Effects of vitamin E on mixed lymphocyte cultures

When lymphocytes from individuals ingesting vitamin E were used as responding cells in the mixed lymphocyte culture (MLC) the proliferative response was normal indicating that T lymphocyte reactivity to allogeneic antigen was undisturbed. However, the use of lymphocytes from individuals receiving vitamin E as stimulating cells in the MLC resulted in a diminished proliferative response suggesting that large doses of vitamin E may effect B cells and/or macrophages as they interacted with T lymphocytes.     

In vitro activation of human suppressor cells: relative quantitation and specificity of suppressor cells generated in primary and secondary mixed lymphocyte cultures

Human suppressor cells activated in primary and secondary mixed lymphocyte cultures (MLC) were quantitatively assessed for their ability to suppress the proliferative response of fresh responding lymphocytes to the original sensitizing (specific suppression) and third party, unrelated (non-specific suppression) alloantigens. Maximal levels of both specific and non-specific suppressor cell activities generated in primary MLC were found in the population of cells harvested on day 7 which corresponded to the peak of proliferation in bulk culture and decayed upon further culture reaching a nadir concomitant with the end of proliferation in primary bulk MLC. Maximal reactivation of the suppressor cells was accomplished by restimulation in secondary MLC with the original sensitizing alloantigens. Assessment of suppressor cell activity revealed that specific rechallenge with the original stimulating alloantigens in secondary MLC resulted in significant increases in both the levels of suppressor cell activity and in the specificity of action of the suppressor cell population as compared to suppressor cells harvested from primary MLC on day 7. Both primary and secondary MLC activated suppressor cells were capable of suppressing the induction of cytolytic lymphocytes in primary MLC. Suppression was not simply the result of altered kinetics of the fresh micro MLC response.     

IN VITRO FUNCTION OF A T-CELL CHRONIC LYMPHOCYTIC LEUKEMIA WITH SUPPRESSOR CELL PHENOTYPE

Abstract Surface membrane studies of cells from a atient with T-cell chronic lymphocytic leukemia T-CLL) showed that they had an OKT3+, OKT4^--, OKT8+ phenotype which IS consistent with tbe phenotype of normal circulating peripheral blood suppressorlcytotoxic T-lymphocytes. The cells were HLA-DR', anti-Tac^-, Smlg- and E rosette+. Parallel studies in vitro showed that the T-CLL cells had a marked suppressive effect on both the mixed lymphocyte culture (MLC) proliferative response and the activation of cytotoxic T-lymphocytes. The T-CLL cells failed to respond or stimulate in MLC and were not directly cytotoxic to allogeneic peripheral blood lymphocyte targets. Both the suppression of the MLC and the cytotoxic responses depended on the presence of the T-CLL cells and not T-CLL culture supernatants. The T-CLL cells did not suppress the production of polyclonal antibody and did not produce interleukin-2 to levels above normal control lymphocytes.     

IL-10 mediates immunosuppression following primary infection with Toxoplasma gondii in mice

Suppression of the host immune response by Toxoplasma gondii has been observed in both human and experimental murine infection. In this study, inbred mice were infected with T. gondii. At day 7 post-infection, the lymphoproli-ferative response to both mitogen and superantigen as well as parasite antigen were found to be significantly depressed. Using a transwell system, it was determined that the reduced proliferative response was due to soluble factor (s) being expressed by splenocytes from the infected mice. Isolation of the splenocytes into an adherent and nonadherent population suggested that both macrophages and T cells were able to produce at least one soluble factor. Tissue culture supernatant derived from the splenocytes of the infected mice contain increased levels of IL-10, whereas measurable IL-2 levels could not be quantitated. At day 7 post-infection, both a biologic assay for IFN-γ in culture supernatant and the expression of IFN-γ mRNA in the splenocytes were reduced. Antibody to IL-10 was able to partially neutralize (almost 50%) the in vitro immune downregulation of the tissue culture supernatant. Anti-IL-10 in combination with a nitric oxide (NO) antagonist was able to reverse the inhibitory activity of the culture supernatant by 85%. Since IL-10 is a potent antagonist of IFN-γ, it may represent a critical cytokine involved in mediating T. gondii induced immunosuppression in the infected host.     

B lymphocyte function in B cell chronic lymphocytic leukaemia

S ummary . B-enriched lymphocyte populations from patients with chronic lymphocytic leukaemia (CLL) were compared to B-enriched lymphocyte populations from normal age-matched controls for their ability to stimulate a proliferative response and to generate cytotoxic cells in allogeneic mixed lymphocyte cultures (MLC). The proliferative responses of MLC_s were less than normal when the stimulating cells originated from B cells of patients with CLL. The degree of stimulation provided by the B cells from the patients with CLL inversely correlated with the peripheral white cell count. Lymphocytes from patients providing a poor stimulatory signal in the MLC tended to have less'Ia-like'antigen than lymphocytes giving a moderate level of stimulation. B lymphocytes from patients with CLL which stimulated poorly in the MLC also failed to generate specific cytotoxic cells even when provided with a normal proliferative trigger. These data suggest that B lymphocytes from cases of CLL with markedly elevated leucocyte counts may have a diminished concentration of both'Ia-like'and serum defined antigens.     

In vitro stimulation of peripheral blood lymphocytes with allogeneic lymphocytes or phytomitogens in patients with insulin-dependent diabetes mellitus (author's transl)]

The lymphocyte transformation response to the allogeneic lymphocytes (mixed lymphocyte culture, MLC) was determined in nineteen well-controlled insulin-dependent diabetics (IDD) and nineteen matched normal subjects. All possible combinations between lymphocytes from the patients and controls were mixed in both one-way and two-way MLC. From the results of one-way MLC, the stimulatory capacity (SC) and responding capacity (RC) of IDD lymphocytes were compared with those of normal lymphocytes as follows: (1) Nm leads to N: 10,538 +/- 3,937 N; normal lymphocytes (2) Nm leads to D: 8;466 +/- 5,387 D; IDD lymphocytes (3) Dm leads to N: 7,562 +/- 3,088 m; mitomycin-treated stimulating lymphocytes (4) Dm leads to D: 7,102 +/- 4,873 (leads to; stimulatory direction, results; M +/- SD cpm) IDD lymphocytes showed a marked depressive function as stimulators (SC, (1) -- (3)), but the RC of IDD lymphocytes was unchanged ((1) -- (2)). Phytomitogen-response was studied simultaneously for the same responding lymphocytes (N, D) of MLC. IDD lymphocytes exhibited significantly decreased responses to phytohemagglutinin P, pokeweed mitogen and concanavalin A.     

Trophoblast modulation of maternal allogeneic recognition.

Human syncytiotrophoblast cell membranes prepared by differential ultracentrifugation were extracted with 3 M KCl, solubilized in 1% deoxycholate, and chromatographically separated into two peaks by passage through a column of Bio-Gel P-200. Previous reports from this laboratory have shown that the first peak (PI) is serologically the same as a group of trophoblast membrane antigens tentatively designated as TA1. Microgram amounts of P1 protein were found to completely inhibit the mixed lymphocyte culture (MLC) reaction but had no suppressive effect on lymphocyte responses to the lectins phytohemagglutinin or pokeweed mitogen. Control P1 membrane fractions identically prepared from human erythrocytes and liver powder had no inhibitory effects on either MLC reactions or lymphocyte responses to mitogens. Dose response experiments with P1 from 10 different placentae showed total inhibition of MLC by all preparations when used between 25 and 50 microgram/0.2-ml MLC mixture, but some P1 fractions inhibited significantly at much lower concentrations. Timed experiments revealed that MLC suppression was maximal when P1 was added within 12 hr after culture initiation and that no effect could be found with addition after 48 hr. We have previously shown that TA1 is a lymphocyte product of allogeneic responses, and the present results indicate that P1 proteins are themselves involved in the biology of lymphocyte responses to allogeneic cells. Pregnancy is one of the few natural circumstances in which a mixing of allogeneic cells occurs in vivo, and the presence of P1 proteins at the operational interface in the host-parasite relationship of human pregnancy suggests that this trophoblast membrane constituent is involved in the modulation of maternal allogeneic responses.     

MLC-activation by acute lymphatic leukemia blasts

Summary The MLC-activating potential of 25 ALL blasts (16 common ALL, 6 T-ALL, 3 not identified) was investigated. Mitomycin-treated leukemic blasts or X-irradiated lymphocytes were cultured with heparinized whole blood from different healthy donors. MLC activation by blast cells was expressed as percentage of MLC activation by X-irradiated lymphocytes. Leukemic blasts showed a heterogeneous pattern of MLC activation, ranging from 2 % to 245 %. Eleven out of 25 cases of ALL poorly stimulated the MLC (2% to 33% response). Twelve ALL stimulated a normal response (50% to 120%); and 2/25 ALL stimulated a supranormal response (more than 200 %). Four of six cases of T-ALL stimulated the MLC as efficiently as irradiated lymphocytes, 2/6 were among the poor stimulators.Most poor stimulator blasts had, however, normal MLC-activating properties if, instead of whole blood, isolated lymphocytes were used as the responding cells. The poor activation of lymphocytes by some leukemic blasts in whole blood appeared to be associated with impaired release of blastogenic factor (s) during the MLBC. No evidence for active suppressor mechanisms was found. The significance of the MLC-activating properties of leukemic blasts for the classification and immunotherapeutic use of ALL is discussed.

---

Abbreviation MLC Mixed lymphocyte culture - ALL Acute lymphocytic leukemia - MLBC Mixed lymphocyte-blast cell culture - HTLA Human Thymus Lymphocyte Antigen - DR-Ag D-related Antigen Supported by Fonds zur Förderung der wissenschaftlichen Forschung in Österreich     

Selective removal of alloreactive cells from haematopoietic stem cell grafts: graft engineering for GVHD prophylaxis.

One of the main goals in allogeneic bone marrow transplantation is the abrogation of graft-versus-host disease with the preservation of antileukaemia and antiviral activity. We have established a novel system for the selective removal of alloreactive lymphocytes from donor grafts while retaining an effective allogeneic response to third-party stimulator cells. Initial feasibility studies were done with unrelated HLA-mismatched pairs and then extended into the matched setting. Mononuclear cells from HLA-matched donors were cocultured with irradiated recipient cells prestimulated with cytokines (gamma-IFN and TNF-alpha) in a modified mixed lymphocyte culture (MLC). Alloreactive donor lymphocytes were identified by expression of CD69, an early activation marker and selectively removed by paramagnetic bead sorting. The remaining 'non-alloreactive' lymphocytes were tested in proliferative assays against the original matched recipient and to a third-party donor. A mean depletion of proliferative capacity to 11.5 +/- 9.9% of the original matched recipient response was achieved while the residual third-party response was largely preserved at 77.8 +/- 20.9% which should translate into improved immune reconstitution and preservation of antiviral activity. The non-alloreactive lymphocytes could also possess functional antileukaemia activity. Moreover, the alloreactive cells are easily recoverable in this selective T cell depletion strategy for cryopreservation and ready for immediate access as therapeutic donor lymphocyte infusions in cases of frank relapse post transplant.     

A study of enhancing effects of monocyte culture supernatant on polymorphonuclear neutrophils-chemiluminescence--comparison between normal controls and collagen disease patients]

Priming effect of monocyte culture supernatant on polymorphonuclear neutrophils (PMN)-chemiluminescence (CL) was studied in patients with systemic lupus erythematosus (n = 11) and mixed connective tissue disease (n = 4). In normal controls, N-formyl-methionyl-leucyl-phenylalanine (FMLP) induced PMN-CL was enhanced when PMN were previously incubated for 15 minutes with monocyte culture supernatant (MS) or T lymphocyte culture supernatant (TS) or T lymphocyte culture supernatant (TS). The enhancing effect of MS on PMN-CL was greater than that of TS. This enhancing effect of MS was inhibited by adding of dexamethasone (1 microgram/ml) during the culture. Recombinant human TNF also enhanced PMN-CL as well as MS. When compared the enhancing effects of MS between patients and normal controls, that of patients under corticosteroid therapy (average prednisolone dose 39.5 mg/day) was reduced significantly. Thus, we concluded that the cytokines from monocyte contributed PMN phagocytosis of invading microorganisms, and that this monocyte-mediated PMN phagocytosis was suppressed partly by corticosteroids in collagen disease.     

Inhibition of specific cell-mediated cytotoxicity by monoclonal antibodies to human T cell antigens

Human T lymphocyte subpopulations recently have been defined by monoclonal antibodies that recognize cell-surface antigens selectively expressed on functionally distinct T cell subsets. The majority (approximately 90%) of the peripheral blood sheep erythrocyte-rosette-forming cells carry the OKT3 antigen. Helper cells are OKT4⁺, whereas cytotoxic/suppressor cells are OKT5⁺ and OKT8⁺. We investigated the effect of several monoclonal antibodies recognizing T cell antigens on certain proliferative responses of T cells and on the effector phase of the specific T cell-mediated cytotoxicity generated in mixed lymphocyte culture (MLC). In the absence of added complement, (i) OKT3 and OKT4 monoclonal antibodies inhibited the proliferative response to phytohemagglutinin (PHA), (ii) OKT3 monoclonal antibody inhibited the proliferative response to allogeneic cells in MLC, and (iii) OKT3 monoclonal antibody significantly and regularly inhibited the effector phase of the specific T cell-mediated cytotoxicity against allogeneic targets (P < 0.001) in a concentration-dependent manner. The OKT5 and OKT8 monoclonal antibodies, again in the absence of complement, inhibited moderately the specific cell-mediated cytotoxicity. This inhibition was observed in some experiments only. Inhibition of the specific cytotoxicity by these antibodies also was observed in secondary responses. In contrast, again in the absence of added complement, none of these antibodies had an effect on the nonspecific cytotoxicity generated in MLC against the K562 targets. The OKT4 antibody in the absence of added complement had no effect on either the specific or nonspecific cytotoxicity. Furthermore, treatment with OKT3 or OKT8 antibody and complement completely abrogated the specific T cell-mediated cytotoxicity but had no effect on the natural killer-like cytotoxicity against the K562 cells. Treatment with the OKT4 antibody and complement had no effect. These results suggest that (i) the T5/T8 and T3 antigens, present on cytotoxic T lymphocytes, may be involved directly or indirectly in the antigen recognition step(s) or the lytic mechanism of T cell-mediated lympholysis; and (ii) nonspecific cytotoxicity against the K562 targets generated in MLC is mediated by cells phenotypically different than those that mediate specific cytotoxicity.     

Impaired response to alloantigens in HIV positive hemophilic patients

The alloantigen responses of hemophilic patients was evaluated using a unidirectional mixed lymphocyte culture (MLC). The results of this study indicate that both the ability of peripheral blood mononuclear cells (PBMC) to proliferate in response to alloantigens and the capacity to stimulate the MLC are impaired in hemophilic patients infected with the human immune deficiency virus (HIV). These defects cannot be overcome by addition of exogenous interleukin-2 (IL-2) and are not related to the absence of IL-2 receptors (IL-2R). Lack of response was evident in PBMC from HIV+ patients with a high proportion of IL-2R+ cells. The number of IL-2 cells was similar in HIV+ and HIV- individuals, and higher than that of normal controls. Impaired MLC proliferation was not related to the occurrence of clinical symptoms. Apparently, MLC is a useful procedure for distinguishing the cell mediated immunity defects associated with HIV infection and unrelated to replacement therapy in hemophilia patients.     

Evidence for immunosuppression by high-dose gammaglobulin

High-dose immunoglobulin therapy, which is useful for the treatment of idiopathic thrombocytopenic purpura, autoimmune neutropenia, childhood epilepsy, and Kawasaki disease is postulated here to act by immunosuppressing the patients. Evidence provided here shows that the immunoglobulin inhibits phytohemagglutinin (PHA) stimulation, mixed lymphocyte culture (MLC) response, natural killer assay, antibody-dependent cell-mediated cytotoxicity, and cell-mediated lympholysis. These assays were all inhibited by IgG concentrations that exceeded normal levels by 1.5-2 times. It is also shown that Fc fragments were 100-1000 times more effective than intact IgG in inhibiting the PHA and MLC responses. Thus, it is likely that the Fc portion of immunoglobulin functions as an inhibitor of cellular immunity.     

Immune status in Crohn's disease. 2. Originally unimpaired primary cell mediated immunity in vitro.

One-way mixed lymphocyte cultures (MLC) were performed with peripheral blood lymphocytes of 21 patients with Crohn's disease (CD) not receiving salizylazosulphapyridine, steroids or azathioprine, seven patients with inflammatory bowel disease other than CD and ulcerative colitis, and 46 age- and sex-matched normal control subjects. The group of CD patients consisted of 11 patients with newly diagnosed, short-standing and so far untreated CD (group CD 1) and 10 patients previously treated with drugs and with mostly long-standing CD (group CD 2). Results showed that the MLC responsiveness was similar in all Crohn's disease groups, normal subjects and diseased controls. While there was no correlation between MLC responsiveness and either disease activity or disease duration when compared singly, those CD 2 patients who had highly active and/or very long-standing disease did exhibit a depressed MLC responsiveness as compared with that of normal subjects (p less than 0.001), CD 1 patients who had both inactive and short-standing disease (P less than 0.05), and diseased controls (0.1 greater than or equal to P greater than 0.05). The stimulatory capacity did not differ significantly between the CD groups and normal subjects or diseased controls; the latter, however, stimulated poorly compared with normal subjects (P less than 0.05). In accordance, an inverse relationship between the magnitude of the stimulatory capacity and the disease activity was found in the CD patients as a whole. These data suggest that there is no depression of the in vitro primary cell mediated immune response as a predisposing factor for CD or as an early event associated with the pathogenesis of CD.     

Effect of HBV antigens and their immune complexes on the PHA induced lymphocyte proliferation. Modulatory influence on interleukin-2 activity

Studies were undertaken to evaluate immunomodulating properties of Hepatitis B virus (HBV) preparations: HBsAg, HBeAg and their complexes: HBsAg-IgG and HBeAg-IgG, on PHA-induced lymphocyte proliferation. Cells were obtained from blood of healthy individuals, serologically negative for HBV markers. HBV preparations were purified from sera of children with HBV-mediated glomerulonephritis. Suppression of lymphocyte proliferation observed in the presence of HBsAg and HBsAg-IgG complexes was irreversible. However, the suppressive effect of HBeAg and HBeAg-IgG was abolished when these preparations were removed from the culture. Addition of exogenous interleukin-2/IL-2/reversed only the suppressive effect of HBeAg-IgG which was constantly present in the culture. The inhibition of lymphocyte proliferation correlated well with the decreased level of IL-2 activity in cultures with HBV-preparations. Experiments performed using ultracentrifugation indicated that HBV preparations, especially HBsAg and HBsAg-IgG, may bind to IL-2 and inactivate it in supernatants. The experiments indicate that HBV antigens, as well as other viral products, can inhibit lymphocyte proliferative response to the mitogen. Furthermore, we suggest that this inhibition may occur via suppression of IL-2 synthesis.     

Studies on drug-induced allergic intrahepatic cholestasis--hepatic cholestasis induced in dogs by lymphocyte culture supernatant.

When peripheral lymphocytes from patients with drug-induced allergic intrahepatic cholestasis were stimulated with a specific drug in vitro in the presence of a liver microsome fraction or soluble liver specific antigen fraction, lympholine production was seen in many cases. By the injection of culture supernatant of stimulated lymphocytes into the mesentery vein of dogs, cholestasis was induced in the liver, chiefly in the central zones of lobules. However, no cholestasis could be observed in dogs administered the supernatants of lymphocyte cultures prepared from normal individuals in the presence of drugs. Moreover, only slight swelling of the hepatocytes was observed in the liver when normal lymphocytes were stimulated with PHA-P and culture supernatant was injected into the mesentery vein of dogs. These results suggest that sensitized lymphocytes may produce a factor (or factors) by stimulation with a specific drug-carrier and this factor (or factors) causes cholestasis in the liver.     

Cell-mediated immunity in sarcoidosis: effect of corticosteroids.

The paradoxical skin response in sarcoidosis is a delayed hypersensitivity skin response to an antigen such as PPD which can be elicited only when corticosteroid is present and not when it is absent. Approximately half the tuberculin-negative patients with sarcoidosis are paradoxical responders. In vitro culture of lymphocytes of 10 paradoxical responders with sarcoidosis demonstrated that a response to purified protein derivative of tuberculin (PPD) could be elicited in every patient on at least one occasion both by thymidine uptake and by macrophage migration inhibition factor (MIF) production. The response, when present, was generally at a lower level that that of normal control lymphocytes. No factor was found in the serum which could explain the low activity of the sarcoid lymphocytes in cross-over experiments with normal cells and serum. Hydrocortisone added to the in vitro lymphocyte cultures abolished lymphocyte transformation with respect to both thymidine uptake and MIF production. Varying the conditions of lymphocyte exposure to hydrocortisone, dose, duration of culture and conditions of exposure to hydrocortisone (before and after antigen) failed to enhance lymphocyte stimulation at each and every attempt. Cultures of lymphocytes obtained after oral administration of prednisone were inhibited just as if hydrocortisone had been added in vitro. However, at this time the skin responses of six out of seven patients showed delayed hypersensitivity to previously injected PPD. It is suggested that in addition to the lymphocyte defect paradoxical responders have a defect in their ability to express delayed hypersensitivity. The effect of corticosteroids is to aggravate the lymphocyte defect but in many patients the skin defect is overcome or corrected.     

Functional T cells in rabbits gut mucosal lymphocytes.

Functional studies of Peyer's patch and gut mucosa lymphocytes were performed in mixed lymphocyte culture reaction (MLC), an in vitro measure of the capacity of T cells to recognize and respond to cell surface alloantigens. The data demonstrate the functional effectiveness of both these mucosal lymphocyte populations in one-way MLC, both as responding as well as stimulating cells. Both the responding and stimulating capacities of these cells in MLC were almost the same as observed for lymphocytes obtained from either spleen or thymus. We conclude that functional T cells exist in both rabbit gut lamina propria and Peyer's patches with approximately the same quantitative capacity to respond in the MLC.     

Demonstration of direct involvement of cytokines in graft-versus-host reactions using an in vitro human skin explant model.

A human skin explant model was used to investigate the role of cytokines in graft-versus-host reactions (GVHR). Responder cells from HLA mismatched mixed lymphocyte cultures (MLC) produced GVHR (Grades I-III) in skin explant assays. Cell-free supernatants from these experiments also induced similar histopathological changes in the skin. The greatest degree of correlation between the GVHR observed with responder cells and the supernatant was shown with CD4 enriched MLC (p less than 0.001). Supernatants were assayed for tumour necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) and CD4 enriched MLC populations produced high levels of these cytokines. These results correlated with the grade of GVHR observed in skin explant assays. The GVHR produced by the supernatant alone could be inhibited by both anti-IFN gamma and anti-TNF alpha polyclonal antibodies. The results suggest that TNF alpha and IFN gamma are directly involved in tissue damage during graft-versus-host disease in allogeneic transplant in man.     

Modulation of in vitro myelopoiesis by alloreactive T cell clones

Summary Regulatory effects of mixed lymphocyte culture (MLC)-derived CD4+ human T cell clones on granulocyte-macrophage colony (CFU-GM) formation by normal bone marrow (BM) were studied in an initial attempt to establish an in vitro model for the negative feedback control of myelopoiesis by alloactivated T cells. This is likely to be of clinical significance in the aberrant control of haematopoiesis during some cases of graft-versus-host disease (GVHD) after allogeneic BM transplantation. Whilst 5 such alloproliferative clones generally failed to suppress CFU-GM, the majority of clones with natural killer (NK)-like activity [10], or those with suppressive activity in MLC [2], regularly and strongly suppressed in this system, reinforcing the view that certain T cells may have potent negative regulatory effects on haematopoiesis.This work was supported by Deutsche Forschungsgemeinschaft SFB 120 (Projects A1, A2a and C2)