CD4+ T cells from (New Zealand Black x New Zealand White)F1 lupus mice and normal mice immunized against apoptotic nucleosomes recognize similar Th cell epitopes in the C terminus of histone H3

We have previously reported that peptide 88-99 of histone H4 represents a minimal T cell epitope recognized by Th cells from nonautoimmune BALB/c (H-2(d/d)) mice immunized with nucleosomes. In this study, we tested a panel of overlapping peptides spanning the whole sequences of H4 and H3 for recognition by CD4(+) T cells from unprimed (New Zealand Black (NZB) x New Zealand White (NZW))F(1) lupus mice (H-2(d/z)). None of the 11 H4 peptides was recognized by CD4(+) T cells from (NZB x NZW)F(1) mice. In contrast, these cells proliferated and secreted IL-2, IL-10, and IFN-gamma upon ex vivo stimulation with H3 peptides representing sequences 53-70, 64-78, and 68-85. Peptides 56-73 and 61-78 induced the production of IFN-gamma and IL-10, respectively, without detectable proliferation, suggesting that they may act as partial agonist of the TCR. Th cells from unprimed BALB/c mice and other lupus-prone mice such as SNF(1) (H-2(d/q)) and MRL/lpr (H-2(k/k)) mice did not recognize any peptides present within the H3 region 53-85. We further demonstrated that immunization of normal BALB/c mice with syngeneic liver nucleosomes and spleen apoptotic cells, but not with nonapoptotic syngeneic cells, induced Th cell responses against several peptides of the H3 region 53-85. Moreover, we found that this conserved region of H3, which is accessible at the surface of nucleosomes, is targeted by Abs from (NZB x NZW)F(1) mice and lupus patients, and contains motifs recognized by several distinct HLA-DR molecules. It might thus be important in the self-tolerance breakdown in lupus.     

B and T cell responses to the spliceosomal heterogeneous nuclear ribonucleoproteins A2 and B1 in normal and lupus mice

Autoantibodies directed against spliceosomal heterogeneous nuclear ribonucleoproteins (hnRNPs) are a typical feature of rheumatoid arthritis, systemic lupus erythematosus, and mixed-connective tissue disease. With the aim of investigating a potential pathogenic role of these Abs, we have studied the Ab response to A2/B1 hnRNPs in different murine models of lupus. The specificity of anti-A2/B1 Abs was tested with a series of 14 overlapping synthetic peptides covering the region 1-206 of A2 that contains most of the epitopes recognized by patients' Abs. A major epitope recognized very early during the course of the disease by Abs from most of MRL lpr/lpr mice but not from other lupus mice and from mice of different MHC haplotypes immunized against B1 was identified in residues 50-70. This peptide contains a highly conserved sequence RGFGFVTF also present in other hnRNPs and small nuclear ribonucleoproteins. Abs reacting with a second A2 epitope identified in residues 35-55 were detectable several weeks later, suggesting an intramolecular B cell epitope spreading during the course of the disease. We identified several T cell epitopes within the region 35-175 that generated an effective Th cell response with IL-2 and IFN-gamma secretion in nonautoimmune CBA/J mice sharing the same MHC haplotype H-2k as MRL/lpr mice. None of the peptides stimulated T cells primed in vivo with B1. Because Abs to peptide 50-70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that within the protein, this segment contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response.     

Antigen-specific therapy of murine lupus nephritis using nucleosomal peptides: tolerance spreading impairs pathogenic function of autoimmune T and B cells

In the (SWR x NZB)F1 mouse model of lupus, we previously localized the critical autoepitopes for nephritogenic autoantibody-inducing Th cells in the core histones of nucleosomes at aa positions 10-33 of H2B and 16-39 and 71-94 of H4. A brief therapy with the peptides administered i.v. to 3-mo-old prenephritic (SWR x NZB)F1 mice that were already producing pathogenic autoantibodies markedly delayed the onset of severe lupus nephritis. Strikingly, chronic therapy with the peptides injected into 18-mo-old (SWR x NZB)F1 mice with established glomerulonephritis prolonged survival and even halted the progression of renal disease. Remarkably, tolerization with any one of the nucleosomal peptides impaired autoimmune T cell help, inhibiting the production of multiple pathogenic autoantibodies. However, cytokine production or proliferative responses to the peptides were not grossly changed by the therapy. Moreover, suppressor T cells were not detected in the treated mice. Most interestingly, the best therapeutic effect was obtained with nucleosomal peptide H416-39, which had a tolerogenic effect not only on autoimmune Th cells, but autoimmune B cells as well, because this peptide contained both T and B cell autoepitopes. These studies show that the pathogenic T and B cells of lupus, despite intrinsic defects in activation thresholds, are still susceptible to autoantigen-specific tolerogens.     

Identification and characterization of T helper epitopes in the nucleoprotein of influenza A virus

By using a series of overlapping synthetic peptides that cover more than 95% of the amino acid sequence of nucleoprotein (NP) of influenza A/NT/60/68 virus, five Th cell epitopes in B10.S (H-2s), BALB/c (H-2d), CBA (H-2k), and B6 (H-2b) mice have been identified. The specificity of Th cell recognition of epitopes is largely dependent on the H-2 haplotype of the responding mouse strain. However, two out of the five Th epitopes defined could be recognized by mice of more than one haplotype, implying that the primary sequence of protein antigens could also influence the selection of dominant T cell epitopes by the immune system. Immunization of B10.S mice with peptide 260-283 generated strong Th cell response against type A influenza viruses. In the other three strains of mice tested, priming with helper peptides induced a stronger antipeptide than antiviral T cell response. However, the low responsiveness to virus in these mice could be partially overcome by immunization with a mixture of several helper peptides. The Th epitopes are defined by the ability of the peptides to stimulate class II MHC restricted CD4+ T cells to proliferate and to produce IL-2 in vitro. When compared with the known epitopes on NP recognised by class I restricted CD8+ cytotoxic T cells, it appears that Th and cytotoxic T cell epitopes are nonoverlapping. The AMPHI and Motifs methods were employed to analyze the sequence of NP and predict the potential dominant sites in the molecule. The predictions are compared with the experimental data obtained and the implications discussed.     

The I-Abm12 mutation, which confers resistance to experimental myasthenia gravis, drastically affects the epitope repertoire of murine CD4+ cells sensitized to nicotinic acetylcholine receptor.

Susceptibility to experimental autoimmune myasthenia gravis (EAMG), which is induced in mice by injection of purified Torpedo nicotinic acetylcholine receptor (TAChR), is influenced by the I-A locus products, which restrict presentation of AChR Th epitopes. The bm12 mutation of the I-Ab molecule in the C57BL/6 strain, which is highly susceptible to EAMG, yields the EAMG resistant mutant B6.C-H-2bm12 (bm12). We investigated here the consequences of the bm 12 mutation on the CD4+ response to the TAChR alpha subunit. Upon immunization with TAChR, CD4+ cells became sensitized to TAChR and anti-AChR antibodies were produced in both bm12 and C57BL/6 strains. Overlapping synthetic peptides, corresponding to the complete sequence of TAChR alpha subunit, were used to identify Th epitopes. CD4+ cells from C57BL/6 mice recognized peptides T alpha 150-169, T alpha 181-200, and T alpha 360-378. CD4+ cells from bm12 mice did not respond to any synthetic sequence. Upon injection of the three C57BL/6 Th epitope peptides, either individually or as a pool, CD4+ cells from C57BL/6 mice recognized each peptide and TAChR. Therefore they recognized epitopes similar or identical to those originated from TAChR processing. CD4+ cells from bm12 mice injected with the same peptides responded to T alpha 360-378 strongly, to a lesser extent to T alpha 181-200, never to peptide T alpha 150-169. Only CD4+ cells sensitized against the T epitope peptide T alpha 181-200 responded to TAChR. We tested if lack of response to T alpha 150-169, and the low response to T alpha 181-200, was due to inability of the I-Abm12 molecule to present the T epitope peptides. bm12 and C57BL/6 APC were used to present the T epitope peptides to specifically sensitized CD4+ cells from C57BL/6 mice. All T epitope peptides were presented by bm12 APC, although T alpha 150-169 was presented less efficiently than by C57BL/6 APC. Resistance to EAMG induced by the bm12 mutation may be due to the change in the epitope repertoire of AChR-specific Th cells, and lack of recognition of otherwise immunodominant Th epitopes. For at least one epitope this might be due to absence of potentially reactive, specific CD4+ clones.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

The Dermatophagoides pteronyssinus group 2 allergen contains a universally immunogenic T cell epitope

The use of T cell epitope-containing peptides for the induction of anergy in allergen sensitization is limited by genetic restriction that could be circumvented by using universally immunogenic epitopes. We attempted to identify such epitopes on Dermatophagoides pteronyssinus group 2 allergen (Der p 2), a major allergen of D. pteronyssinus T cells from BALB/c (H-2(d)), C57BL/6 (H-2(b)), C3H (H-2(k)), and SJL (H-2(s)) mice that were immunized with rDer p 2, recognized an immunodominant region encompassing residues 21-35. A synthetic 21-35 peptide (p21-35) induced strong dose-dependent in vitro T cell proliferation with cells of the four mouse strains and required processing for MHC class II presentation. Substitution of Ile(28) with Ala resulted in reduction of T cell proliferation in each strain. Ile(28) could represent an important MHC class II anchoring residue for T cell response to p21-35. An immunodominant T cell epitope of Der p 2 therefore behaves as a universal epitope and could be a suitable candidate for T cell anergy induction.     

MHC class II-bound self peptides from autoimmune MRL/lpr mice reveal potential T cell epitopes for autoantibody production in murine systemic lupus erythematosus

The systemic lupus erythematosus-like syndrome in MRL/lpr mice involves high-titered IgG autoantibodies, particularly antinuclear Abs that target histones, DNA, and RNA particles. Although T cell help is required for the generation of antinuclear Abs, the epitopes recognized by such helper T cells are unknown. To address this question, we isolated and sequenced self peptides bound by MHC class II molecules from MRL/lpr mice. We identified a number of peptides that are not seen in similar preparations from nonautoimmune C3H animals. The "abnormal" peptide donors include histone, a protein component of a small nuclear ribonucleoprotein, ribosomal proteins, and RNA processing enzymes. We postulate that the peptides from these donors are T cell epitopes required for the generation of the most frequent antinuclear Abs specificities seen in MRL/lpr mice.     

DM peptide-editing function leads to immunodominance in CD4 T cell responses in vivo

DM functions as a peptide editor for MHC class II-bound peptides. We examined the hypothesis that DM peptide editing plays a key role in focusing the in vivo CD4 T cell responses against complex pathogens and protein Ags to only one, or at most a few, immunodominant peptides. Most CD4 T cells elicited in the wild-type BALB/c (H-2d) mice infected with Leishmania major predominantly recognize a single epitope 158-173 within Leishmania homologue of activated receptor for c-kinase (LACK), as is the case when these mice are immunized with rLACK. Using DM-deficient (DM-/-) H-2d mice, we now show that in the absence of DM, the in vivo CD4 T cell responses to rLACK are skewed away from the immunodominant epitopes and are diversified to include two novel epitopes (LACK 33-48 and 261-276). DM-/- B10.BR (H-2k) mice showed similar results. These results constitute the first demonstration of the role of DM peptide editing in sculpting the specificity and immunodominance in in vivo CD4 T cell responses.     

The murine B cell repertoire is severely selected against endogenous cellular prion protein

Abs to the prion protein (PrP) can protect against experimental prion infections, but efficient Ab responses are difficult to generate because PrP is expressed on many tissues and induces a strong tolerance. We previously showed that immunization of wild-type mice with PrP peptides and CpG oligodeoxynucleic acid overcomes tolerance and induces cellular and humoral responses to PrP. In this study, we compared Ab and T cell repertoires directed to PrP in wild-type and PrP knockout (Prnp o/o) C57BL/6 mice. Animals were immunized with mouse PrP-plasmid DNA or with 30-mer overlapping peptides either emulsified in CFA or CpG/IFA. In Prnp o/o mice, Abs raised by PrP-plasmid DNA immunization recognized only N-terminal PrP peptides; analyses of Ab responses after PrP peptide/CFA immunization allowed us to identify six distinct epitopes, five of which were also recognized by Abs raised by PrP peptides/CpG. By contrast, in wild-type mice, no Ab response was detected after PrP-plasmid DNA or peptide/CFA immunization. However, when using CpG, four C-terminal peptides induced Abs specific for distinct epitopes. Importantly, immune sera from Prnp o/o but not from wild-type mice bound cell surface PrP. Abs of IgG1 and IgG2b subclasses predominated in Prnp o/o mice while the strongest signals were for IgG2b in wild-type mice. Most anti-PrP Th cells were directed to a single epitope in both Prnp o/o and wild-type mice. We conclude that endogenous PrPC expression profoundly affects the Ab repertoire as B cells reactive for epitopes exposed on native PrPC are strongly tolerized. Implications for immunotherapy against prion diseases are discussed.     

Localization of T cell epitope regions of chicken ovomucoid recognized by mice

We localized the T cell epitope regions of chicken ovomucoid (OVM), a potent egg allergen, with the overlapping pin-peptides covering the entire sequence of OVM and three strains of mice with different haplotypes. In C3H/He (H-2k) mice, the T cells recognized relatively broad regions on OVM; the dominant regions were 49-93 and 97-114 residues, and the subdominant regions were 7-21, 37-48, 94-96, 115-123 and 145-177 residues. In contrast, a more limited number of T cell epitope regions were localized in BALB/c (H-2d) and C57BL/6 (H-2b) mice. The T cells from BALB/c mice recognized 100-114 and 157-171 residues, and the T cells from C57BL/6 mice recognized only 157-180 residues. These results were confirmed by using peptides separately synthesized and purified on the putative epitope regions. The roles of the carbohydrate moieties and cysteine residues involved in the disulfide bridges of OVM were also examined, and we found that they were not important in recognition by the T cell/antigen presenting cell.     

Identification of epitopes within the circumsporozoite protein of Plasmodium vivax recognized by murine T lymphocytes.

The murine cellular immune response to the circumsporozoite (CS) protein of Plasmodium vivax was characterized using five synthetic peptides, some of which we identified as corresponding to T cell epitopes. The peptides P308-320, P344-355 and P353-364 were immunogenic, inducing a genetically restricted proliferative response, due to the activation of CD4+ T cells. The peptide P308-320 was recognized only by the lymphocytes of B10 (H-2b) mice. The other two peptides were recognized by primed lymphocytes of H-2a and H-2k mice. Of interest was the finding that one of these peptides, P353-364, induced a proliferative response of a large percentage of immune outbred Swiss mice. Our data provide evidence that, at least in mice, there is recognition of multiple T cell epitopes within the major surface antigen of P. vivax sporozoites.     

Selective modulation of CD4+ T cells from lupus patients by a promiscuous, protective peptide analog

A peptide encompassing residues 131-151 of the spliceosomal U1-70K protein and its analog phosphorylated at Ser140 were synthesized as potential candidates for the treatment of patients with lupus. Studies in the MRL/lpr and (NZB x NZW)F1 lupus models have demonstrated that these sequences contain a CD4+ T cell epitope but administration of the phosphorylated peptide only ameliorates the clinical manifestations of treated MRL/lpr mice. Binding assays with soluble HLA class II molecules and molecular modeling experiments indicate that both peptides behave as promiscuous epitopes and bind to a large panel of human DR molecules. In contrast to normal T cells and T cells from non-lupus autoimmune patients, we found that PBMCs from 40% of lupus patients selected randomly and CFSE-labeled CD4+ T cells proliferate in response to peptide 131-151. Remarkably, however, we observed that phosphorylation of Ser140 prevents CD4+ T cells proliferation but not secretion of regulatory cytokines, suggesting a striking immunomodulatory effect of phosphorylated analog on lupus CD4+ T cells that was unique to patients. The analog might act as an activator of regulatory T cells or as a partial agonist of TCR.     

A dominant Th epitope in influenza nucleoprotein. Analysis of the fine specificity and functional repertoire of T cells recognizing a single determinant.

The sequence 260-283 of the nucleoprotein (NP) of influenza A virus is an epitope recognized by virus-immune lymph node cells from CBA (H-2k), B6 (H-2b), and B10.S (H-2s) mice. Further analysis shows that there are at least two Th epitopes within this sequence: the one close to the N-terminal (p260-273) is recognized by T cells from CBA and B6 mice while that close to the carboxyl-terminal (p270-283) is a dominant Th determinant in B10.S mice. The fine specificity of the recognition of this epitope by NP-specific T cell clones is also studied. When B10.S mice were infected intranasally or i.v. with live influenza virus, or immunized by different ways with various Ag preparations, P270-283 persistently emerged as a dominant T cell epitope. Immunization of B10.S mice with peptide p270-283 induces T cells with different in vivo functions including class II-restricted cytotoxicity, cognate help for Ag-specific antibody synthesis and delayed type hypersensitivity. This may have important implications for the understanding of the differentiation and classification of subsets of CD4+ T cells. The corresponding sequence of the NP of an equine influenza virus, A/Eq/Prague/56, which has a substitution (leucine to proline) at position 283, was not recognized by the lymph node cells from mice primed with either A/Okuda or A/Eq/Prague. However, the peptide, p270-283(E), representing this sequence induced T cell responses to both human and equine viruses. The data are discussed with respect to the development of viral vaccines.     

Novel engineered trastuzumab conformational epitopes demonstrate in vitro and in vivo antitumor properties against HER-2/neu

Trastuzumab is a growth-inhibitory humanized Ab targeting the oncogenic protein HER-2/neu. Although trastuzumab is approved for treatment of advanced breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive and has a limited duration of action, necessitating repeated administrations of the mAb. Active immunotherapy with conformational B cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide Abs. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the Ag-binding region of HER-2 spans residues 563-626 that comprises an extensive disulfide-bonding pattern. To delineate the binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion "promiscuous" T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformational peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell proliferation, with 563-598 and 597-626 showing greater reactivity. All epitopes were immunogenic in FVB/N mice with Abs against 597-626 and 613-626 recognizing HER-2. The 597-626 epitope was immunogenic in outbred rabbits eliciting Abs which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their Ab-dependent cell-mediated cytotoxicity. Moreover, immunization with the 597-626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. These results suggest the peptide B cell immunogen is appropriate as a vaccine for HER-2-overexpressing cancers because the resulting Abs show analogous biological properties to trastuzumab.     

Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.     

Cell-mediated immune responses to vaccine peptides derived from the circumsporozoite protein of Plasmodium falciparum

T cell epitopes residing within vaccine candidate peptides have been identified by delayed-type hypersensitivity (DTH) responses in mice. The recombinant sporozoite vaccine candidate, R32tet32, contains at least two T epitopes, one located within the repeat region and another in the tet tail. When C57BL/6 (H-2b) and BALB/c (H-2d) mice were sensitized intradermally with R32tet32 or the truncated protein R32LR emulsified in CFA and challenged 5 days later with R32tet32, only H-2b mice recognized a T epitope located within the major repeat sequence (NANP) and encoded by four or less repeats. H-2d mice responded solely to the T epitope located on the tet tail. Ear swelling was maximal at 48 h and revealed a histologic pattern characteristic of DTH. CD4+ T cell lines derived from immunized animals demonstrated the ability to mediate local DTH, proliferate, and secrete lymphokines in response to stimulation with Ag. High dose i.v. administration of R32tet32 in C57BL/6 and BALB/c mice before intradermal sensitization with R32tet32 revealed that DTH responses were suppressed only in BALB/c mice. Further experiments localized the suppressive determinant to the tet tail. Collectively, these data indicate that DTH may prove to be a useful method to characterize the biologic activity of T epitopes, furthermore they suggest that candidate vaccine peptides should be tested for suppressive activity before inclusion in a vaccine.     

A recombinant malaria protein that can induce Th1 and CD8+ T cell responses without antibody formation.

Inbred strains of mice were immunized with p190-3, a 38-kDa recombinant protein derived from p190, a major merozoite surface Ag of the malaria parasite Plasmodium falciparum. Ag-specific proliferative T cell responses were obtained in H-2b, H-2d, and H-2k mouse strains. Surprisingly, mice of the H-2b haplotype (e.g., C57BL/6) did not give a measurable antibody response to the recombinant protein administered in Freund's adjuvant, but CD8+/CD4- as well as CD4+/CD8- T cells specific for p190-3 could be obtained after in vivo priming and in vitro selection with Ag. Distinct epitopes of p190-3 recognized by the CD8+ and CD4+ T cells from C57BL/6 mice were identified. The CD8+ T cells could kill H-2b APC in the presence of the appropriate epitope-containing peptide. The p190-3-specific CD4+ cells isolated from C57BL/6 mice were of the Th1 type. In contrast, Th2 cells, but no CD8+ T cells were present in a p190-3-specific line from BALB/c mice, which give good antibody responses to p190-3.