Epitope repertoire of human CD4(+) T cells on the A3 domain of coagulation factor VIII.

Severe hemophilia A patients treated with factor (F)VIII may develop antibodies (Ab) that block FVIII function (inhibitors). Autoimmune inhibitors may develop in subjects without congenital hemophilia, and cause acquired hemophilia. Hemophiliacs without inhibitors and healthy subjects may also have small amounts of antiFVIII Ab. FVIII-specific CD4(+) T cells induce antiFVIII Ab synthesis. Here, we have examined their epitope repertoire in hemophilia patients and healthy subjects. We used overlapping synthetic peptides, spanning the sequence of the FVIII A3 domain, to challenge blood CD4(+) T cells in proliferation assays. The epitopes recognized in hemophilia A patients with or without inhibitors, acquired hemophilia patients, or healthy subjects overlapped, yet had characteristic differences. Most members of one or more study groups recognized the sequence regions 1691-1710, 1801-1820, 1831-1850, and 1941-60. In the proposed three-dimensional structure of the A3 domain, these sequences are largely exposed to the solvent and flanked by flexible sequence loops: these are structural features characteristic of 'universal' CD4(+) T epitopes. Hemophilia A patients with inhibitors recognized prominently only the sequence 1801-1820, which overlaps a known inhibitor binding site. This is consistent with the possibility that CD4(+) T cells recognizing epitopes within residues 1801-1820 have a role in inducing inhibitor synthesis. In contrast, CD4(+) T cells sensitized to sequences 1691-1710 and 1941-60, which are recognized by healthy subjects and hemophilia A patients without inhibitors, might curb inhibitor synthesis.

Recognition of coagulation factor VIII by CD4+ T cells of healthy humans.

Hemophilia A patients treated with coagulation factor (F)VIII may develop an anti-FVIII immune response. Anti-FVIII antibodies may occur also in healthy subjects. To understand the extent to which an immune response to FVIII occurs in healthy subjects, we investigated the proliferative response of blood CD4+ T cells from 90 blood donors to FVIII and to pools of overlapping synthetic peptides spanning the sequences of individual FVIII domains (A1-A3, C1-C2). Most subjects responded to FVIII and several FVIII domains. Men had stronger responses to FVIII than women, and older subjects than younger subjects. The domain-induced responses were weaker than the FVIII-induced responses, yet their intensity in individual subjects correlated with that of the response to FVIII. We examined whether Th1 and/or Th2 cells responded to FVIII in 68 subjects, by determining the CD4+ T cells that secreted interferon-gamma (IFN-gamma) or interleukin (IL)-5 after stimulation with FVIII: 25 subjects had FVIII-specific IFN-gamma-secreting cells, and seven of them had also FVIII-specific IL-5-secreting cells. None had only IL-5-secreting cells. Thus, a CD4+ T cell response to FVIII, which first involves Th1 cells, is common among subjects with a normal procoagulant function.

Human CD4+ T-cell epitope repertoire on the C2 domain of coagulation factor VIII.

Approximately 25% of severe hemophilia A patients develop antibodies (Ab) that neutralize the procoagulant function of factor (F)VIII (inhibitors). Autoimmune FVIII inhibitors may develop in individuals without congenital FVIII deficiency and cause acquired hemophilia. Low titers of anti-FVIII Ab may be present in hemophilia A patients without inhibitors and in healthy blood donors. FVIII-specific CD4+ T-cells drive the synthesis of anti-FVIII Ab. We examined the epitope repertoire of CD4+ T-cells from 15 healthy subjects, 10 hemophilia A patients without inhibitors, 11 hemophilia A patients with inhibitors, and six acquired hemophilia patients. Blood CD4+ T-cells were challenged in proliferation assays with a panel 16 overlapping synthetic peptides, spanning the sequence of the FVIII C2 domain. The sequence region 2291-2330 contained the most frequently and strongly recognized peptides in each of the four subject groups. Crystallographic B factor data and the location of these peptides within the three-dimensional structure of the C2 domain confirm that this region has a high degree of solvent exposure and flexibility within the peptide backbone, which are structural features typical of immunodominant universal CD4+ epitopes. Furthermore, this sequence region overlaps inhibitor-binding sites, suggesting that CD4+ T-cells recognizing peptide sequences within this region might be involved in inhibitor synthesis. The sequence regions 2191-2210 (recognized strongly by each study group except hemophilia A patients with inhibitors) and 2241-2290 (recognized primarily by acquired hemophilia patients and healthy subjects) share the same structural features, and also overlap inhibitor-binding sites. Although similar, there appear to be important differences in the CD4+ epitope repertoires of congenital and acquired hemophilia patients.

CD4+ T cells specific for factor VIII as a target for specific suppression of inhibitor production.

The studies we reviewed here have begun to clarify the complex cellular mechanisms involved in the immune response to fVIII, and the circumstances under which fVIII inhibitors develop. Further characterization and comparison of the immune response to fVIII in both hemophilia patients and healthy subjects will help to further elucidate these mechanisms. The murine hemophilia model will hopefully provide further insights into the mechanisms of inhibitor formation, and prove to be a suitable tool for the design and testing of therapeutic strategies aimed at preventing the development of fVIII inhibitors.

Sensitization of CD4+ T cells to coagulation factor VIII: response in congenital and acquired hemophilia patients and in healthy subjects.

Antibodies (Ab) that inhibit factor VIII (fVIII) may develop in patients with hemophilia A and rarely in individuals without congenital fVIII deficiency (acquired hemophilia). Synthesis of fVIII inhibitors requires CD4+ T cells. We investigated the proliferative response of blood CD4+ cells from 11 patients with congenital or acquired hemophilia and 12 healthy subjects, to recombinant human fVIII, and to pools of overlapping synthetic peptides spanning the sequences of individual fVIII domains. All patients had CD4+ cells that responded to fVIII. The intensity of the responses fluctuated over time: several patients had brief periods when they did not respond to fVIII. All healthy subjects had transient CD4+ responses to fVIII, that were significantly lower than those of hemophilia patients. Also, healthy subjects responded to fVIII less frequently and for shorter periods than hemophilia patients. All patients and healthy subjects recognized several fVIII domains: the A3 domain was recognized most strongly and frequently. The transient sensitization of CD4+ cells to fVIII in healthy subjects suggests that inadequate tolerization of CD4+ cells to fVIII, due to lack of endogenous fVIII, is an important factor in the development of clinically significant anti-fVIII antibodies in hemophilia A.

T cell recognition of muscle acetylcholine receptor in ocular myasthenia gravis.

We examined the proliferative response of blood CD4(+) cells to muscle acetylcholine receptor (AChR) subunits and the epitope repertoire of the epsilon and gamma subunits, in ocular myasthenia gravis (oMG) patients and healthy subjects. oMG patients seldom recognized all subunits. The frequency and intensity of recognition was the same for all subunits, irrespective of the disease duration. The responses in oMG were lower than in generalized myasthenia gravis. Healthy subjects had frequent, low responses to one or more subunits. oMG patients recognized several epitopes on the gamma and epsilon subunits, that partially overlapped those recognized in gMG. The subunits and epitopes recognized by individual oMG patients changed over time. Thus, oMG patients have minimal and unstable sensitization of anti-AChR CD4(+) cells, in agreement with their low and inconsistent synthesis of anti-AChR antibody.

Universal epitopes for human CD4+ cells on tetanus and diphtheria toxins.

Previous studies suggested that tetanus and diphtheria toxoids (TTD and DTD, respectively) contain "universal" epitopes for human CD4+ cells (residues 632-651 and 950-969 of TTD and 271-290, 321-350, 351-370, 411-430, and 431-450 of DTD). To investigate whether CD4+ cells of 100 randomly selected subjects recognized those sequences, the proliferation of CD4+ cell-enriched blood lymphocytes to TTD and DTD and individual synthetic universal epitopes was measured. CD4+ cells of 98 subjects recognized both toxoids, those of 1 subject only TTD, and those of 1 only DTD. The TTD peptides and DTD peptides 271-290 and 331-350 were recognized by >/=80% of the toxoid-sensitized subjects. The other DTD sequences were recognized by 63%-71% of subjects. DR-homozygous subjects recognized several universal epitopes less frequently than did DR-heterozygous subjects. The intensity of responses to the epitope peptides correlated with that to TTD or DTD, consistent with recognition of the peptides by CD4+ cells specific for the cognate toxoid.

CD4+ T cell response to factor VIII in hemophilia A, acquired hemophilia, and healthy subjects.

These studies have begun to clarify the complex cellular mechanisms involved in the immune response to factor VIII. Although vigorous sensitization of CD4+ cells occurs in healthy subjects, the absence of clinically significant levels of inhibitor antibodies is likely related to the prompt down-regulation of the immune response. It may also be possible that the specific epitope repertoire recognized by CD4+ cells plays a role in the outcome of the immune response to factor VIII. Further characterization and comparison of the CD4+ repertoire in healthy subjects with that of hemophilia patients with and without inhibitors will help clarify which mechanism explains the absence of productive inhibitor synthesis in certain individuals. Also, it might identify CD4+ epitopes recognized by T helper cells that are essential for inhibitor synthesis. Additional studies to further characterize the role of Th1 and Th2 cells in the immune response to factor VIII may also be needed for the design of novel therapeutic strategies aimed at preventing inhibitor development.

Response of CD4+ T cells from myasthenic patients and healthy subjects of biosynthetic and synthetic sequences of the nicotinic acetylcholine receptor.

We investigated the suitability of pools of overlapping synthetic peptides spanning the complete alpha 1 subunit sequence of the human muscle acetylcholine receptor (AChR) (alpha 1 pool) or the extracellular domain (residues 1-218, alpha 11-218 pool), and of biosynthetic alpha 1 constructs from E. coli, as stimulants of human CD4+ cells from myasthenia gravis (MG) patients and healthy subjects. A construct corresponding to residues alpha 11-209 was obtained as solubilized inclusion bodies (ib alpha 11-209), or purified by SDS gel electrophoresis (pur alpha 11-209). A second construct included the extracellular, cytoplasmic and carboxylterminal domains plus histidine residues, and was obtained as inclusion bodies (ib alpha 1NoTrans) or purified by gel permeation and histidine tag affinity chromatography (pur alpha 1NoTrans). A biosynthetic extracellular domain of the neuronal AChR alpha 7 subunit (ib alpha 71-206) isolated from E. coli as inclusion bodies served as control for bacterial contaminants. We used ib alpha 11-209, pur alpha 11-209 and peptide pools to propagate CD4+ lines from two MG patients. The lines obtained using pur alpha 11-209 and the peptide pools recognized the peptide pools and alpha 1 constructs tested well, but ib alpha 71-206 poorly or not at all. These lines recognized peptides known to form CD4+ epitopes in these patients. The ib alpha 11-209 lines recognized ib alpha 11-209 and ib alpha 71-206 strongly, but recognized poorly pur alpha 11-209 and the alpha 11-218 pool. We propagated T-cell lines from a healthy subject using pur alpha 11-209 and ib alpha 11-209. The pur alpha 11-209 line recognized pur alpha 11-209 and the alpha 11-218 pool, but not ib alpha 11-209 or ib alpha 71-206. The ib alpha 11-209 line recognized ib alpha 11-209 and ib alpha 71-206, but not pur alpha 11-209 or the alpha 11-218 pool. We tested blood CD4+ cells from six MG patients and eight healthy subjects with ib alpha 11-209, pur alpha 11-209, the alpha 11-218 pool and--in the healthy subjects--also ib alpha 71-206, ib alpha 1NoTrans and pur alpha 1NoTrans. In both populations, the alpha 11-218 pool elicited low and sporadic responses, while the constructs elicited clear responses that were frequently higher for ib alpha 11-209 than pur alpha 11-209. The responses to ib alpha 71-206 were strong and comparable to those to ib alpha 11-209, ib alpha 1NoTrans, and pur alpha 1NoTrans. These results indicate that even purified constructs from E. coli contain bacterial contaminants recognized by CD4+ cells. They should not be used to test unselected blood CD4+ cells, because they may evoke strong CD4+ responses to the bacterial antigens. Purified recombinant sequences may be suitable for propagation of CD4+ cell lines, if the specificity of the lines can be verified using different antigen preparations. Short synthetic peptide sequences can be safely used for propagation of specific CD4+ cells. Although they are poor stimulants for unselected blood CD4+ cells, the low responses they elicit are probably due to these cells.

Epitope repertoire of human CD4+ T cells on tetanus toxin: identification of immunodominant sequence segments.

Sequence regions of tetanus toxin-forming CD4+ cell epitopes in 8 HLA-disparate subjects were identified. Overlapping synthetic peptides corresponding to the complete tetanus toxin sequence were used to test, in a proliferation assay, unselected blood CD4+ cells or CD4+ cell lines propagated by stimulation with tetanus toxoid. The CD4+ cell lines recognized most peptides recognized by the blood CD4+ cells and they recognized additional peptides. Their responses were stronger than those of unselected blood CD4+ cells. Two peptides were recognized by all subjects: one largely overlapped a tetanus toxin sequence region previously identified as a "universal" T cell epitope. Thirteen other peptides elicited a CD4+ cell response in 6 or 7 of the 8 subjects, and another 10 elicited responses in 5 subjects.

Epitope repertoire of human CD4+ lines propagated with tetanus toxoid or with synthetic tetanus toxin sequences.

The use of synthetic antigen sequences allows propagation in vitro of T cell lines and clones specific for rare antigens, or for individual epitopes. In the present study we investigated the extent of similarity of the epitope repertoire of CD4+ T cell line specific for the antigen tetanus toxin (TTX), propagated with the complete molecule of tetanus toxoid (TTD), and with the synthetic TTX peptides. We propagated from two healthy subjects CD4+ T cell lines specific for TTD, by cycles of stimulation in vitro with TTD or with pools of overlapping synthetic peptides, 20 residues long and overlapping by five residues, corresponding to all or part of the tetanus toxin (TTX) sequence. One pool corresponded to the complete TTX sequence (peptide pool). Two other pools corresponded to residues 1-305 of the TTX light chain and 476-780 of the TTX heavy chain (peptide minipools). The peptide pool-propagated lines recognized TTD vigorously, at levels comparable with those of the TTD-propagated lines. They recognized several peptides, most of which were also recognized by the TTD-propagated line from the same subject. They also recognized to a low extent a few peptides not recognized by the corresponding TTD-propagated line, which might contain cryptic epitopes. The TTD-propagated lines recognized also several peptides that did not elicit a detectable response by the lines propagated with the complete peptide pool. The peptide minipool propagated lines recognized most of the peptides recognized by the TTD-propagated lines. They also recognized several peptides that did not elicit a measurable response of the TTD-propagated line from the same subject, which might contain cryptic epitopes. Very few peptides recognized by the TTD-propagated line did not evoke a response from the peptide minipool propagated lines. To verify that the response to the TTD molecule of the lines propagated with the peptide pools reflected the response of clones recognizing different epitopes produced upon in vitro processing of the TTD molecule, we propagated from each of the two subjects CD4+ T cell lines by stimulation with individual peptide recognized by the TTD-specific lines of that subject (13 peptide-specific lines from subject #1, and 15 from subject #2). All lines responded to the presence of TTD to an extent comparable to the response induced by the same concentration of the relevant peptide, demonstrating that propagation by synthetic epitope sequences allows expansion of T cell clones specific for epitopes which result from processing of the complete TTD molecule. Therefore, whereas the use of very large pools of synthetic antigen peptides for propagation of antigen specific human CD4+ cell lines might lead to loss of clones recognizing less immunogenic sequence regions, peptide pools comprising a relatively limited number of synthetic sequences allow propagation of the majority of the antigen specific T cell clones. The use of peptide pools, and especially of limited peptide pools, results in propagation of polyclonal T cell lines having a more diverse repertoire than the lines propagated by stimulation with the complete antigen molecule. The T clones propagated by the use of the short peptide sequences, which are not expanded when the complete antigen molecule is used, may recognize poorly processed, cryptic epitopes. This approach may be adopted to propagate and detect minor clonal populations, recognizing less immunogenic parts of the antigen.

Epitopes for human CD4+ cells on diphtheria toxin: structural features of sequence segments forming epitopes recognized by most subjects.

The sequence regions of diphtheria toxin (DTX) recognized by CD4+ T cells of seven healthy humans of different major histocompatibility complex haplotypes were identified. Overlapping synthetic peptides, screening the DTX sequence, were used to test in proliferation assays unselected blood CD4+ cells, or DTX-specific CD4+ lines propagated by stimulation with DTX of blood mononuclear cells. Blood CD4+ cells and DTX-specific CD4+ lines gave consistent results. Although each subject had an individual pattern of peptide recognition, six peptide sequences (residues 271-290, 321-340, 331-350, 351-370, 411-430 and 431-450) were recognized by all subjects. In the native DTX molecule, these sequence regions are flanked by sequence loops exposed on the DTX surface. They overlap uncharged segments of the DTX sequence. These structural properties may be general requirements for immunodominance in CD4+ cell sensitization in humans.

A nicotinic acetylcholine receptor regulating cell adhesion and motility is expressed in human keratinocytes.

Acetylcholine is synthesized and released by human epidermal keratinocytes and modulates the adhesion and motility of these cells. To understand the molecular basis of the effects of acetylcholine on keratinocytes, we investigated the presence, pharmacology, structure, and function of nicotinic acetylcholine receptors in human epidermal keratinocytes. Patch-clamp studies indicated that keratinocytes express acetylcholine receptors with ion gating and pharmacologic properties similar to those observed so far only in neurons, and containing the alpha 3 subunit. Specific binding of the receptor-specific ligand 125I-kappa-bungarotoxin revealed approximately 5500 binding sites per cell on undifferentiated keratinocytes in cell cultures and approximately 35,400 binding sites per cell on mature keratinocytes freshly isolated from human neonatal foreskins. Antibody binding and polymerase chain reaction experiments demonstrated the presence of alpha 3, beta 2, and beta 4 nicotinic receptor subunits. Binding of subunit-specific antibodies indicated that nicotinic receptors were associated with the suprabasal keratinocytes in epidermis and localized to the cell membranes of differentiated keratinocytes in cell cultures. Acetylcholine and the nicotinic agonist nicotine increased cell-substrate and cell-cell adherence of cultured keratinocytes and stimulated their lateral migration. The specific antagonists kappa-bungarotoxin and mecamylamine caused cell detachment and abolished migration. Thus, a nicotinic receptor expressed in keratinocytes may mediate acetylcholine control of keratinocyte adhesion and motility.

Immunoregulatory CD8+ cells recognize antigen-activated CD4+ cells in myasthenia gravis patients and in healthy controls.

CD8+ cells inhibiting the response of CD4+ cells exist in rodents, recognizing epitopes unique to a CD4+ clone (Ids) or expressed by all activated CD4+ cell (ergotypes). Stimulation of CD8+ cells recognizing ergotypes shared by all Ag-activated CD4+ cells would be useful for treatment of diseases involving undesirable CD4+ responses to ill defined Ags, such as many autoimmune diseases and allergies. As a first step toward demonstrating the existence of anti-ergotype CD8+ immunoregulatory cells in humans, we investigated here whether CD8+ cells recognizing Ag-activated CD4+ cells exist in autoimmune and healthy humans. CD4+ cells specific for human muscle acetylcholine receptor, tetanus, or diphtheria toxoids were propagated from patients with myasthenia gravis patients and healthy controls. Ag-activated CD4+ cells were irradiated and used as Ag to test the response of CD(4+)-depleted CD(8+)-enriched PBMC (CD8+ PBMC) from myasthenic patients and controls and to propagate short-term CD8+ cell lines from CD8+ PBMC. In both patients and controls CD8+ PBMC and CD8+ lines responded vigorously to autologous Ag-activated CD4+ cells. The CD8+ lines responded equally well to the Ag-activated CD4+ cells of different Ag specificity, suggesting that they recognized CD4+ ergotypes. They did not seem to respond to CD4+ cells activated by PHA. The CD8+ cells recognized class I-restricted epitopes, as their response to activated CD4+ cells was suppressed by anti-class I Ab. CD8+ cells recognizing Ag-activated CD4+ were present cells in the controls for 5 to 12 wk after immunization. In myasthenic patients, CD8+ cells recognizing activated anti-acetylcholine receptor CD4+ cells seemed to be always present.

Effects of fermentation on product consistency.

A variety of different fermentation processes has been successfully employed to produce consistent protein-based biopharmaceuticals from genetically engineered animal cells. Chinese hamster ovary (CHO) cells were genetically modified to produce recombinant human soluble CD4, tissue plasminogen activator (tPA) or erythropoietin (EPO). Soluble CD4 was collected from extended perfused fermentations of several months' duration, during which some quantitative loss of DNA copy level, mRNA expression level, and fermentation titer were observed. In one extended run, a novel contaminant appeared in intermediates purified from later harvests. However, in all cases, the final soluble CD4 product was consistent in terms of purity and potency. Evaluation of genetic stability for tPA examined both biological traits at the cellular level as well as potency, purity and structure of product derived from cells at various levels of in vitro age; no significant cell age effects were observed. Similarly, evaluation of the EPO product showed that genetically-determined and process-determined traits such as potency, tryptic peptide mapping, and sialylation were consistent from lot to lot. These data exemplified how process design, process validation, and in-process and quality control assays can be used effectively to ensure the consistency of recombinant products derived from cell culture fermentations.

Effect of induction temperature on the production of malaria antigens in recombinant E. coli.

As part of a process development campaign, studies have been conducted to determine the influence of induction temperature on the expression of two different malaria antigens, RN1 and RT2. Single-step temperature inductions, in which growth at 32.0 degrees C is followed by a shift in temperature to a desired setpoint, show that there exists an optimum duration and temperature of induction which is product specific. Between an induction temperature of 39.5 and 44.5 degrees C RN1 yield is constant at ca. 0.20 g/g total soluble protein (TSP). RT2 yield approaches 0.20 g/g TSP only at elevated induction temperatures. The optimum temperature of induction for RN1 production is 39.5 degrees C, whereas, that for RT2 production is 41.0 degrees C. Above the optimum temperature of induction antigen concentration decreases owing to decreases in biomass. Furthermore, the maximum concentration of these two antigens differ by a factor of four. With increasing temperature of induction the extent of proteolysis of the products also appears to increase.