Lymphocyte-stimulating Activity Research Articles

Differentiation of Human Hematopoietic Progenitor Cells Into Dendritic Cells In Vitro Under Stimulation of PPAR-Gamma

The differentiation of dendritic cells (DCs) in vitro is not yet under full control and although PPAR-γ stimulation may interfere with DC differentiation from cord blood progenitors, the expression of PPAR-γ by different precursors and the effect of PPAR-γ stimulation on DC differentiation are poorly known. To address these issues, CD14+ monocytes, CD34+ progenitors and CD133+ progenitors were isolated from adult healthy donors and cultured with cytokines; rosiglitazone (1 μmol/l) was used to stimulate PPAR-γ. All precursors generated large, HLA-DR+ DCs, a proportion of which, highest when starting from CD133+ precursors expressed the Langerhans cell marker CD207/langerin; many cells expressed the connective tissue DC marker CD209/DC-SIGN, even together with CD207, and some cells contained Birbeck granules. Only CD133+ precursors expressed PPAR-γ mRNA appreciably; the DCs from these precursors contained a higher proportion of Langerhans like cells and caused 25% less stimulation of lymphocyte proliferation when generated in the presence of rosiglitazone. In conclusion, the phenotype of DCs differentiated in vitro does not match exactly that of DCs in vivo; PPAR-γ is expressed by freshly isolated CD133+ precursors; PPAR-γ agonists can direct DC differentiation from CD133+ precursors towards Langerhans type cells and inhibit the lymphocyte stimulating activity of the generated DCs.

Induction of Dendritic Cell Maturation and Activation by a Potential Adjuvant, 2-Hydroxypropyl-β-Cyclodextrin.

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) is a chemically modified cyclic oligosaccharide produced from starch that is commonly used as an excipient. Although HP-β-CD has been suggested as a potential adjuvant for vaccines, its immunological properties and mechanism of action have yet to be characterized. In the present study, we investigated the maturation and activation of human dendritic cells (DCs) treated with HP-β-CD. We found that DCs stimulated with HP-β-CD exhibited a remarkable upregulation of costimulatory molecules, MHC proteins, and PD-L1/L2. In addition, the production of cytokines, such as TNF-α, IL-6, and IL-10, was modestly increased in DCs when treated with HP-β-CD. Furthermore, HP-β-CD-sensitized DCs markedly induced the proliferation and activation of autologous T lymphocytes. HP-β-CD also induced a lipid raft formation in DCs. In contrast, filipin, a lipid raft inhibitor, attenuated HP-β-CD-induced DC maturation, the cytokine expression, and the T lymphocyte-stimulating activities. To determine the in vivo relevance of the results, we investigated the adjuvanticity of HP-β-CD and the modulation of DCs in a mouse footpad immunization model. When mice were immunized with ovalbumin in the presence of HP-β-CD through a hind footpad, serum ovalbumin-specific antibodies were markedly elevated. Concomitantly, DC populations expressing CD11c and MHC class II were increased in the draining lymph nodes, and the expression of costimulatory molecules was upregulated. Collectively, our data suggest that HP-β-CD induces phenotypic and functional maturation of DCs mainly mediated through lipid raft formation, which might mediate the adjuvanticity of HP-β-CD.

Human dendritic cell differentiation from monocytes

Dendritic cells (DCs) induce, sustain, and regulate immune responses and are essential mediators of immunity and tolerance. These cells are specialized to capture antigens, process and present them to T cells to initiate immune responses towards pathogens and tumours. DCs are heterogeneous for origin, anatomical localization, phenotype and function. They differentiate from haematopoietic stem cells-derived precursors, migrate from sites of antigen uptake to lymphoid organs, mature and interact with lymphocytes. Langerhans cells (LCs) are immature DCs, are located in stratified squamous epithelia of the epidermis and mucosae. LCs express CD1a, langerin/ CD207, E-cadherin and have intracytoplasmic Birbeck granules. We wonder if the PPAR (peroxisome proliferator activated receptor)-gamma stimulation can give rise to LCs in vitro starting from CD14+ monocytes, as occurs with CD133+ haematopoietic precursors (1). Circulating CD14+ monocytes have been isolated by immunomagnetic separation and cultured with GM-CSF, IL-4, TNF-α and TGF-β for 7 days. The complete maturation was induced by further culture for 48 h with the same cytokines, but the concentration of the TGF-β was increased (2). Rosiglitazone was used to stimulate PPAR-γ from the start of the culture. The cells were analysed by phase contrast, electron microscopy, immunofluorescence, flow cytometry and mixed lymphocyte reaction. All cultures generated mature cells that expressed MHCII intensely, both with and without rosiglitazone, and had lymphocyte stimulating activity. The obtained DCs express the DC-SIGN, such as connective tissue DCs, more intensely with than without rosiglitazone. Some of these cells were CD1a+ and among them a small part was CD207+, i.e. they were LC-like, however they never contained Birbeck granules. The percentages of cells CD1a+ and CD207+ were slightly higher without rosiglitazone. The results indicate that upon addition of rosiglitazone the in vitro differentiation of monocytes to LCs is hampered, at variance with what happens with cultures of CD133+ precursors. For this reason, experiments have been planned with CD34+ haematopoietic stem cells, because the generation of different types of DCs is a necessary step to gain control on these cells for medical purposes.

Dendritic cells with lymphocyte-stimulating activity differentiate from human CD133 positive precursors

AbstractCD133 is a hallmark of primitive myeloid progenitors. We have addressed whether human cord blood cells selected for CD133 can generate dendritic cells, and Langerhans cells in particular, in conditions that promote that generation from CD34+ progenitors. Transforming growth factor-β1 (TGF-β1) and anti–TGF-β1 antibody, respectively, were added in some experiments. With TGF-β, monocytoid cells were recognized after 7 days. Immunophenotypically immature dendritic cells were present at day 14. After 4 more days, the cells expressed CD54, CD80, CD83, and CD86 and were potent stimulators in mixed lymphocyte reaction; part of the cells expressed CD1a and langerin, but not Birbeck granules. Without TGF-β, only a small fraction of cells acquired a dendritic shape and expressed the maturation-related antigens, and lymphocytes were poorly stimulated. With anti–TGF-β, the cell growth was greatly hampered, CD54 and langerin were never expressed, and lymphocytes were stimulated weakly. In conclusion, CD133+ progenitors can give rise in vitro, through definite steps, to mature, immunostimulatory dendritic cells with molecular features of Langerhans cells, although without Birbeck granules. Addition of TGF-β1 helps to stimulate cell growth and promotes the acquisition of mature immunophenotypical and functional features. Neither langerin nor Birbeck granules proved indispensable for lymphocyte stimulation.

Cladosporium Species-Related Hypersensitivity Pneumonitis in Household Environments

Home-related chronic hypersensitivity pneumonitis (HP) is sometimes difficult to discriminate because patients do not have an obvious history of antigen exposure. We report two HP cases which developed in an office area and in a home: a 47-year-old woman with acute-onset HP and a 72-year-old woman with chronic HP followed up as idiopathic pulmonary fibrosis following isolation of Cladosporium cladosporioides and Cladosporium herbarum, respectively. Lymphocyte stimulating activity and antibody titer to these fungi were increased in these patients. Since Cladosporium spp. and several other fungi are present ubiquitously in our living environment, it is difficult to eliminate the antigen from the patients' environment to control the disease. Cladosporium spp. can be key antigens in inducing chronic HP in the home environment.

Anaphylactic Shock Caused by Exposure to Sea Anemones

Since the first report of a dog that developed severe systemic symptoms in response to a second injection of sea anemone toxin by Richet and Portier in 1902, no clear human cases of anaphylaxis related to exposure to sea anemones has been reported in the literature. A 24-year-old man with an episode of local urticaria on his first contact with a sea anemone (Stichodactyla haddoni), developed dyspnea, severe urticaria and hypotension on exposure to water containing the dead bodies of the organism. To study whether this reaction was mediated by antigen-specific IgE, we performed a histamine release test with blood, Western blotting with serum and lymphocyte proliferating test with peripheral blood mononuclear cells of the patient, for the homogenate of sea anemones. The homogenate of sea anemones induced histamine release from the blood of the patient, but it also induced histamine release from the blood of control subjects. Moreover, it also caused hemolysis of blood of all donors. However, Western-blotting demonstrated the presence of an 86 kd protein-specific IgE in the serum of the patient. Protein antigen(s) in sea anemones may cause anaphylactic shock under the influence of the cytolytic effects and/or lymphocyte-stimulating activity elicited by the toxin of sea anemones.

Fusokine Interleukin-2/Interleukin-18, a Novel Potent Innate and Adaptive Immune Stimulator with Decreased Toxicity

To redress the immune imbalances created by pathologies such as cancer, it would be beneficial to create novel cytokine molecules, which combine desired cytokine activities with reduced toxicities. Due to their divergent but complementary activities, it is of interest to combine interleukin-2 (IL-2) and IL-18 into one recombinant molecule for immunotherapy. Evaluation of a fusokine protein that combines murine IL-2/IL-18 shows that it is stable, maintains IL-2 and IL-18 bioactivities, has notably reduced IL-2 associated toxicities, and has a novel lymphocyte-stimulating activity. An adeno-viral expression system was used to explore the biology of this "fusokine". Inclusion of the IL-18 prosequence (proIL-18) increases the expression, secretion, and potency of this fusokine. In vivo gene transfer experiments show that Ad-IL-2/proIL-18 dramatically outdoes Ad-IL-2, Ad-proIL-18, or the combination of both, by inducing high rates of tumor rejection in several murine models. Both innate and adaptive effector mechanisms are required for this antitumor activity.

Glycoproteins Contained within Soamsan, a Traditional Oriental Medicine, are the Main Class of Active Ingredients Responsible for the Medicine-induced Immune Stimulation

In our previous study, Soamsan, a traditional Oriental medicine, was shown to enhance the induction of antigen-specific immune responses, and it was speculated that the enhancing activity might be closely associated with glycoproteins contained within the medicine. To elucidate this speculation, protein samples from each component, used in the preparation of Soamsan, were obtained and their immune stimulating activities were tested with mouse splenocytes. All the samples markedly enhanced the lymphocyte proliferation and cytokine secretion by the mouse splenocytes. In particular, the enhancement was significantly higher with the protein sample treatments than with those of the original crude sample. Furthermore, the pronase E- and NaIO4-mediated inhibition of splenocyte-stimulation activity of the protein samples clearly supported that glycoproteins are the main class of active ingredients responsible for the lymphocyte stimulating activity of the samples. Consequently, our findings suggest that glycoproteins might have a pivotal role in Soamsan-mediated immune modulation, although the in vivo effect of the glycoproteins should be further elucidated.

Selective modulation of superantigen-induced responses by streptococcal cysteine protease.

Streptococcal pyrogenic exotoxin (Spe) B, a streptococcal cysteine protease, is believed to be important in group A streptococcal (GAS) pathogenesis. The present study examined the effect of SpeB on the activity of superantigenic exotoxins secreted by M1T1 GAS isolates. The proliferative response of human lymphocytes to culture supernatant (SUP) from an SpeB(+) isolate increased significantly (P<.05) when the isolate was grown with N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]-agmatine, a cysteine protease inhibitor. The lymphocyte-stimulating activity of SUP from a spontaneous SpeB(-) variant or SpeB(-) knockout (DeltaSpeB) mutant was also significantly higher than that of SUP from the SpeB(+) parent isolate (P<.001). The addition of recombinant SpeB to the DeltaSpeB mutant reduced the lymphocyte response to a level comparable to that with the SpeB(+) isolate. SpeB affected superantigens that stimulate cells expressing T cell receptor Vbeta (TCRBV)-4, TCRBV7, and TCRBV8 but not those that stimulate TCRBV2. SpeB has a selective proteolytic effect on GAS superantigens.

Purification, characterization, and modification of T lymphocyte-stimulating polysaccharide from spores of Ganoderma lucidum.

The hot-water extract of the spores of Ganoderma lucidum was shown to have a stimulating effect on concanavalin A-induced mitogenic activity of T lymphocytes. Bioassay-guided separation led to the isolation of a polysaccharide with potent T lymphocyte-stimulating activity by ethanol fractionation, anion-exchange, and size-exclusion chromatography. Based on the composition and methylation analyses, periodate oxidation, Smith degradation, and NMR spectroscopy, the native polysaccharide was shown to be a beta-D-(1-->3)-glucan with branches of terminal glucosyl residues substituted at C-6 of the glucose residues in the main chain. The branching ratio is approximately 20%. A series of sulfated or carboxymethylated derivatives were prepared and their structural features were elucidated by chemical and spectral analyses. The solution conformation and T lymphocyte proliferation effect of the glucans before and after derivatization were compared and discussed. The data obtained indicate that the introduction of ionic groups would significantly affect the original conformation of the native glucan in aqueous solution and further affect T lymphocyte-stimulating activity. The triple-helical structure of the glucans, the nature of the ionic groups, and the density of negative charge were considered to be closely related to this activity.

Purification of the Major Allergen of Red Soft Coral (Dendronephthya nipponica)

Red soft coral (RSC; Dendronephthya nipponica, a marine coelenterate) causes spiny lobster fishermen living along the Pacific coast of Miyazaki Prefecture in Japan to develop occupational allergies, such as conjunctivitis, rhinitis, dermatitis and bronchial asthma. The aim of this study was to purify and to characterize RSC allergen, which causes occupational asthma in spiny lobster fishermen. The allergic responsiveness of spiny lobster fishermen to RSC was examined. The examinations included specific IgE production, skin test responses, lymphocyte stimulation tests and specific IgG production. We found that RSC has a strong sensitizing activity in humans at a molecular weight of 10 kD or more, while it has no IgE-producing activity at a molecular weight of less than 10 kD. Neither the nonatopic controls nor the atopic non-coral-allergic controls exhibited any RAST-binding activity to any fraction. For the purification and the identification of this new allergen component, repeated gel filtration of the RSC extract was performed on a Sephacryl S-200 column, followed by gel filtration on a Superose-6 column. The purified major allergen component Den n 1, which is separated on a Mono-Q column, showed intradermal responses, lymphocyte stimulating activity and specific IgG-producing activity in RSC-induced bronchial asthma patients. The 53-kD component was electroblotted on a polyvinylidene difluoride membrane. The N-terminal amino acid sequence of this new allergen component (Den n 1) was determined as Asp-Asp-Ile-Asn-Arg-Tyr-Ala-Phe-Asp-Asn-Lys-Ile-Asn- Asp-Lys-Leu-Phe-Asp-His-Trp-Gln-Ser.

2-Acetylaminofluorene-produced selective cytotoxic damage of a ductal compartment and its repair in the submandibular gland of rats.

Salivary glands of rodents are rarely affected with spontaneous and induced malignancies. This may be linked with low physiologic cell renewal and the infrequency of cytolethal actions by xenobiotics. The genotoxic 2-acetylaminofluorene, carcinogenic for other organs, causes acute damage in salivary ducts. In the submandibular glands the damage is limited to the granulated convoluted tubules. They produce and release regulatory peptides including epidermal growth factor and nerve growth factor. The partial chemical sialoadenectomy is repaired by sequential cell proliferation in the basal cell layer of interlobular ducts and in dilated intralobular ducts (day 4 and 6), in intermediate duct-like structures (day 6 and 8), and lastly in acini (day 8 and 12). This is associated with a transient loss of structural characteristics of striated ducts and acini (up to day 6) and of the immunoreactivity for S-100 protein (up to day 4). Actin immunoreactivity at the acinar base is increased from day 6 to 20. Analogous to the late postnatal differentiation of the granulated convoluted tubules, their structural characteristics and immunoreactivity for epidermal growth factor do not recover within 20 days. The acute lesion of the endocrine ductal segment is suggested to be causally involved with other systemic effects following treatment with 2-acetylaminofluorene. First, hypophagia with loss of body, liver and thymus weight may result from disturbed saliva production. Second, previous studies have shown a mitotic burst of the biliary epithelium and bloodborne lymphocyte-stimulating activities. Either effect could be brought about by regulatory peptides (see above), probably after elevated circulatory release from necrotic granulated convoluted tubules.

Regulatory activity of Kupffer cells in acute blood loss

The scavenger function of Kupffer cells and the erythropoietinlike, granulocyte-macrophage colony-stimulating, and leukocyte- and lymphocyte-stimulating activities of extracts of Kupffer cells obtained before and at different times after acute massive blood loss were experimentally evaluated on mice. Extracts of Kupffer cells from normal mice are shown to exhibit all types of the studied activities. Acute blood loss reduces the scavenger function of Kupffer cells during the first few hours, especially erythropoietinlike activity. The activities return to normal levels 5 days after blood loss and, after a relatively stable period, they rise again to the end of the recovery period. This is accompanied by an increase in the number of phagocytizing cells and is evidently related to the renewal of their population.

Major histocompatibility complex class II binding site for streptococcal pyrogenic (erythrogenic) toxin A.

Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of "superantigens" produced by Staphylococcus aureus and Streptococcus pyogenes and its T lymphocyte stimulating activity is involved into the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have produced and characterized recombinant SPEA molecules in Escherichia coli. These molecules are indistinguishable from natural SPEA in both T cell stimulatory and HLA class II binding activities. Human class II molecules are more efficient than mouse class II molecules in presenting SPEA to T cells. In binding tests to major histocompatibility complex class II-positive cells SPEA competes with staphylococcal enterotoxin B and A but not with toxic shock syndrome toxin-1.

Mutations affecting MHC class II binding of the superantigen streptococcal erythrogenic toxin A.

Streptococcal pyrogenic exotoxin A (SPEA) is an important pathogenicity factor of group A streptococci. It is a member of the family of 'superantigens' produced by Staphylococcus aureus and Streptococcus pyogenes, and its T lymphocyte stimulating activity is involved in the pathogenesis of certain diseases caused by pyogenic streptococci. In this study we have generated nine mutant SPEA molecules by substituting amino acids in the regions of homology between different streptococcal and staphylococcal superantigens. An additional mutant was created by deletion of the 10 N-terminal amino acids. The mutants were expressed as fusion proteins. Several mutations led to a loss of function due to a loss of class II-binding activity. Such loss mutations did not cluster to a certain region of the SPEA molecule. Rather, even a substitution of neighboring amino acids had opposite effects. None of the loss mutations affected the binding of neutralizing mAb and all loss mutants could be precipitated in Ouchterlony tests by a polyclonal anti-SPEA serum. We conclude that the functional activities of SPEA, and probably of other superantigens as well, cannot be attributed to a defined region of the molecule.

Endocrine control of the immunosuppressive activity of the submandibular gland

Extracts of the submandibular gland (SMG) of rats contain fractions that stimulate the in vitro proliferation of Con A-treated lymphocytes. One of the stimulatory fractions was also shown to induce in vivo immunosuppression in rats and mice in several experimental models. Since many other biologically active factors of the SMG had been found to be hormone dependent, we investigated the effects on the immunosuppressive factor of hypophysectomy (Hx) and of hormonal reconstitution in male Fischer rats. Hx induced a marked atrophy of the SMG together with an almost complete disappearance of both the in vitro lymphocyte-stimulating activity and the in vivo immunosuppressive activity, the latter assayed with the contact sensitivity reaction in mice. The treatment of the Hx rats with pituitary hormones demonstrated that prolactin (PRL), thyroid stimulating hormone (TSH), and luteinizing hormone (LH) induced a significant reconstitution of these biological activities, growth hormone led to the recovery of the lymphocyte-stimulating activity but not of the immunosuppressive activity, while follicle-stimulating hormone, and adrenocorticotropic hormone did not induce any recovery of these biological activities. In view of the positive results obtained with TSH and LH further experiments were done to compare the effects of thyroid and sex hormones with those of PRL. The results demonstrated that testosterone and thyroid hormones induced significant recovery of the lymphocyte-stimulating and the immunosuppressive activity. The combination of these two hormones with PRL produced the most effective results. On the other hand, estrogens and progesterone had no significant effects. These results confirm the effectiveness of androgens and thyroid hormones in stimulating the production of biologically active factors by the SMG. Moreover, they demonstrate that PRL, a hormone not previously considered to increase the activity of the SMG, stimulates the production of immunoregulatory factors in Hx animals.

Relationship between enterotoxic- and T lymphocyte-stimulating activity of staphylococcal enterotoxin B.

The enterotoxins produced by Staphylococcus aureus cause a gastrointestinal intoxication probably via their action on intramucosal neuronal cells. Staphylococcal enterotoxins are also the most powerful mitogens known, activating CD3+ T lymphocytes of several species in a clonally variable and MHC class II-dependent fashion. We examined a possible relationship between enterotoxic and mitogenic activity of staphylococcal enterotoxin serotype B (SEB). We used a monoclonal anti-Id directed against the combining site of an anti-SEB mAb. This anti-Id failed to elicit an enteric response by itself but could block the enteric response in monkeys to a 6000-fold excess of SEB. The anti-Id was mitogenic, however, for human and monkey T cells, triggering a fraction of CD4+ and CD8+ T cells. Not all SEB-reactive T cells were activated by the anti-Id. The anti-Id bound to T cells with a similarly low affinity as did SEB. Additional evidence for a separation of enterotoxic and mitogenic activity comes from studies with carboxymethylated SEB. Although this modified SEB had lost its enterotoxic activity, it was as mitogenic as the unmodified molecule. These results support the notion that the enteric reaction to SEB is not mediated via its effect on T lymphocytes. We conclude that SEB and anti-Id might bind to a common structure of different receptors on T cells and target cells in the intestinal mucosa, probably peripheral sensory neurons.

In vitro effect of human growth hormone on lymphocyte transformation and lymphocyte growth factors secretion.

Cultures of human blood peripheral lymphocytes were performed in the presence or absence of human growth hormone, and also of phytohemagglutinin and normal human serum 10%. After incubation for 48 h, the supernatants were tested for their ability to promote the uptake of [3H]thymidine into lectin-activated lymphocytes. Supernatants from lymphocyte-free control samples, treated in the same manner, were assayed under the same experimental conditions. Variance analysis of the different dose-response relationships was performed. The results of these in vitro experiments confirm that physiological levels of GH inhibit the lectin-induced lymphoproliferation and that lymphocytes secrete an 'activity' able to stimulate the incorporation of [3H]thymidine into lectin activated lymphocytes. Furthermore we show that: 1) Secretion of this lymphocyte-stimulating activity is increased by physiological levels of GH; 2) This lymphocytic secretion is not radioimmunoassayable IGF-I; 3) Using fast protein liquid chromatography (FPLC), this activity appears in fractions with various molecular weights.

Brugia malayi antigens associated with lymphocyte activation in filariasis.

Filarial parasites induce immune response in humans which are still poorly characterized. To define the antigens responsible for inducing lymphocyte responses, fast protein liquid chromatography was used to fractionate the antigens of Brugia malayi adult worms which were then tested on lymphocytes from patients with filariasis and normal controls. From an anion exchange column (Mono Q), three peaks of lymphocyte-stimulating activity were eluted which were further fractionated by gel filtration (Superose-12). Peak I induced both a proliferative response as well as the production of filaria-specific antibody in patient lymphocytes. Peak II, capable of inducing only a proliferative response (without antibody production) in patient lymphocytes, was a glycoprotein with phosphocholine as one of the antigenic determinants. Peak III induced proliferative responses in both patient and normal lymphocytes and thus appears to be mitogenic. Two-dimensional gel electrophoresis was then used to identify changes in the major cellular proteins associated with the activation of patient lymphocytes by these partially purified antigens. Stimulation of patient lymphocytes with peak I resulted in increased synthesis of immunoglobulin heavy, light, and J chains. Further, these were the only major secreted proteins found in the culture supernatants. Peak II resulted in quantitative changes in proteins associated with T and not B lymphocyte stimulation. Further analysis of these antigens should help to elucidate the mechanism of host-parasite interaction at both the cellular and molecular levels.

A defined fragment of bacterial protein I (OmpF) is a polyclonal B-cell activator.

Protein I from the outer membrane of Escherichia coli and other members of the family Enterobacteriaceae is a potent mitogen and polyclonal B-lymphocyte activator. To determine the part of the polypeptide responsible for biological activity, we cleaved the molecule into defined polypeptide fragments of approximate molecular weights 24,000, 15,000, 9,000, 7,000, and 3,000 by using the cyanogen bromide method. The fragments were purified by gel permeation chromatography and by preparative polyacrylamide gel electrophoresis. They were investigated for mitogenicity and for the induction of immunoglobulin synthesis in lymphocyte cultures from several inbred mouse strains. The fragment of molecular weight 24,000 turned out to be a potent polyclonal B-lymphocyte activator comparable to native protein I. The low-molecular-weight fragments exhibited only marginal effects. Neither purified T lymphocytes nor thymocytes were activated. Our results show that a defined fragment of protein I is responsible for its lymphocyte-stimulating activity.