RESUMEN
Hemophilia A patients treated with coagulation factor VIII (FVIII), and also some healthy subjects, may develop anti-FVIII antibodies (Ab), whose synthesis is driven by FVIII-specific CD4+ T cells. Some Ab block the procoagulant function of FVIII (inhibitors). Many inhibitors recognize epitopes on the FVIII A2 domain. Here, we have sought to identify A2 epitopes recognized by CD4+ T cells. We tested the proliferative response of CD4+ blood lymphocytes (BL) from hemophilia patients and healthy subjects, to overlapping synthetic peptides spanning the A2 domain sequence. Many A2 peptides induced proliferative responses of CD4+ BL from one or more subjects. The peptide-induced responses were strongest in hemophilia patients with inhibitors, weakest in healthy subjects. A2 peptides comprising residues 371-400, 621-650 and 671-690 elicited frequent and strong responses in hemophilia A patients, and especially in those with inhibitors. Healthy subjects recognized frequently only the sequence 371-400. A three-dimensional model of the A2 domain suggests that these CD4+ epitope sequences have structural features typical of 'universal' CD4+ T epitopes.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Factor VIII/inmunología , Hemofilia A/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Estudios de Casos y Controles , Proliferación Celular , Mapeo Epitopo , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factor VIII/química , Hemofilia A/sangre , Anticuerpos/química , Enfermedades Autoinmunes , Sitios de Unión , Linfocitos T CD8-positivos/química , Proliferación Celular , Epítopos/química , Femenino , Hemofilia A/inmunología , Humanos , Masculino , Modelos Moleculares , Péptidos/química , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Factor VIII/metabolismo , Adulto , Factores de Edad , Anciano , Antígenos/metabolismo , Donantes de Sangre , División Celular , Coagulantes/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-5/metabolismo , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Péptidos/química , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Factores Sexuales , Linfocitos T/metabolismo , Células TH1RESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factor VIII/metabolismo , Sitios de Unión , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/química , Estudios de Casos y Controles , División Celular , Epítopos de Linfocito T/inmunología , Factor VIII/inmunología , Femenino , Hemofilia A/sangre , Hemofilia A/tratamiento farmacológico , Humanos , Linfocitos/metabolismo , Masculino , Modelos Moleculares , Péptidos/química , Estructura Terciaria de ProteínaRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factor VIII/inmunología , Hemofilia A/inmunología , Isoanticuerpos/biosíntesis , Cooperación Linfocítica , Animales , Especificidad de Anticuerpos , Autoanticuerpos/inmunología , Desensibilización Inmunológica , Modelos Animales de Enfermedad , Epítopos/química , Epítopos/inmunología , Factor VIII/química , Factor VIII/uso terapéutico , Estudios de Factibilidad , Hemofilia A/terapia , Humanos , Tolerancia Inmunológica , Terapia de Inmunosupresión/métodos , Interleucina-4/deficiencia , Interleucina-4/genética , Interleucina-4/fisiología , Isoanticuerpos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miastenia Gravis/inmunología , Miastenia Gravis/terapia , Fragmentos de Péptidos/inmunología , Estructura Terciaria de Proteína , Receptores Colinérgicos/inmunología , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Mice genetically deficient in factor VII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-gamma and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-gamma, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis.
Asunto(s)
Factor VIII/inmunología , Hemofilia A/sangre , Ratones Mutantes/inmunología , Animales , Anticuerpos/análisis , Anticuerpos/sangre , Células Productoras de Anticuerpos/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , División Celular/efectos de los fármacos , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Epítopos/análisis , Factor VIII/administración & dosificación , Factor VIII/farmacología , Hemofilia A/inmunología , Humanos , Inmunoglobulina G/sangre , Inyecciones Intravenosas , Interleucina-10/metabolismo , Ratones , Bazo/citología , Bazo/inmunología , Factores de TiempoRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factor VIII/inmunología , Hemofilia A/inmunología , Adulto , Anciano , Presentación de Antígeno , Células Cultivadas , Femenino , Humanos , Activación de Linfocitos , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Músculos/inmunología , Miastenia Gravis/inmunología , Receptores Colinérgicos/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos/inmunología , Células Cultivadas , Femenino , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Músculos/metabolismo , Fragmentos de Péptidos/inmunología , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismo , Células TH1/inmunologíaRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Toxina Diftérica/inmunología , Epítopos de Linfocito T , Toxina Tetánica/inmunología , Adulto , Anciano , Femenino , Humanos , Activación de Linfocitos , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factor VIII/inmunología , Hemofilia A/inmunología , Anticuerpos , Epítopos , Humanos , Tolerancia Inmunológica , Biblioteca de Péptidos , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). The s.c. administration to C57B1/6 mice of synthetic AChR CD4+ epitopes, before and during AChR immunization, reduced the epitope-specific CD4+ responses and the anti-AChR Ab synthesis, and prevented EMG. The s.c. administration of solubilized AChR had effects similar to those of peptide treatment. Sham-tolerized mice had only Th1 anti-AChR cells, whereas peptide-treated mice had also Th2 cells, and Th2-induced anti-peptide Ab. Established EMG was not affected by s.c. peptide treatment, whereas it worsened after s.c. administration of solubilized AChR.
Asunto(s)
Epítopos/inmunología , Miastenia Gravis/inmunología , Miastenia Gravis/prevención & control , Receptores Colinérgicos/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , División Celular/efectos de los fármacos , División Celular/inmunología , Modelos Animales de Enfermedad , Epítopos/farmacología , Tolerancia Inmunológica/inmunología , Inmunización , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Solubilidad , Células Th2/inmunología , TorpedoRESUMEN
Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). We investigated EMG in interleukin (IL)-4 knock out B6 (KO) mice, that lack Th2 cells. EMG was more frequent in KO than in wild type B6 mice. KO and B6 mice developed similar amounts of anti-AChR antibodies. They were IgG2a and IgG2b in KO mice, IgG1 and IgG2b in B6 mice. CD4+ cells from KO and B6 mice recognized the same AChR epitopes. Nasal administration of synthetic AChR CD4+ epitopes reduced antibody synthesis and prevented EMG in B6, not in KO mice. Thus, Th2 cells may have protective functions in EMG.
Asunto(s)
Antígenos CD4/farmacología , Interleucina-4/genética , Miastenia Gravis/inmunología , Miastenia Gravis/prevención & control , Receptores Colinérgicos/inmunología , Administración Intranasal , Animales , Autoanticuerpos/inmunología , Autoanticuerpos/farmacología , Enfermedades Autoinmunes/inmunología , Bungarotoxinas/farmacología , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Proteínas del Sistema Complemento/análisis , Epítopos/inmunología , Inmunoglobulina G/análisis , Interleucina-4/deficiencia , Interleucina-4/inmunología , Ratones , Ratones Noqueados , Mutagénesis/inmunología , Unión Neuromuscular/química , Unión Neuromuscular/inmunología , Bazo/citología , Bazo/inmunología , Células TH1/química , Células TH1/inmunología , Células Th2/química , Células Th2/inmunología , TorpedoRESUMEN
Interaction of the tertiary amines, arecolone, eserine (physostigmine), (+)-epibatidine, and (+/-)-epibatidine, with Torpedo nicotinic acetylcholine receptor-enriched membrane vesicles was investigated to characterize their action on the receptor, using stopped-flow thallium (I)-flux spectrofluorimetry. Arecolone, (+)-epibatidine, and (+/-)-epibatidine were agonists with activation constants of 390, 19, and 39 microM, respectively. Eserine was not an agonist but rather an antagonist for agonist-induced activation of the receptor with an inhibition constant of approximately 150 microM. The choice of the fluorescent dye used (entrapped within the membrane vesicles) was critical for interpretation of the effects of eserine. With 1,3,6,8-pyrene tetrasulfate (PTS), eserine appeared to act as an agonist. However, it was shown that such an effect was caused by rapid diffusion of the uncharged form of the amine across the membrane followed by direct interaction with PTS rather than eserine-induced cation transport. The use of a different fluorescent dye, 8-aminonaphthaline-1,3,6-trisulfate, with which eserine does not interact allowed demonstration of the action of eserine as an antagonist rather than as an agonist.
Asunto(s)
Aminas/química , Fisostigmina/química , Receptores Nicotínicos/química , Membranas Sinápticas/química , Vesículas Sinápticas/química , Animales , Arecolina/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Polarización de Fluorescencia , Colorantes Fluorescentes/química , Naftalenos/química , Agonistas Nicotínicos/química , Pirenos/química , Piridinas/química , Espectrometría de Fluorescencia , TorpedoRESUMEN
We have identified sequence regions of the human muscle acetylcholine receptor epsilon subunit recognized by CD4+ T cells from myasthenia gravis patients. We tested the proliferative response in vitro of blood CD4+ cells from 18 myasthenic patients and 5 controls, to individual overlapping synthetic peptides spanning the epsilon subunit sequence. All patients recognized a complex epitope repertoire. The peptides recognized by the CD4+ cells included sequence regions of the epsilon subunit that were diverged as compared to the homologous sequences of the other receptor subunits. Recognition of epitopes formed by sequence regions unique to the epsilon subunit suggests a direct role of this subunit in sensitizing the CD4+ cells. Several epsilon subunit peptides were recognized by many patients. Thus the epsilon subunit, like other acetylcholine receptor subunits, forms 'universal' CD4+ epitopes. The healthy subjects recognized some epsilon subunit peptides sporadically and at a low level.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Miastenia Gravis/inmunología , Receptores Colinérgicos/inmunología , Adulto , Anciano , Enfermedades Autoinmunes/inmunología , Química Encefálica/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos/inmunología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
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.