Differential regulation of activation, clonal expansion, and antibody secretion in human B cells

Limiting dilution analysis, hemolytic plaque assay, and ELISA procedures were used to study the recruitment, clonal expansion, and antibody secretion in human TNP-specific B cells activated in the presence of TNP-ovalbumin (TNP-OA), pokeweed mitogen (PWM), or regulatory T cells. TNP-OA-responsive, hapten-specific PFC precursor cells occupy approximately 0.5% of all sIgM+/sIgD+ B cells in cord blood, bone marrow, peripheral blood, and tonsil. The PWM-responsive, hapten-specific PFC precursor pool is 70 to 90% smaller and does not express sIgD. Antigen-reactive B cells go through a minimum of three divisions in culture (six to nine PFC per clone), and antibody secretory rates of about 10(4) molecules IgM/cell/hr are achieved. In contrast, PWM-induced clone sizes were at least 60 PFC per clone, with antibody secretory rates of approximately 6 to 7 X 10(4) molecules IgM/cell/hr. Addition of high-dose carrier-primed suppressor T cells to limit dilution cultures reduced PFC precursor cell recruitment by up to 99%. However, in the few clones escaping from suppression, both clonal expansion and antibody secretory rates were much higher than in suppressor cell-free cultures, generating 30 to 60% of the antibody secreted in controls but with consequently much more restricted clonal diversity. When limiting dilution cultures were compared with standard microcultures of 2 X 10(5) cells, both clonal expansion and antibody secretory rates were much lower than expected, with a culture efficiency calculated to be 10 to 20% of that in low-density cultures. Our data suggest that the B cell subsets activated by antigen and by mitogen differ in their abilities for clonal expansion and antibody secretion. The hapten-specific and -responsive B cell family is expressed early in ontogeny, and in adults it is distributed evenly throughout the body. These limiting dilution experiments revealed that the primary effect of regulatory T cells is a drastic reduction in clonal diversity, and much less a mere reduction in overall response magnitude.     

Beta 2-adrenoceptor stimulation increases the number of antigen-specific precursor B lymphocytes that differentiate into IgM-secreting cells without affecting burst size.

Previous studies have shown that early addition of a beta 2-adrenergic agonist to whole splenocyte cultures immunized with SRBC induced an increase in the number of cells secreting Ag-specific antibody. Because of the low frequency of Ag-specific B lymphocytes in these cultures, it has been difficult to determine the cellular mechanism by which this increase is produced. To gain insight into this cellular mechanism, the present study was designed to evaluate the responsiveness of TNP-specific B lymphocytes cultured at both high density and limiting dilution with keyhole limpet hemocyanin (KLH)-specific, IL-4-producing Th lymphocytes, TNP-KLH, and the beta 2-adrenergic agonist, terbutaline. The results showed that a maximal twofold increase in both the number of anti-TNP IgM-secreting cells and the amount of anti-TNP IgM secretion occurred in terbutaline-exposed lymphocytes after 5 days of bulk culture. This response occurred in a concentration-dependent manner and was inhibited by concomitant culture with beta-adrenoceptor antagonists. No appreciable change was measured in the level of either IgG1 secretion in terbutaline plus Ag-exposed bulk cultures or MHC class II expression on terbutaline plus Ag-exposed TNP-specific B lymphocytes as compared with Ag alone. These data raised the possibility that beta 2-adrenoceptor stimulation induced either the differentiation of a larger proportion of TNP-specific B lymphocyte precursors into anti-TNP IgM-secreting cells, or the extensive proliferation of a constant number of TNP-specific B lymphocyte precursors, or both. Limiting dilution results showed that beta 2-adrenoceptor stimulation induced a twofold increase in the number of TNP-specific B lymphocyte precursors that differentiated into anti-TNP IgM-secreting cells, without affecting the number of anti-TNP IgM-secreting cells produced by each precursor clone.     

Immobilized anti-CD3-induced T cell growth: comparison of the frequency of responding cells within various T cell subsets.

Human T cells can be divided into subsets based on the expression of CD29, CD45RA, CD45RO, LFA-3, or CD11a. It has been suggested that the subset of CD4+ T cells that expresses high densities of CD29, CD11a, CD45RO, and LFA-3 contains "memory" T cells, whereas the subset of cells that expresses CD45RA contains "naive" T cells. In order to obtain a more complete picture of the functional capacities of human naive and memory CD4+ and CD8+ T cell subsets, highly purified T cells were activated with a uniform stimulus and responses were examined in bulk cultures and under limiting dilution conditions. T cell activation was achieved with an immobilized mAb to the CD3 molecular complex, 64.1. In bulk cultures, immobilized 64.1 stimulated a vigorous response. Moreover, the number of cells entering the cell cycle, the magnitude of the [3H]thymidine incorporation, and the growth of the cells over 6 days in culture by naive and memory CD4+ T cells was comparable. To delineate the frequency of responsive cells in each subset more precisely, cells were cultured with immobilized 64.1 at limiting dilution and the precursor frequency of responding cells was assessed by examining wells microscopically for visible growth. Immobilized 64.1 was able to induce some T cells from each subset to grow in the complete absence of AC, when exogenous IL2 was present. The number of responding CD4+ and CD8+ cells was comparable. The percentage of naive cells responding in each population was approximately three times greater than the frequency of memory cells. IL4 could also support the growth of immobilized 64.1-activated CD4+ T cells, but the frequency of responding cells was much lower than that supported by IL2. The vast majority of the IL-4 responsive CD4+ cells resided within the naive cell subset. The data indicate that the response of CD4+ and CD8+ naive and memory T cell subsets to immobilized anti-CD3 depends on the density of responding cells. Naive T cells have an enhanced capacity to grow when cultured in the absence of other T cells or accessory cells. This ability may facilitate their expansion during primary immune responses.     

Frequency of human B cells that differentiate in response to anti-CD3-activated T cells

Activation of T cells by mAb to the CD3 molecular complex induces the differentiation of many more Ig-secreting cells (ISC) from resting human B cells in bulk cultures than do other modes of polyclonal B cell activation. In the current experiments, a limiting dilution assay was used to demonstrate that this increase in ISC generation reflects an increased frequency of responding B cells. Highly purified B cells were cultured at densities of between 1000 cells and 0.5 cell per microwell with fresh, mitomycin C-treated T cells (T mito) or T cell clones stimulated by immobilized mAb to CD3. After 5 days in culture, the number of wells containing ISC was determined, and the frequency of responding B cells was calculated. The proportion of B cells responding to anti-CD3-stimulated T cells was very large (10.7 +/- 2.8%) and greatly surpassed that induced by other polyclonal activators. B cells cultured with anti-CD3-stimulated T cell clones responded better than did those cultured with T mito. The addition of exogenous IL-2 or IL-6 to cultures supported by activated T mito enhanced the frequency of responding B cells, whereas IL-4 did not increase the generation of ISC and inhibited the augmentation of B cell responses induced by IL-2. Supplementation of cultures with mitomycin C-treated B cells as accessory cells had less of an effect. The addition of both accessory cells and IL-2 markedly increased B cell responsiveness, with precursor frequencies of 60 to 80% noted. In some experiments, cultures were carried out for 7 to 14 days and supernatants were analyzed for IgM, IgG, and IgA secretion. B cells activated by anti-CD3-stimulated T cells produced all three Ig isotypes. When the classes of Ig produced by single B cells were examined, it was observed that the stimulation of individual B cell precursors led to the production of multiple Ig isotypes, suggesting that isotype switching occurs in these cultures. These results demonstrate that under optimum culture conditions, T cells stimulated with immobilized anti-CD3 can activate the majority of human peripheral blood B cells to produce Ig and induce isotype switching by many.     

The effect of age on the antigen-presenting mechanism in limiting dilution precursor cell frequency analysis

We have utilized limiting dilution analysis (LDA)2 to compare the intrinsic precursor cytotoxic T lymphocyte (pCTL) frequency for influenza-plus-self in young and old C57BL/6 mice. Under conditions of excess interleukin 2 (IL-2) and antigen presenting cells (APC) derived from spleens of mice matched in age to those being tested, we found more than a twofold difference in pCTL frequency between young and old animals. However, there was no difference in pCTL frequency between the two age groups if antigen was presented to the old responder cells on spleen cells derived from young mice. The apparent decrease in pCTL frequency in old mice by standard LDA may in fact be due to a defect in the antigen processing and/or presentation mechanism of old spleen cells. We conclude that the age-associated defective CTL activity previously reported by us and by others may be due at least in part to a defect in the antigen presentation mechanism of aging mice.     

Antigen concentration determines helper T cell subset participation in IgE antibody responses.

A recently developed in vitro system for antigen-stimulated primary and secondary murine IgE antibody responses has been used to define (a) the relative participation of the Th1 and Th2 cell-derived lymphokines IFN-gamma and IL-4, respectively, in such responses, and (b) the role of antigen concentration in determining functional helper T cell activity. These studies confirm that IL-4 and IFN-gamma exert regulatory effects on IgE synthesis, but the nature and extent of their respective effects on primary and secondary IgE responses differ. Thus, primary IgE responses are considerably more sensitive to and dependent on IL-4 than are secondary IgE responses since (1) anti-IL-4 monoclonal antibody totally inhibited primary IgE responses, but only partially affected secondary responses; and (2) exogenously added IL-4 could stimulate primary IgE responses to optimal antigen concentrations, but had no effect on secondary IgE production. Likewise, antigen-stimulated primary IgE responses are about eightfold more sensitive than are secondary responses to the inhibitory effects of IFN-gamma. Studying the effect of antigen dose on the quantity of IgE antibody produced revealed that although IFN-gamma could be detected by ELISA in cultures exhibiting high-dose antigen-dependent diminution of IgE production, anti-IFN-gamma monoclonal antibody could not reverse this phenomenon. Thus, IFN-gamma is not solely responsible for decreased IgE synthesis associated with high-dose antigen exposure. IL-4 activity was detected in the fluid from cultures stimulated with low, but not high, levels of antigen. Moreover, addition of exogenous IL-4 restored IgE production to normal levels in cultures exposed to high antigen concentrations. Therefore, it appears that high levels of antigen result in selective stimulation of Th1 cells which produce IFN-gamma, and diminished activation of IL-4-producing Th2 cells. These results help explain observations regarding the influence of antigen dose on the generation of experimental and clinical IgE antibody responses in vivo.     

Synergy of helper factors in the differentiation of in vivo-preactivated antigen-specific human B cells

After in vivo immunization with antigen, B cells appear in the peripheral blood which can be induced in vitro by nonspecific factors found in mixed lymphocyte culture supernatants (MLC-SN) to differentiate and secrete antibody specific for the immunizing antigen. In order to further delineate the nature of the factors involved in the differentiation of these in vivo-activated B cells, various helper factors, including interleukin 1 and interleukin 2 (IL-1 and IL-2), B-cell growth factor (BCGF), and B-cell differentiation factor (BCDF) were added separately and in combination to cultures of these preactivated B cells. T-cell-depleted fractions of peripheral blood mononuclear cells were obtained from normal individuals immunized in vivo with keyhole limpet hemocyanin. MLC-SN alone, without the addition of antigen, selectively triggered an antibody response specific for the antigen used to immunize in vivo in the absence of a polyclonal B-cell response. In order to obtain responses equal to those seen with MLC-SN, a combination of BCGF, IL-2, and BCDF was required, although any two factors partially reconstituted the response. Exogenous IL-1 had the least effect but was suppressive in the presence of optimal concentrations of monocytes. Thus, for maximal in vitro differentiation of in vivo-preactivated B cells, a combination of at least three helper factors is required and acts in a synergistic manner to induce antigen-specific antibody responses.     

High frequency of precursors of anomalous killer cells in human peripheral blood: evidence for T-cell regulation

The frequency of precursors (P) of the anomalous killer (AK) cells able to kill a melanoma target cell line without prior sensitization was determined by limiting dilution analysis. The frequencies obtained from the peripheral blood mononuclear cells of six healthy individuals ranged from 1/250 to 1/750, which was considerably higher than those of alloreactive cytolytic T-lymphocyte (CTL) precursors induced in the same cultures (range 1/900 to 1/7500). The presence of phytohemagglutinin (PHA) inhibited the appearance of both CTL and AK in bulk cocultures, and in limiting dilution analysis the presence of the lectin resulted in multiphasic cell dose-response curves rather than linear single hit responses for both types of precursor cells. The results suggest that AK-P are under the same type of regulation as are CTL-P.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. II. Mechanisms, specificity, and cellular analysis of 2,4,6-trinitrophenol (TNP)-specific cytolytic response priming by intravenous versus subcutaneous injection with TNP-modified syngeneic cells

We have examined the underlying mechanisms accounting for the enhanced in vitro TNP-specific cytotoxic T-lymphocyte (CTL) response following the parenteral injection of syngeneic hapten-modified lymphoid cells. Augmented CTL activity noted following parenteral injection (iv vs sc) of 2,4,6-trinitrophenol-modified syngeneic spleen cells (TNP-SC) is most apparent when limiting numbers of TNP-modified stimulator cells are used in the in vitro sensitization phase. Enhanced CTL responses seen following sc and iv priming is due to distinct mechanisms. Spleen and lymph node (LN) cells from sc primed mice were found to contain significant levels of radioresistant helper activity upon coculture with either viable normal spleen cells in bulk culture or with thymocytes as the source of precursor CTLs in a limiting dilution assay. The helper activity was found to be mediated by a Lyt 1+2- T cells. In addition, Lyt 2-depleted spleen and LN cells from sc primed BALB/c mice could restore the ability of tolerant spleen cells from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-injected BALB/c mice to generate TNP-specific CTLs. Conversely, Lyt 2-depleted spleen and LN cells from iv primed mice provided no measurable helper activity either in bulk culture or in the limiting dilution assay and did not restore the ability of TNBS-tolerant BALB/c spleen cells to generate TNP-specific CTLs. CTL priming via the iv route was found to be completely antigen specific as iv injection of either 2,4-dinitrophenol (DNP)- or fluorescein isothiocyanatel (FITC)-modified cells caused no enhanced CTL activity. Priming via the sc route exhibited a unique specificity pattern as it was shown that sc injection of both TNP-SC and DNP-SC, but not FITC-SC, resulted in enhanced TNP-specific CTL responses. CTL T-helper (Th)-cell induction via the sc route was correlated with (1) the presence of H-2 I region determinants on the inducer cells as the sc injection of TNP-modified erythrocytes led to no enhanced CTL responses or CTL Th activity (while iv injection of TNP-erythrocytes did lead to enhanced CTL responses without detectable helper activity) and (2) the detection of both hapten-specific T-cell proliferation and Interleukin 2 (IL-2) production upon restimulation in culture. We conclude that the sc injection of TNP-SC leads preferentially to an increase of specific Lyt 1+ helper activity, while iv injection leads preferentially to an apparent expansion of Lyt 2+ prelytic effector CTLs.     

Long-lasting deficit of functional T cell precursors in human bone marrow transplant recipients revealed by limiting dilution methods

We have applied limiting dilution methods suitable for the estimation of mitogen-reactive helper (pHTL) and cytotoxic (pCTL) T cell frequencies to the analysis of immune function in patients 1 mo to 6 yr after allogeneic bone marrow transplantation (BMT). Although the majority of these patients have regained normal levels of Leu-3+ (helper) and Leu-2+ (killer/suppressor) cells by 6 to 12 mo after BMT as assessed by cytofluorimetry, the fraction of these cells that can function in limiting dilution cultures is substantially below normal levels in nearly all patients. Although some BMT patients eventually recover normal frequencies of pCTL and pHTL, values typically remain greatly depressed even in patients transplanted as many as 4 to 6 yr previously. In contrast, recovery of precursors able to proliferate (without expressing either helper or cytotoxic function) in response to phytohemagglutinin (PHA) and interleukin 2 occurs in many patients by 1 yr after transplant. In spite of the decreased frequency of functional precursor cells found after BMT, each precursor is capable of giving rise to the same amount of function at limiting dilutions as that produced by cells from normal controls. In many BMT patients, proliferation in conventional PHA-stimulated cultures returns to near-normal levels even though precursor frequencies remain low. The limiting dilution method is sensitive to residual immune dysfunction in BMT recipients not easily quantitated by other, more conventional techniques.     

Mucosal mast cells and nematode infection: strain-specific differences in mast cell precursor frequency revisited

Mucosal mast cells (MMC) play an important role in the immune response against selected species of intestinal nematode. The kinetics with which different strains of inbred mice resolve infection with Trichinella spiralis correlates with their ability to mount MMC responses in the intestinal mucosa. Homologues of MMC that express and constitutively secrete abundant amounts of the granule chymase, mouse mast cell protease-1 (mMCP-1), can be generated in vitro from bone marrow cultures supplemented with interleukins-3 and -9, stem cell factor and transforming growth factor-beta1. Using the enhanced growth characteristics of these MMC homologues, a novel limiting dilution assay for mast cell precursor (MCp) frequency has been developed. The assay is highly specific, in that cultures containing mast cells are identified with mMCP-1 specific antibody, and almost three-fold more sensitive than previously published systems. MCp frequencies were compared in BALB/c and C57/BL10 strains of mice that, respectively, respond rapidly and slowly to infection with T. spiralis. MCp frequency (1/378 bone marrow cells) was significantly greater in BALB/c than C57/BL10 mice (frequency: 1/751). Similarly the rate of growth of MMC homologues and the production of mMCP-1 was significantly greater in BALB/c than in C57/BL10 bone marrow cultures.     

Priming of peripheral lymph node B cells with TNP-Ficoll: role of lymphokines in B cell differentiation.

We have previously shown that peripheral lymph node (PLN) B lymphocytes of adult DBA/2J mice failed to make an antibody response to type 2 antigen TNP-Ficoll, but exhibited a good antibody response to type 1 antigen TNP-Brucella abortus. In the present study we wanted to find out whether the unresponsiveness of PLN B cells to TNP-Ficoll is due to defects in the early activation and proliferation stage or in the final differentiation stage of B cells. Therefore, we have used a two-step protocol of in vivo immunization of mice with TNP-Ficoll and the subsequent in vitro challenge with TNP-Brucella abortus and studied the anti-TNP plaque-forming cell (PFC) responses. The results indicate a three- to sixfold increase of PFC responses in PLN cell cultures derived from TNP-Ficoll-primed animals compared to saline control mice. This increased antibody response was TNP-specific as 93% of the PFC's were inhibited by TNP-lysine. Limiting dilution experiments confirm that the increase in anti-TNP PFC response from the TNP-Ficoll-primed animals was indeed due to an increase in TNP-specific precursor B cells. Further, the addition of rIL-5 or rIL-6 induced anti-TNP PFC in the TNP-Ficoll-primed and in control PLN cell cultures in the presence of antigen. However, in primed PLN cells lymphokines alone were sufficient to restore anti-TNP PFC response. In conclusion, our results show that in PLN, the TNP-Ficoll can induce proliferation of hapten-specific B cells but not final differentiation. These primed PLN B cells mature into antibody-secreting cells upon stimulation with TNP-BA or lymphokines.     

Early loss of precursors of CTL and IL 2-producing cells in the development of neonatal tolerance to alloantigens

Bulk culture and limiting dilution analysis (LDA) were used to follow the ontogeny of the tolerant state in CBA/ HT6T6 mice neonatally tolerized to allogeneic histocompatibility antigens. Advantage was taken of the fact that the lymph nodes (LN) of young mice show immunocompetence before spleen cells do, allowing analysis of actual reactivity as early as 1 wk of age. At 1 wk, the LN cells of mice tolerized i.v. showed a loss of CTL reactivity in bulk culture specific for the tolerizing antigens; a corresponding specific decrease was seen in the frequency of CTL precursors (CTLp). At the same age, however, proliferative responses and interleukin 2 (IL 2) production in MLC were nonspecifically depressed in the tolerized animals. LDA of IL 2 producer precursor frequency (IL- 2Tp ) showed that there was a nonspecific loss of 50% of functional alloreactive IL- 2Tp , accompanied by a larger specific decrease of 90% in the frequency of IL- 2Tp responding to the injected alloantigens. These characteristics of the tolerant state persisted through at least 4 wk of age. Neither the proliferative nor CTL response deficiencies could be overcome by the addition of Con A supernatant containing IL 2. Mixing experiments failed to show evidence of suppressor cell involvement in the loss of the proliferative response. Our results indicate that the specific loss of alloreactivity after tolerization is due to clonal inactivation or deletion of both CTLp and IL- 2Tp , which is obvious as early as 7 days of age. In addition, the differences in the specificity of the clonal inactivation between CTLp and IL- 2Tp suggest the existence of independent mechanisms for tolerization.     

Frequencies of IL-2- and IL-4-secreting T cells in naive and antigen-stimulated lymphocyte populations

The relative frequencies of IL-2-and IL-4-secreting precursors in naive and Ag-primed populations were investigated by using limiting dilution analysis. Cells capable of IL-4 production in lymphoid populations freshly isolated from mice were rare in comparison with those producing IL-2 when the cells were stimulated by nominal Ag, by alloantigens, or by mitogens. One cycle of in vitro Ag restimulation and rest, however, enabled us to detect high proportions of IL-4-secreting cells among keyhole limpet hemocyanin-primed lymph node cells. With cultures set up at monoclonal cell doses, it was shown that IL-2 and IL-4 are secreted by separate precursor populations at this stage of their development. The IL-4-secreting cells were further shown to be dependent upon the presence of IL-2, either secreted by separate precursors or exogenously added, for the production of detectable amounts of IL-4. Analysis of the frequencies of helper cells producing both IL-2 and IL-4 at various stages of the in vivo immune response and the requirements for their growth and differentiation should give a better understanding of the relative contributions of each cell type.     

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4⁺ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.     

Virus-specific memory T cells are Pgp-1+ and can be selectively activated with phorbol ester and calcium ionophore

Memory lymphocytic choriomeningitis virus (LCMV)-immune cytotoxic T-lymphocyte precursors (CTLp) can be stimulated to proliferate and to mediate specific cytotoxic activity following incubation with phorbol myristate acetate (PMA), calcium ionophore (CaI), and interleukin 2 (IL-2). This protocol can be used to selectively induced virus-specific CTL activity under both bulk culture and limiting dilution conditions, in the absence of added antigen. There is no concurrent stimulation of alloreactive CTLp. Proliferation of the effector Lyt-2+ population in medium containing PMA and CaI requires L3T4+ cells, which can be replaced by adding IL-2, and the development of cytotoxicity is totally IL-2 dependent. The LCMV-specific memory T cells are also characterized by the expression of the Pgp-1 (Ly24) glycoprotein. The availability of this marker, together with the capacity to selectively stimulate primed CTLp in the absence of antigen, should greatly facilitate the analysis of T-cell memory in virus infections.     

Significant frequency of cytotoxic T lymphocyte precursor cells specific for TNP-modified allogeneic cells in normal lymphocytes

It was tested whether the cytotoxic T-lymphocyte precursor (CLP) repertoire in normal mice is biased toward recognizing foreign antigen in association with self H-2 as opposed to allogeneic H-2. The frequencies of CLPs in normal mice (H-2b,k,d) specific for TNP-modified syngeneic and TNP-modified allogeneic cells have been compared by limiting dilution analysis. Normal spleen cells were cultured at a limiting dilution with TNP-modified (TNP-self) or TNP-modified allogeneic (TNP-allo) stimulator cells. Cultures were split into four aliquots and assayed against TNP-self, TNP-allo, unmodified syngeneic, and unmodified allogeneic Concanavalin A blast targets and classified for cytotoxic activity directed against TNP-self, TNP-allo, and allo H-2 determinants. In disagreement with our expectations from the literature, the frequencies of CLPs in H-2b and H-2d responder cells recognizing TNP-modified H-2k were higher than the frequencies of CLPs recognizing TNP-self. There was no clear preference for TNP-self in the case of H-2b responder and H-2d allogeneic cells, nor vice versa. Only in the case of H-2k responder cells was there a distinct preference for TNP-self. The significance of a considerable number of TNP-specific, allo H-2-restricted CLPs in normal lymphocytes is discussed.     

Reciprocal regulatory effects of IFN-gamma and IL-4 on the in vitro development of human Th1 and Th2 clones.

The effects exerted on the in vitro development of purified protein derivative (PPD)-specific or Dermatophagoides pteronyssinus group I (Der p I)-specific T cell lines (TCL) and T cell clones (TCC) by IL-4 or IFN-gamma addition or neutralization in human PBMC cultures were examined. PBMC from two normal individuals, which were stimulated with PPD and then cultured in IL-2 alone, developed into PPD-specific TCL and TCC able to produce IFN-gamma and IL-2 but not IL-4 and IL-5 (Th1-like). IFN-gamma or anti-IL-4 antibody addition in bulk cultures before cloning did not influence the PPD-specific TCL profile of cytokine production. In contrast, the addition of IL-4 resulted in the development of PPD-specific TCL and TCC able to produce not only IFN-gamma and IL-2 but also IL-4 and IL-5. PBMC from one atopic Der p I-sensitive patient, which were stimulated with Der p I and then cultured in IL-2 alone, developed into Der p I-specific TCL and TCC able to produce IL-5 and large amounts of IL-4 but no IFN-gamma (Th2-like). The addition in bulk cultures, before cloning, of either IFN-gamma or anti-IL-4 antibody markedly inhibited the development of Der p I-specific T cells into IL-4- and IL-5-producing TCL. Accordingly, the development into Der p I-specific Th2-like TCC was significantly reduced by the addition of IFN-gamma in bulk culture and was virtually suppressed by the presence of both IFN-gamma and anti-IL-4 antibody. These data suggest that the presence or the absence of IL-4 and IFN-gamma in bulk cultures of PBMC before cloning may have strong regulatory effects on the in vitro development of human CD4+ T cells into Th1 or Th2 clones.     

Establishment of continuous cultures of thy1.2+, Lyt1+, 2-T cells with purified interleukin 3

IL-3 is a recently described lymphokine that induces the expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in an early T-cell precursor. We demonstrate that purified IL-3 an be used to establish continuous cultures of a discrete subpopulation of T cells with virtually 100% efficiency from normal, unstimulated splenic lymphocyte populations. Once established over a period of approximately 4-6 weeks, the cultures can be readily cloned either in soft agar or by limiting dilution. All of the established lines are Lyt1+, 2- la-, lg-, Tdt-, 20 alpha SDH+, a phenotype characteristic of helper T cells; they are therefore distinct from continuous cultures of T cells established with IL-2. although initiation of these cell lines was absolutely dependent on IL-3, once established all of the cell lines were independent of exogenously added lymphokines for their growth in vitro. However, all of the cells lines were found to constitutively produce IL-3 at high levels. None of the cell lines constitutively produced lL-2, but could be readily induced to produce this lymphokine by treatment with phorbol-myristic acetate. The ability to produce lL-3 and lL-2 is a further indication that all the cell lines are helper T cells. The possible mechanisms by which lL-3 allows the specific differentiation and/or amplification of T cells of helper phenotype in tissue culture are discussed.     

The human MHC-restricted cellular response to herpes simplex virus type 1 is mediated by CD4+, CD8- T cells and is restricted to the DR region of the MHC complex

The nature of the in vitro human cytotoxic T-cell responder population to HSV type 1 (HSV-1) was studied. In 5-day HSV-1-stimulated cultures that contained MHC-restricted activity, two phenotypically distinct populations of cells were present that were capable of lysing HSV-1-infected B cell lines in a 5-h 51Cr-release assay. The first was CD4+, CD8-, CD16- cell typical of class II-restricted T cells, whereas the other population bore a CD4-, CD8-, CD16+ NK-cell phenotype. Elimination of the NK cell fraction from bulk cultures by using anti-CD16 plus C frequently resulted in cell populations that killed in an Ag-specific, HLA-DR-restricted fashion. In some cases the anti-CD16-pretreated cultures retained a killing population that was unrestricted to MHC products. In no instance were any cytotoxic T cells that were restricted to class I Ag in evidence. Limiting dilution analysis of precursor frequency indicated that about 1 in 4000 to 1 in 8000 cells from peripheral blood are specific for HSV-1 in seropositive individuals. Comparisons of HLA class I-matched and HLA class II-matched targets with the autologous target by using limiting dilution analysis yielded results entirely consistent with those obtained in the bulk culture assay system.