IgE formation in the rat following infection with Nippostrongylus brasiliensis: I. Proliferation and differentiation of IgE-bearing cells

Sprague-Dawley rats were infected with Nippostrongylus brasiliensis larvae, and IgE formation was studied. Before infection, the serum IgE level was less than 0.4 μg/ml. The IgE level began to increase from the 10th day of infection, reached its maximum (50–100 μg/ml) at the 14th day and gradually declined. Reinfection of the rats resulted in an increase of the serum IgE level within 7 days. The IgE antibody response to N. brasiliensis antigens did not parallel the increase of IgE synthesis. In most animals, the antibody became detectable in the serum at the 21st day when the total IgE level already began to decrease. The animals showed a secondary IgE antibody response upon reinfection. Both mesenteric lymph nodes and spleen cell suspensions were examined for the presence of IgE-bearing cells (IgE-B cells) and IgE-forming cells by fluorescent antibody technique. The IgE-bearing lymphocytes became detectable in the mesenteric lymph nodes and spleen at the 8th day of infection. The proportion of the IgE-B cells in nonadherent cell population gradually increased and reached maximum at the 14th day; about 20% of immunoglobulin (Ig)-bearing cells in the mesenteric lymph nodes and 10% of Ig-bearing cells in spleen bore IgE on their surface. Evidence was obtained that these lymphocytes synthesized IgE. The IgE-forming cells were detected in both mesenteric lymph nodes and spleen of the infected animals. The number of IgE-forming cells was greater in the mesenteric lymph nodes than in spleen, indicating that the regional lymph nodes are the major source of serum IgE in the N. brasiliensis-infected animals.     

Ontogeny of IgE-bearing lymphocytes in the rat

IgE-bearing lymphocytes were detected by immunofluorescence in the spleen of neonatal Hooded Lister strain rats within 24 hr after birth. The same cells were detected in the bone marrow as early as the 4th day after birth. Both fetal spleen and liver obtained 1 day before birth contained IgM-bearing cells but no detectable IgE-bearing cells. The proportion of IgE-bearing cells in the spleen and bone marrow increased during the neonatal period and reached an adult level within 3 to 4 weeks after birth. In adult Hooded Lister rats, IgE-bearing cells were 3 to 6% of total spleen cells and 1.5 to 2.2% of bone marrow cells. Most of the IgE-bearing cells from bone marrow cells. Most of the IgE-bearing cells from both newborn and adult animals carried IgM determinants on their surface. Capping experiments showed that epsilon chain determinants and mu chain determinants belonged to separate molecules. IgG2a-bearing lymphocytes were detected in the neonatal spleen as early as the 4th day after birth, but a significant number of these cells was not detected in the bone marrow until the 4th week. In newborn spleen the percentage of IgE-IgM double bearing cells was higher than that of IgG2a-bearing cells.     

The effect of colony stimulating factor on the synthesis of ribonucleic acid by mouse bone marrow cells in vitro.

The effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on the synthesis of RNA in liquid cultures of mouse bone marrow, spleen, thymus, peritoneal, peripheral blood leukocytes and lymph node cells was investigated. GM-CSF appeared to stimulate RNA-synthesis in syngeneic bone marrow cells within ten minutes of adding it to the culture. In the presence of GM-CSF bone marrow cultures maintained their initial rate of RNA synthesis for approximately ten hours. GM-CSF had no apparent effect on the uptake of 3H-uridine into bone marrow cells. This stimulation was still observed in the presence of puromycin and cycloheximide, but was abrogated by actinomycin D. The magnitude of the stimulation was not affected by the density of cells between 1 and 20 x 10(6) cells/ml but was slightly smaller at 0.1 and 40 x 10(6) cells/ml. Increasing concentration of GM-CSF (up to 2 X 105 units per ml) led to increased stimulation of RNA synthesis in bone marrow cells, but a significant stimulation could be detected at concentrations as low as 800 units/ml. GM-CSF did not significantly stimulate RNA synthesis in spleen, thymus, mesenteric or subcutaneous lymph node cells. However a small stimulation was observed in peripheral blood leukocytes and peritoneal cells. Autoradiographic studies showed that GM-CSF stimulated RNA synthesis in blast cells, myelocytes, metamyelocytes and polymorphs. Nucleated erythroid cells showed no increased labeling with GM-CFS. Labeling in lymphoid-like cells was highly variable but the level of labeling did not appear to be influenced by GM-CSF.     

Effect of Anti-Ia Serum In Vitro on Lipopolysaccharide-Induced Proliferative Responses of Murine Bone Marrow and Spleen Cells

The in vitro proliferative response of murine bone marrow cells and spleen cells to bacterial lipopolysaccharide (LPS) and the effect of anti-Ia serum on the response were studied. The incorporation of [³H]thymidine into cells prepared from bone marrow increased in the presence of LPS, but the addition of anti-Ia serum to the cultures reduced the incorporation. Pretreatment of bone marrow cells with anti-Ia serum and complement did not abolish the ability of the cells to respond to LPS, while the same pretreatment destroyed this ability in spleen cells. These results suggest that cultures of Ia-negative bone marrow cells generate Ia-positive cells during the culture period, and the Ia-positive cells are responsive cells to LPS. The proliferative response of 1- or 2-week-old spleen cells was easily suppressed by anti-Ia serum when compared with that of 4-week-old spleen cells. Furthermore, the responses of spleen cells obtained from γ-irradiated and syngeneic bone marrow cell-reconstituted mice were prominently suppressed by anti-Ia serum in comparison with that of normal adult spleen cells. These findings suggest that LPS-responsive lymphocytes in the developmental stage are quite sensitive to anti-Ia serum. The effect of anti-Ia serum on the maturation of bone marrow-derived lymphocytes was discussed.     

Specific activation of lymphocytes against acetylcholine receptor in the thymus in myasthenia gravis

In 13 patients with myasthenia gravis, spontaneous in vitro production of antibody to acetylcholine receptor (AChR) by thymic cells was observed in seven patients, by bone marrow cells in nine, by peripheral blood cells (PBL) in six, and by lymph node cells in nine. The rate of anti-AChR production in culture closely correlated with the serum anti-AChR level. Specific activity of the immunoglobulin (Ig) G spontaneously produced (anti-AChR/total IgG) was about 10-fold higher in the thymus than in bone marrow, peripheral blood, or lymph node cultures. Pokeweed mitogen (PWM) enhanced anti-AChR production only by PBL. With neither thymus nor lymph node cells did PWM stimulate anti-AChR production, although it greatly enhanced total IgG production. In bone marrow, it depressed both, and it appeared that the anti-AChR was derived from long-lived plasma cells that may be responsible for delaying the fall of serum anti-AChR levels after thymectomy. The results suggest that AChR-specific cells are selectively activated in the thymus, and this may help to explain the benefits of thymectomy in myasthenia gravis.     

Complement-dependent stimulation of normal lymphocytes by immune complexes.

Normal rabbit lymphocytes were stimulated to proliferate in vitro by antibody-antigen complexes. Stimulation was dependent upon C activity. Heat-inactivation or zymosan-treatment of the serum used in culture caused a 75 to 100% loss of responsiveness to the complexes. Serum-free culture or cultures with less than 1% serum supported only low levels of stimulation, but responsiveness reappeared proportionally with increased serum concentration. The low level dose-dependent responses seen in the absence of active C may have been due to C carried over with the cells or to stimulation independent of C. Aggregated rabbit gamma-globulin tested over a broad dose range failed to stimulate normal lymphocytes more than minimally whether or not C was present. Stimulation with immune complexes was sustained by C4-deficient guinea pig serum, indicating participation of the alternative C pathway. Normal rabbit lymphocytes from peripheral blood, bone marrow, spleen, and lymph node proliferated in response to rabbit antibody-antigen complexes. Normal thymocytes were consistently unresponsive to the complexes.     

Potentiation of bone marrow colony growth in vitro by the addition of lymphoid or bone marrow cells

Colony formation and growth in vitro by C57B1 mouse bone marrow cells were analysed following stimulation by a standard dose of serum colony stimulating factor. Under restricted conditions, colony crowding was observed to potentiate colony growth rates. The addition of thymic or lymph node lymphoid cells or nonviable bone marrow cells also potentiated colony growth. Extensive reutilisation of nuclear material by bone marrow colony cells was observed when labeled lymphoid and bone marrow cells were added to the culture system. The results provide evidence that lymphocytes can exert trephocytic effects on proliferating hematopoietic cells.     

Stromal cells derived from spleen or bone marrow support the proliferation of rat natural killer cells in long-term culture.

Rat nylon wool nonadherent bone marrow cells were propagated for up to 75 days in co-culture with stromal cells derived from either spleen or bone marrow. Interleukin (IL) 1 enhanced the ability of spleen stroma to support the long-term culture of natural killer (NK) cells, ostensibly by inducing these support cells to synthesize other cytokines. Flow cytometry studies indicated that the nylon wool separation procedure enriched the concentrations of mature NK cells from 7.9% to 38.1% for splenocytes and from 3.8% to 19.5% for bone marrow cells. Analyses of the adherent zones of suspended nylon screen NK cell cultures revealed substantial numbers of large granular lymphocytes that expressed NK 323+/MOM/3F12/F2- phenotypes. The presence of both mature and immature cells of the NK lineage in this matrix was inferred by the presence of both IL-2 receptor (IL-2R) positive and IL-2R negative, and OX-8+ and OX-8- NK 323+ cells over the greater than 4-month experimental period. Suspended nylon screen cultures displayed a greater potential for producing cytolytic cells than either co-cultures of bone marrow nonadherent cells on stroma monolayers or suspension cultures. The large granular lymphocytes produced in suspended nylon screen cultures could be transformed into active killers of YAC-1 targets by IL-2. In contrast to bone marrow nonadherent cells, more splenic nylon-wool-passed cells displayed a mature NK phenotype, but their proliferative potential and ability to be transformed into cytolytic cells by IL-2 decreased rapidly in culture. In the suspended nylon screen culture system, NK cells migrate from the underlying stroma in stages as they mature, retain their cytolytic potential, and manifest a capacity for self-renewal. Cultured cells were routinely dissociated into single cell suspensions via enzyme treatment and were reinoculated onto "fresh" nylon screen/stromal cell templates after passage through nylon wool columns. These co-cultures continued to generate cytolytic cells in numbers greater than those of the initial inoculum.     

Further evidence for the existence of B-lymphocyte subpopulations.

We have studied the homing properties of B lymphocytes by using ⁵¹Cr-labeled lymphoid cells obtained from athymic, nu/nu mice, and animals made T-lymphocyte deficient by thymectomy and lethal irradiation followed by reconstitution with syngeneic bone marrow. Comparison was made to the patterns of distribution observed when cell preparations containing normal numbers of T and B lymphocytes were migrated. A small but significant percentage of labeled lymphocytes from lymph nodes, spleen, Peyer's Patches, and bone marrow of T-cell-deficient animals was shown to be lymph node seeking. Secondary transfers of lymph node cells from primary recipients caused enrichment of this lymph node-seeking population. Treatment of T-lymphocyte-deficient lymphoid cell preparations with neuraminidase reduced the percentages of cells homing to the lymph nodes. The data showed that B lymphocytes exhibit unique homing properties when injected into normal recipients. In addition, direct comparison of the homing patterns of B lymphocytes prepared from spleen and lymph nodes of athymic mice revealed differences suggesting that these lymphoid organs contained unique mixtures of at least two different kinds of B cell. The evidence supports the notion that the B-lymphocyte populations contain at least two subpopulations, one of which possesses the ability to home to lymph nodes.     

Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues

The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding.Cell suspensions from CBA mice were exposed to ¹²⁵I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin-¹²⁵I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin-¹²⁵I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin-¹²⁵I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti-θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice.The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ-bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.     

Potentiation of the T lymphocyte response to mitogens IV. Serum-free production and testing of macrophage soluble products.

Serum-free cultures of activated macrophages generate conditioned media containing both potentiating and inhibitory activities for the lectin-induced transformation of syngeneic thymocytes, lymph node cells, or spleen cells, also cultured in serum-free medium. Exhaustive dialysis of macrophage conditioned medium (MCM) eliminates its inhibitory activity. At mitogenic doses of lectin, the dialyzable material enhances the potentiating activity exerted by macro-molecular factors at low and optimal concentration of MCM. The inhibitory effect of intermediate concentrations of nondialyzed MCM on [³H]thymidine uptake can be reversed if the cells are washed and pulsed in fresh medium, and thus is artefactual. On the other hand, high doses have a real inhibitory effect on proliferative response of transformed lymphocytes. Rat MCM is not mitogenic for any of the target lymphocytes tested. Its effect is observed both in primary cultures of lymphocytes and secondary cultures of blast cells.     

Induction and long-term maintenance of Thy-1 positive T lymphocytes: Derivation from continuous bone marrow cultures

In the present study we investigated the presence of T-lymphocyte progenitors in the long-term murine bone marrow culture system described by Dexter: mature Thy-1 antigen-bearing T lymphocytes are lost in these cultures after a few days. By culturing nonadherent cells from such cultures in the presence of a supernatant of concanavalin A-stimulated spleen cells, a source of T-cell growth factor, we found that Thy-1 positive blast cells proliferated together with a second population of Thy-1 negative cells. These two populations of cells have been maintained in long-term in vitro cultures by passaging the cells in fresh conditioned medium at regular intervals. Moreover, we have been able to establish pure cultures of the Thy-1-bearing blast cells after separating them from the non-T cells using their adherence property to plastic surfaces. Long-term cultures of T lymphocytes can thus be established from long-term marrow cultures as well as from the spleen, thymus or fresh bone marrow.     

T cells in inductive and effector compartments of the intestinal mucosal immune system of nonhuman primates differ in lymphokine mRNA expression, lymphokine utilization, and regulatory function.

To define further the basis of T cell function in the inductive and effector limbs of the normal intestinal immune system, the capacity of mucosal lymphocytes to produce and use lymphokines and their effects on regulation of Ig production were determined in normal nonhuman primates. Northern blots of RNA from mitogen-activated lamina propria T cells contained more mRNA for IL-2 and IFN-gamma than did mesenteric lymph node T cells. In comparison with lymphocytes from peripheral sites, there was high expression of IL-4 and IL-5 mRNA in both mesenteric lymph node and lamina propria T cells. In studies of lymphokine utilization, T cells from lamina propria had high IL-2-induced but no IL-4-induced proliferative responses. In contrast, mesenteric lymph node T cells had high IL-4-induced and lower IL-2-induced proliferative responses compared with lamina propria T cells. Lamina propria T cells had higher helper activity in PWM-stimulated cultures and exhibited less inhibition by IL-4 than did mesenteric lymph node T cells. These data and previous studies suggest that T cells in an inductive site such as the mesenteric lymph node are a mixed population containing both "naive" cells with low potential for IFN-gamma and IL-2 production and differentiated cells with high potential for IL-4 and IL-5 production. In contrast, the data suggest that T cells in the effector compartment of the lamina propria are comprised primarily of differentiated "memory" cells that produce high levels of IL-2, IFN-gamma, IL-4, and IL-5, have high helper activity, and have a more limited ability to proliferate in response to lymphokines such as IL-4.     

Mitogenic factor from inbred guinea pigs. I. Isolation of the factor

Methods are described for the reproducible elicitation of mitogenic factor (MF) from antigen-sensitive lymphocytes of inbred strains of guinea pigs. The use of inbred animals minimizes complications due to histocompatibility factors. Each of several antigens tested was effective. Mitogenic factor is released in vitro as early as 6 hr after stimulation of lymphocytes by antigen. It was obtainable from serum-free cultures in which medium RPMI-1640 was used; this should facilitate isolation of MF. The addition of 5 mMl-cysteine to cultures substantially improved the yield of MF. MF was obtained from cultures of lymph node cells of highly purified small lymphocytes, which indicates that the small lymphocyte is the source of MF in the guinea pig. It was shown that MF can induce mitosis as well as blast transformation in non-immune lymph node cells. MF from a given strain of guinea pig is capable of stimulating lymphocytes of another strain.     

Immune mechanism of rats on Nippostrongylus brasiliensis in vitro. II. The influence of lymphocytes and peritoneal cells

Nippostrongylus were collected from the intestines of rats 6 days p.i. and kept under sterile conditions in cultures. Serum, lymphocytes and peritoneal cells of immune or non-infected animals were added in various combinations to the culture media. The culture media were changed 2-3 times in an experimental period of 10 days, resp. serum and cells were added. The lymphocytes were isolated from the peripheral blood or from the mesenterial lymph nodes whereas the mononuclear cells were obtained from the peritoneal cavity. Serum and lymphocytes from the peripheral blood both from immune and non-infected rats, had no increased lethal effect on Nippostrongylus. The highest lethality rate of adults (65-68%) was achieved in cultures with peritoneal cells and lymphocytes from the lymph nodes of sensitized rats. Serum of infected or non-infected animals had no influence on adult Nippostrongylus in cultures with these cell combinations. In the controls without any cell-supplements the survival rate of the parasites was up to 88%.     

Dietary effect of guar gum and its partially hydrolyzed product on the lipid metabolism and immune function of Sprague-Dawley rats

The dietary effect of the water-soluble dietary fibers (WSDF), guar gum, partially hydrolyzed guar gum (PHGG), glucomannan, highly methoxylated (HM) pectin, on the serum lipid level and immunoglobulin (Ig) production of Sprague-Dawley rats was compared with that of water-insoluble cellulose. Although serum total cholesterol and triglyceride levels were significantly lower in the rats fed with WSDF than in those fed with cellulose, a decrease in the level of phospholipids was only observed in the rats that had been fed on guar gum or glucomannan. In addition, all WSDF feeding enhanced IgA productivity in the spleen and mesenteric lymph node lymphocytes, although the increase in serum IgA level was only observed in the rats fed on WSDF, and not on PHGG. When mesenteric lymph node lymphocytes were cultured in the presence of various concentrations of guar gum or glucomannan, no significant increase in Ig production was apparent. These data suggest that WSDF indirectly enhanced the Ig production of lymphocytes, and that serum lipid reduction and IgA production-enhancing activities of WSDF were dependent on their molecular sizes.     

Dietary gamma-linolenic acid dose-dependently modifies fatty acid composition and immune parameters in rats.

gamma-linolenic acid (GLA) has been reported to improve several inflammatory disorders through regulation of eicosanoid production. However, since GLA is a precursor of arachidonic acid, it may bring about increasing tissue arachidonic acid levels with subsequent pro-inflammatory events. To explore this possibility, we examined the effect of high-dose GLA acid on the fatty acid profile of immune cells, leukotriene B4 production by peritoneal exudate cells and immunoglobulin productivity of mesenteric lymph node lymphocytes of Sprague-Dawley rats. Male rats were fed 10% fat diets containing graded levels, 0, 20, 40 and 60% of GLA for 3 weeks. The results showed the distinction in activity of metabolizing GLA between immune cells and liver. Thus, in immune cells such as mesenteric lymph node and spleen lymphocytes and peritoneal exudate cells, more dihomo-gamma-linolenic acid was found than in the liver. Leukotriene B4 production by peritoneal exudate cells was significantly suppressed when fed the highest level of GLA suggesting a lower risk of allergic reaction. Moreover, immunoglobulin productivity in mesenteric lymph node lymphocytes was promoted by dietary GLA. The present study indicates that a high dose of GLA may exert anti-inflammatory effects through suppression of leukotriene B4 release and strengthening of gut immune system, thus ameliorating allergic reaction.     

Augmentation of erythroid burst formation by the addition of thymocytes and other myelo-lymphoid cells

Bone marrow from barrier-sustained specific pathogen-free (SPF) CBA and C57BL/6 mice gave relatively low numbers of BFU-E colonies in methylcellulose culture, as compared to conventional mice. Addition of thymocytes to the marrow cultures increased the yield of BFU-E colonies more than fourfold in SPF mice but only 1.5-fold in conventional mice. Colony size was also increased. Increased yield of BFU-E colonies was also obtained by co-culture of bone marrow with lymph node cells or with bone marrow or spleen cells from 900R whole-body-irradiated mice. The effect appeared to be cellular rather than humoral. It was not reproduced by conditioned medium from thymus or pokeweed mitogen stimulated spleen cells. The helper effect of thymus cells was eliminated or reduced by freezing and thawing, or by 48 hours of incubation after irradiation. Treatment of bone marrow cells in vitro with anti-theta serum and complement did not decrease the number of BFU-E colonies. The putative helper cells appear not to be T cells, were non-adherent to the plastic culture dish, and were cortisone resistant and radioresistant. The low BFU-E colony yield from SPF mouse marrow is presumed to be largely the result of deficiency of these non-T helper cells in SPF bone marrow, rather than of BFU-E progenitor cells.     

In vitro alloreactivity of mouse bone marrow lymphocytes

Before characterizing alloreactive cells of the bone marrow, it was necessary to reevaluate the alloantigen response in this tissue. The results of previous studies using the parental-F₁ system in the mixed-lymphocyte reaction (MLR) are open to question because of the recently documented proliferation of F₁ stimulator cells (W. H. Adler, T.Takiguchi, B. Marsh, and R. T. Smith,J. Immunol. 105, 984, 1970; P. F. Piguet, H. K. Dewey, and P. Vassalli, J. Exp. Med. 146, 735, 1977). The culture system was optimized for measuring the MLR of bone marrow lymphocytes enriched on sucrose density gradients. The proliferative response of the enriched fraction (BML) to 2000-R irradiated allogeneic spleen cells was three times as high as the response of unfractionated bone marrow. For maximal responses, antigen concentration had to be twice as high for the BML as for the lymph node, and in a time course study the highest [³H]TdR uptake occurred on Day 3 in BML cultures and on Day 5 in the LN. In lymph node semiallogeneic cultures stimulator cell proliferation can be disregarded, while semiallogeneic BML MLR err significantly on the high side. When BML were matched with allogeneic stimulator cells at the H-2 locus, they gave good MLR responses, provided there was a minor Mls histocompatibility locus difference, while in the lymph node the response was greatly diminished in similar mixtures. The differences in the BML and lymph node alloantigen responses with respect to antigen concentration, kinetics and susceptibility to F₁ and Mls stimulation, suggest that the bone marrow alloantigen response is mediated by a cell population that is different than alloresponsive cells in the lymph node.     

Development of IgE-forming cells in vitro from rat mesenteric lymph node cells.

Mesenteric lymph node cells from normal rats and rats infected with Nippostrongylus brasiliensis (Nb) were cultured with pokeweed mitogen (PWM) or Nb antigen, and the development of IgM-, IgG2a-, or IgE-containing cells was assessed by immunofluorescence. Normal lymph node cells stimulated with PWM developed into both IgM- and IgE-containing cells, whereas similar stimulation of cells from Nb-infected rats resulted in the development of IgM-, IgG2a-, and IgE-containing cells. The in vitro plasma cell response to PWM was dependent on the presence of T lymphocytes. Lymph node cells from Nb-infected rats responsed to Nb antigen and developed into plasma cells of IgM, IgG, and IgE classes. The response was antigen specific and required antigen-primed T cells. Depletion of IgE-bearing cells or IgM-bearing cells before stimulation with either PWM or Nb antigen diminished the level of IgE forming cell development, suggesting that IgE-IgM double bearing cells are precursors of IgE-forming cells. The distribution of the three isotypes among the If-forming cells that developed in response to PWM was influenced by the source of both B and T cells. When B cells from Nb-infected rats were employed as a source of precursors, T cells from infected animals were more effective than normal T cells for the development of IgE-forming cells, whereas the latter cells were more effective for the development of IgG2a-forming cells than T cells from infected animals.