Transfer factor—Transfer of tuberculin cutaneous sensitivity in an allogeneic and xenogeneic monkey model

Tuberculin cutaneous sensitivity can be transferred from rhesus monkeys, sensitized to tuberculin using BCG, to rhesus and cynomolgus recipients with viable or disrupted leucocytes, and with a dialysed lysate preparation from 3 × 10⁸ leucocytes. Dialysable transfer factor (TF) using the lyophilized aqueous dialysate of a leucocyte lysate prepared by the freeze-thaw method did not give an active material. Modifications to the preparative method, in that leucocytes from fresh blood were disrupted gently by mechanical shearing forces, the lysate dialysed against a balanced salt solution and injected without lyophilization, yielded an active preparation. Transferred tuberculin (PPD) skin reactivity, confirmed by biopsy, was always less than the reactivity of the donor monkey and lasted for approximately 2–3 months. Although donor monkeys showed good in vitro lymphocyte transformation response to antigen stimulation, recipient monkeys which became skin test positive did not have a concomitant blastogenic response to PPD.     

The effect of hyposensitization on alternaria-induced lymphocyte blastogenesis

Twenty-one atopic children were treated with alternaria hyposensitization therapy. Leukocytes from these children were cultured with and without alternaria and phytohemagglutinin prior to treatment, and after 6 and 12 months of treatment. Patients demonstrating positive immediate cutaneous hypersensitivity showed significant lymphocyte transformation prior to hyposensitization whereas the skin test-negative patients did not. The skin test-negative patients developed significant lymphocyte transformation after 6 months of hyposensitization. Twelve months after the initiation of therapy both skin test-negative and skin test-positive patients showed a decreased lymphocyte reactivity, and there was no longer any significant change in the concomitant phytohemagglutinin responses indicating that hyposensitization had a specific effect on alternaria-induced in vitro lymphocyte reactivity. Plasma factors were found to have modulating effects on the lymphocyte reactivity of hyposensitized patients.     

Peripheral blood lymphocyte subpopulations in sarcoidosis.

We have determined the numbers of thymus-derived (T) and bone marrow-derived (B) lymphocytes in the peripheral blood of 20 patients with sarcoidosis and 15 healthy controls. T cells were estimated from the number of lymphocytes forming rosettes in vitro with unsensitized sheep red blood cells, and B cells were enumerated by immunofluorescent assesssment of membrane-bound immunoglobulins. The total lymphocyte count was lower in patients with sarcoidosis owing to a depletion of T lymphocytes from the blood. Nonetheless, the relative and absolute numbers of B lymphocytes were significantly increased. These alterations in lymphocyte subpopulations did not show any consistent correlation with the duration of the disease, clinical stage, activity, or treatment. Changes in the subpopulations may be related to both decreased cellular immunity and increased reactivity of the antibody-forming system as commonly seen in sarcoidosis.     

Two categories of lymphocyte unresponsiveness to phytohemagglutinin

Peripheral lymphocytes from healthy subjects, sarcoidosis and influenza patients were studied in vitro by measurement of the tritiated thymidine uptake of unstimulated and phytohemagglutinin. (PHA) stimulated cells. When the mitogen induced metabolic response is defined as the ratio between thymidine uptake by stimulated and unstimulated cells (stimulation index), PHA responsiveness was significantly decreased in both diseases and varied inversely with the level of isotope incorporated by unstimulated cells (p = 0.0002). The uptake of isotope by unstimulated cells from influenza patients was significantly increased (p = 0.0001). Isotope incorporation by mitogen stimulated cells from the same patients did not differ significantly from controls (p = 0.0925). In contrast, the impaired PHA responsiveness of lymphocytes from sarcoidosis patients was associated with levels of isotope incorporation in unstimulated cell cultures similar to those observed in healthy controls (p = 0.6444). These observations suggest that two different mechanisms may be responsible for low lymphocyte PHA stimulation indices associated with disease states. Methods are presented for minimizing variation of replicate observations and identification of both categories of lymphocyte unresponsiveness.     

Lymphocyte suppression associated with alpha globulin. Changes in cellular immunity

Sensitization of guinea pigs to 2,4-dinitrofluorobenzene is accompanied by increases in alpha globulins determined electrophoretically. During sensitization, lymphocyte responses were measured in vitro by mitogen induced ³H-thymidine uptake in whole blood cultures and in vivo by dermal skin reactivity. Following 5 days of dinitrofluorobenzene sensitization alpha globulins were elevated and lymphocyte transformation to phytohemagglutinin, pokeweed mitogen, and concanavalin A was significantly suppressed. When the alpha globulins returned to normal levels following sensitization, lymphocyte responses returned to pretreatment values. Antigen induced lymphocyte responsiveness was also suppressed concomitant with elevations in alpha globulins. Tuberculin sensitive guinea pigs responded poorly to PPD in vitro and in vivo during DNFB sensitization. It is suggested that increases in alpha globulins detected during the development of cellular immunity are associated with immunosuppression.     

Lymphocyte transforming factor in sarcoidosis

Lymphocyte transforming factor (LTF) was induced by tuberculin in cultures of lymphocytes from tuberculin-sensitive healthy subjects. In the presence of tuberculin, lymphocytes from nonsensitive patients with sarcoidosis responded to LTF as did cord blood lymphocytes. The LTF activity correlated to the tuberculin skin sensitivity of the healthy adult donors. Less LTF activity was produced by lymphocytes from tuberculin-sensitive patients with sarcoidosis than by those from the tuberculin-sensitive controls.     

Suppression of in vitro lymphocyte responsiveness to purified protein derivative by measles virus. A reexploration of the phenomenon.

Clinical measles and measles vaccination have classically been associated with transient in vivo impairment of delayed hypersensitivity-type responses, especially skin test reactivity to purified protein derivative (PPD). In vitro data appeared to substantiate this in vivo observation by the demonstration of suppression of lymphocyte responsiveness to PPD by measles. Utilizing a measles preparation which has been recently demonstrated to elicit specific blastogenesis of sensitized human lymphocytes in vitro, we have reexplored the question of in vitro suppression of lymphocyte responsiveness to PPD by this virus. In contrast to previous reports, this study demonstrates that the addition of both measles and PPD to lymphocyte cultures can have a variable effect on lymphocyte responsiveness to PPD alone. This effect varies from marked inhibition to enhancement beyond a summation effect. The response is different for each lymphocyte donor and is dose related but cannot be predicted on the basis of combinations of high or low concentrations of either antigen. Purified, attenuated measles virus (Enders' strain), which uniformly suppressed in vitro lymphocyte reactivity when tested alone also demonstrated a significant dose related enhancement of the response to PPD alone. The present data suggest a reconsideration of the supposed importance of transient diminution of skin test reactivity during measles infection or immunization.     

In vitro study of macrophages from patients with sarcoidosis

Cultures of leukocytes from patients with sarcoidosis were stimulated with Kveim antigen or PPD-S and the macrophage reactions were compared with those obtained under similar conditions with leukocyte cultures from healthy tuberculin reactors. The non-stimulated control cultures of Kveim-positive sarcoid patients showed signs of macrophage activation, namely an increase in cytoplasmic pyroninophilia in small and enlarged macrophages, together with a higher than normal rate of lymphoblast transformation. In the presence of Kveim antigen this activation was more marked, even in cases where the blast response to the antigen was of low magnitude. Stimulation with tuberculoproteins produced a slight activation of macrophages only in cultures from Kveim reactors who had also shown positive tuberculin reactions.These observations suggest that the study of lymphocyte transformation together with that of macrophage activation will improve the immunocytological assessment of sarcoidosis.     

Cell-mediated immunity in Cryptococcosis

Cell-mediated immune responses in patients who had recovered from cryptococcosis were compared to those of healthy subjects. Cryptococcal patients were mildly lymphopenic but showed no defect in percentage of thymus-derived lymphocytes. One-third had positive delayed skin test reactions to cryptococcal antigen. Their skin test reactivity to two commonly used noncryptococcal antigens was less intense than healthy control subjects. Strongly positive and specific lymphocyte transformation occurred in the presence of an extract of Cryptococcus neoformans (cryptococcin) in half of the patients. In contrast, few healthy subjects had positive transformation responses to cryptococcin. One patient who was followed sequentially through treatment of cryptococcal meningitis acquired strong cryptococcin reactivity during the course of treatment. Cellular immunologic response to cryptococcin identifies many subjects who have had C. neoformans exposure, and may be of value for assessing immunologic status of patients undergoing therapy. These studies also indicate that most patients with cryptococcosis have a degree of deficiency in cell-mediated response to fungal antigens even when a specific underlying disease process cannot be identified.     

Depression of lymphocyte transformation and exacerbation of Behcet's syndrome by ingestion of english walnuts

Extracts of English and black walnuts, almonds and pecans stimulated increased incorporation of tritiated thymidine by cultured leucocytes from both normals and patients with Behcet's syndrome. English walnut extract also stimulated newborn cord blood leucocytes, indicating it to be a nonspecific stimulant. Within 2 days after ingestion of English walnuts by normals and patients with Behcet's syndrome, their in vitro lymphocyte reactivity to both the walnut extract and Candida antigens was significantly decreased. This inhibition of lymphocyte transformation may be more prolonged in the patients than in normal subjects and was associated with an increase in frequency and severity of their symptoms. Hypotheses relating the mechanism of action of these two apparently disparate phenomenon are discussed.     

Human eosinophil stimulation promoter lymphokine: Production by antigen stimulated lymphocytes and assay with a new electro-optical technique

A human lymphokine, eosinophil stimulation promoter (ESP), was shown to be produced by mononuclear leukocytes in response to non-helminthic antigens. Supernatants of tuberculin-stimulated cultures of mononuclear leukocytes from tuberculinskin test reactive humans contained non-dialyzable ESP activity; ESP could not be demonstrated in the supernatants of tuberculin stimulated cultures from skin test negative donors. Thus, human ESP, like murine ESP, is a soluble lymphokine product of sensitized mononuclear cells. ESP activity was also demonstrated in supernatants of mononuclear leukocyte cultures stimulated with streptococcal antigens. ESP selectively enhanced migration of eosinophils. ESP production could be blocked by puromycin, and its activity was diminished by prior incubation with eosinophil-rich granulocytes. Correlation of antigen stimulated ESP activity with skin test reactivity establishes ESP as another in vitro correlate of delayed hypersensitivity in man. A video method of data collection and analysis is described which greatly facilitates in vitro studies of ESP activity. The method is applicable to the study of other lymphokines which effect cellular migration.     

Tuberculin response of lymphocytes from human skin test nonreactors; evidence for in vitro primary sensitization of T lymphocytes.

Tuberculin-purified protein derivative (PPD) is a B-lymphocyte mitogen in a variety of experimental animals. Although peripheral blood mononuclear cells (PB MNC) from healthy human tuberculin responders consistently responded to PPD by increased incorporation of [³H]thymidine, cell fractionation studies showed this to be due to T-lymphocyte rather than B-cell blastogenesis. Moreover, utilizing thymidine suicide experiments, the T-lymphocyte response could be categorized as antigenic rather than nonspecific mitogenic reactivity. Kinetic studies revealed a delayed peak of PPD-induced thymidine incorporation in PB MNC from tuberculin skin test-negative as compared to skin test-positive donors. This suggested in vitro primary sensitization of T lymphocytes to PPD, which was corroborated in experiments demonstrating tuberculin reactivity of human umbilical-cord blood lymphocytes.     

Interspecies transfer by "immune" RNA of lymphocyte proliferative response to specific antigen

Ribonucleic acid (RNA) extracted from the lymph nodes of BCG sensitized cattle transferred tuberculin sensitivity to normal guinea pig lymphocytes as indicated by increased incorporation in vitro of ³H-thymidine in response to Purified Protein Derivative (PPD). The RNA treated lymphocytes were unresponsive to a nonspecific antigen, histoplasmin. Ribonuclease treatment of the RNA abolished its ability to transfer tuberculin reactivity and RNA extracted from the lymph nodes of normal cattle was also ineffective.     

Leukocyte procoagulant activity in man: an in vitro correlate of delayed-type hypersensitivity

Human mononuclear leukocytes generate cell-bound procoagulant activity (LPCA) after incubation with an antigen (mumps or tuberculin) to which the donor was previously sensitized. An inhibitor of coagulation appears to be liberated into the extracellular culture fluid during incubation of leukocytes with the sensitizing antigen. Removal of this activity before measuring LPCA resulted in a reliable test that correlated directly with delayed skin reactivity. The assay was particularly sensitive in that cells from weakly sensitized donors who reacted only to high doses of tuberculin (100 TU) in the delayed skin tests produced detectable LPCA in vitro. By contrast cells from weakly sensitized donors did not react to PPD in the lymphocyte blast transformation test or the direct macrophage migration inhibition factor test. The LPCA assay correlated closely with the blast transformation and MIF tests in which cells were used from more strongly sensitized donors who reacted in skin tests with lower doses of tuberculin (1 or 10 TU). The assays were antigen-specific in that cells from donors sensitive to mumps antigen but not to tuberculin reacted only to mumps antigen in vitro. The assay was extremely reproducible; cells from individual donors reacted to the same extent over a period of 8 mo). We propose that the assay system reported here represents an improved method for the measurement of cell-mediated immunity in vitro because it requires fewer donor cells, is technically simpler, and is more sensitive than previously described methods.     

Echinococcus granulosus: host lymphocyte transformation by parasite antigens.

Lymphocyte transformation, measured by in vitro tritiated thymidine incorporation, and indirect hemagglutination tests were carried out on hydatid patients and normal individuals using sheep and human hydatid fluid or scolex antigens. The hydatid patients showed statistically significant lymphocyte transformation with human and sheep hydatid fluid or scolex antigens when compared to normal individuals. The indirect hemagglutination tests resulted in high titers of antibody with sheep or human hydatid fluid antigens, while very low titers were obtained with scolex antigens. Unlike in the indirect hemagglutination test, the source of the antigen, scolex or fluid, was not of consequence in the lymphocyte transformation test. Furthermore, there was no correlation between the results of the serologic and lymphocyte transformation tests, since some patients with very high lymphocyte stimulation indices produced low indirect hemagglutination titers and vice versa. Similar results were obtained from rabbits which were immunized with sheep hydatid fluid or scolex extracts. The skin tests were of the immediate type of hypersensitivity reactions. Delayed skin reactions did not occur in spite of the presence of sensitized lymphocytes in the blood of the immunized rabbits.     

In Vivo Transfer of cellular immunity to primates with transfer factor prepared from human or primate leucocytes

Dialyzable transfer factor (TF_d) prepared from a human donor and transfer factor (TF) from baboon whole cell lysates was administered to 3 species of nonhuman primates: baboons, cebus monkeys and marmosets. In vivo transfer was evaluated with in vivo skin test and in vitro blastogenic responses to multiple antigens. Transfer of cellular reactivity in all three nonhuman primate species was demonstrated with both human TF_d and baboon TF. A cumulative conversion rate of 45% for skin test responses and 65% for lymphocyte blastogenesis was demonstrated following human TF_d injection while conversion was 17% and 33% respectively following baboon TF. Specificity was supported by the absence of conversion to TF donor negative antigens. There were no signficant differences observed between the 3 recipient primate species.     

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.     

Dipetalonema viteae: in vitro blastogenesis of hamster spleen and lymph node cells to phytohemagglutinin and filarial antigens

Hamsters of the randomly bred LAKZ and inbred LSH strains were infected with Dipetalonema viteae, and the in vitro responses of lymph node and spleen lymphocytes to male and female worm antigens and phytohemagglutinin (PHA) were measured by a [³H]-thymidine-uptake assay at various times after infection. The PHA response remained unchanged at the level of controls in infected LAKZ hamsters while LSH hamsters showed a depressed response to the mitogen during late infection. Stimulation of lymph node cells by filarial antigens was maximal in both strains of hamsters at Week 4 postinfection, almost reaching values obtained in PHA stimulated cultures. A similar high lymphocyte transformation reaction was measured after the injection of dead third stage larvae. During transient microfilaremia, when antibody titers reached a maximal level, the lymphocyte reactivity to filarial antigens decreased drastically and only occasionally was demonstrated in hamsters 20 and 30 weeks after infection. No correlation between lymphocyte reactivity and parasitological findings (worm load or intensity and duration of microfilaremia) could be demonstrated. The cellular unresponsiveness to filarial antigens was further analyzed in chronically infected LAKZ hamsters. No suppressor cells could be found in lymphocyte suspensions of nonresponding hamsters. A challenge infection did not restore lymphocyte reactivity. Serum of chronically infected hamsters caused marked inhibition when added to filarial antigen-sensitive lymphocytes. Lymphocytes from hamsters with a mixed D. viteae and Schistosoma mansoni infection responded as well to soluble schistosomal egg antigens at Week 30 of a D. viteae infection as lymphocytes from hamsters infected with S. mansoni alone. The humoral immune response to schistosomal antigens, however, was significantly lower in animals with a mixed infection.     

Contact sensitivity in rabbits sensitized with dinitrophenylated Mycobacterium tuberculosis

The conjugation of Mycobacterium tuberculosis with DNFB results in the formation of a haptenated preparation that induces the formation of contact sensitivity when administered subcutaneously. This contact sensitivity can be measured in vivo by topical application of the free chemical and in vitro by lymphocyte transformation. The antigens suitable for the in vitro detection are those preparations obtained by the haptenation of cell membranes. Haptenation of serum proteins, of homologous and heterologous origin, does not produce antigens suitable for in vitro assay. The antigen requirements for the in vitro transformation assay of contact sensitivity are similar for adjuvant induced sensitivity as well as for free chemical induced sensitivity.     

Linkage of type I interferon activity and TNF-alpha levels in serum with sarcoidosis manifestations and ancestry

Background

Both type I interferon (IFN), also known as IFN-α and tumor necrosis factor alpha (TNF-α) have been implicated in the pathogenesis of sarcoidosis. We investigated serum levels of these cytokines in a large multi-ancestral sarcoidosis population to determine correlations between cytokine levels and disease phenotypes.

Methods

We studied serum samples from 98 patients with sarcoidosis, including 71 patients of African-American ancestry and 27 patients of European-American ancestry. Serum type I IFN was measured using a sensitive reporter cell assay and serum TNF-α was measured using a commercial ELISA kit. Clinical data including presence or absence of neurologic, cardiac, and severe pulmonary manifestations of sarcoidosis were abstracted from medical records. Twenty age-matched non-autoimmune controls were also studied from each ancestral background. Differences in cytokine levels between groups were analyzed with Mann-Whitney U test, and correlations were assessed using Spearman''s rho. Multivariate logistic regression models were used to detect associations between cytokines and clinical manifestations.

Results

Significant differences in cytokine levels were observed between African- and European-American patients with sarcoidosis. In African-Americans, serum TNF-α levels were significantly higher relative to matched controls (P = 0.039), and patients with neurologic disease had significantly higher TNF-α than patients lacking this manifestation (P = 0.022). In European-Americans, serum type I IFN activity was higher in sarcoidosis cases as compared to matched controls, and patients with extra-pulmonary disease represented a high serum IFN subgroup (P = 0.0032). None of the associations observed were shared between the two ancestral groups.

Conclusions

Our data indicate that significant associations between serum levels of TNF-α and type I IFN and clinical manifestations exist in a sarcoidosis cohort that differ significantly by self-reported ancestry. In each ancestral background, the cytokine elevated in patients with sarcoidosis was also associated with a particular disease phenotype. These findings may relate to ancestral differences in the molecular pathogenesis of this heterogeneous disease.