Comparison of allergenic potentials of timothy (Phleum pratense) pollens from different pollen seasons collected in the Belgrade area

We investigated extracts of timothy grass pollen from four seasons (1989, 1990, 1991, and 1994) by protein content, SDS-PAGE, immunoblot, RAST, RAST inhibition, and crossed immunoelectrophoresis. Extract of the pollen from 1991 showed the lowest yield in quantitative assays. SDS-PAGE, crossed immunoelectrophoresis, RAST, and RAST inhibition expressed approximately comparable patterns for all extracts except that from 1991. Obviously, the quality of grass pollens, as shown for some ragweed ( Ambrosia elatior ) pollens depend on year of collection. Our findings are partially in agreement with some earlier examinations of the quality of timothy pollen from different pollen seasons.     

Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense) pollen allergens.

In order to establish a test system for grass pollen allergy based on the use of recombinant allergens we chose timothy grass (Phleum pratense), a widely spread grass, as a model. From a lambda gt11 cDNA expression library that we had constructed from pollen RNA of timothy grass (P. pratense), we had obtained with serum IgE from a grass pollen-allergic individual 60 IgE-binding clones. By differential testing with sera from different grass pollen-allergic patients, we selected three distinct clones encoding Phl p I (group I), Phl p V (group V) and profilin from timothy grass, which when used together allowed the diagnosis of grass pollen allergy in 97 out of 98 tested grass pollen-allergic patients employing a simple plaque lift technique. This recombinant test based on plaque lifts containing allergen-beta-galactosidase fusion proteins was compared with IgE immunoblots using crude pollen protein extracts from timothy grass. Both methods were in good agreement with RAST scores and clinical data, and proofed to be useful for the diagnosis of grass pollen allergy. Our results further indicate that a limited panel of only two recombinant grass pollen allergens, Phl p I and Phl p V, together with the plant panallergen profilin could be sufficient for the diagnosis and possibly immunotherapy of grass pollen allergy.     

Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11

BACKGROUND: Allergy to grass pollen is typically associated with serum IgE antibodies to group 1 and/or group 5 allergens, and additionally often to one or several less prominent allergens. Most of the grass pollen allergens identified to date have been characterized in detail by molecular, biochemical and immunological methods, timothy grass being one of the most thoroughly studied species. However, a 20-kDa allergen frequently recognized by IgE antibodies from grass pollen allergics has so far escaped cloning and molecular characterization. OBJECTIVE: To clone and characterize the 20 kDa timothy grass pollen allergen Phl p 11. METHODS: Phl p 11 cDNA was cloned by PCR techniques, utilizing N-terminal amino acid sequence obtained from the natural allergen. Phl p 11 was expressed as a soluble fusion protein in Escherichia coli, purified to homogeneity and used for serological analysis and to study Phl p 11 specific induction of histamine release from basophils and skin reactivity in sensitized and control subjects. RESULTS: Phl p 11 cDNA defined an acidic polypeptide of 15.8 kDa with homology to pollen proteins from a variety of plant species and to soybean trypsin inhibitor. The sequence contained one potential site for N-linked glycosylation. Serological analysis revealed that recombinant Phl p 11 shared epitopes for human IgE antibodies with the natural protein and bound serum IgE from 32% of grass pollen-sensitized subjects (n = 184). Purified recombinant Phl p 11 elicited skin reactions and dose-dependent histamine release from basophils of sensitized subjects, but not in non-allergic controls. CONCLUSION: As the first representative of group 11 grass pollen allergens, Phl p 11 has been cloned and produced as a recombinant protein showing allergenic activity. One-third of grass pollen-sensitized subjects showed specific IgE reactivity to recombinant Phl p 11, corresponding in magnitude to a significant proportion of specific IgE to grass pollen extract.     

Design of an optimally-diagnostic skin test solution for diagnosis of sensitivity to timothy grass (Phleum pratense) pollen

Background Although most of the common allergen extracts that are used for diagnosis of type 1 hypersensitivity are now well standardized, this gives no assurance that they are within the concentration range that gives the best chance of a true diagnosis. Objective The objective of this study was to identify the most appropriate concentration range of timothy grass pollen Phleum pratense extract to diagnose sensitivity to this pollen correctly through skin-testing. Methods Dilutions of a well-standardized extract were made and used to skin test ‘true’ positive and ‘true’ negative populations of subjects as identified by case history, challenge tests and radioallergosorbent test (RAST). Weal diameters were measured and the data were submitted to receiver operating characteristics (ROC) analysis. For any particular weal size cut–off, the optimal diagnostic concentration (ODC) range was thus calculated. Results A 3 mm weal diameter cut-off was chosen as an appropriate size for routine diagnosis. Therefore the ODC range at this diameter was used to establish a product target concentration and specification for formulation of the diagnostic reagent. This method of allergen extract standardization can lead to a true-biological unitage that can be used for labelling purposes. Conclusion The optimun concentration range at which to formulate an allergen extract, in terms of an in vitro immunologically based assay, can be determined by carrying out ROC analysis of the results of clinical studies as described in this communication. Diagnostic units (DU), are now used by us for labelling of such final formulations which conveys the information that the product is at the most appropriate concentration for diagnosis.     

The international collaborative study establishing the first international standard for timothy (Phleum pratense) grass pollen allergenic extract

A selected candidate international reference preparation of timothy grass (Phleum pratense)-pollen extract was studied together with two other freeze-dried timothy pollen allergenic extracts in a multinational study. The collaborators used RAST inhibition, histamine release, quantitative immunoelectrophoresis (crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis and rockets), isoelectric focusing, and other methods. The total allergenic potencies measured in RAST inhibition were evaluated for validity of linearity and parallel-line response. The relative concentrations of some important individual allergenic components were measured. The relative potencies for the total allergenic activity and the timothy components studied in each preparation were expressed relative to the selected candidate. This preparation was established in 1983 by the World Health Organization expert committee on biologic standardization as the international standard for timothy grass-pollen extracts with assigned units of 100,000 IU per ampule.     

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen Phl p 13 from timothy grass pollen (Phleum pratense)

BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.     

Characterization of immunoglobulin E responses in Balb/c mice against the major allergens of timothy grass (Phleum pratense) pollen

BACKGROUND: Grass pollen, such as that from timothy grass (Phleum pratense), represents a major cause of type I allergy. OBJECTIVE: To characterize the IgE immune response and to identify the major allergens eliciting an IgE response in a mouse model using pollen extract of P. pratense for sensitization, in order to assess analogies to human hyperreactivity and to gain information on the allergenic potential as determined by the IgE-reactivity kinetics of defined allergens. METHODS: Balb/c mice were sensitized with pollen extract or with purified natural allergens. Serum IgE levels, the induction of specific IgE antibodies and immediate hypersensitivity were monitored by ELISA, Western blot and a skin test, respectively. RESULTS: The sensitized mice mounted a strong IgE response and showed IgE-reactivity first against Phl p 5a and 5b, then Phl p 4 and 13 and lastly against Phl p 6. No IgE response was mounted against Phl p 1. However, all purified fractions examined (Phl p 5a, 5b, 6 and 1) induced specific IgE and showed similar kinetics of IgE induction as pollen extract (first Phl p 5a and 5b, then Phl p 6). Skin test experiments demonstrated positive reactivity only in sensitized mice. CONCLUSION: The IgE reactivity induced by the major allergens in Balb/c mice was very similar to that found in allergic patients, with the exception of Phl p 1. The kinetics of the specific IgE response was comparable using either pollen extract or the purified major allergens, indicating that the intrinsic properties of the allergens are of importance rather than their proportionate amounts in pollen extract. This model should prove to be suitable for investigations regarding the mechanisms of induction and manifestation of timothy grass pollen allergy and for the evaluation of therapeutic strategies.     

Studies on allergen and allergoid preparations from purified timothy (Phleum pratense) pollen extracts. III. Comparative investigations by skin prick tests and nasal provocation tests

The allergenic activities of allergen (GEN) and allergoid (GOID) preparations from partially purified timothy pollen extract were investigated in quantitative skin prick tests and nasal provocation tests. In skin prick test GOID yielded significantly less activity than GEN. Approximately 90 times more protein of GOID than of GEN was necessary to elicit wheals of the same mean size as 1 mg/ml histamine control solution (1 HEP). In nasal provocation the patients seemed to react differently with respect to their nasal sensitivity to GOID. One group of patients showed equal sensitivity to GOID and GEN, whereas the second group showed higher thresholds to GOID. These differences in nasal sensitivity to GOID could be of practical value to predict the optimal dosage schedule for the treatment of patients with GOID.     

Timothy grass (Phleum pratense L.) pollen as allergen carriers and initiators of an allergic response

Contrary to indoor allergen exposure (e.g. house dust mite), there is no reliable quantitative association between pollen exposure and symptoms of allergic diseases. Therefore we studied localization and release of major allergens from timothy grass (Phleum pratense L.) pollen using different methods and pollen grain sources. Localization of major allergens Phl p 5 and Phl p 1 was visualized by field emission scanning electron microscopy after anhydrous fixation and immunogold silver staining in a three-dimensional reconstruction; Phl p 5 was found in the cytoplasm and on the exine, Phl p 1 in the intine. No allergens were found inside the starch granules. Allergen liberation from pollen grains was studied in vitro under physiological conditions (30 min, 37 degrees C) at pH 6. 0, 7.4 and 9.0. Besides total protein measurements in the supernatant, major allergens were determined by immunoblot, Phl p 5 was quantitated by ELISA. There were striking differences in total protein and major allergen release between freshly collected and commercially available grass pollen grains as well as among freshly collected pollen between rural meadows and areas near high-traffic roads. There was a significantly different release of total protein being lowest in supernatants from commercially available pollen grains (rural/traffic vs. commercial, p<0.001), and of Phl p 5 major allergen (rural>traffic>commercial, p<0.005). Therefore, allergen bioavailability seems to be an important parameter in order to establish reliable dose-response relationships for the outdoor allergen response. Pollen grains incubated in aqueous protein-free buffer solution were also found to secrete significant amounts of eicosanoids namely prostaglandin E2 and leukotriene B4. Pollen grains thus do not act only as allergen carriers but also might have important implications on early events as initiators of allergy.     

Intratracheal instillation of cytoplasmic granules from Phleum pratense pollen induces IgE- and cell-mediated responses in the Brown Norway rat

BACKGROUND: Release of cytoplasmic granules from grass pollen upon contact with water is thought to be an important source of airborne allergens. OBJECTIVES: To investigate the humoral and cellular responses to intratracheal instillation of Phleum pratense (timothy grass) pollen cytoplasmic granules (PCG) in the Brown Norway rat. METHODS: PCG were purified from timothy grass pollen by filtration through 5-microm-mesh filters. Rats were sensitized (day 0) and challenged (day 21) intratracheally with purified PCG suspended in saline (6 x 10(6) PCG/rat). Rats were then challenged 4 weeks later (1.5 x 10(6) PCG/rat). Blood samples, bronchial lymph nodes and lungs were collected from the rats 4 days after the second challenge. PCG-specific IgE and IgG1 levels and specificity were determined by ELISA and Western blotting. Pollen, pollen extract and PCG-induced proliferation of lymph node cells were monitored by [(3)H]-thymidine incorporation in a lymph node assay. Histopathological examination was carried out on the lungs. RESULTS: Specific IgE and IgG1 were present in the sera. Cultured lymph node cells proliferated in the presence of pollen, pollen extract and PCG. Western blots showed that all major pollen allergens are recognized by IgE and IgG1 from PCG-treated rats. Histopathological examination revealed features of a mild allergic reaction. CONCLUSIONS: In our rat model of allergy, purified timothy grass PCG instillation induced specific antibodies and lymph node cell responses, comparable to those obtained with intact pollen.     

Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy

BACKGROUND: Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. OBJECTIVE: We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. METHODS: Brown-Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 microm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. RESULTS: An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. CONCLUSIONS: It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.     

The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50–60 kDa is comprised of two major allergens: Phl p 4 and Phl p 13

BACKGROUND: More than 70% of the patients allergic to grass pollen exhibit IgE-reactivity against the high molecular mass fraction between 50 and 60 kDa of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allergen, Phl p 13, has recently been purified and characterized at the cDNA level. OBJECTIVE: The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE-binding frequency and capacity. METHODS: Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects allergic to grass pollens were used to determine specific IgE-binding frequency to Phl p 4 and Phl p 13. IgE-binding of allergens was quantified by ELISA measurements. Pre-adsorption of sera with purified allergens and subsequent incubation of nitrocellulose-blotted timothy grass pollen extract was performed to determine whether or not Phl p 4 and Phl p 13 represent the whole high molecular mass allergen fraction. Proteolytic stability of both allergens was investigated by addition of protease Glu-C. RESULTS: More than 50% of 300 patients displayed IgE-binding with both allergens. Clear differences concerning the immunological properties of Phl p 4 and Phl p 13 were confirmed by individual IgE reactivities. Quantification of specific IgE for both allergens revealed comparable values. For complete inhibiton of IgE-binding in the high molecular mass range preincubation of sera with both allergens was necessary. Interestingly, inhibition of strong reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 kDa double band, but in addition a 'background smear'. Whilst undenatured Phl p 4 was resistent to proteolytic digestion with Glu-C, native Phl p 13 was degraded rapidly. CONCLUSION: Phl p 4 and Phl p 13 are immunologically different and must both be considered as major allergens. They are judged to be important candidates for potential recombinant therapeutics that may provide a basis for improved immunotherapy.     

Group V allergens in grass pollens. I. Purification and characterization of the group V allergen from Phleum pratense pollen, Phl p V

An allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated by a combination of copper chelate affinity chromatography and ion exchange chromatography. Phl p V binds IgE from serum of grass-sensitized donors as revealed in immunoelectrophoretic techniques and in SDS-PAGE immunoblot, and luminescence immunoassay (LIA) inhibition experiments indicate that the allergen represents a significant part of the IgE binding capacity of the extract. In immunoelectrophoresis, Phl p V is revealed as a single precipitate. However, molecular weight studies show that Phl p V consists of at least two isoforms with similar immunochemical properties, but with different molecular size. After SDS-PAGE treatment purified Phl p V is identified as two IgE-binding components, Phl p Va and Phl p Vb, with molecular weights 33 and 29 kD. After HPLC gel filtration, Phl p Va and Phl p Vb are identified in the major 30-kD eluate. After Sephadex G75 gel filtration of whole pollen extract, Phl p V is identified in fractions corresponding to molecular weights 47 and 25 kD. The 47-kD fraction corresponds to Phl p Va/Phl p Vb as seen in SDS-PAGE, while the 25-kD component presumably corresponds to a degradation product present in whole pollen extract. The NH2-terminal sequence of Phl p V, corresponding to approximately 10% of the molecule, has been determined. The sequence shows minor variations in some residues and contains besides many alanine residues also hydroxyproline; the sequence reveals no homologies to any known NH2 terminal sequence of other proteins. The amino acid composition, revealing 26 mole % alanine and no cysteine, does not show any similarities to other known amino acid compositions of allergens. From the amino acid composition determination and an immunoelectrophoretic comparison, Phl p V is estimated to represent 6% (w/w) of the whole pollen extract.     

Modifications of Phleum pratense grass pollen allergens following artificial exposure to gaseous air pollutants (O(3), NO(2), SO(2))

BACKGROUND: Air pollution is frequently proposed as a potential cause of the increased incidence of allergy in industrialised countries. Our objective was to investigate the impact of the major gaseous air pollutants on grass pollen allergens. METHODS: Timothy grass pollen was exposed to ozone (O(3)), nitrogen dioxide (NO(2)) and sulphur dioxide (SO(2)) alone or in combination. Allergen contents were analysed by 2-dimensional immunoblot using grass pollen-sensitive patient sera. RESULTS: For O(3)-treated pollen, immunoblotting showed an acidification of allergens Phl p 1b, Phl p 4, Phl p 5 and Phl p 6 and an IgE recognition decrease in Phl p 1, Phl p 2, Phl p 6 and Phl p 13. NO(2) exposure induced a decrease in Phl p 2, Phl p 5b and Phl p 6 recognition, and SO(2) treatment induced a decrease in Phl p 2, Phl p 6 and Phl p 13 recognition. Moreover, samples treated with a mix of NO(2)/O(3) or NO(2)/SO(2) showed a higher decrease in allergen content, compared with samples treated with only one pollutant. The O(3) acidification was also observed with the NO(2)/O(3) mix. CONCLUSION: Exposure of pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts. This decrease could be due to a mechanical loss of allergens from the altered pollen grains and/or post-translational modifications affecting allergen recognition by IgE.     

Heterogeneity of commercial timothy grass pollen extracts

Background The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources.
Objective To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity.
Methods Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients.
Results The allergen extracts showed broad variations in protein compositions and amounts (24.1–197.7 μg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32–384 ng/mL; Phl p 2: 1128–6530 ng/mL, Phl p 5: 40–793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients.
Conclusions Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.     

Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data

BACKGROUND: The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. OBJECTIVE: To compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. METHODS: A population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. RESULTS: Ninety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. CONCLUSIONS: The use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.     

Antigen B-specific helper factor from timothy grass pollen: isolation and partial characterization.

Spleen cells from LAF1 mice primed with photo-oxidized antigen B exhibit helper T-cell activity when assayed in an adoptive transfer system with spleen cells from mice primed with timothy pollen extract and treated with anti-thy 1.2 serum and complement. These helper cells, when placed in culture with antigen B, secrete a soluble antigen B-specific helper factor (THF) that requires the presence of normal spleen cells or cells that do not adhere to nylon wool in order to exhibit T cell-replacing activity. The THF has been partially purified over an immobilized antigen adsorbent. The eluted THF exhibits a mol. wt of 65,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and contains 5% carbohydrate by weight. The THF is I-Ak-positive and Ig-negative.