemed to have only a marginal effect on proliferation of the splenocytes. Furthermore, although both VL1-9 and VL1-15 were recognized when used on ⁵¹Cr-labelled P815 target cells by the CTLs induced, only VL1-15 proved consistently more effective in stimulating this induction of cytotoxic effector cells (Fig. 8b).

Figure 8.

The role of CD4⁺ T helper cells in the induction of CD8⁺ cytotoxic T lymphocytes (CTLs) (a) Proliferative responses of normal BALB/c splenocytes in the presence of 2C3 idiopeptide. The I^A-binding regions, as predicted by protean software (see Fig. 1), were incorporated into the VL1-9 peptide (corresponding to the CDR1 region of the 2C3-Ig light chain), and the resulting larger VL1-15 peptide was used to stimulate naïve BALB/c splenocytes in vitro. This stimulation was abolished when CD4⁺ T cells were removed. *Statistically significant difference (P < 0·05). (b) Cytotoxicity assay performed using VL1-9- and VL1-15-stimulated splenocytes on 2C3 and appropriate peptide-loaded P815 target cells.

The effects of CTLs induced with VL1-15 peptide on 2C3 cell proliferation and its antibody secretion

It was of interest to determine whether VL1-stimulated CD8⁺ T cells would suppress the growth and/or function of autoreactive B cells, such as 2C3 hybridoma cells which secrete cross-reactive DNA-binding anti-phthalate immunoglobulin. Following co-incubation with CTLs (generated using VL1-15) in 96-well plates, the growth of 2C3 cells was monitored by Trypan blue dye exclusion as well as in [³H]TdR proliferation assays, and its immunoglobulin secretion was quantified on DNA-coated ELISA plates. As shown in Fig. 9(a,b), CD8⁺ cytolytic T cells inhibited the growth of 2C3 cells and also suppressed their ability to secrete 2C3-Ig mAb. VL1-15-stimulated splenocytes from NZB/W F1 mice exerted no suppressive effects on either the growth of 2C3 cells or the DNA-binding property of secreted 2C3-Ig.

Figure 9.

Proliferation assay to assess the effects of primed and unprimed splenocytes on 2C3 cell proliferation and antibody secretion. Mice were immunized with phthalate–keyhole limpet haemocyanin (KLH) two or three times before harvesting the spleens (see the Materials and methods) (a) Inhibition of 2C3 cell growth in the presence of splenocytes stimulated with VL1-15 peptide. (b) Enzyme-linked immunosorbent assay (ELISA) for immunoglobulin in 2C3 culture supernatant performed on DNA-coated microtitre plates.

Discussion

An understanding of how the immune system remains unresponsive to self-antigens, while retaining the capacity to fight pathogenic organisms, is central to any attempt to selectively control immunopathological manifestations. Self-tolerance has been attributed to two types of mechanisms. The first type is dependent on the physical elimination (deletion)^12,33 or functional elimination (anergy)^1,34 of autoreactive cells. The second type is dependent on the presence of T cells able to suppress potentially pathogenic autoreactive cells that have been neither clonally deleted nor rendered anergic.^35,36

The existence of a subpopulation of T cells that specialize in the suppression of autoimmune responses was originally postulated in the early 1990s.^37,38 However, the cellular and molecular mechanisms responsible for these suppressive phenomena were never clearly characterized, with the result that interest in the field of suppressor T cells gradually dwindled. Nevertheless, it is becoming increasingly apparent that a defined subset of T cells from syngeneic healthy donors can prevent the development of autoimmune disease on transfer to lymphopenic recipients.³⁹ A broad body of experimental evidence supports the existence of professional cells in the normal immune system, which exhibit a downregulatory function in the prevention of autoimmunity by a variety of effector mechanisms. Thus, suppressor T cells have been reborn as regulatory T (T_R) cells, as described by Maloy et al.⁴⁰ These specialized T cells reside mainly within a minor subpopulation of CD4⁺ T cells that express the CD25 marker.^41,42

Although CD4⁺ CD25⁺ T_R cells have been shown to play an important role in the maintenance of self-tolerance, this does not necessarily preclude the existence of other regulatory T cells. Recently, another regulatory population of T cells – MHC class I-restricted CD8⁺ T cells – has been described by several groups.^43–45 In multiple sclerosis, clonal depletion or downregulation of autoreactive T cells was shown to occur following T-cell vaccination, with the induction of anti-idiotypic B- and T-cell responses.^14,46 In this study, we describe a similar CD8⁺ T-cell-induced response to expression of the Vκ 1 germline gene that has been implicated in autoimmunity.⁴⁷ These CD8⁺ CTLs are generated not only during exposure in vitro to the idiopeptides (VL1) corresponding to the first CDR of Vκ1 gene, but also in vivo when the immunogen is a protein conjugate of phthalate, an environmental factor. Interestingly, these CTLs recognize VL1-peptide-loaded target cells, in the context of the K^d allele, and exert inhibitory effects in vitro on the growth and antibody secretion of a prototype autoreactive B-cell hybridoma, 2C3. The cytolytic activity of these VL1 idiopeptide-specific CTL can be blocked by anti-idiotypic anti-CD8, as well as by anti-MHC immunoglobulin. Although it is not clear how anti-Id immunoglobulin exerts inhibitory effects on CTL-mediated killing, this has been consistently observed in many studies, as previously reported.^{19–21,31,32} This phenomenon can be explained on the basis of affinity differences of an epitope for MHC and Ab molecules. The peptide dissociated from MHC molecules could be removed by anti-Id immunoglobulin affecting CTL recognition because the affinity of a peptide for MHC-binding sites is much lower than that for the antigen-binding sites on an antibody.⁴⁸ In our study, the in vivo depletion of CD8⁺ T cells in BALB/c mice virtually abolishes the activity of these anti-idiotypic CTLs. This suggests to us that these Vκ1-induced CTLs are essentially autoregulatory T cells that are present in BALB/c mice, but not in NZB/W F1 mice. The usage of this gene has been shown to be elevated in many autoimmune responses.^15,33,47

It has been postulated by Filaci et al. that multiple CD8⁺ T-suppressor cell subsets act together with the CD4⁺ T-regulatory cell subpopulations in the regulation of the immune homeostasis.⁴⁵ Our study was undertaken to investigate whether T helper cells would facilitate induction of such CTLs. As there are several MHC class II-binding motifs in the Vκ1 gene sequence (Table 1), the inclusion of one such motif, which is contiguous with the CDR1 region, further activates regulatory CTLs, indicating recruitment of T helper cells. The nature and characteristics of these CD4⁺ T helper cells (including CD4⁺ CD25⁺ regulatory T cells) in the context of CTL activity are currently under investigation.

Autoreactive antibodies are known to arise in many individuals and in many experimental models. As idiotypic regions are unique and are usually most immunogenic among immunoglobulin domains, it is not surprising that there are instances when anti-idiotypic responses have been shown to occur.^13,14,31 However, such responses do not always lead to autoreactivity, except in susceptible strains. It is reasonable to expect that a resistant strain would have mechanisms to downregulate such autoreactivity before it causes pathological changes or a related malaise. How exactly the immune system discriminates autoantibody idiotype from other idiotypes, is not well understood. It is possible that the autoantibody idiotype mimicks a non-self offending agent,⁴⁹ thereby sending a danger signal (molecular pattern recognition) to the immune system. The latter then triggers counteractive regulatory idiotype/anti-idiotype processes, such as the induction of Id-specific CTLs in resistant strains. Another possibility is that the concentration of autoantibody idiotype in normal serum, which is usually very low, exceeds certain threshold levels as a result of activation and overcomes low zone tolerance. Susceptible strains may lack this regulatory network.

The induction of idiotype-specific CD8⁺ CTLs in non-lupus-prone BALB/c mice to prevent an autoreactive humoral response (in particular to the widely used chemical, phthalate) is, to our knowledge, the first such report. The induction of idiotype-specific regulatory CTLs, reported here, appears to be a counter-measure to prevent the growth and propagation of autoreactive B cells. Whether similar mechanisms operate in other resistant strains of mice is currently being pursued.