A self-Ia reactive T cell clone directly stimulates every hundredth B cell and helps antigen-specific B cell responses

A self-I-A reactive T cell clone has been isolated that proliferates in the presence of irradiated syngeneic spleen cells in mouse serum. The cells directly stimulate approximately every hundredth B cell, which includes small B cells, into both proliferation and antibody formation in the absence of added antigen. There is evidence to suggest that cells of this type are generally present in murine T cell populations. The cells may recognize self-I-A as such. We failed to obtain evidence, but cannot exclude that they recognize an antibody idiotype in addition. The clone also augments antigen-specific antibody responses in vivo and in vitro in the presence of antigen. In the in vitro response to a T-independent TI-2 antigen, the T cells in co-culture with B cells induced specifically the production of IgG1 antibodies, thus mimicking the function of major histocompatibility complex-restricted, antigen-specific helper T cells. However, IgG1 production was not observed when splenic T cells were added to this system. Thus, self-I-A reactive T cells, generally inactive in the system, may locally help B cell responses to antigens which the T cells do not themselves recognize. In such instances one might attribute to the T cells antigen or idiotype-binding specificities which the cells in reality do not display.     

IgG- and IgE-mediated antigen presentation on MHC class II

IgG- and IgE-antibodies have the ability to enhance the production of antibodies directed against the antigen they are specific for. It has been suggested that the mechanism behind IgG- and IgE-mediated feedback enhancement is the ability of these isotypes to induce a more potent antigen-specific T helper cell response, increasing the chances that antigen-specific B cells receive the T cell help they require to become antibody-producing cells. With emphasis on the murine system, we will here focus on the ability of IgG and IgE to capture antigen and facilitate presentation of antigenic peptides to T helper cells. Whether this mechanism underlies feedback enhancement of antibody responses to these antigens will be discussed.     

Evaluation of accessory cell heterogeneity. II. Failure of dendritic cells to activate antigen-specific T helper cells to soluble antigens

The activation of antigen-specific T cells requires Ia⁺, antigen-presenting accessory cells (AC). Dendritic cells (DC) and macrophages (M?) isolated from spleen and peritoneal exudate were tested as AC for the activation of T helper cells and the induction of T cell proliferation. The cell separations to obtain DC and splenic M? were performed by discontinuous bovine serum albumin gradients, adherence on petri dishes and rosetting with opsonized sheep erythrocytes. DC as well as the M? were able to induce antigen-specific T cell proliferation, but only the M? and not the DC activated antigen-specific T helper cells which help B cells for antibody production to soluble antigens. Keyhole limpet hemocyanin-specific T cells repeatedly stimulated with DC and antigen also did not express helper activity. The failure of DC to induce T helper cells was not due to the activation of a suppressor pathway. Thus, dendritic cells, although very efficient as AC in the induction of various T cell functions, are not able to activate T helper cells required for carrier-specific T-B cooperation and therefore cannot be the sole accessory cells. Based on these results and on previous data using Ia⁺ tumor cell line as AC, we confirm the existence of functional AC heterogeneity.     

Differential effects of superantigen-induced “anergy” on priming and effector stages of a T cell-dependent antibody response

The in vitro T cell nonresponsiveness or anergy to restimulation with staphylococcal enterotoxin B (SEB) following the in vivo injection of the superantigen is well characterized. Here we use mice transgenic for a V_β8.2⁺ T cell receptor (TcR) (reactive with SEB) to establish a large population of anergic T cells in vivo. As expected, peripheral T cells from the SEB injected transgenic mice failed to proliferate or produce interleukin (IL)-2 following restimulation with the superantigen in vitro. However, in this system superantigen reactivity could be restored by either addition of exogenous IL-2, or stimulation with immobilized anti-TcR antibody. To evaluate the effects of superantigen-induced anergy in vivo, SEB-injected or noninjected control transgenic mice were immunized and boosted with the T cell-dependent antigen tetanus toxin (TT). SEB injection of the V_β8.2⁺ transgenic mice 5 days prior to the TT immunization inhibited the anti-TT antibody response as measured over a 100-day period, whereas injection of a superantigen which does not interact with the V_β8.2⁺ TcR (such as SEA) did not. Furthermore, SEB injection of control nontransgenic mice did not interfere with the induction of a high titer anti-TT antibody response. In contrast to the inhibition seen when SEB was given prior to TT immunization, injection of transgenics with SEB either after the priming TT immunization or after the recall booster injection did not significantly influence the titers of anti-TT antibodies produced. These results demonstrate that the establishment of peripheral T cell anergy to superantigens inhibits the specific antigenic priming of helper T cells in vivo, but does not prevent primed T cells from helping B cells to mount an effective antibody response.     

Endogenous retroviral superantigen presentation by B cells induces the development of type 1 CD4+ T helper lymphocytes

The endogenous retroviral superantigen, minor lymphocyte stimulating antigen (Mls 1^a, encoded by Mtv-7), when presented by highly purified B cells induced the development of a highly polarized population of T helper (Th)1 cells from naive peripheral CD4⁺ T cells in vitro. Immobilized anti-Vβb6⁺ antibodies similarly generated highly polarized, largely Vβ6⁺, Th 1 populations in vitro. In the presence of exogenous interleukin-4, both stimuli were capable of generating Th 2, rather than Th 1 populations. Mls 1^a presentation by B cells in vivo led to the development of an equally polarized Th 1 population. Using monoclonal antibodies against interferon-γ and transforming growth factor-β it was demonstrated that maximal Th 1 development with either stimulus in vitro was dependent on the endogenous production of these two cytokines. Thus, our results demonstrate that the retroviral encoded superantigen, Mls 1^a, drives the development of Th 1 cells both in vitro and in vivo, and they suggest that B cell presentation does not, in itself, lead to the generation of Th 2 cells.     

Synergistic effect of interleukin-2 and a vaccine of irradiated melanoma cells transfected to secrete staphylococcal enterotoxin A

We have previously reported that immunization of mice with melanoma cells transfected to secrete the superantigen, Staphylococcal enterotoxin A (SEA), increased the production of antibodies to the B700 melanoma antigen, stimulated the production of endogenous interleukin 2 (IL-2), activated the expression of CD4, CD8 and CD25 T cell markers and enhanced NK cell activity. Now we show that immunization of mice with a vaccine of irradiated sea-transfected melanoma cells coupled with IL-2 therapy was even more effective in inhibiting the growth of primary melanoma tumors and the development of lung metastases than was the irradiated melanoma cell vaccine alone or IL-2 alone. The morphological and immunological effectiveness of the therapy was dose-dependent on IL-2.     

Mimicking the humoral immune response in vitro results in antigen-specific isotype switching supported by specific autologous T helper cells: generation of human HIV-1-neutralizing IgG monoclonal antibodies from naive donors

Molecular and cellular requirements for antigen-specific isotype switch of human B cells have been investigated by mimicking signaling occurring in germinal centers. Peripheral blood mononuclear cells from healthy seronegative blood donors were first primary immunized in vitro, using a synthetic immunogen containing both a T and B cell epitope, which generated specific IgM-secreting B cells. We used the apex of the V3 loop of gp120 as B cell epitope linked to a promiscuous T helper epitope from tetanus toxin. In parallel, CD4⁺ T helper cell clones specific for the T epitope of the immunogen were established. In a secondary in vitro stimulation period, we co-cultured the antigen-specific T and B cells on CD32-transfected fibroblasts, together with an anti-CD40 monoclonal antibody. This resulted in isotype switching and human antigen-specific, IgG-secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen-specific human B cells derived from this primary and secondary in vitro immunization were subsequently subjected to electrofield-induced somatic cell hybridization and hybridomas secreting human anti-V3 IgG monoclonal antibodies were isolated. One human antibody was further characterized and shown to be specific for the immunizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HIV-1, achieving a 50% neutralization at 0.46 μg/ml.     

Antigen specificity and frequency of autologous and allogeneic helper T cells in the in vitro production of antibody against influenza virus by human blood lymphocytes

The frequency of antigen-specific helper T cells in human peripheral blood mononuclear cells was determined by limiting dilution analysis of specific in vitro antibody responses to influenza virus A/X-31 (A-H3N2). Limiting numbers of irradiated E rosette-forming (E⁺) (T) cells were added to a constant number of syngeneic non-rosette-forming (E^?) (“B”) cells and their ability to support production of antibody to influenza virus determined. The frequency of T helper cells was then calculated by a Poisson distribution analysis and found to range between 0.78 × 10^?5 and 2.86 × 10^?5. Specific antibody production can be obtained in this system from allogeneic combination of E^? and E⁺ cells provided that the added E⁺ cells are irradiated to abrogate alloactivated T suppressor effects. Limiting dilution analysis applied to determine the frequency of helper T cells under these conditions showed that in most, but not all, cases the frequency of T helper cells was higher in allogeneic combinations. This result could be interpreted as showing an increase in the number of activated specific T helper cells, due perhaps to a positive allogeneic effect. On the other hand, it could also be explained by the alloactivation of an entirely different subset of nonspecific helper cells, or by the production of a nonspecific allogeneic helper factor. The specificity of T cell help in allogeneic combinations was therefore examined by adding limiting numbers of E^? cells to a fixed number of E^? cells and simultaneously challenging with two non-cross-reacting influenza viruses A/X-31 and B/HK. Under these conditions, individual cultures produced antibody to A/X-31, B/HK or both. The frequency of cultures producing antibody to both viruses was as predicted for the chance occurrence of specific helper T cells for both antigens being present in the same culture. The fact that a significant number of individual cultures made antibody to only one antigen in both autologous and allogeneic combinations showed that T cell help was antigen-specific in both situations. Thus, for human in vitro antibody responses, antigen-specific T cell help can be obtained across a major histocompatibility (complex) barrier.     

Selected populations of alloreactive T cells contain helper T cells but lack ThId,an antigen-specific helper T cell required for dominant production of the T15 idiotype

Isolated, alloreactive T cell populations were primed with protein carriers in vivo and tested for their ability to provide help for an anti-phosphorylcholine (PC) antibody response and for production of the T15 idiotype. It was found that alloreactive T cell populations would support anti-PC antibody responses but did not selectively activate B cells capable of producing the T15 idiotype that normally dominates such responses. This failure to help for the production of the T15 idiotype was shown to be due to the absence of an antigen-specific helper T cell that is required for dominant idiotype production (ThId). These studies suggest that ThId cells have recognition structures for antigen and for self idiotype, but lack recognition structures for major histocompatibility complex-encoded antigens.     

H-2-restricted T-B cell interactions involved in polyspecific B cell responses mediated by soluble antigen

Maturation of polyspecific B cells to Ig secretion was induced in response to the interaction of helper T cells with soluble antigen. The in vitro propagated, keyhole limpet hemocyanin (KLH)-primed C57BL16 T cells used in these experiments proliferated in response to antigen presented on I-A-compatible antigen-presenting cells and were depleted of alloreactive cells. These T cells induced a strong polyspecific plaque-forming cell response from normal C57BL/6 B cells over a wide range of KLH concentrations (100 μg/ml to 0.01 μg/ml). Culture conditions were established whereby H-2-restricted T cell-macrophage interactions were nonlimiting, allowing a direct analysis of H-2-restricted T-B cell interactions involved in polyspecific B cell induction. The requirements for B cell induction/amplification were dependent on the size of the responding B cell population. Small B cells were activated only at high concentrations of KLH and required a direct H-2-restricted interaction with KLH-primed helper T cells. In contrast, large B cells were induced/amplified over the full range of KLH concentrations tested in an H-2-unrestricted manner, limited only by the H-2-restricted, antigen-dependent activation of T helper cells. The requirement for high antigen concentration in the activation of small B cells is proposed to reflect a requirement for antigen binding to the B cell surface via nonspecific interactions. This binding was not in itself stimulatory, but expression of antigen in conjunction with B cell surface Ia antigens provided a focus for a direct interaction with Ia-restricted, antigen-specific T helper cells (or factors). In contrast, the H-2-unrestricted induction/ amplification of large B cells was mediated solely as a consequence of the helper T cell activation which occurred at the lowest concentrations of KLH tested. T helper cell activation is proposed to lead to the production of non-antigen-specific, non-Ia-restricted factors supporting the continued growth of blasted B cells. Thus, while an H-2-restricted T cell-macrophage interaction is an obligate requirement for polyspecific B cell responses, the requirement for a direct H-2-restricted T-B cell interaction varies with the pre-existing state of activation of the responding B cell population.     

Antigen-specific plaque-forming cell response of human cord blood lymphocytes after in vitro stimulation by T cell-dependent antigens

Mononuclear cells isolated from human cord blood (CBL) of full-term neonates were stimulated in vitro with a dose range of T cell-dependent antigens, i.e. ovalbumin or sheep erythrocytes, and tested for the capacity to mount an antigen-specific plaque-forming cell (PFC) response. Both of the antigens used induced in CBL a PFC response with the same kinetics of PFC formation and of the same magnitude as found in cultures of adult peripheral blood lymphocytes (PBL). However, optimal PFC responses in CBL were obtained at a hundredfold lower concentration of the antigens compared with the optimal antigen doses for the induction of a PFC response in adult PBL. This phenomenon was further investigated with respect to the antigen dose dependency of the activation of neonatal B cells and neonatal regulatory T cells. The induction of a PFC response in CBL at antigen concentrations that were suboptimal for adult PBL showed a correlation with the particular antigen dose requirements for the activation of B cells and T helper cells in neonates. Furthermore, the findings suggest that the decrease of the PFC response in CBL stimulated with supraoptimal doses of antigen was not caused by the induction of unresponsiveness at the B cell level or by interference of pregnancy-associated substances with the PFC response, but was rather the result of the activation of antigen-specific T suppressor cells. Neonatal T suppressor cells were activated at antigen concentrations that generated T helper activity in the adult. Thus, although neonatal B cells possess the intrinsic capacity to mature into antigen-specific PFC, the conditions for effective activation of neonatal T cells regulating the B cell response differ from those for the activation of adult regulatory T cells.     

Stimulation of Human Peripheral Lymphocytes via CD3 and Soluble Antigen Abrogates Specific Antibody Production by Reducing Memory B Cell Numbers

Human B cells are polyclonally activated in vitro by T cells stimulated with immobilized anti-CD3 monoclonal antibodies. We have analysed the effect of CD3 ligation on the production of antigen-specific antibodies, using peripheral blood lymphocytes from tetanus toxoid vaccinated blood donors. High levels of antigen-specific antibodies were obtained after stimulation with anti-CD3 antibodies for 7 days. Addition of a soluble recall antigen did not affect the total amount of Ig produced, but dramatically decreased the antigen-specific response. The addition of IL-2, IL-4, anti-CD40 or anti-CD28 antibodies or the removal of antigen did not restore the B cell response. Analysis using limiting dilution of B cells showed that the frequency of antigen-specific memory B cells decreased significantly in cultures stimulated with antigen. The antigen-specific B cell response could be completely restored only if the soluble antigen was cross-linked on the surface of the B cells. These results suggested that peripheral memory B cells were eliminated or anergized in the presence of soluble antigen.     

Constraints in T-B cooperation related to epitope topology on E. coli beta-galactosidase. I. The fine specificity of T cells dictates the fine specificity of antibodies directed to conformation-dependent determinants

Experiments to test the relationship between the epitopes on a protein antigen recognized by T and B cells in their collaboration to produce antibody cannot rely solely on hapten-carrier models. In the present work we used E. coli beta-galactosidase, a molecule whose tertiary and quaternary epitopes have been well characterized, as the model antigen. T helper cells were raised by stimulating mice with the intact or the denatured molecule or with any of several beta-galactosidase cyanogen bromide peptides. In a series of in vitro helper T cell assays we confronted the various T populations with B cells preimmunized with the native antigen, and we tested their capacity to help production of (a) binding antibodies and (b) antibodies directed to single conformational epitopes, characterized by their capacity to protect the enzyme from heat denaturation or to activate defective beta-galactosidase. According to our results, (a) equivalent T cell help can be provided by T helper cells primed with native or denatured antigen, even for the production of "conformational" antibodies; (b) one of the peptides (CB-18) is most efficient in raising help for binding antibodies; and (c) two peptides (CB-20 and CB-21) rank highest in priming T helper cells for the eventual production of protecting and activating antibodies, respectively. Thus, not every beta-galactosidase-specific T helper cell is useful in providing help to B cells specific for any particular epitope on the molecule, but rather preferential pairings exist, possibly governed by a proximity rule.     

Enhancement of specific immunoglobulin production in SCID-hu-PBL mice after in vitro priming of human B cells with superantigen.

Priming of human mature B cells in vitro with staphylococcal enterotoxin A (SEA) prior to transplantation of the B cells into severe combined immunodeficiency (SCID) mice, together with human T-helper cells, resulted in higher and more uniform concentrations of serum IgG in the mice. This indicated that a large number of B cells had become activated, which was supported by the finding that SEA priming resulted in production of immunoglobulin displaying a more normal kappa/lambda ratio than was obtained in the absence of SEA priming. However, IgM concentrations were not affected by SEA priming. Immunization of mice, transplanted with SEA-primed B cells, with both primary and secondary antigens resulted in a high specific IgG response to both types of antigen. The elevated levels of specific antibodies were not merely the consequence of an unspecific stimulation of B cells caused by SEA, as the ratio of specific antibody to total IgG was much higher in animals receiving SEA-primed B cells. Thus, a co-operative effect on immunoglobulin production of stimulating B cells via surface immunoglobulin and help delivered by SEA-activated T-helper cells was indicated. A specific antigen-dependent IgM response to a secondary antigen was observed as well, but was, in contrast to the IgG response, not influenced by SEA priming of B cells. No IgM antibodies with reactivities to the primary antigens were detected in the SCID sera at any time-point after immunization. The results thus indicate that SEA might replace T-cell epitopes in antigens and efficiently recruit an abundance of T-cell help to B cells, resulting in enhanced production of specific IgG antibodies.     

In vitro stimulation prior to fusion generates antigen-binding human-human hybridomas

Production of useful human monoclonal antibodies has been limited by the inability to reliably generate and isolate antigen-specific B cells by in vivo immunization. An in vitro culture system employing antigen and mitogen to stimulate lymphocytes derived from solid lymphoid organs has been developed. Human tonsilar or splenic lymphocytes were stimulated in vitro with antigen and mitogen in short term culture and then fused with either of two enzyme deficient human B cell lines. This approach appears to expand antigen-specific B cell clones prior to fusion resulting in the production of a significant number of antigen-binding human hybridoma antibodies. The system has been effective in the production of human monoclonal antibodies following stimulation with KLH-ARS, a soluble antigen, and intact group B streptococcus, a particulate antigen. Hybridomas have been produced by fusion with two distinct parental human B cell lines supporting the previously reported observation that human B lymphoblastoid cell lines representing different stages of B cell differentiation may be useful fusion partners. The utility of the in vitro stimulation system in producing human-human hybridomas secreting antibody directed against two distinct classes of antigens establishes this approach as a generally useful method for the production of human monoclonal antibodies.     

Subcellular antigen localization in commensal E. coli is critical for T cell activation and induction of specific tolerance

Oral tolerance to soluble antigens is critically important for the maintenance of immunological homeostasis in the gut. The mechanisms of tolerance induction to antigens of the gut microbiota are still less well understood. Here, we investigate whether the subcellular localization of antigens within non-pathogenic E. coli has a role for its ability to induce antigen-specific tolerance. E. coli that express an ovalbumin (OVA) peptide in the cytoplasm, at the outer membrane or as secreted protein were generated. Intestinal colonization of mice with non-pathogenic E. coli expressing OVA at the membrane induced the expansion of antigen-specific Foxp3⁺ T_regs and mediated systemic immune tolerance. In contrast, cytoplasmic OVA was ignored by antigen-specific CD4⁺ T cells and failed to induce tolerance. In vitro experiments revealed that surface-displayed OVA of viable E. coli was about two times of magnitude more efficient to activate antigen-specific CD4⁺ T cells than soluble antigens, surface-displayed antigens of heat-killed E. coli or cytoplasmic antigen of viable or heat-killed E. coli. This effect was independent of the antigen uptake efficiency in dendritic cells. In summary, our results show that subcellular antigen localization in viable E. coli strongly influences antigen-specific CD4⁺ cell expansion and tolerance induction upon intestinal colonization.     

T-cell responses to minor histocompatibility antigens

We have investigated the helper and cytotoxic T-cell response to minor histocompatibility antigens and generated long term antigen-specific cell lines to them. Antigen-specific activity was selected for by regular restimulation with irradiated cells bearing the antigens in the presence of interleukin 2, so that alloreactivity to other cell surface antigens was gradually lost. Helper T cells cultured over several months were active in vivo and in vitro, but the culturing method eventually selected for cytotoxic T cells at the expense of helper T cells, with concomitant changes in the proportions of cells expressing the Lyt phenotypes. Individual long term cultures of cytotoxic T cells specific for minor histocompatibility antigens were restricted by either H2K or D but not both. Helper T cells to minor histocompatibility antigens derived directly from primed F1 mice did not show restriction to the priming parental haplotype. This is consistent with antigen reprocessing by the F1 antigen presenting cells such that populations of helper T cells restricted by both parental H-2 haplotypes were primed. F1 cytotoxic T cells were restricted to the parental H-2 haplotype used for in vitro boosting, irrespective of which H-2 was used for in vivo priming.     

Major histocompatibility complex-restricted and unrestricted T helper cells recognizing minor histocompatibility antigens of B cell surfaces

The present experiments analyze the functional properties of helper T cells specific for "minor" histocompatibility antigens. T cells from C3H/HeJ mice, primed in vivo and highly enriched in vitro for reactivity to membrane antigens of C3H/Tif B cells, specifically proliferate, and provide polyclonal help to splenic B cells from strains carving a variety of different H-2 haplotypes on C3H or BALB backgrounds, while failing to respond to cells carrying the same H-2 haplotypes on C57BL or A backgrounds. Since it has been previously demonstrated (A.A. Augustin and A. Coutinho, J. Exp. Med. 1980. 151: 587) and B cell activation in this system strictly requires direct, specific recognition of B cell surface antigens by helper cells and does not result from the production of soluble "mitogenic" or "nonspecific helper factors", it is concluded that this phenomenon represents specific, major histocompatibility complex (MHC)-unrestricted T cell help. In addition, it has now been found that expression of helper activity requires viability of the helper cells and is partially radiation-sensitive. Lack of MHC restriction is not a general property of specific helper cells which directly recognize B cell "minor" antigens, since BALB.C3H anti-C3H/Tif T cells appear to be restricted by H-2 in their polyclonal helper activity. The helper activity mediated by specific anti-"minor", H-2 restricted helper cells could not be inhibited by anti-VH antibodies, and the inhibition obtained with anti-Ia antibodies appeared to operate at the level of B cell induction, rather than at the level of helper cell activation.     

T helper responsiveness in human Loa loa infection; defective specific proliferation and cytokine production by CD4+ T cells from microfilaraemic subjects compared with amicrofilaraemics

The proliferation and cytokine profiles of peripheral blood mononuclear cells (PBMC) from microfilaraemic (Mf⁺) subjects infected by Loa loa in response to antigens of several parasitic stages were compared with those from amicrofilaraemic (Mf⁻) individuals. While a strong lymphoproliferative response and consistent levels of both Th1 (IL-2, interferon-gamma (IFN-γ)) and Th2 (IL-4, IL-5) type cytokines were observed in response to adult worm (AW) and microfilariae (Mf) antigen in Mf⁻ individuals, Mf⁺ subjects were characterized by a T cell unresponsiveness, including proliferation, cytokine production and IL-2 mRNA expression. Conversely, T cell responsiveness to mitogens and non-specific antigen were similar in the two endemic populations. Depletion of lymphocyte subpopulations indicated that T CD4⁺ were mainly involved in the specific cellular response. In contrast to other cytokines, IL-10 was produced in response to all parasitic stages, in both Mf⁺ and Mf⁻ patients. Neutralization of IL-10 did not restore cytokine production in Mf⁺ patients, while B7 mRNA expression was similar between Mf⁺ and Mf⁻ subjects in response to Mf antigen, suggesting that IL-10 was not the only factor responsible for T cell unresponsiveness. Mf⁺ patients have lower Mf antigen-specific IgG levels compared with Mf⁻, and there is a significant correlation between Mf antigen-specific antibodies and IL-5 responses. These findings suggest that Mf⁻ status is correlated with T helper responsiveness, including proliferation and production of both Th1- and Th2-type cytokines, whereas Mf⁺ status is characterized by unresponsiveness of the same cell population, induced and/or maintained by microfilariae.     

B cell hyperactivity and abnormalities in T cell markers and immunoregulatory function in a patient with nucleoside phosphorylase deficiency.

We describe a 2 year old girl with nucleoside phosphorylase (PNP) deficiency, who had low blood T cell numbers and T lymphocyte blastogenic response to mitogens, hypergammaglobulinaemia, high titres of antibodies to many common antigens, various autoantibodies, a monoclonal IgM-kappa protein, an increased frequency of mature Ig containing blood B cells and a high production of Ig in vitro in unstimulated cultures. E rosetting cells showed faint or no immunofluorescence staining with monoclonal antibodies directed against T cell membrane antigens. In vitro Ig production in response to pokeweed mitogen was defective, and no T cell helper or suppressor activity was observed. It is suggested that the immunoregulatory deficiency might have caused the B cell hyperactivity.