Suppression of immune cell function in vitro by recombinant human transforming growth factor-beta

The influence of recombinant human transforming growth factor-beta (rHuTGF-beta) on B-cell function and antigen-specific T-cell responses in vitro was investigated. The addition of 0.1 ng/ml of rHuTGF-beta to cultures of peripheral blood mononuclear cells (PBMC) stimulated with tetanus toxoid resulted in a 50% inhibition of the PBMC proliferative response as determined by [3H]thymidine incorporation. Further, rHuTGF-beta at 0.37 ng/ml caused a greater than 50% reduction in the number of immunoglobulin G-secreting cells among PBMC induced by pokeweed mitogen. rHuTGF-beta also inhibited the generation of allospecific cytotoxic T lymphocytes (CTL) in the mixed-lymphocyte reaction but had no effect on the cytolytic function of CTL generated in the absence of exogenous HuTGF-beta. The results indicate additional immunoregulatory activities for HuTGF-beta and suggest that this factor may play an important role in the regulation of the antigen-dependent immune response.     

Report of a patient with T-cell deficiency and normal B-cell function: A new immunodeficiency disease with response to transfer factor

An 11-yr-old boy had recurrent fevers and pulmonary infections since early childhood and, at age 7, had disseminated varicella with bilateral pneumonitis. A female sibling, age 1, died during this period of time with varicella pneumonia. Two years later, an immunological evaluation showed severe deficits in cellular immunity with skin anergy and very low or poor in vitro lymphocyte proliferative responses to mitogens, allogeneic cells and specific antigens. Quantitation of peripheral T-cells by spontaneous rosette formation was also low—40–45% (normal 61%). On the other hand, B-cell immunity seemed to be completely normal. Serum immunoglobulins and the immunoglobulin receptors on peripheral lymphocytes were normal. The patient produced specific antibodies upon antigen challenge (immunization) and after natural infection. Following transfer factor therapy, conversion of skin reactivity and clinical improvement occurred. No changes were seen in in vitro lymphocyte function with transfer factor therapy. Immunologic reconstitution persisted for 6 mo, after which the patient responded again to the administration of transfer factor. Although this patient has several characteristics in common with Nezelof's syndrome, the patient described in this report appears to represent a distinct clinical entity of primary isolated T-cell deficiency and normal B-cell immunity. The normal B-cell immune system, and the clinical and immunological response to transfer factor therapy, differentiates our patient from the syndrome of thymic dysplasia with immunoglobulin synthesis (Nezelof's syndrome).     

Evaluation of IgM,IgG, IgA,IgD, and IgE secretion by human peripheral blood lymphocytes in cultures stimulated with pokeweed mitogen and Staphylococcus aureus Cowan I

Sheep erythrocytes coated with staphylococcal protein A were used as target cells in a reverse hemolytic plaque assay. Monospecific antisera to human immunoglobulins G, M, A, D, and E were used to detect each of these classes in cultures of human peripheral blood mononuclear cells stimulated with the polyclonal B-cell activators pokeweed mitogen and Staphylococcus aureus (Cowan I). Both mitogens induced substantial increases in the numbers of cells actively secreting immunoglobulins; in time-course experiments, the peak response was seen on Day 5. The numbers of cell secreting IgM, IgE, and IgD were usually higher in cultures stimulated with S. aureus than with pokeweed mitogen.     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

Nature of T-cells resident in spleens of thymectomized,lethally irradiated,bone marrow-reconstituted mice

Adult thymectomized, lethally irradiated, bone marrow-reconstituted (ATxXB) mice that had been weakly primed with SRBC or HRBC between thymectomy and irradiation were shown to retain antigen-specific immunological memories for at least 1–5 months after bone marrow reconstitution. This could be shown by anamnestic antibody response in vivo as well as by proliferative response of the spleen cells to the test antigens in vitro. Spleen cells taken from ATxXB mice showed a reduced but significant proliferative response to nonspecific T-cell mitogens, in particular to Con A, in vitro. Treatment of the donor bone marrow cells used for reconstitution of ATxXB mice with anti-Thy 1.2 sera + C′ did not affect the generation of immunological memories nor the magnitude of the proliferative response of spleen cells to nonspecific T-cell mitogens in vitro, indicating that the cells responsible for such functions were host derived. Finally, the antibody-forming capacity of spleen cells derived from SRBC-primed ATxXB mice to the test antigen in vitro was completely abrogated by exposure to 450 R, whereas the helper function of the same cell suspension remained unaffected even after exposure to 1000 R. Implication of these findings on the nature of T cells resident in spleens of ATxXB mice was discussed.     

Studies of murine lymphocytes and alloantigens: I. Ly-6 mitogen and MLR responses

Antisera to the mouse lymphocyte surface alloantigens Ly-6.1 and Ly-6.2 were used to further study the functional distribution of these antigens. After selective depletion with antiserum + rabbit complement (RC), lymph node or spleen cells from Ly-6 congenic (C3H and C3H.B6-Ly-6^b) and noncongenic strains of mice were tested for: (a) their proliferative responses to T- and B-cell mitogens; and (b) their proliferative responses to alloantigens, or ability to stimulate in the MLR. Lymphoid cells required in the proliferative responses to the mitogens leucoagglutinin, concanavalin A (Con A), lipopolysaccharide (LPS), and pokeweed mitogen (PWM) were Ly-6⁺. Lymph node responder cells in the mixed lymphocyte reaction (MLR) were also Ly-6⁺, whereas spleen stimulator cells were Ly-6^?. Treatment of lymph node cells with anti-Ly-6 sera in the absence of RC had no specific blocking effect on the response to any of these mitogens. The studies indicate that the Ly-6 antigen is a potentially valuable marker for distinguishing between functionally distinct Ly-1⁺ T-cell subsets.     

Role of monocytes in pokeweed mitogen-induced differentiation of human peripheral blood lymphocytes

Separate stimulation (“pulsing”) method of different cell populations with pokeweed mitogen (PWM) was used to study the regulatory role of monocytes in the PWM-induced plaque-forming cell response of human peripheral blood lymphocytes. T cells, B cells, and monocytes were separated, pulse-stimulated with PWM, extensively washed, and cocultured with unstimulated cell populations without additional PWM. Pulse-stimulated T cells helped unstimulated B cells to differentiate into immunoglobulin-secreting cells. This generation of helper T cells by PWM-pulsing was enhanced by monocytes in the presence of free PWM, as well as by PWM-pulsed monocytes in the absence of free PWM. A coculture of pulse-stimulated B cells and unstimulated T cells produced more substantial B-cell differentiation than the coculture of stimulated T cells and unstimulated B cells. Further enhancement of the latter response was obtained when B cells were pulse-stimulated in the presence of monocytes. However, pulse-stimulated B cells did not differentiate in the absence of T cells, and monocytes were unable to replace this T-cell function. It appears that there are several pathways by which PWM induces B-cell differentiation and in each, monocytes play an enhancing role.     

A series of murine interleukin molecules which stimulate both murine and human lymphocytes: I. Production by Phytolacca americana (pokeweed) lectin 2 (Pa-2)-stimulated thymus and thymus-derived cells

Cell-free supernatant fluid, from cultures of Phytolacca americana (pokeweed) lectin 2 (Pa-2)-pulsed murine spleen or thymus cells, contains factors which induce cultured lymphocytes to differentiate into IgM-secreting cells (assayed by a reverse plaque technique) and to proliferate (measured by the incorporation of tritiated thymidine) without the addition of mitogen. The factors in this supernatant fluid responsible for these activities have been designated as lymphocyte stimulating factors (LSF). LSF showed no genetic restrictions related to the major histocompatability complex; LSF made in one strain of mice worked in other strains. Indeed, LSF is not restricted by species barriers; human peripheral blood mononuclear cells were also stimulated by murine LSF to proliferate and differentiate into immunoglobulin-secreting cells without further addition of antigen or mitogen. Maximum production of LSF was achieved within 12 hr of culture and was independent of cell division. In contrast to TRF, no further production of LSF was detectable after 24 hr of culture. Unlike T-cell growth factor, this material stimulated increased mitosis of thymic, T, and B lymphocytes without the addition of mitogen or antigen. LSF also stimulated polyclonal B-cell differentiation into IgM-secreting cells. Maximal numbers of immunoglobulin-secreting cells were generated when LSF was added at the initiation of the culture. Indeed, unlike TRF, LSF needed to be present only during the first 6 hr of culture to achieve maximum stimulation, and did not require the presence of antigen. The production of LSF by a T-cell population in the spleen was shown by two independent methods. Spleen cells treated with anti-Thy 1 plus complement failed to produce detectable levels of LSF. On the other hand, purified populations of surface immunoglobulin-negative spleen cells produced LSF. Furthermore, the subset of thymocytes responsible for LSF production was the small population (approximately 10%) of cells in the thymus, which are not agglutinated by peanut agglutinin.     

Progressive immune dysfunction in cats experimentally infected with feline immunodeficiency virus.

Within 6 months of infection with the Petaluma isolate of feline immunodeficiency virus, specific-pathogen-free domestic cats exhibited a decrease in the percentage and number of circulating CD4+ lymphocytes and in the CD4+/CD8+ T-cell ratio, along with a marginally significant depression of pokeweed mitogen-induced lymphocyte proliferation in vitro. There was no loss of responsiveness to concanavalin A during this stage, and the cats were capable of mounting a satisfactory antibody response to a T-dependent, synthetic polypeptide immunogen. The pokeweed mitogen response deficit became clearly demonstrable by 11 to 12 months postinfection. A decline in the lymphocyte proliferative response to concanavalin A and a diminished ability to mount an in vivo antibody response to the T-dependent immunogen evolved by 25 to 44 months postinfection. Virus infection did not affect the ability of cats to mount an antibody response to a T-independent synthetic polypeptide immunogen. These data indicate that feline immunodeficiency virus produces a slowly progressive deterioration of T-cell function but does not affect the ability of B cells to recognize and respond to a T-independent antigenic stimulus.     

Induction of human immunoglobulin secretion. II. T lymphocyte dependency and radiosensitivity of T-cell help for induction of B-cell differentiation by Staphylococcus aureus strain Cowan I

The induction of peripheral blood B lymphocytes to mature to immunoglobulin-secreting cells (ISC) when stimulated by Staphylococcus aureus strain Cowan I was found to be T helper cell-dependent (J. Immunol.127, 1044, 1981). The nature of T help was studied in B- and T-cell separation and reconstitution experiments. T helper cells for Cowan I were very radiosensitive (D₃₇ < 500 rad) in comparison to helpers for pokeweed mitogen (PWM) (D₃₇ > 2000 rad). PWM synergized with Cowan I in induction of ISC, and helper T cells for dual stimulation were also radioresistant. The ratio of T to B cells was found to be critical in judging reactivity of donors for both PWM and Cowan I. T cells stimulated with PWM, but not Cowan I, produced T cell-replacing factors essential for Cowan I-induced maturation of B cells. Irradiation of T cells prior to PWM stimulation increased the level of such helper factors. Poor responders to Cowan I, as judged by mononuclear cell cultures, had apparently few helpers for the bacterial stimulant, compared to high responders. Cowan I helper T-cell activity did not appear to be due to protein A leaking from the bacteria and stimulating T cells. In all these experiments, induction of ISC by Cowan I was completely dependent on T cells or factor, providing a good model for investigation of B-cell differentiation regulated by a unique subset of radiosensitive T helper cells.     

The effect of ceramic fibers on the immune system

Male Sprague-Dawley rats were treated by intratracheal instillation with 1 mg/animal of refractory ceramic fibers. Intratracheal exposure to ceramic fibers led to significant changes of immune response. Results of proliferative activity of spleen lymphocytes showed significantly decreased proliferative activity of T-cells in response to mitogens phytohemagglutinin and concanavalin A in animals given ceramic fibers in comparison with control rats. Similarly, T-dependent B-cell response to pokeweed mitogen was significantly suppressed. Spontaneous proliferative activity of lymphocytes in non-stimulated spleen cell cultures did not differ in exposed and control rats. No significant changes were found among groups in percentage of phagocytic blood polymorphonuclear leukocytes and percentage of cells with respiratory burst.     

Activation, proliferation, and differentiation of circulating B cells in autoimmune thyroid disease

Several studies of thyroid autoantibody production in vitro have been reported with the use of pokeweed mitogen, but the conclusions that have resulted regarding the immunoregulation of B cell function in thyroid disease are difficult to interpret due to the relatively nonphysiologic nature of pokeweed mitogen stimulation. We have therefore examined the responses of circulating B cells in Graves' disease and Hashimoto's thyroiditis by using a combination of lymphokines and other stimuli that act at various stages of the B cell cycle. In patients with autoimmune thyroid disease, nonspecific B cell proliferation and differentiation into IgG-secreting cells were both normal. However, a previously unsuspected heterogeneity among patients was found in their ability to produce autoantibodies in vitro. B cells of certain patients produced maximal autoantibody in response to pokeweed mitogen, some in response to Staphylococcus aureus Cowan strain I, and some in response to the lymphokines contained in the supernatants of stimulated T cell cultures. There was no correlation between serum autoantibody levels and those achieved in vitro. Attempts to stimulate antibody production by autoantigen (thyroglobulin) were unsuccessful, even when B cells were cultured with purified autologous OKT4+ T cells to avoid potential suppressor effects in the OKT8+ population. However, OKT4+ T cells enhanced pokeweed mitogen-driven autoantibody production. Our results show that several different functional stages of B cells exist in the circulation of patients with autoimmune thyroid disease, and that circulating B cells from such patients do not manifest a uniform response to B cell stimulators. This is presumably the result of differences in migration of circulating B cells and in their level of activation at the major sites of autoantibody production, such as the thyroid gland itself. In the light of these findings, caution is required in interpreting the results obtained from studies of circulating B cells as a means of elucidating the pathophysiology of autoimmune thyroid disease.     

Isolation and characterization of membrane receptors for pokeweed mitogens from mouse lymphocytes.

The major glycoproteins that bind pokeweek B-cell mitogen (Pa-1) and pokeweed T-cell mitogen (Pa-2) were isolated and identified from bone-marrow-derived lymphocytes (B-cells) and thymus-derived lymphocytes (T-cells) of C3H/He mice. The surfaces of the cells were 125I-labelled by using the enzyme lactoperoxidase, and the plasma membranes were isolated from the 125I-labelled cells. These membranes were solubilized with Triton X-100 and subjected to affinity chromatography on the affinity adsorbent prepared by coupling mitogen Pa-1 or Pa-2 to activated Sepharose 4B. The glycoproteins specifically eluted with di-N-acetylchitobiose from the affinity adsorbents were analysed according to their mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate. These glycoproteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes, but they were not detected in the eluate from the T-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were found to be major receptor sites for the pokeweed mitogens on both B-cells and T-cells. However, mitogen Pa-1 (B-cell) has a stronger affinity to Ia antigens than does mitogen Pa-2 (T-cell).     

In vitro proliferation of blood,spleen and thymus leukocytes from the turtle Mauremys caspica

Cell-mediated immune responses is commonly evaluated by cell proliferation assays. Mitogens are known to induce a vigorous proliferative response in lymphoid cells from mammals but relatively fewer studies have investigated mitogen-mediated lymphoproliferation in non-mammalian animals. In the present work, we incubated spleen, thymus and blood leukocytes with phytohaemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS) and pokeweed mitogen (PWM), by different times of incubation (96 and 120 h) and at different concentrations. Our results show that the optimal mitogen concentrations inducing proliferation on leukocytes from Mauremys caspica were 20 microg/ml PHA, 1 microg/ml Con A, 12.5 microg/ml LPS and 1/150 dilution PWM. The optimal time of incubation was dependent on the type of leukocytes (peripheral blood leukocytes, splenic leukocytes or thymic cells) and the mitogen utilized.     

Direct evidence for the response of B and T cells to pokeweed mitogen

Chicken spleen cells containing chromosomally marked thymus derived (T) and bursa-derived (B₂) cells were evaluated for their ability to respond to pokeweed mitogen (PWM), concanavalin A (Con A), and to anti-immunoglobulin serum during a 4-day culture period. The results indicate that soluble PWM induces a proliferative response of B₂ cells in addition to a predominant T cell response. The PWM-induced B₂ cell proliferative response was clearly detected only at 4 days after culture initiation. Soluble Con A did not induce detectable proliferation of B₂ cells and stimulated T cells exclusively. In contrast, anti-immunoglobulin serum was a specific stimulant for B₂ cells under the culture conditions used.     

The kinetics of suppression of proliferation of oxidized murine lymphoid cells by sodium borohydride reduction.

Murine splenic lymphoid cells are stimulated to proliferate following mild oxidation with sodium periodate. To assess the class of cells responding, we used periodate treatment alone or in association with concanavalin A, a T-cell mitogen, or lipopolysaccharide (LPS), primarily a B-cell mitogen. Brief periodate treatment followed by culturing with concanavalin A gave no additive proliferative response to that seen using concanavalin A alone, while culturing periodate-treated cells with LPS gave approximately an additive response. Furthermore, periodate failed to stimulate spleen cells from neonatally thymectomized mice while LPS produced significant stimulation of proliferation, suggesting that periodate is stimulating a class of T lymphoid cells or a subpopulation of T cells. Studies were performed to determine an optimal concentration of borohydride which would suppress proliferation in lymphoid cells initially oxidized with periodate. It was observed that 2 mM borohydride would suppress proliferation of oxidized cells yet permit a normal response of these cells to another T-cell mitogen, concanavalin A. Higher concentrations of borohydride, from 3 to 5 mM, would also suppress proliferation of oxidized cells but would interfere with the ability of these cells to respond to concanavalin A, perhaps due to cell damage. Studies were performed to determine when it was possible to suppress periodate-induced mitogenesis by reducing with borohydride at various times after the initial oxidation. It was observed that 2 mM borohydride treatment could suppress stimulation through 8 hr after the original periodate oxidation and that from 12 hr through 20 hr after the initial periodate oxidation, borohydride was incapable of inhibiting proliferation. Additional studies demonstrate that optimal mitogenesis induced by periodate or concanavalin A is contingent upon a serum factor.     

Mitogen induction of deoxyuridine triphosphatase activity in human T and B lymphocytes

Deoxyuridine triphosphatase (dUTPase; deoxyuridine diphosphohydrolase; EC 3.6.1.23) activity during mitogen stimulation was investigated in human T-cell and B-cell enriched mononuclear leucocyte fractions as well as in a mixed lymphocyte population. The dUTPase activity was very low in the resting peripheral blood lymphocytes. A remarkable enhancement of enzymatic activity was observed when cells were stimulated with different mitogens; T-cells and non-separated lymphocytes with phytohaemagglutinin, and the B-cell enriched fraction with pokeweed mitogen. There was a positive correlation between dUTPase activity and the enhancement of macromolecule synthesis (protein and RNA). In particular, a highly significant correlation was observed between dUTPase activity and DNA synthesis in the three human lymphocyte populations studied. This supports the view that the enzyme dUTPase may have a significant role in cellular proliferation. The physiological role of the enzyme is discussed.     

Antigen-induced in vitro lymphocyte blastogenesis in the rabbit: a T-cell response.

In vitro lymphocyte stimulation of sensitized rabbit lymphocytes to specific antigen (ovalbumin) was found to depend on thymic-dependent lymphocytes. This conclusion is based on an enhanced response upon enrichment with T lymphocytes by passage of lymphocytes through nylon wool, and on the elimination of the response after treatment of lymphocytes with complement and an antiserum to rabbit thymus cells prepared in a goat. Specificity of the antiserum was demonstrated by elimination of in vitro T-cell function and retention of in vitro B-cell functions.     

Inhibition of the lymphocyte blastogenic response to antigen by serum-free culture supernatants of leukemic B cells

Suppressive factors were detected in culture supernatants of the guinea pig B-cell L₂C leukemia. Dialyzed culture supernatants (DCS) inhibited the blastogenic response of sensitized lymph node cells (LNC) to a wide dose range of the sensitizing antigen (ovalbumin or PPD) but failed to inhibit the proliferative response to PHA or Con A. In addition, DCS inhibited the response of blast cells to preformed T-cell growth factor (TCGF). The inhibitor(s) in DCS was noncytotoxic, heat stable (30 min at 80 °C), resistant to treatment with trypsin, and exerted its effect subsequent to activation of sensitized LNC by antigen. Washing of DCS-treated cells restored normal reactivity to a subsequent antigen challenge. The target cell for the inhibitor may be cells responding to amplification signals produced by activated T cells. KCl (3 M) extracts of L₂C cells behaved like DCS in inhibiting only antigen responses. Both undialyzed culture supernatants (UCS) and leukemic sera inhibited mitogen, allogeneic, and antigen-stimulated proliferative responses by greater than 80%. These soluble factors may participate in the depression of cell-mediated immunity associated with lymphoid malignancies.     

Human helper-T-cell function does not require T4 antigen expression

The relationship between immunoregulatory T-cell function and the expression of T-cell subset-specific differentiation antigens was examined using a phenotypically anomalous human T-cell line (TCL), termed H-1. H-1 cells were found to express T11, extremely high levels of T3, but no T4 nor T8 antigen. Despite their lack of T4 antigen expression, H-1 cells could be activated by coculture with pokeweed mitogen (PWM), anti-T3 antibody, or autologous B cells to provide potent help for B-cell differentiation into plaque-forming cells (PFC). In contrast, H-1 cells did not suppress the PFC response triggered by PWM-activated T4+ cells. These results demonstrate that the expression of the T-cell subclass-specific differentiation antigen, T4, is not required for a T cell to become activated and to implement the program for helper function. In addition, enhanced expression of T3 on the T4-, T8-, H-1 cell surface may reflect a compensatory upregulation of the T3/Ti receptor complex on T cells which are deficient in these nonpolymorphic associative recognition structures.