Attempt to restore lymphocytes with defective T-cell function by mixing with normal cells in vitro

Blood leukocytes from a patient with T-cell immunodeficiency failed to respond to stimulation with phytohemagglutinin (PHA) in vitro although ample lymphocytes were present. Leukocytes from this patient were mixed in various proportions with normal leukocytes, and the mixtures were stimulated with PHA and cultured for three days. There was no restoration of PHA responsiveness of the patient''s cells in the presence of normal cells. This would indicate that the defect in the patient''s cell population was not due to the absence of any facilitating factor or cooperative function that could be provided by normal T cells or other blood leukocytes. Since in thymic aplasia the patient''s own cells are rapidly restored to normal PHA responsiveness by an unknown facilitating factor after thymic transplantation, we suggest that this mixed-cell culture method might differentiate between patients who need a bone marrow transplant and those who will respond to a thymus transplant.     

Clinical studies on cell-mediated immunity in gestational trophoblastic disease: nonspecific cellular immunocompetence in patients with hydatid mole and trophoblastic neoplasia

The nonspecific cell-mediated immunocompetence of 51 patients with gestational trophoblastic disease (GTD), including 16 patients with hydatid mole (HM), 24 with nonmetastatic trophoblastic neoplasia (NTN) and 15 with metastatic TN (MTN), was studied with the use of both in vitro and in vivo parameters of cell-mediated immunity (CMI) such as lymphocyte blastogenic response to phytohemagglutinin (PHA), subpopulation constitution, and delayed cutaneous hypersensitivity responses to 2,4-dinitrochlorobenzene (DNCB) and to purified protein derivative of tuberculin (PPD). The pretreatment cell-mediated immune status of patients with HM developing no malignant sequelae was shown to be essentially similar, in terms of both in vitro PHA and in vivo DNCB reactivities, to that of normal women and of patients with benign gynecological diseases. In patients with TN, however, there was a significant depression in the blastogenic lymphocyte response to PHA before evacuation of the mole, which was persistently demonstrated after uterine evacuation and more marked throughout the course of disease in patients with MTN, than in those with NTN, with a tendency to return to normal in remission. Moreover, patients with TN had a significant depletion of T lymphocytes as determined by rosette-forming cell procedures before treatment, which was most evident in patients with MTN. Plasma from the MTN patients was also shown to have an inhibitory effect on PHA responsiveness of lymphocytes from normal women. There was an increased incidence of impaired reactivity to DNCB in patients with TN (higher in MTN than in NTN), compared with HM and benign diseases, while no such difference in incidence was observed in response to PPD. On the basis of these findings, a preliminary characterization of altered immunocompetence in patients with TN and its mechanism are discussed.     

Immunologic effects of interleukin 2 in primary immunodeficiency diseases

Five children with primary deficiencies of T cell function were studied to assess the effects of highly purified exogenous Interleukin 2 (IL 2) on their in vitro T cell responses. The lymphocytes from one child with Nezelof's T cell deficiency demonstrated absence of endogenous IL 2 production and improved proliferative responses to mitogen or alloantigen in the presence of exogenous IL 2. Moreover, during in vitro mixed lymphocyte culture in the presence of exogenous IL 2, his lymphocytes were able to develop into cytotoxic effector cells. A second child with Nezelof's syndrome demonstrated a different type of defect. The lymphocytes from this child had less impairment of endogenous IL 2 production. Although IL 2 increased the proliferation of his cells in response to PHA, similar augmentation was not seen after stimulation with OKT3 or alloantigen. In cell-mediated cytotoxicity assays, after mixed lymphocyte culture, natural killer-like activity was strongly boosted in the cultures that contained IL 2, but T cell-mediated cytotoxicity was not. The lymphocytes from three patients with severe combined immunodeficiency did not show improved proliferative responses in the presence of IL 2. Thus, only one of the five patients demonstrated the combination of defective endogenous IL 2 production, but preservation of the ability to respond appropriately to exogenous IL 2. This child may therefore have suffered from a T cell defect pathophysiologically similar to that seen in nude or aged mice.     

Effect of phytohemagglutinin on T-lymphocytes isolated from human blood]

T--enriched lymphocyte fraction from normal donors was isolated from heparinized peripheral blood on Ficoll-Urotrast gradients. The mitogenic response of T-lymphocytes in culture was 3 times as high as that of unseparated mononuclear cells. The intensity of blast transformation to PHA did not correlate with the increase of T-lymphocytes in culture. These data suggest that cells of not T-origin may be the limiting factor during mitogenic response of T-lymphocytes to PHA.     

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In a previously healthy 13-year-old girl with disseminated blastomycosis, immunodeficiency was considered because of lymphopenia and the slow response of her lung disease to therapy with amphotericin B. Cellular immunity was found to be profoundly impaired, with absent delayed cutaneous hypersensitivity to several common antigens, a decreased count of thymus-dependent lymphocytes in the peripheral blood and a greatly diminished in-vitro proliferative response of lymphocytes to phytohemagglutinin (PHA). Humoral immunity was intact. Two additional types of therapy were assessed: subcutaneous injection of transfer factor was associated with an unsustained increase in lymphocyte counts and a positive cutaneous response to PHA but no clinical change; parenteral alimentation to ensure an adequate energy intake was associated with rapid clinical improvement, the development of delayed hypersensitivity to four additional antigens, and the return of lymphocyte counts and proliferative response to normal. These findings suggest that increased energy intake rather than transfer factor therapy was responsible for the child''s recovery, and they emphasize the importance of adequate nutrition in the maintenance of intact cellular immunity.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

Immune function in aged mice. I. T-cell responsiveness using phytohaemagglutinin as a functional probe.

The loss of cell-mediated immunity with age was assessed by a detailed analysis of the in vitro response of murine lymphocytes to the well-defined probe of T-cell function, PHA (phytohaemagglutinin). The reduced mitogenic activity of lymphoid cells from old mice compared with young mice could not be explained in terms of a shift in kinetics of the responding cells. Removal of macrophages, which are known to exert a regulatory effect on T-cell function, failed to reverse the poor response of old lymphoid cells. Furthermore, no evidence was found for a role of soluble inhibitors released by either lymphocytes or macrophages in the decreased response of old cells. Not only were old cells less efficient in producing such factors, but in addition, they responded less well to them than did young cells. Taken together, these observations implied that the defect in PHA responsiveness of old cells is due to a disturbance in the T cells themselves rather than to any extracellular influences. The total number of T cells, assessed by labelling with anti-Thy-1 serum was comparable in old and young animals. Selective depletion of a subpopulation of PHA-reactive cells was excluded by direct quantitation of PHA-binding cells. Thus, 25% of small lymphocytes from the spleens of old mice bound ¹²⁵I-labelled PHA ([¹²⁵I]PHA) compared with 15% in the case of young mice. To show that the cells binding PHA were those reacting to it, a suicide technique was used. Spleen cells pretreated with [¹²⁵I]PHA failed to respond to subsequent challenge with the specific mitogen, but could mount a normal response to a control (B-cell), mitogen, LPS (lipopolysaccharide). When PHA cultures were carried out in the presence of colchicine, fewer cells from old mice were found to react to the mitogenic signal. In the absence of evidence for depletion of precursor cells, the conclusion was reached that the T-cell defect in old mice is more likely to be qualitative than quantitative, perhaps due to metabolic or structural abnormalities preventing lymphocyte transformation and/or proliferation.     

Immunologic reconstitution, autoimmunity, and T-cell immune deficiencies.

A 17-month-old female with combined immunodeficiency, a proliferative response to allogeneic cells in MLC, and circulating B lymphocytes (50%, all with surface immunoglobulin M) was treated with transfer factor (TF). After six doses of transfer factor, serum levels of IgG increased from 234 mg/dl to 1071 mg/dl and a lymphocyte proliferative response was detected to pokeweed mitogen. However, no change in T-cell function was observed. Clinically, she did well, but after the eighth dose of transfer factor, she developed the nephrotic syndrome. Renal biopsy suggested immune complex glomerulonephritis. Subsequently following treatment for Pneumocystis carinii, a 20-day cultured thymus was transplanted intraperitoneally. Two weeks after thymus transplant, the patient started to develop delayed skin reactivity and in vitro mitogen responsiveness. Six weeks posttransplant, the PHA and pokeweed mitogen responses were approximately 60% of normal, but the Concanavalin A response had disappeared. Subsequently she developed a Coombs positive hemolytic anemia, a cutaneous vasculitis, and thrombocytopenia. There was no evidence of GVH disease. Multiple autoimmune manifestations developed in association with transfer factor therapy and transplantation of cultured thymus tissue. T-Cell deficiencies are predisposed to autoimmune diseases because of imbalances of regulatory T-cell function. These imbalances of regulatory T-cell function may occur especially following partial immune reconstitution. The changes in B-cell function during transfer factor therapy may have resulted from enhanced T-cell-B-cell interaction. This immunopotentiation effect of transfer factor together with partial T-cell reconstitution may have led to B-cell hyperresponsiveness and autoimmune disease.     

Thymocyte-stimulating factor from peripheral blood cells.

Human peripheral blood leukocytes (HPBL) produce a thymocyte-stimulating factor (TSF-HPBL) that enhances the phytohemagglutinin (PHA) and concanavalin A (Con A) responsiveness of murine thymocytes. This activity is considerably specific for thymocytes. TSF-HPBL is not mitogenic by itself. Experiments with cell cultures pretreated with carbonyl iron particles showed that phagocytic cells are not involved in the production of mouse and rat TSF but are involved in the production of TSF-HPBL. The dose-response profile to PHA of murine thymocytes cultured in the presence of TSF-containing supernatants is similar to that of mature, immunocompetent spleen cells. TSF-HPBL, however, does not enhance the PHA responsiveness of murine thymocytes at low (<0.25 μg/microwell) concentrations of mitogen. TSF enhances the PHA and Con A responsiveness of the high-density subpopulations of thymocytes isolated on a Ficoll-Hypaque gradient. In general, the enhancing effect of TSF-HPBL on these subpopulations of thymocytes is smaller than that exerted by TSF. While supernatants containing TSF confer to thymocytes the ability to participate in a mixed lymphocyte reaction, this effect is not exerted by supernatants containing TSF-HPBL. A factor enhancing the PHA and Con A responsiveness of murine thymocytes is also produced by murine peripheral blood leukocytes (TSF-MPBL). This factor, similarly to TSF-HPBL, is produced by phagocytic cells and does not confer to murine thymocytes the ability to participate in a mixed lymphocyte reaction. Human T-cell lines do not enhance the PHA or Con A responsiveness of murine thymocytes. TSF-HPBL has a molecular weight of about 30,000 daltons, as measured by Sephadex filtration. Its half-time of inactivation as 56 °C is 162 ± 8 min.     

Epstein-Barr virus antibodies in patients with ataxia-telangiectasia and other immunodeficiency diseases

An unusual antibody response to the Epstein—Barr virus (EBV) has been noted in patients with ataxia—telangiectasia. Of a group of 16 such patients 8 were found to have antibodies in their serum to the EBV viral capsid antigen (VCA), and 4 of them also had antibodies to the EBV early antigen (EA); antibodies to the nuclear antigen (EBNA), however, were absent in 3 of the 8. The antibody pattern persisted for more than 2 years in the patients available for follow-up study. In comparison, of 24 patients with various other immunodeficiency syndromes 9 were found to have EBV-VCA antibodies in their serum, but none of the 9 had EA antibodies and 3 lacked EBNA antibodies. Two other groups of subjects, all of whom had EBV-VCA and EBNA antibodies in their serum late after an EBV infection, were also studied; 82 had infectious mononucleosis and 55 were healthy and had no such history. EA antibodies were detected in 45 of the first group during the acute phase of the illness but persisted in only 6 of the 68 who were followed up for more than 2 years, and they were detected in only 7 of the second group.All eight lymphoblastoid cell lines established from the peripheral blood of the four patients with ataxia—telangiectasia who are still available for follow-up study express EBV-VCA, whereas most similar cell lines established from normal individuals express only EBNA. In two of these patients cell-mediated immunity, as assessed from lymphocyte transformation induced by mitogens, was markedly decreased but autologous cell-mediated immune regression of EBV-induced transformation of B (bone-marrow-derived)-lymphocytes was normal. The percentage of T (thymus-derived)-helper cells was greatly decreased in two of the three patients in whom it was measured, and the percentage of T-suppressor cells was greatly increased in one of them, but the percentage of total T-lymphocytes was within normal limits in all three.The possible significance of these findings — in particular, the persistence of EA antibodies and the diminished restriction of expression of EA — in the late development of tumours after an EBV infection in patients with ataxia-telangiectasia deserves careful attention. Finally, the apparent correlation between immunoglobulin deficiency and poor or absent EBNA antibody response warrants further study.     

Abnormalities in lymphocyte proliferation in classical and atypical measles infection

Impaired in vitro lymphocyte responses to PHA stimulation have been observed in seven patients with acute classical measles infection, and three patients with atypical measles. The impairment is evident only at suboptimal concentrations of PHA and disappears in convalescence. Spontaneous DNA synthesis in peripheral leukocytes was elevated in both types of patients, but the classical measles patients demonstrated a delayed return to normal values as compared to the atypical measles group. We failed to demonstrate a serum inhibitory factor as an explanation for the impaired PHA responses of lymphocytes in these patients.     

Dose-dependent hyporreactivity to phytohemagglutinin in systemic lupus erythematosus

The response of peripheral blood lymphocytes to stimulation with optimal and suboptimal doses of PHA was measured in patients with active SLE before initiation of therapy. The [³H]thymidine uptake of SLE patient's lymphocytes was significantly lower than that of their matched controls when cells were stimulated with suboptimal PHA doses in the presence of autologous plasma. A moderate improvement in the PHA response was observed by culturing washed patient's lymphocytes in medium supplemented with pooled normal human plasma, but only in one case the response reverted to normal values. A significant inhibitory effect of SLE plasma on the response of normal donor's lymphocytes to stimulation with low PHA doses, which was independent from the presence of lymphocytotoxic antibodies and persisted after complement inactivation was observed in further experiments.The results indicate that depression of lymphocyte transformation could be demonstrated in patients with active SLE using suboptimal doses of PHA and suggest that this depression may be caused by both a defect in the responding lymphocyte populalation and the presence of inhibitory factor(s) in SLE plasma.     

The role of the thymus in maturational development of phytohemagglutinin and pokeweed mitogen responsiveness

A sensitive culture system for measuring lymphocyte transformation under physiological conditions by thymidine incorporation into DNA has been developed to study mouse and chick cell responses to mitogens. Both phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulated thymus and spleen lymphocytes. Reduced but definite responses were obtained with lymph nodes, but negligible response with bone marrow cells.Thymocytes of newborn mice did not respond to PHA, but responded well to PWM. PHA responsiveness of thymocytes increased with aging until 12 weeks of postnatal life and then decreased in older animals. The level of background thymidine incorporation increased with advancing age. Spleen cells of 2-week-old mice were transformed by PHA and PWM, but in contrast to mouse thymus there was no decrease in older animals.Neonatal thymectomy of mice reduced the response of spleen cells to both PHA and PWM, especially in younger animals. The reduction was almost complete in the case of the PHA response, but only partial with the PWM response. Spleen cells from bursectomised chickens, checked for absence of B cell function, still responded well to both PWM and PHA.The results suggest PHA is a marker for T-lymphocytes in a certain “mature” stage of differentiation. PWM appears to stimulate a wider spectrum of cells.     

Glucocorticosteroid modification of lymphocyte blastogenesis in tumor-bearing hosts

The present study was done to evaluate glucocorticosteroid modulation of the individual cells contributing to tumor-induced dualistic (macrophage and suppressor T-cell) suppression in mitogen-induced lymphocyte proliferation. Steroid-sensitive T cells from normal mice showed a characteristic decrease in phytohemagglutinin responsiveness in the in vitro presence of 0.1 μg hydrocortisone sodium-21 succinate (HC), while tumor-bearing host (TBH) T cells demonstrated little or no decrease. This correlates well with kinetic study results demonstrating decreased HC-mediated suppression of mitogen-induced TBH T-cell DNA synthesis at 8 to 10 days posttumor cell inoculation. Hydrocortisone-treated cells from mice with advanced tumors had significantly higher degrees of blastogenesis than untreated TBH cells. In the optimal presence of both macrophages (Mφ) and HC, TBH cells had significantly greater blastogenesis than their normal counterparts. Alone, Mφ or HC depressed normal host PHA responsiveness. Experiments were carried out to determine if the increased blastogenesis was due to the additional Mφ presence in TBH. In vivo administration of methylprednisolone acetate demonstrated that steroids can inhibit tumor growth. A significant delay in tumor appearance occurred when the steroid was administered 4 to 7 days after tumor transplant perhaps due to the measurable lack of suppressor T cells. These studies indicated that steroid suppression was directed against a sensitive suppressor T cell and possibly acts by preventing its differentiation. The role of the Mφ in the afferent limb of the cell-mediated immune response seems less clear, but the results suggest nonspecific inhibition of TBH T-cell response to blastogenic stimulus.     

Direct endolymphatic instillation (ELI) of BCG-Tumor cell mixture

Summary A method of active immunization by direct Endolymphatic Instillation (ELI) of a mixture of BCG and Autologous Irradiated Tumor Cells (AITC) is described. ELI was performed in 36 patients with advanced breast cancer, melanoma and some other solid tumors. The procedure was well tolerated and was not associated with any untoward systemic side effects other than those often encountered in conventional BCG immunotherapy. Exposure of patients to AITC by the direct endolymphatic route proved to be superior to immunization by the intradermal route as judged by the subsequent induction of cutaneous Delayed Hypersensitivity Reaction (DHR) toward AITC. When employed as the first immunization attempt ELI was effective in 5 of 6 patients. It led to invigoration of preexisting weak DHR in 6 of 8 patients. In 15 of 22 patients conversion to a strongly positive cutaneous DHR to AITC was achieved by ELI, where previous attempts of immunization by the intradermal route had failed to elicit a positive response. Acquisition of positive cutaneous response to AITC following ELI was found to correlate with favourable prognosis in terms of longer median survival. The effect of ELI was particularly notable with respect to enhancement of in vitro lymphocyte stimulation by PPD antigen, but was not followed by increased reactivity of other in vitro parameters of cell-mediated immunity such as peripheral lymphocyte count, proportion of E-rosette forming cells, and PHA lymphocyte stimulation.Abbreviations used: ELI — Endolymphatic Instillation; AITC — Autologous Irradiated Tumor Cells; BCG — Bacillus Calmette-Guérin; DHR — Delayed Hypersensitivity Reaction; PPD — Purified Protein Derivative (tuberculin); PHA — Phytohemagglutinin; SI — Stimulation Index; E-RFC — E-Rosette Forming Cells; PBL — Peripheral Blood Lymphocytes.     

Serial immune testing in surgically resected lung cancer patients

Summary Immunoregulation of phytohemagglutinin (PHA) responsiveness by glass-adherent cells and prostaglandin-synthesizing cells was serially monitored in the peripheral blood mononuclear cells (PBMC) of surgically resected stage I and stage II lung cancer patients entered on a trial of adjuvant immunotherapy. The relationship between immunoregulatory cell function, immunocompetence, and disease relapse was determined. Immunoregulatory activity was measured in PHA-stimulated cultures in the presence and absence of 2 g/ml indomethacin and in the presence and absence of glass-adherent cells. In each instance of disease relapse seen, an increase in immunoregulatory cell function to a level significantly different from normal was observed 3 months prior to or coincident with clinical confirmation of disease recurrence. This was usually associated with a decline in PHA responsiveness. In the patients who did not relapse, the levels of PHA responsiveness and immunoregulatory function persisted within normal limits throughout the course of study. Percentages and numbers of leukocytes and leukocyte subsets and delayed cutaneous hypersensitivity were also monitored in this study, but could not be consistently correlated with early changes in clinical disease status. These data suggest that the development of indomethacin-sensitive and glass-adherent suppressor cells may precede and predict for tumor recurrence in surgically resected lung cancer patients.     

In vivo and in vitro evaluation of cell-mediated immunity in patients with paracoccidioidomycosis

The cellular immune response to specific and nonspecific agents was investigated. both in vivo and in vitro, in 19 patients with paracoceidioidomycosis. In addition, the immunologic study of an investigator aceidentally inoculated with P. brasiliensis was included in this study. Nearly half of the patients showed depressed cell-mediated immune responses, as evaluated by intradermal tests with an antigenic preparation from P. brasiliensis (P.b.Ag.), ubiquitous antigens, and by the ability to develop sensitization to 2,4-dinitrochlorobenzene. A similar proportion of impaired responses was observed when the patients' lymphocytes were cultured with phytohemagglutinin (PHA). C'. albicans antigen and P.h.Ag. A factor was detected in the plasma of some patients which reduced the ability of patients' and normal lymphocytes to undergo blastic transformation. A positive correlation was found between the ability to develop delayed cutaneous hypersensitivity reactions to P.b.Ag. and other ubiquitous antigens, normal in vitro responsiveness to PHA and the absence of humoral blastogenic inhibitory factor. The inhibition of leukocyte migration, but not lymphocyte transformation, correlated positively with delayed hypersensitivity. The percentage of T lymphocytes was significantly reduced in the group of patients, being the absolute number and percentage of B cells bearing receptors tor complement normal. Two polar immunological patterns emerged. One characterized by positiveness in the skin test to P.b.Ag. and lack of significant abnormalities in cellular immunity, and another anergic to P.b.Ag., with cell mediated immunity severely depressed. Between the two polar groups, there were patients with intermediary patterns of immune response. This paper also includes the results obtained with the administration of transfer factor and levamisole to some of the patients.     

HIV and other lentiviral infections cause defects in neutrophil chemotaxis, recruitment, and cell structure: immunorestorative effects of granulocyte-macrophage colony-stimulating factor

Patients with HIV infection exhibit deficits in bacterial and fungal clearance, and possibly depressed innate immunity. In this study, we observed that neutrophils from HIV-infected patients have a profound defect in chemotaxis in response to endogenous (IL-8) and bacterial (fMLP) chemoattractants, which was directly correlated with peripheral CD4(+) lymphocyte levels but not plasma viral load. A similar chemotactic defect was observed in the feline immunodeficiency virus (FIV) model of HIV infection. Intravital microscopy of FIV-infected animals revealed marked impairment in the in vivo recruitment of leukocytes; specifically integrin-dependent neutrophil adhesion and emigration induced by bacterial products. Treatment of FIV-infected animals with GM-CSF re-established both neutrophil recruitment (rolling, adhesion, and emigration) and in vitro chemotaxis to the levels seen in uninfected animals. This restoration of neutrophil responses was not due to GM-CSF-mediated priming. Rather, HIV and FIV infections resulted in defective neutrophil development, with an ensuing reduction in neutrophil granularity and chemotactic receptor expression. GM-CSF therapy restored neutrophil granularity, implying restoration of normal neutrophil development. Together, our findings underscore the fundamental defects in innate immunity caused by lentivirus infections, while also indicating that GM-CSF may be a potential immunorestorative therapy for HIV-infected patients.     

Regulation of the immune response in autoimmune NZB/NZW F1 mice. I. The spontaneous generation of splenic suppressor cells.

The lymphoproliferative responses of rat peripheral blood lymphocytes to phytohemagglutinin (PHA) were studied following treatment with single or multiple doses of cyclophosphamide. A dose-dependent lymphocytopenia was observed with both regimes. The remaining lymphocytes had decreased responses to PHA. Serum collected 24 hr after a single injection of cyclophosphamide and used at a concentration of 5% enhanced the response of cells from normal or cyclophosphamide-treated rats. Serum collected after a course of treatment did not have this effect, but it lacked the marked suppressive activity, at a concentration of 20%, which was shown by normal rat serum. The enhancing activity was not dialysable. Doses of cyclophosphamide adequate to abolish primary antibody production to sheep erythrocytes did not totally abrogate responsiveness to PHA. Thus, the pattern of immunological defects in cyclophosphamide-treated rats consisted of decreased primary antibody production, lymphocytopenia with a decreased response of the remaining lymphocytes to PHA, and diminution of serum suppressive activity.     

In vitro assay for responsiveness of lymphocytes to transfer factor by a new leukocyte migration inhibitory test.

Transfer factor (TF) causes nonimmune lymphocytes to produce leukocyte migration inhibitory factor (LMIF) in the presence of purified protein derivative (PPD). The activity of TF was measured by leukocyte migration inhibitory test (LMIT). The LMIT was a modification of the conventional agarose droplet method. To express the activity of LMIF quantitatively and simply, LMIF titer was introduced. The LMIF titer was obtained from the combination of two factors, LMIF dilution and cell migration diameter, and therefore this made the LMIT much more sensitive as compared to the conventional LMIT. The responsiveness of lymphocytes from acute lymphoblastic leukemia (ALL) and from cell-mediated immunodeficiency in children to TF was assayed by LMIT. In ALL, the lymphocyte responsiveness was poor in relapse but improved with remission. The responsiveness was remarkably well in 3 patients with cell-mediated immunodeficiency. This method appears useful for the in vitro evaluation of responsiveness of lymphocytes to TF.