Phytohemagglutinin Response Research Articles

Response of sheep lymphocytes to PHA: quantitation by nuclear volume measurement and cell counts.

Phytohemagglutinin response of peripheral blood lymphocytes (PBL) of sheep was studied. Assessment of proliferative response was performed by determination of nuclear volumes and cell counts in cultures from 14 sheep and by incorporation of tritiated thymidine in cultures in four additional sheep. PBL of sheep were found to transform and proliferate with PHA similarly to human peripheral blood lymphocytes with minor differences. Quantitation of the proliferative response by determining the cell count and nuclear volumes provided more information on cell kinetics in culture than the commonly used isotope-labeled thymidine incorporation method.

Age-dependent changes in human peripheral blood B cells and T-cell subsets: Correlation with mitogen responsiveness

Lymphocyte and T-cell subpopulations were quantitated in 64 healthy, ambulatory adults, 33 women and 31 men, ranging in age from 22 to 83. Phytohemagglutinin (PHA) responsiveness was assessed simultaneously in 19 of these subjects and statistically correlated with T-cell subpopulations. B cells declined significantly in proportions and numbers with age ( P < .0005) and this significance was maintained when men and women were separately considered. There was no significant change with age for males, females, or for the group as a whole when absolute and relative numbers of T G cells were statistically evaluated by regression analysis. An inverse correlation of percentages of T M cells with age was shown by regression analysis both in the entire sample ( P < 0.00001) and when the sexes were separately considered ( P < 0.01 in males; P < 0.005 in females). When absolute numbers of T M cells were evaluated by regression analysis significant decreases were found in the entire group ( P < 0.005). However, this resulted primarily from changes in the male cohort as the P value for females was not significant. No correlation was found between PHA responsiveness and either the relative or absolute number of T G or T M cells.

Mixed lymphocyte culture, phytohemagglutinin stimulation, and matching grade: clinical relevance in renal transplantation from living related donors.

Mixed lymphocyte cultures (MLC), including phytohemagglutinin (PHA) stimulation and HLA-A and HLA-B loci typing of donor and recipient, were performed on 70 renal allograft recipients and relevant family members. Graft survival was correlated retrospectively with matchnd PHA response index (PHARI), in order to assess the clinical relevance of each variable singly or jointly. Overall graft survival was significantly associated with SI (log10) (P less than 0.001) and matching grade (P = 0.027). No significant association with either PHARI or II was detected (P greater than 0.10). In addition, the product of the last two indices--the lymphocyte response index (LRI)--was not found to be related to graft survival (P greater than 0.10). Survival of grafts with one-haplotype identity was significantly associated with SI (P = 0.002). Survival in this group was not found to be related to II, PHARI, or LRI, considered either alone or jointly (P greater than 0.10). Grafts with two-allele-identical grafts, whereas PHARI did not differ. SI and matching grade were related significantly to graft survival and appeared to be the most important variables.

The role of insulin in the response of murine T lymphocytes to mitogenic stimulation in vitro.

The ability of insulin to influence the responsiveness of murine T lymphocytes in a culture system containing a serum substitute was documented. The presence of insulin was found to enhance the concanavalin A (Con A) reactivity of the lymphocytes. Once the cells were activated by a short-term exposure to Con A, insulin was capable of replacing Con A for the continued stimulation of the cells. This was true both for lymphocyte proliferation and for the generation of nonspecific cytotoxic T lymphocytes. The presence or absence of insulin was not found to influence the phytohemagglutinin responsiveness of the T lymphocytes. Possible reasons for the observed results are discussed in relation to a proposed model for lymphocyte activation.

Replication of rubella virus in human mononuclear blood cells.

Rubella virus was capable of replicating in both unstimulated and phytohemagglutinin-stimulated cultures of human mononuclear blood cells. Monocyte-derived macrophages were the main cell type responsible for viral replication. The susceptibility of macrophages increased during cultivation. Phytohemagglutinin-stimulated lymphocytes were able to support replication to a limited degree. No viral replication was detected in unstimulated lymphocytes. Both stimulation and viral replication in phytohemagglutinin-treated lymphocyte cultures were enhanced by the addition of murine macrophages. Human leukocyte interferon depressed the production of virus in these combined cultures. The finding that rubella virus is able to replicate in human lymphocytes as well as in macrophages may contribute to understanding the mechanisms of the suppressive effect of the virus on in vitro lymphocyte phytohemagglutinin responsiveness and in vivo immune functions.

Lynestrenol: a progesteronelike agent with immunostimulatory properties.

Lynestrenol, a synthetic progesteronelike drug, was assessed for its influence upon human leukocyte tests in vitro and in vivo. Lynestrenol markedly enhanced the lymphocyte response to phytohemagglutinin, the mixed lymphocyte culture, the active T-rosette test, the autologous red cell rosettes, and the number of nonadherent cells in the leukocyte adherence inhibition test. All these results indicate a stimulatory effect upon human T-cell function. Lynestrenol also increased monocyte phagocytosis. Lynestrenol delayed the appearance of malignant tumors in hamsters. Finally, lynestrenol, given to lung cancer patients, increased their T-cell functions as evaluated by skin tests. T-cell rosettes, and phytohemagglutinin response. Lynestrenol does possess immunostimulatory properties.

Non-T, non-B acute lymphocytic leukemia cells enhance the mitogen induced proliferation of normal lymphocytes

Peripheral blood and bone marrow mononuclear cells (MNC) from patients with untreated non-T, non-B acute lymphocytic leukemia (ALL) were assessed for capacity to respond to the mitogens phytohemagglutinin (PHA), pokeweed (PWM) and staphylococcal enterotoxin A (SEA). At low mitogen concentrations considerable reactivity was observed to PHA and SEA, but not to PWM by patient MNC including specimens comprised of at least 90% leukemic cells. The responding cells were identified in cell separation experiments and by surface marker analysis as residual normal T-cells. The non-T, non-B leukemic cells enhanced the mitogen induced proliferation of responding lymphocytes as determined by co-culture experiments in which 5 × 10 3 or 10 4 MNC from normal donors were cultured with feeder layers of 3 × 10 5 mitomycin C treated MNC from autologous or unrelated normal donors or from ALL patients. In contrast to MNC feeder layers from normal donors, feeder layers of non-T, non-B leukemic cells enhanced normal lymphocyte reactivity to PHA and SEA but not to PWM as determined by 3H-thymidine uptake and cell survival. The potentiating effect of non-T, non-B leukemic cells was not mediated entirely by an allogeneic interaction as shown by the demonstration that frozen-thawed non-T, non-B leukemic cells enhanced the mitogen response of autologous MNC obtained during remission. Mitomycin treated feeder layers of leukemia cells from patients with acute myelogenous leukemia as well as bone marrow MNC from a normal donor or from lymphoma patients without bone marrow involvement failed to enhance PHA and SEA reactivity of normal lymphocytes. In addition, leukemia cells from patients with T-cell ALL did not potentiate normal lymphocyte reactivity in contrast to non-T, non-B ALL cells, providing a functional distinction between these two subtypes of ALL.

Effects of washing on phytohemagglutinin responsiveness of lymphocytes from irradiated patients.

The peripheral lymphocytes from irradiated patients generally have a reduced capability to respond to phytohemagglutinin (PHA). Whether a relationship exists between PHA-responsiveness and chromosome aberration frequencies was examined by washing the lymphocytes with culture medium. The results indicate that the defect in lymphocyte activation in patients receiving radiation therapy was caused by some reversible changes in the lymphocyte membrane directly associated with radiation exposure rather than by a radiation induced suppressor substance secondarily acting on the lymphocyte membrane.

Effects of age on cellular immune responses in BALB/cJ mice: Increase in antibody-dependent T lymphocyte mediated cytotoxicity

Two in vitro cell-mediated immune (CMI) responses of splenic lymphocytes of BALB/cJ mice of various ages (1.25, 2.5, 4, 10.5 and 30 months) were examined. These were the antibody-dependent lymphocyte-mediated cytotoxicity (ADLMC) responses, mediated by the K cell subclass of T lymphocytes, and phytohemagglutinin (PHA) induced mitogenesis, mediated by the theta (θ) positive T lymphocytes. ADLMC responses increased with age, culminated at 10.5 months, then slowly declined. Lymphocytes of young mice (2.5 or 4 months of age) were significantly more responsive to PHA than lymphocytes of mature (10.5 months) or aged adults (30 months) ( p < 0.01), indicating progressive suppression of this type of CMI response with age, as observed previously. The difference in time of maturation and extent of suppression of ADLMC and PHA responses was not influenced by changes in numbers of specific effector cells, since percentages of these did not change with age (as measured by immunofluorescence and rosetting techniques). The increase with age of ADLMC responses may be due to deregulation of earlier suppressor activity which allows the K cell to be fully responsive, but other mechanisms may be involved.

Predicting Fatal Sepsis in Burn Patients

The high morbidity after severe thermal insult is believed to be related partially to a resultant decrease in immunocompetence. We tested the ability of phytohemagglutinin (PHA) and Concanavalin (Con A) to stimulate lymphocyte transformation in 17 patients with moderate to severe thermal injury (greater than 25% BSA). The patients acted as their own controls and the per cent change in their mitogen response was measured over time. Eight acutely burned patients who subsequently developed severe sepsis (Group I) had decreased ability (mean, 12% of normal) to proliferate in response to PHA, and six of these died of severe sepsis. The depressed response appeared 4 to 7 days postinjury and predated clinical evidence of sepsis by 2 to 4 days. Cells from four patients who had mild infectious complications (Group II) demonstrated greatly augmented mitogen responses (mean + 243%) approximately 7 to 10 days postinjury. Five burn patients whose clinical course was sepsis free (Group III) exhibited only minimal changes in their mitogen responses (mean +30%). Although the Con A responses of the patients' cells corresponded less to their pathology, Group I patients whose cells exhibited depressed PHA responsiveness also had diminished Con A responses. Group II patients' cells also showed increases in Con A-induced stimulation. Group III patients, who had only slightly augmented PHA responses, had minimal decreases of the Con A-induced lymphocyte transformation. Many severely burned patients develop septicemia as a result of their large wound surfaces. The appearance of decreases in mitogen-induced proliferation, however, appears to characterize those patients who will be unable to handle the septic challenge.

Suppression of Phytohemagglutinin Response by Fungi from a “Leukemia” House

Fungal isolates from the house of a husband and wife who both developed acute myelomonocytic leukemia were assayed for effects on the in vivo response to phytohemagglutinin in guinea pigs. Skin responses to intradermal phytohemagglutinin were measured following injections of sterile fungal extracts. Isolates of Penicillium canescens, Curvularia, Fusarium sambucinum, Fusarium equiseti and Trichoderma koningii from the leukemia-associated house depressed the responses to phytohemagglutinin, but none of the fungal isolates obtained from a nearby control house depressed responses to phytohemagglutinin. Such environmental agents may contribute to development of malignancy by suppression of immune responses.

The lymphoproliferative response during human experimental gingivitis

The lymphocyte blastogenic responses to antigen preparations of bacteria from dental microbial plaque and to other non‐oral stimulants were longitudinally assayed in eitht adult subjects using the experimental gingivitis model. During the 28 days of the experimental period, these subjects abstained from all oral hygiene procedures which let to accumulatio of increased amounts of microbial dental plaque and subsequent gingivitis. In general, all subjects demonstrated a significant increase in the lymphoproliferative response to all doses of phytohemagglutinin (PHA) during the experimental period. The subjects were divided into 2 groups based on rate of plaque accumulation. Slow plaque formers exhibited a gradual linear increase in blastogenic response to bacterial antigen preparations throughout the experimental period. In the group characterixed by rapid accumulation of plaque, a marked early increase in blastogenic responses to antigens of Actinomyces viscosus, Actinomyces naeslundii, Bacteroides melaninogenicus, and Treponema denticola was observed. These same subjects, however, demonstrated a reduction in lymphocyte response to the above antigens during the later phase of the experimental period, when gingivitis was most severe. This decrease, in light of the similtaneous increase in PHA response, suggests the existence of a homeostatic mechanism to control the immune response during episodes of increased antigen load.

Sensitivity of lymphocytes to prostaglandin E2 increases in subjects over age 70.

We examined the sensitivity of lymphocytes from different age groups to inhibition by prostaglandin E2. Phytohemagglutinin-stimulated cultures of peripheral blood mononuclear cells from 12 healthy subjects over the age of 70 were much more sensitive to inhibition by exogenously added prostaglandin E2 than were cells from 17 young controls (ID50 congruent to 10 nM for the subjects over 70 vs. greater than 3 micronM for the young controls). The more senstivie lymphocytes from a subject over 70 were to prostaglandin E2, the lower was his or her response to phytohemagglutinin (r = 0.75, P less than 0.01). The mean responses to phytohemagglutinin of the peripheral blood mononuclear cells from the subjects over 70 were significantly depressed compared to the young controls. Addition of indomethacin, a prostaglandin synthetase inhibitor, to the cultures resulted in an increase in [3H]thymidine incorporation of 140 +/- 16% in the cells of the subjects over 70 vs. a 36 +/- 3% increase in the young controls (mean +/- SEM, P less than 0.001). The mean phytohemagglutinin response of the subjects over 70 was 40% of the control response without indomethacin. With addition of indomethacin the response of subjects over 70 rose to 72% of control. Thus, increased sinsitivity to prostaglandin E2 appears to be responsible in part for the depressed mitogen response of peripheral blood mononuclear cells from healthy subjects over 70.

Concurrent Responses of Peripheral Blood and Splenic Mononuclear Cells to Antigenic and Mitogenic Stimulation in Human Hepatosplenic Schistosomiasis

Lymphocyte reactivity and its control was assessed in human hepatosplenic schistosomiasis, a disease in which host hypersensitivity may contribute to long-term morbidity. Antigen- and mitogen-induced incorporation of [3H]thymidine was evaluated in peripheral blood and splenic mononuclear cells of 15 patients at the time of splenectomy. The response to phytohemagglutinin (PHA) was depressed in the peripheral blood of 42% and in the spleen cells of 70% of these patients. Significant stimulation was noted, however, upon culture of blood with soluble schistosome egg antigen (SEA) in 80% and of spleen cells in 100% of the patients whose respective responses to PHA were depressed. The contribution of adherent cells to the overall response of mononuclear cells was evaluated by depletion techniques. A significant and specific decrease in the response of the resulting thymus-derived (T) lymphocyte-enriched splenic mononuclear cells to SEA was noted. These studies suggest preferential preservation of the response of circulating and splenic lymphocytes to SEA despite impairment of PHA reactivity in human hepatosplenic schistosomiasis, which may be causally related to the advanced disease of this group of patients. Moreover, activity of helper adherent cells was consistently restricted to the splenic T-lymphocyte response induced by the specific antigen SEA.

Immunosuppressive activity of BCG: effects of adjuvant disease, lymphocyte subpopulations, and homing of thoracic duct cells in rats

Administration of BCG by various dosage schedules suppressed adjuvant disease in rats. BCG administration produced an initial increase, followed by a depression, of the phytohemagglutinin response of purified blood lymphocytes. An increase in absolute and relative numbers of bursa-equivalent (B)-cells followed BCG administration, concurrent with a decrease in the phytohemagglutinin responsiveness. With adjuvant alone, there was a diminution in phytohemagglutinin response and an increase in number of B-cells; the latter occurred immediately after adjuvant injection and also when the generalized disease appeared. When both BCG and adjvant were present, parallel increases of phytohemagglutinin responsiveness and B-cell numbers resulted. The pattern of tissue localization of radioactively labeled thoracic duct cells from normal or BCG-treated donors given to normal, BCG-treated, adjuvant-injected, and BCG-treated + adjuvant-injected syngeneic recipients indicated significantly greater homing to the thymus and decreased localization to the bone marrow when BCG had been given to either donors or recipients. When labeled thymus cells were used, only the decreased bone marrow localization was noted. These observations suggest that the suppressive effect of BCG may be mediated through modification of the lymphocyte recirculation pattern, possibly resulting from alterations in lymphocyte recognition sites.

Suppressor cells and immunodeficiency in (NZB × NZW)F1 hybrid mice

Old (15-20 month) male (NZB x NZW)F1 (B/W) mice have severely impaired spleen cell reactivity to phytohemagglutinin (PHA), a mitogen which stimulates mainly T lymphocytes. Spleen cells from old mice markedly suppressed the PHA response of splenocytes from young (3-4 month) B/W males. Similar suppressor activity was not present in the spleens of old mice of four nonautoimmune strains. The suppressor activity of old B/W spleen cells was mediated by a nonphagocytic, radioresistant, mononuclear leukocyte. Although this cell was eluted in the "T lymphocyte" fraction of nylon wool colums, it was not sensitive to treatment with anti-Thy-1 antiserum and complement. Suppressor activity was lost after 18 h incubation at 37 degrees C in tissue culture medium. Supernatants of these overnight cultures had no suppressive effect on fresh young B/W spleen cells. Old B/W spleen cells suppressed PHA reactivity more than concanavalin A or lipopolysaccharide reactivity. Kinetic studies demonstrated an increasing suppression with time over 72 h of culture. This study demonstrate that the severely impaired PHA reactivity of old B/W mice is mediated, at least in part, by active suppression.

Identity of mononuclear cells which compromise the resistance of trauma patients

The presence of aberrant activation of suppressor T lymphocytes and inhibitory monocytes (ø) was examined in the Ficoll-Hypaque-purified mononuclear cells from 18 thermally injured and 20 splenectomy patients. The cells were either depleted of T cells by the E-rosette method in the burn patients or of ø by filtration over Sephadex G-10 columns in the splenectomy patients. Undepleted and depleted cells were then examined for their phytohemagglutinin (PHA) response and ability to suppress the “one-way” mixed leukocyte response (MLR) of normal individuals. Undepleted cells from burn patients had depressed PHA responses and gained suppressive activity by 5 to 8 days after injury, suppressing the normal MLR an average of 57.5% compared with only 14.0% suppression by T-depleted cells ( P < 0.01). The PHA response of splenectomy patients' cells was markedly depressed to 22% of normal at 12 to 16 days after operation, compared to a supranormal (mean 113%) response after removal of ø by filtration ( P < 0.01). These data demonstrate the activation of suppressor T cells after thermal injury and an increase in inhibitory monocytes after splenectomy. Such regulatory cells may have a major role in decreasing the immunocompetence of these patients.

An Immunosuppressive Serum Factor in Widespread Cutaneous Dermatophytosis

A patient had a widespread dermatophytosis and a serum factor that specifically suppressed his T-cell rosette formation and the phytohemagglutinin responsiveness of his lymphocytes. The patient was also unable to mount a delayed hypersensitivity reaction to a standard anergy panel, although he did exhibit an immediate type hypersensitivity reaction to the trichophytin skin test antigen. Although many immunosuppressed patients succumb to severe bacterial and fungal infections, it is our contention that in certain cases the infectious process itself may generate a serum factor that is capable of inducing immunosuppression, thereby rendering the patient susceptible to further spread of the infection.

Effect of thymocyte-stimulating factor-containing supernatants on the surface antigens of murine thymocytes

Incubation of murine thymocytes in thymocyte-stimulating factor (TSF)-containing supernatants causes a four- to fivefold increase in the expression of the H-2 k and H-2 d antigens and a similar decrease in the expression of the TL antigen (in TL + strains) on the surface of these cells. Experiments with antisera directed toward the private H-2K and H-2D antigens showed that TSF-containing supernatants cause approximately the same increase in the expression of the H-2K and H-2D antigens of thymocytes of the d and b haplotypes. With thymocytes of the k haplotype, only an increase in the expression of the H-2D antigens takes place, while no significant increase was found for the H-2K antigens. TSF-containing supernatants cause no significant change in the expression of the following antigens on the surface of thymocytes: Thy-1.1, Thy-1.2, Ly-1.2, Ly-2.2, Ly-6.2, Th-B, Ia-1,2,3,7, and G IX. A factor similar to murine TSF, produced by human peripheral blood leukocytes, does not affect appreciably the expression of the H-2 antigens on the surface of murine thymocytes. The factor(s) causing the increased expression of the H-2 antigens on the surface of murine thymocytes appears to be produced by T lymphocytes. The factor(s) is eluted from a Sephadex G-100 column in at least two broad peaks with molecular weights of 300,000 and 90,000-25,000. Most of the activity enhancing the expression of the H-2 antigens is lost at pH 2, while most of it is maintained at pH 11.5 and at 56 °C. On the basis of these properties, it is concluded that the factor under study is probably different from the factor enhancing the phytohemagglutinin responsiveness of thymocytes.

ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY TESTS FOR IMMUNOSUPPRESSION MONITORING OF TRANSPLANT PATIENTS

The antibody-dependent cell-mediated cytotoxicity (ADCC) effector activity and phytohemagglutinin (PHA) response of kidney transplant patients were monitored by a new whole blood microassay that permitted performance of 20 tests using only 1 ml of blood. Eleven patients were followed on as many as 55 different occasions with these tests. The ADCC activity of dialysis patients was essentially the same (78% chromium release as compared with 83% in normal controls). However, patients with kidney transplants undergoing immunosuppression had approximately one-half the chromium release (40%). Daily fluctuation in the chromium release could not be used as a predictor of subsequent rejection. The PHA response was as much as 50-fold less in the immunosuppressed patients. Both values also tended to be slightly higher among patients who were doing well than those who were doing poorly. This may reflect the need for greater immunosuppression in the rejecting patients.