The involvement of T cell receptor peptide-specific regulatory CD4+ T cells in recovery from antigen-induced autoimmune disease.

Experimental allergic encephalomyelitis (EAE) is a prototype for CD4+ T cell-mediated autoimmune diseases. Immunization with myelin basic protein (MBP) in B10.PL mice results in EAE, and a majority of animals recover permanently from the disease. Most MBP-reactive encephalitogenic T cells recognize an immunodominant NH2-terminal peptide, Ac1-9, and predominantly use the T cell receptor (TCR) V beta 8.2 gene segment. Here we report that in mice recovering from MBP-induced EAE, peripheral T cells proliferate in response to a single immunodominant TCR peptide from the V beta 8.2 chain (amino acids 76-101), indicating natural priming during the course of the disease. Cloned T cells, specific for this TCR peptide, specifically downregulate proliferative responses to Ac1-9 in vivo and also protect mice from MBP-induced EAE. These regulatory T cells express CD4 molecules and recognize a dominant peptide from the TCR variable framework region of V beta 8.2, in the context of the major histocompatibility complex class II molecule, I-Au, and predominantly use the TCR V beta 14 gene segment. This is the first demonstration of the physiological induction of TCR peptide-specific CD4+ T cells that result from MBP immunization and that are revealed only during the recovery from disease. The downregulation of disease-causing T cells by TCR peptide-specific T cells offers a mechanism for antigen-specific, network-induced recovery from autoimmune disease.

The X-Y-Z scheme of immunocyte maturation. IV. The exhaustion of memory cells.

A set of conditions has been described under which primed rabbit lymph nodes produce a secondary antibody response upon in vivo stimulation with a large dose of antigen, but are subsequently "exhausted;" that is, lymph node cultures prepared at intervals following the booster injection cannot be re-stimulated to display tertiary responses. Rabbits given 100-fold less antigen in the booster inoculum were able to give a tertiary response upon in vitro challenge. The system used permits neither induction nor continuation of a primary response to BSA in vitro. Since it could be demonstrated that no memory cells were generated by the booster injection within the intervals between in vivo injection and culture, the tertiary response in nonexhausted nodes must have been due to residual memory cells which remained untriggered by the in vivo booster injection. The unresponsive state was not caused by antibody feedback. These results are interpreted to mean that a population of memory cells can be exhausted by a supraoptimal dose of antigen, rendering the node temporarily incapable of further response. This implies that long-lived memory is not due to asymmetric division of memory cells. The source and fate of memory cells is discussed with regard to this evidence.

Induction and reversal of immune paralysis in vitro.

Induction of the immune response can only be completed after antigen is removed from the cellular environment. Primed rabbit lymph node fragments were cultured in vitro with 5 mg/ml BSA. If antigen was removed from the fragments 2 hr later, they produced a normal anti-BSA response, which was first evident 5 days later. If antigen removal was delayed for 3 days, the onset of the response was postponed for 2 to 3 days. Pulses with BUDR marked the periods of cell proliferation in both sets of cultures, and established that the postponement of antibody production was preceded by a postponement in the wave of proliferation among precursors of antibody forming cells. The similarity in avidity of antibody-containing fluids from normal and postponed cultures support the idea that the same cell population produced the response in each case. It was concluded that a reversible state of paralysis could be instituted in antigen-responsive cells, and this state did not depend upon cell-killing. The widespread incidence of temporary paralysis as an early aspect of the immune response was discussed.

The X-Y-Z scheme of immunocyte maturation. V. Paralysis of memory cells.

A concentration of 5 mg/ml bovine serum albumin (BSA) prevents the in vitro elicitation of a secondary response in primed rabbit popliteal lymph nodes, if it is left in contact with the node fragments for the first 6 days of culture. No antibody formation can be detected at any time during the culture period in most cases, although occasional fragments are resistant to inhibition. Reducing the exposure time to the first 3 days of culture delays the peak of the antibody response. The inhibition is antigen specific. Reconstruction experiments demonstrate that the inhibition is not due to antigen masking of the antibody. Even shortly after optimal stimulation, the addition of 5 mg/ml BSA to the fragments was not able to prevent a normal antibody response. The implications of these findings are that (a) a high antigen concentration suspends the memory cell in a reversibly paralyzed state, (b) memory cells have a heterogeneous susceptibility to inhibition, (c) once induced, the antibody response cannot be inhibited by antigen overloading, (d) unresponsiveness in a primed animal can be due to either exhaustion of the memory cell population or paralysis of the memory cell.

Dominant determinants in hen eggwhite lysozyme correspond to the cryptic determinants within its self-homologue, mouse lysozyme: implications in shaping of the T cell repertoire and autoimmunity.

We have studied the mouse lysozyme (ML) peptide-specific T cell repertoire in mice of five different major histocompatibility complex (MHC) class II haplotypes. 14 ML peptides were tested in a lymph node T cell proliferation assay. Upon immunization of diverse mouse strains with native ML, there was no response to any of the ML peptides tested. However, nine peptides were immunogenic, although there was no consistent pattern of reactivity toward any peptide among these strains. Thus, an autoreactive T cell repertoire directed against cryptic self(ML)-determinants exists, and it is different in mice of different MHC haplotypes. Moreover, our results demonstrate that crypticity is MHC associated and not merely a structural attribute of the determinant. On comparison of the pattern of response of various peptides of ML and that of its foreign homologue, hen eggwhite lysozyme (HEL) in H-2k, H-2b, and H-2d strains of mice, a striking correlation was evident. The stretches of amino acid sequences of determinants within HEL that were dominant in each of these three strains, almost exactly overlapped in position with those of the cryptic ML determinants against which self-reactivity was demonstrated in the same strain. These results demonstrate that the dominance-crypticity relationship between HEL and ML resulting from differential processing of these two proteins is critical in determining the response to HEL rather than the degree of sequence difference between them. These observations have important implications in the shaping of the T cell repertoire for foreign proteins and in the pathogenesis of autoimmunity.

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. II. Only certain T helper or T suppressor cells are relevant in particular regulatory interactions.

11 cyanogen bromide (CB) peptides, comprising 70% of the large protein, Escherichia coli beta-galactosidase (GZ), were studied for their ability to induce T suppressor (Ts) cells capable of strongly suppressing the in vitro anti-fluorescein (FITC) response to GZ-FITC. Only CB-2 (amino acid residues 3-92) and CB-3 (residues 93-187) were found to bear such Ts-inducing epitopes. In examining the specificity of T helper cell (Th) targets susceptible to CB-2 and CB-3-specific Ts, it appeared that only nearly Th targets could be suppressed. Thus, CB-10-primed Th were not suppressed by either Ts; even CB-3-primed Ts did not suppress CB-2-specific Th, although CB-2-specific Ts were effective. Furthermore, analysis of the suppression pattern revealed a hierarchical use of potential epitopes on native GZ in triggering functional regulatory T cells. A dominant Th epitope near the amino terminus of GZ tops a hierarchy of potential Th, most of which are never engaged. The dominant determinant seems to exist on the peptide CB-2-3 (residues 3-187), and presumably is destroyed by its cleavage at Met 92; the Th cells that it induces are suppressible by each of the Ts-inducing peptides. In the GZ system, where the native antigen is quite large, the interactions between Th and Ts are highly circumscribed. This may be attributable to the topology of antigen fragments produced during processing; any relevant fragment must bear at least a Ts- and Th-reactive determinant to permit intercellular regulation. A final implication of these results is that, not only does the existence of a Th-inducing determinant depend on its being an appropriate distance from a B cell epitope, but the existence of Ts-inducing determinants likewise depends on the existence of a neighboring Th-B cell association.

Idiotypic repertoire of anti-hen eggwhite lysozyme antibodies probed with hybridomas. Selection after immunization of an IdX marker common to antibodies of distinct epitope specificity.

A panel of hybridoma antibodies obtained from lymphoid cells that were fused during a primary response ("early") or a secondary response ("late") gave results concordant with analysis of conventional, in vivo-produced anti-lysozyme idiotypes: early antibodies did not display the predominant anti-hen eggwhite lysozyme idiotype (IdX-HEL), whereas late antibodies all displayed IdX-HEL. Furthermore, individual late hybridomas could each remove the entire anti-IdX-HEL activity by absorption, whereas early hybridomas could not. The epitope specificities of the hybridomas in both the early and late populations were heterogenous. We conclude that epitypic specificity in the response to HEL is determined independently from idiotypic specificity and that the predominant idiotype is selected for during the maturation of the anti-lysozyme response.

Induction of tolerance to one determinant on a synthetic peptide does not affect the response to a second linked determinant. Implications for the mechanism of neonatal tolerance induction.

To investigate the mechanism underlying neonatal T cell tolerance, we used synthetic peptides to induce tolerance. We found that induction of tolerance to one determinant on a 23-amino acid peptide did not affect the response to an adjacent determinant on the same peptide. There was no evidence of suppression of the response to the second determinant. Furthermore, even small peptides near the minimal size for a determinant, which would be very unlikely to possess a suppressor T cell-inducing determinant as well as a proliferative T cell-inducing determinant, could induce tolerance. These studies provide in vivo experiments supporting clonal inactivation as the mechanism of neonatal tolerance to immunogenic peptides.

Ia molecule-associated selectivity in T cell recognition of a 23-amino-acid peptide of lysozyme.

Ak- or Ek-restricted T cells, generated by immunization with a 23-amino-acid peptide of hen egg-white lysozyme (amino acid 74-96), showed a strict correlation between the minimal peptide determinant recognized and the Ia molecule restricting recognition. All Ak-restricted clones obtained from six independently derived lines recognized determinants contained within peptides 74-86, while Ek-restricted clones recognized determinants within 85-96. This correlation was true whether B10.A mice (Ak, Ek) were immunized with peptide 74-96 or with each of the two smaller peptides (74-86 or 85-96). Furthermore, a T cell response could be obtained to peptide 74-86, but not to peptide 85-96 in B10.A (4R) mice, which express only the Ak molecule. Thus, an Ia molecule-associated selectivity exists in the choice of T cell determinants even within this small 23-amino-acid peptide antigen. Significant differences were noted, however, in the boundaries of the minimal peptide determinants recognized within peptide 74-96 by Ak- or Ek-restricted T cells, in comparison to those recognized by Ab-restricted T cells. These results indicate that interaction of the same peptide with distinct Ia molecules results in recognition of unique aspects of the antigenic determinants by the T cell receptor.

In a small multideterminant peptide, each determinant is recognized by a different V beta gene segment.

Given the vast potential for diversification of the T cell receptor (TCR) repertoire and the fact that V(a) beta mice exist in the wild, it would have been predicted that in spite of the absence of 10 TCR V beta gene segments, V(a) beta mice would still have been able to produce an antigen-specific T cell response to all determinants. We have recently shown that Vb beta mice, with a wild-type TCR V beta repertoire, respond to peptide 110-121 of sperm whale myoglobin, with a majority of T cells expressing TCR V beta 8.2 and restricted to a hybrid I-A(d)/I-E(d) major histocompatibility complex molecule, and a smaller number of T cells expressing TCR V beta 8.1 and restricted to the I-A(d) molecule. However, V(a) beta mice, lacking members of the TCR V beta 8 gene family, responded only with I-A(d)-restricted T cells. Thus, it appeared that the I-A(d)-restricted response was less constrained, or more plastic. We now show that the two separate panels of I-A(d)-restricted T cell hybrids derived from V(a) beta or Vb beta mice in fact recognize distinct determinants within the same peptide 110-121. The determinant recognized by V(a) beta T cells is NH2 terminal (core: 110-118) with an absolute requirement for the residue Ala-110 for a successful interaction with TCRs. On the other hand, Vb beta T cells recognize the COOH-terminal region (core: 112-118) on the same peptide with an absolute requirement for COOH-terminal residue 118. In the dominance hierarchy displayed by the three distinct determinants of peptide 110-121, V(a) beta mice cannot recognize the two most dominant: the hybrid I-A(d)/I-E(d)-restricted determinant and the COOH-terminal, I-A(d)-restricted determinant. They instead respond with T cells specific for a third, distinctly NH2-terminal determinant. Our results show a strict association between recognition of a particular specificity and TCR V beta usage. This evidence suggests that even when a small peptide induces a heterogenous group of TCR V beta S, this need not be considered evidence for plasticity. Rather, at the level of individual determinants within the peptide, the results can point in the opposite direction, towards serious constraints in recognition at the level of V beta expression.

Neonatal peptide exposure can prime T cells and, upon subsequent immunization, induce their immune deviation: implications for antibody vs. T cell-mediated autoimmunity.

Neonatal exposure to antigen is believed to result in T cell clonal inactivation or deletion. Here we report that, contrary to this notion, neonatal injection of BALB/c mice with a hen egg lysozyme peptide 106-116 in putative "tolergenic" doses induced a T cell proliferative and an immunoglobulin G (IgG) antibody (Ab) response of both T helper cell 1 (Th1)- (IgG2a, IgG2b, and IgG 3) and Th2-dependent (IgG1) isotopes. Upon subsequent challenge with the peptide in complete Freund's adjuvant in adult life, although this neonatal regimen suppressed proliferation and the production of Th1 cytokines (interleukin[IL]-2 and interferon gamma), Th2 cytokine (IL-5, IL-4, and IL-10) secretion was increased, and the serum levels of Th1- and Th2-dependent isotypes of peptide-specific Ab remained elevated. The in vitro proliferative unresponsiveness in Th1 cells could be reversed by Abs to Th2 cytokines (IL-4 and IL-10). Thus, neonatal treatment with a peptide antigen induces T cell priming including production of IgG Abs of both Th1- and Th2-dependent isotypes. Upon subsequent peptide exposure, the peptide-specific T cell responses undergo an effective class switch in the direction of Th2, resulting in T cell proliferative unresponsiveness. Accordingly, this shift towards increased Ab production to autoantigen could be deleterious in individuals prone to antibody-mediated diseases. Indeed, neonatal treatment with a self-autoantigenic peptide from an anti-DNA monoclonal Ab (A6H 58-69) significantly increased the IgG anti-double-stranded DNA Ab levels in lupus-prone NZB/NZW F1 mice, despite suppressing peptide-specific T cell proliferation. This adverse clinical response is in sharp contrast to the beneficial outcome of neonatal treatment with autoantigens in Th1-mediated autoimmune diseases, such as autoimmune encephalomyelitis, as reported by others. A Th1 to Th2 immune deviation can explain the discordant biological responses after the presumed induction of neonatal tolerance in autoantibody- vs. Th-1 mediated autoimmune diseases.

Fine specificity of regulatory T cells. II. Suppressor and helper T cells are induced by different regions of hen egg-white lysozyme in a genetically nonresponder mouse strain.

We have examined the ability of two purified peptide fragments derived from hen (chicken) egg-white lysozyme (HEL); N-terminal, Co-terminal peptide (a.a. 1--17:cys 6--cys 127:120--129) and mixed disulfide LII peptide (LII) (a.a. 13--105) to induce antigen-specific suppression or help in B10 (H-2b) nonresponder and B10.A (H-2a) responder mice. An anti-HEL primary in vitro antibody response can be obtained in either strain by stimulation with HEL coupled to erythrocytes (RBC). Preimmunization with HEL-complete Freund's adjuvant-(CFA) or N-C-CFA-induced suppression of the anti-HEL PFC response to HEL-RBC in spleen cell cultures from B10 mice, whereas helper activity was demonstrated in cultures from B10.A mice similarly immunized. LII-CFA priming elicited helper cells in both C57BL/10 Sn (B10) and B10.A/SgSn (B10.A) mice. The genetic nonresponsiveness of B10 mice to HEL can therefore be attributed to the activation of suppressor T cells by a limited portion of the molecule (e.g., N-C) which prevent the potential response directed against other epitopes on the same molecule (e.g., LII). One manifestation of major histocompatibility complex gene activity appears to be the intramolecular selection of different antigenic determinants leading to activation of functionally different T-cell subpopulations.

Distinct recognition phenotypes exist for T cell clones specific for small peptide regions of proteins. Implications for the mechanisms underlying major histocompatibility complex-restricted antigen recognition and clonal deletion models of immune response gene defects.

Using synthetic peptides as antigens, it was found that T cell clones of a given haplotype specific for 13-16 amino acid peptides could be clearly distinguished by the varied influence of amino acid substitutions on recognition. This was true for different antigenic determinants within peptides 81-96 and 74-86 of hen egg-white lysozyme, recognized in the context of the I-Ab and I-Ak molecules, respectively. Considerable complexity was demonstrated in the induced T cell repertoire specific for apparently single determinants, which implies that diversity of T cell recognition approaches that for B cells. The implications of the degeneracy of T cell recognition are discussed in the context of mechanisms through which Ia molecules restrict recognition and theories of Ir gene defects.

Recognition of multiple peptide cores by a single T cell receptor.

We present evidence that a single T cell clone can recognize at least five different overlapping peptides, each with its distinct core structure, in the context of the same major histocompatibility complex (MHC) molecule. Distinct core residues are crucial for triggering the T cell receptor (TCR) in each case. These results suggest that the TCR (a) has multiple sets of contact residues for alternative peptide-MHC ligands, the binding to any one of which can trigger the cell; and/or (b) is able to attach to the peptide-MHC complex in more than one orientation. In this sense, the TCR is a multisubsite structure capable of being stimulated by a variety of peptide ligands associated with the same MHC molecules.

Differential major histocompatibility complex-related activation of idiotypic suppressor T cells. Suppressor T cells cross-reactive to two distantly related lysozymes are not induced by one of them.

B10 (H-2b) mice are genetic nonresponders to hen egg-white lysozyme (HEL) and the distantly related human lysozyme (HUL). However, anti-HEL or anti-HUL primary antibody responses in vivo or in vitro can be obtained in B10 mice by immunization with the appropriate lysozyme coupled to erythrocytes. T cells able to suppress either anti-lysozyme plaque-forming cells (PFC) response are induced in B10 mice after immunization with HEL-complete Freund's adjuvant (CFA) or HUL-CFA. This cross-reactivity of HEL and HUL in the induction and the expression of suppressive activity is in marked contrast to their very low cross-reactivity at the PFC level. These results suggest that either HEL or HUL can stimulate a suppressor T cell which recognizes a particular epitope present on both lysozymes. Suppressor cells induced by HEL or HUL bear the same predominant idiotype found on the majority of anti-HEL antibodies, and on the small proportion of anti-HUL antibodies cross-reactive with HEL. B10.Q (H-2q) mice are responders in vivo to HEL-CFA, but not to HUL-CFA. In contrast to B10, HEL-CFA priming in B10.Q micr induces helper cells whereas HUL-CFA priming induces suppressor cells. These suppressor cells are cross-reactive with HEL and are fully able to suppress HEL-specific helper cells. The presence of HEL-specific suppressor cell precursors in B10.Q mice which are not activated by HEL, seems to implicate differential choice by the antigen presenting system as a basis for Ir gene control, rather than the absence of a regulatory cell type from the T cell repertoire.

Unresponsiveness to a self-peptide of mouse lysozyme owing to hindrance of T cell receptor-major histocompatibility complex/peptide interaction caused by flanking epitopic residues.

A self-peptide containing amino acid residues 46-61 (NRGDQSTDYGIFQINSR) of mouse lysozyme (ML) (p46-61, which binds strongly to the A(k) molecule but does not bind to the E(k) molecule), can induce a strong proliferative T cell response in CBA/J mice (A[k], E[k]) but no response at all in B10.A(4R) and CBA/J mice. The critical residues within p46-59 are immunogenic in both B10.A(4R) and CBA/J mice. The critical residues within p46-61 reside between amino acid positions 51 and 59. T cells of B10.A(4R) mice primed with the truncated peptides in vivo cannot be restimulated by p46-61 in vitro. This suggests that T cell receptor (TCR) contact (epitopic) residue(s) flanking the minimal 51-59 determinant within p46-61 hinder the interaction of the p46-61/A(k) complex with the appropriate TCR(S), thereby causing a lack of proliferative T cell response in this mouse strain. Unlike B10.A(4R) mice, [B10.A(4R) x CBA/J]F1 mice responded vigorously to p46-61, suggesting that thymic APC of B10.A(4R) mice do not present a self ligand to T cells resulting in a p46-61-specific hole in the T cell repertoire in B10.A(4R) or the F1 mice. Moreover, APC from B10.A(4R) mice are capable of efficiently presenting p46-61 to peptide-specific T cell lines from CBA/J mice. The proliferative unresponsiveness of B10.A(4R) mice to p46-61 is not due to non-major histocompatibility complex genes because B10.A mice (A[k], E[k]) respond well to p46-61. Interestingly, B10.A(4R) mice can raise a good proliferative response to p46-61 (R61A) (in which the arginine residue at position 61 (R61L/F/N/K), indicating that R61 was indeed responsible for hindering the interaction of p46-61 with the appropriate TCR. Finally, chimeric mice [B10.A(4R)-->B10.A] responded vigorously to p46-61, suggesting that thymic antigen presentation environment of the B10.A mouse was critical for development of a p46-61-reactive T cell repertoire. Thus, we provide experimental demonstration of a novel mechanism for unresponsiveness to a self peptide, p46-61, in the B10.A(4R) mouse owing to hindrance: in this system it is the interaction between the available TCR and the A(k)/p46-61 complex, which is hindered by epitopic residue(s) within p46-61. We argue that besides possessing T cells that are hindered by R61 of p46-61, CBA/J and B10.A mice have developed an additional subset of T cells bearing TCRs which are not hinderable by R61, presumably through positive selection with peptides derived from class II E(k), or class I D(k)/D(d) molecules. These results have important implications in self tolerance, shaping of the T cell repertoire, and in defining susceptibility to autoimmunity.

T cell receptor complementarity determining region 3 length analysis reveals the absence of a characteristic public T cell repertoire in neonatal tolerance. The response in the "tolerant" mouse within the residual repertoire is quantitatively similar but qualitatively different.

All adult BALB/c mice immunized with hen egg white lysozyme (HEL) or its dominant determinant, peptide (p)106-116, mount a T cell response using a "public" Vbeta8.2Jbeta1.5 T cell clone. Neonatal exposure to tolerance-inducing doses of antigen can drastically diminish responsiveness in the draining lymph nodes but not in the spleens of animals challenged as adults with the cognate antigen. To determine the role of T cell deletion or anergy within the mechanisms of observed neonatal "tolerance," we treated neonatal BALB/c mice with HEL and directly followed the characteristic public clone using complementarity determining region 3 length T cell repertoire analysis. Our results confirm that despite intraperitoneal injection of neonates with a high dose of HEL emulsified in incomplete Freund's adjuvant, a strong splenic proliferative response to HEL was observed upon recall. However, the adult splenic T cell response of these neonatally treated mice lacked the usual Vbeta8.2Jbeta1.5 public clone characteristic of HEL-primed BALB/c mice. After challenge with HEL-complete Freund's adjuvant as adults, immunoglobulin (Ig)G2a isotype antibody was drastically reduced, and IgG1 was found to be the predominant anti-HEL IgG isotype expressed, indicating a deviation of cytokine response toward T helper type 2. 5-wk-old mice, nasally instilled with tolerogenic doses of HEL p106-116, also showed significant inhibition of this public T cell expansion. These results demonstrate that during neonatal and adult nasal tolerance induction, deletion/anergy removes the public clone, exposing a response of similar specificity but that is characterized by the T helper type 2 phenotype and a splenic residence.

Donor major histocompatibility complex (MHC) peptides are presented by recipient MHC molecules during graft rejection.

Peptides from donor major histocompatibility complex (MHC) molecules were examined for their activation of allogeneically primed T cells. After immunization with either allogeneic spleen cells or a skin allograft, primed T cells proliferate in response to peptides derived from polymorphic regions of alpha and beta chains of class II allo-MHC molecules. The results demonstrate that presentation of donor-MHC peptides by host-derived antigen-presenting cells is a common event in vivo. Thus, self-restricted T cell recognition of processed alloantigens may play a critical role in transplantation. An in-depth understanding of this response may result in the development of additional molecular therapies to combat allograft rejection.

T cell determinant structure: cores and determinant envelopes in three mouse major histocompatibility complex haplotypes.

T lymphocytes recognize discrete regions on an antigen. The specificity of the T cell responses in three mouse strains of differing major histocompatibility complex (MHC) haplotype to a protein antigen, lysozyme, was analyzed using a series of peptides that walk the antigen in single amino acid steps. These peptide series were synthesized using the pin synthesis system, which was modified to allow the peptides to be cleaved from the pins into a physiological buffer free of toxic compounds. This methodology overcomes many of the problems associated with the production of peptides for screening proteins for antigenic determinants. The T cell determinants for the three strains were markedly different. This result points out the limitations of algorithms predicting determinants without reference to the MHC, and the importance of the empirical methodology. This analysis of the T cell response to lysozyme constitutes the most complete study of reactivity to a foreign protein to date and illustrates many important features of antigen recognition by T cells, e.g., presence of major and minor determinant regions. The outer boundaries of each immunogenic region, the determinant envelope, are difficult to define from recently immunized lymph nodes because of the heterogeneity in T cell recognition. However, core sequences common to all the immunogenic peptides in a continuous sequence can be easily defined.

T cell determinants from autoantibodies to DNA can upregulate autoimmunity in murine systemic lupus erythematosus.

(NZB x NZW) F1 (BWF1) mice develop spontaneous T cell autoimmunity to VH region determinants of syngeneic anti-DNA before the onset of clinical disease. In this study, we characterized the immunogenicity, MHC binding, and lymphokine secretion patterns induced by T cell determinants from the VH region of one such anti-DNA mAb (A6.1) and examined their role in the regulation of autoimmunity. Determinants were identified by proliferation of syngeneic splenic T cells from young, unprimed BWF1 mice in response to overlapping 12-mer peptides representing the entire VH region sequence. Immunization of young BWF1 mice with any of three determinants (A6H 34-45 [p34], A6H 58-69 [p58], and A6H 84-95 [p84]) elicited proliferative responses upon in vitro recall. Upon immunization with the whole A6.1 molecule, however, proliferative responses could be recalled only to the p58 peptide, defining this as immunodominant. The other two peptides (p34 and p84) elicited minimal or no proliferation and could be termed cryptic. Proliferative responses elicited by the cryptic determinants were restricted by a single class II (I-Ed for p34 and I-Au for p84), whereas the immunodominant p58 determinant was restricted by both I-Ed and I-Eu. The cryptic p34 and p84 bound strongly to I-Ed and I-Au, respectively, whereas the immunodominant p58 peptide bound poorly to I-Ed. A6H p84 elicited T cells that secreted lymphokines in a pattern consistent with a Th1-like phenotype, whereas p58 induced a Th2-like cytokine pattern. Immunization with p34 or p84, or adoptive transfer of a p84-reactive T cell line to young BWF1 mice significantly increased IgG anti-DNA levels, accelerated nephritis, and decreased survival. In conclusion, in BWF1 mice, autoreactive T cells recognizing both cryptic and dominant self-determinants on anti-DNA autoantibodies escape deletion or anergy induction. Furthermore, since these cells are spontaneously activated before the onset of clinical disease, they may be involved in the development of the autoimmune process.