Workshop D-3 Mechanisms of Immunological Perturbations

T cell function in burns

Brigham and Women's Hospital/Harvard Medical School, Department of Surgery, Boston, MA., U.S.A.

294. Fundamental decrease of T cell function in thermally injured individuals

MARY L. RODRICK, J. J. WOOD, J. B. O'MAHONY, S. B. PALDER, and J. A. MANNICK

Dysfunction of immune response in thermally injured individuals has been observed both in vivo by anergy to recall skin test antigens and in vitro by depressed lymphocyte blastogenesis assays. Moreover, death in patients surviving the initial injury is most frequently caused by overwhelming sepsis with opportunistic organisms. In a long-term study of immune function in such patients, we have observed decreased in vitro response by lymphocytes to optimal doses of T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), decrease in total T cells (OKT3+) and reversals of helper (OKT4+) and suppressor (OKT8+) cell ratios as well as in vivo anergy. These results appeared to indicate an increase of T suppressor (OKTS+) cell activity. However, other results indicated that this was not the case: (a) production of immunoglobulin G by patient lymphocytes in response to pokeweed mitogen was normal or enhanced (reported to be down regulated by OKTS+ cells), (b) response to high doses of Con A resulting in suppressed proliferative response in normals often did not result in suppression in patients indicating a lack of production of Con A-induced suppressor cells, and (c) further investigation of the number of OKT4+ and OKTS+ cells indicated that the reversal was due to a decrease in OKT4+ cells not an increase in OKTS+ cells. More studies of these patients have shown that initial antibody response to tetanus toxoid (TT) measured as serum antibody was normal in the first 10 days post-immunization, but decreased rapidly compared with the response in normal individuals and although Interleukin-1 production by adherent cells was normal in thermal injury patients, production of IL-2 by T cells was markedly decreased. Further characterization of peripheral blood cells in patients showed markedly increased numbers of immature T cells (OKT6, OKT9 and OKTlO positive). All immune abnormalities were more obvious and prolonged in large thermal injuries and in patients who developed septicemia. Our results indicate that the numerous malfunctions of the immune response in patients not only in T cell-mediated responses, but in humoral antibody production, are caused by abnormal differentiation and regulatory function of T cells and the defect is probably caused more by lack of help than by suppression. The characteristics of this dysfunction bear striking resemblance to acquired immune deficiency syndrome (AIDS) and may represent a temporary form of this disease.

Supported by NIH Grant # SROI GM26016-06.

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T cell function in transplantation

National Institutes of Health, Immunology Branch, National Cancer Institute, Bethesda, Maryland 20205, U.S.A.

295. Effect of donor and/or host T-cell depletions of bone marrow on specific hyporeactivity to skin grafts in mixed allogeneic ally and xenogeneic ally reconstituted animals

SUZANNE T. ILDSTAD, SHERRY M. WREN, and D. H. SACHS

Transplantation between genetically disparate individuals continues to require non-specific immunosuppressants to abrogate the rejection reaction. Models utilizing bone marrow trans­plantation in an attempt to induce specific tolerance in adult animal models have been limited to date by graft-versus-host disease (GVH) and a generalized level of immunoincompetence probably resulting from a failure of appropriate immune cell interactions in the reconstituted host. We have recently shown that reconstitution of lethally irradiated BI0 mice with T cell depleted syngeneic (B 1 0) plus allogeneic (B 1 OD2) or xenogeneic (F344 RAT) bone marrow results in long term survival of animals and specific prolongation of donor-type full thickness tail skin grafts. The effect of selective host and/or donor bone marrow T cell depletion on reconstitution and skin graft hyporeactivity has not been assessed. Reconstitution of recipient mice with untreated syngeneic (host) bone marrow, with or without T-cell depletion of donor­type bone marrow resulted in prompt rejection of donor type allografts or xenografts. Such mixed allogeneically reconstituted animals were repopulated exclusively with host-type cells as demonstrated by trypan blue exclusion micro cytotoxicity assays on peripheral blood lympho­cytes (PBL). In striking contrast, T-cell depletion of the syngeneic bone marrow resulted in specific prolongation of skin allografts or xenografts whether or not allogeneic or xenogeneic bone marrow was T-cell depleted. Such animals exhibited excellent survival and no evidence of GVH disease. In the mixed allogeneic model, skin allografts (BI0D2) survived indefinitely and their appearance was identical whether the donor-type bone marrow was untreated or T-cell depleted. When only syngeneic marrow was T-cell depleted, animals reconstituted exclusively with donor-type cells. When both syngeneic and allogeneic marrow was T-cell depleted, variable percentages of donor and host type elements were detected in the reconstituted host. In preliminary experiments in the xenogeneic model, the MST for skin grafts in recipients in which only syngeneic T-cells were depleted appeared to be shorter (MST = 62 days) than that for animals in which both donor and host T-cells were depleted (MST = 130 days). Mechanisms for this effect are currently under investigation. These results suggest a model for the induction of specific transplantation tolerance with potential application to solid organ transplantation in the clinical setting.

UCLA Medical Center, Div. Ped. Neph., Los Angeles, CA, U.S.A.

296. Spontaneous anti tubular basement membrane antibody (anti­TBM) production by lymphocytes isolated from a rejected allograft

S. JORDAN, R. SAKAI, and S. BARKLEY

The specific reactivity and antibody specificity of infiltrating lymphocytes and mononuclear cells in mediating allograft rejection (AR) is poorly defined. To further define the role of

16th International Leucocyte Culture Conference, Cambridge . 197

antibody-mediated rejection, a rejected allograft from a patient with primary anti-TBM disease was sterilly minced and pressed through a microscreen. The allograft-bound lymphocytes (ABLs) were then isolated by density gradient centrifugation. Using this technique, 8.5 X 106

ABLs were isolated. 1 X 106 washed ABLs/ml were suspended in RPMI 1640 with 20 % FCS and cultured in microtiter plates with media only, or with pokeweed mitogen (PWM) (100 [!g/ culture). The cells were incubated for 6 days and supernatants were collected and assayed for total IgG and IgM by solid phase EIA and reactivity with a normal human kidney target by indirect immunofluorescence techniques (IF). Total IgG production was 200 ng/ml for both spontaneous and (PWM) stimulated cells. No IgM production was detected. IF studies demonstrated IgG-anti-TBM antibodies in the spontaneous supernatants only. IgG antibodies reactive with peritubular capillaries (anti-PTC) were also noted. IgG-anti-TBM antibodies and antibodies reactive with arterioles were subsequently demonstrated by direct immuno­fluorescence techniques in the rejected allograft. The role of anti-TBM antibodies in mediating the AR is unclear, but their presence suggest recurrence of the original disease in the allograft. Anti-PTC antibodies could be important in mediation of the vascular (AR).

lDepartment of Pathophysiology, Institute of Basic Medical Sciences WAM, and 21. Depart­ment of Internal Medicine W AM, L6di, Poland

297. Some mechanisms of cimetidine action on immune system in duodenal ulcer patients

P. MALEd, H. TCHORZEWSKI\ and K. MARKIEWICZ2

Cimetidine, a selective antagonist of histamine Hz-receptor, is therapeutically used to inhibit gastric acid secretion. In addition, it is also thought to act as an immunomodulator. The exact mechanism of cimetidine action on immune system is not yet known.

The aim of the present study was a trial to elucidate some mechanisms of cimetidine action on immune system in duodenal ulcer patients. The following parameters were studied: the suppressor activity of T-Iymphocytes, the generation of Interleukin 1 (IL-1) by macrophages, and of Interleukin 2 (IL-2) by lymphocytes. Changes in the suppressor T-cell activity were studied in 11 patients with duodenal ulcer. Cimetidine was administered intravenously in a dose of 200 mg four times a day for a fortnight. Suppressor T-Iymphocyte activity was determined, using two-step culture, before treatment, after 4 days of the treatment, just before drug withdrawal, and two days and two weeks after the treatment. The investigation on IL-I and IL-2 generation were performed before, during, and one week after cimetidine therapy. IL-I generation was determined by ability of supernatants from two-day cultured adherent cells stimulated by lipopolysaccharide (LPS) to enhance proliferation of PHA-stimulated mice thymocytes. IL-2 generation was determined by ability of supernatants from two-day cultured, PHA -stimulated mononuclear cells to proliferate autologous IS-18-day cultured T­cells (CTC).

Suppressor T-cell activity significantly decreased soon after starting the treatment (p < 0.005), remained low throughout the treatment, and rapidly and significantly (p < 0.005) increased following drug withdrawal. In each of the 5 studied patients IL-I generation diminished, and IL-2 generation enhanced during cimetidine treatment. After drug withdrawal this parameters nearly returned to the values before therapy.

The presented studies showed that cimetidine, a selective Hz-receptor antagonist, deeply changes the mechanisms of immunoregulation in human with duodenal ulcer. Thus, it seems that caution should be exercised, especially in cases of long-term cimetidine therapy of patients with suspected autoimmune disease or predisposed to it. It is also necessary to monitor some of the parameters of cellular immunity. Further studies are needed to explain the role of histamine Hz-receptor in the regulation of the immune response.

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Department of Physiology, McGill University and Department of Pathology, Montreal Children's Hospital, Montreal, Quebec, Canada

298. Macrophage activation in acute and chronic murine graft vs. host disease

F. P. NESTEL, T. A. SEEMAYER, and W. S. LAPP

The prolonged survival of animals undergoing graft vs. host disease (GVHD) associated with generalized immunosuppression may be a consequence of the activation of nonspecific defence mechanisms such as natural killer (NK) cell or macrophage (M0) cytotoxicity. During the early course of GVHD, animals exhibit an increased resistance to tumor transplantation despite marked suppression of both T and B lymphocyte function. We have examined the cytotoxic activity of peritoneal exudate cells in acute and chronic GVHD between days 8 and 30 post GVH induction. AB6F1 mice were treated with either A or B6 lymphoid cells and GVHD was monitored histologically by the appearance of lymphocyte infiltration and lesions in nonlymphoid organs. M0 tumoricidal activity was examined in a 48 hr Indium-Ill radioisotope release assay. Augmentation of NK activity during the early course of GVHD has been previously described and therefore M0 and NK function were differentiated by the use of MDAY-D2 and MDW4 tumor cell variants which are respectively M0 sensitive, NK resistant and M0 resistant, NK sensitive. Cytotoxic activity was not observed either spontane­ously or following in vitro addition of 5 ng/mllipopolysaccharide (LPS). The presence of both macrophage activating factor (MAF) and LPS were required for expression of M0-mediated tumoricidal activity. M0 cytostatic activity mediated by splenic M0 isolated from GVH animals did not however require further in vitro addition of activating reagents. The charac­terization of M0 activation and apparent development of cytostatic activity may provide further insight into survival and graft vs. leukemia observed during GVHD.

Supported by the National Cancer Institute of Canada and the Medical Research Council of Canada.

Department of Pathophysiology, Institute of Basic Medical Sciences W AM, L6di, Poland

299. PMNLs - modulators of alloantigen - induced reactions in vitro and in vivo

K. ZEMAN and H. TCHORZEWSKI

There is evidence that suggest the role of early inflammatory cells in the modulation of different aspects of the immune response. Lymphocytes are often found in inflammatory lesions together with macrophages and polymorphonuclear neutrophils (PMNL). Recently, possible interrelationships between lymphocytes and PMNLs have received much attention. Human neutrophils have been shown to release the specific granule products when those cells are exposed to surface or adherence activators glass in vitro and when they become exudate cells in vivo.

In the present study we have investigated the effect of granulocyte factor (GF) eluted from specific PMNL granules on lymphocyte proliferative responses in the human mixed lympho­cyte reaction in vitro and local graft versus host reaction in vivo.

The results demonstrated that the GF eluated from specific PMNL granules by four times repeated freezing and thawing can diminish the response of lymphocytes in MLR (about 70%). GF secreted by adhering granulocytes inhibited this reaction in about 40%. In vivo experiments have demonstrated that GF had an inhibitory effect on the lymph node enlarge-

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ment during local graft versus host reaction in rats. There was 63.5 % of inhibition. The mechanism of this effects has not been completely elucidated yet.

Inhibition was not dependent on macrophage cooperation. The development of inhibition potency was visiable when GF was incubated with responder cells.

Stable blockade of T suppressor cell functions is suspected because cells from primary MLR modified with GF, tested for suppressor activity in the second MLR containing analogous cells as in the first MLR, did not reveal suppressor cell activity.

AIDS, HTLV

lRuth Ben-Ari Institute of Clinical Immunology, 2Laboratory for Pediatric Research, Kaplan Hospital, and JDepartment of Cell Biology, Weizmann Institute of Science, Rehovot, Israel

300. Immune derangements in clinically healthy male homosexuals

z. BENTWICH!, Y. BERNER!, R. BURSTEIN!, E. GALILI-WEISSTUB!, S. LEVIN2, M. PECHTJ, N. TRAININJ, and Z. T. HANDZELl

A cohort of 160 Israeli male homosexuals (HS) has been screened for clinical signs of Acquired Immune Deficiency Syndrome (AIDS) and their immune state evaluated. None were drug addicts and/or alcoholics. The following immune parameters were studied: Total lymphocyte and T cell subsets, proliferative response to mitogens and to alloantigens, NK activity, interferon system and serum levels of circulating immune complexes (CIC). The results of the study showed that: (a) None of the subjects studied had any clinical signs compatible with AIDS; (b) Marked alterations in number and distribution of T cells, i. e., significant decrease of total TH cells, T cells and TH/Ts ratio compared with normal controls; (c) Impairment of NK activity; (d) Heightened activity of the interferon system, i. e., increased serum levels of a interferon, increased production of induced a and y interferon and presence of antiviral state in the vast majority of the subjects; (e) Increased levels of CIC in 28/160 MHS. These functional immune alterations did not correlate with patterns and/or role in the sexual behavior of the individuals studied. The results of this study show that several functional immune aberrations are commonly found among clinically healthy male homosexu­als in Israel. They cannot be accounted for in this group by drug addiction or alcoholism. Since Israel may still be considered as a «low incidence» area for AIDS, they support the idea that homosexuals are primarily immunodeficient and therefore more prone to develop AIDS.

Department of Pediatrics, Oregon Health Sciences University, Portland, OR 97201, U.S.A.

301. T lymphocyte hyporesponsiveness to Interleukin-2 in the Acquired Immune Deficiency Syndrome

M. s. BORZY and 1. MANSELL

Purified T cell and T helper cell (OKT4+) populations from three male homosexuals with the acquired immune deficiency syndrome (AIDS) characterized by opportunistic infections

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and/or Kaposi's sarcoma and 14 male homosexuals with persistent generalized lymph­adenopathy (PGL), fever and weight loss were assessed for responsiveness to interleukin-2 (IL-2) to explore the role of IL-2 in the T cell proliferative defect seen in AIDS. Short-term exposure to concanavalin A was used to induce cell surface receptors for IL-2 (1), and then varying numbers of T cells and T helper cells were cultured for three days in the presence of a saturating amount of purified, human IL-2 with proliferation estimated by the uptake of a pulse label of tritiated thymidine. T cells from all three AIDS patients showed significantly decreased responsiveness to IL-2 at one or more cell dose as compared to control. Similarly, the mean response to IL-2 of T cells from the PGL patients was also significantly depressed to approximately 30 % of the mean of the control response at all cell doses. T helper cell-enriched populations showed an increased mean response as compared to T cells, and the response varied from 56 % of control at the lowest (5 x 104 cells/culture) cell dose to 77 % at the highest (2 x 105) cell dose. These results suggest that part of the hyporesponsiveness of PGL T cells was due to a decreased number of IL-2 responsive cells; however, since the T helper cell population was depleted of T suppressor (OKT8+) cells, the enhanced responsiveness of the T helper cell population may also be due to the removal of an inhibitory cell population. Analysis of individual PGL patient T cell and T helper cell responsiveness to IL-2 revealed three groups. Group I, four patients (29 %), had both normal T cell and T helper cell responsiveness; Group II, four patients, had either normal T cell or normal T helper cell responsiveness; and Group III, six patients (42 %), had both decreased T cell and T helper cell responsiveness. These groups may represent different stages in the course of PGL. Monitoring of these patients has begun to test the possibility that hypo responsiveness to IL-2 may help to identify those PGL patients who will develop AIDS. Thus, these results demonstrate a T cell proliferative hyporesponsiveness to IL-2 in AIDS patients and in some PGL patients and suggest that responsiveness to IL-2 may identify clinically relevant subgroups among PGL patients.

1. LARSSON, E.-L. 1981. Mechanism of T cell activation. II. Antigen and lectin-dependent acquisition of responsiveness to TCGF is a nonmitogenic, active response of resting T cells. J. Immunol. 126: 1323.

Memorial Sloan Kettering Cancer Center, N.Y. New York Hospital-Cornell Medical Center, N.Y. John Hopkins Medical School, MD. U.S.A.

302. Regulation of natural killer, NK, activity in the acquired immunodeficiency disease syndrome, AIDS

S. CUNNINGHAM-RUNDLES, B. SAFAI, C. E. METROKA, B. RUBIN, and G. HAYWARD

Examination of natural killer, NK, activity was undertaken in patients with AIDS associated Kaposi's sarcoma, AIDS-KS and in patients with unexplained generalized lymphadenopathy, LA, at risk for AIDS to determine the functional strength and regulation of cells of the suppressor/cytotoxic phenotype since this population of T cells is proportionately increased in this setting.

NK activity of PBL and lymph node lymphocytes, LN were assessed against the K562 erythroleukemia tumor target in a short term 51Cr release assay either freshly or following preincubation with interferons, IFN, or interferon inducers. Freshly isolated PBL from these patient populations and spontaneously established cell lines were also used as targets.

Endogenous NK activity of PBL against the K562 target was negative in 33 % of patients with AIDS-KS (40 patients) and negative in 48 % of LA (90 patients) in contrast to 0 % of controls (100). 40 % of AIDS-KS did not have precursor NK cells capable of being activated by a IFN. NK activity of LA patients could be augmented in less than 25 % of cases, was

16th International Leucocyte Culture Conference, Cambridge· 201

observed to be uninducible in patients with strong endogenous activity, and was predictive of AIDS. Activated cytotoxic activity could be generated in short term culture with E. coli or OKT-3 which was associated with the appearance of y IFN in the culture supernatant. y IFN production was independent of NK-like expression. LN cells were unable to mediate endogenous NK activity with or without IFN but could by activated in 8 of 9 cases by E. coJi but rarely by a or y IFN despite strong proliferative response. In view of possible viral aetiology in AIDS, fresh PBL from 13 patients were used as putative NK targets with normal controls serving as source of effector cells. In contrast to the normal control fresh PBL targets, which did not bind SICr 13/13 AIDS cell bound SICr and 4/13 were as sensitive to NK lysis as K562 with comparable spontaneous lysis. These studies were expanded using spontaneously established cell lines which proved to be highly sensitive targets with marked effector cell restriction. The lines were found to contain multiple copies of the Epstein-Barr virus by DNA hybridization analysis.

The studies suggest that modulation of NK cell differentiation and activity in AIDS may occur during the development of response to modified cell surface structures involving interaction of host and viral antigens.

Clinical Laboratory of Laakso Hospital, and Minerva Institute, Helsinki, Finland

303. Decreased helper/suppressor T-lymphocyte ratios in healthy subjects not turning tuberculin positive after Calmette vaccination

T. FORSLUND, T. H. WEBER, MA]-GRET WELIN, P. TANI, and F. FYHRQUIST

Alterations in lymphocyte subsets have been described in many diseases, among others tuberculosis and sarcoidosis. Reduced numbers of T -helper (T H) lymphocytes and a decreased TH/T-suppressor (Ts) cell ratio have been reported in certain immunodeficiency states, most notably in AIDS. In Finland all children are vaccinated against tuberculosis in early infancy and they mostly develop skin test positivity to tuberculin. Those remaining negative are revaccinated at the age of 11-15 years. However, a small number of individuals, below 5 %, remain tuberculin negative to at least 10 TV throughout their lifetimes for unknown reasons. We have examined the lymphocyte subsets in tuberculin negative young females attending a school for nurses in Helsinki and in age-matched tuberculin positive schoolmates. The lymphocyte populations were examined by immunofluorescence, using specific monoclonal antibodies and polyclonal antibodies against immunoglobulins. The total number of lympho­cytes, the numbers of T- and B-Iymphocytes as well as the number of immunoglobulin positive B-Iymphocytes were similar in both groups. But, highly significant differences were recorded in the numbers of TH and Ts lymphocytes and in the TH/Ts ratios.

TH ± SEM cells/ftl Ts ± SEM cells/ftl TH/Ts

Tuberculin - (n = 10) 740 ± 120 840 ± 80 0.89 ± 0.09 Tuberculin + (n = 9) 1040 ± 110 650 ± SO 1.61 ± 0.11 Significance p > 0.001 P > 0.001 P > 0.001

These changes resemble, but are not as grave as those reported in AIDS. However, the examined tuberculin negative young women were healthy nurse students, not reportedly showing any increased susceptibility to infections or other diseases. The fact remains, that this group showed an abnormal response to tuberculin after Calmette vaccination. At the present time we do not know whether the lymphocyte abnormality is hereditary or acquired. Out data indicate the possibility of significant changes in the peripheral blood lymphocyte subpopula­tions of certain individuals, a phenomenon not necessarily connected to any disease.

Supported by the Sigrid Juselius Foundation, Helsinki, Finland.

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Immunology Branch, Centers for Disease Control, Atlanta, GA 30333, U.S.A.

304. Identification of surface antigens on immunoglobulin-secreting cells found in homosexual men with generalized lymphadenopathy

L. S. MARTIN, J. S. McDOUGAL, T. J. SPIRA, and S. LOSKOSKI

One of the abnormalities found in patients with AIDS or homosexual men with generalized lymphadenopathy (a prodrome to AIDS) is the presence of activated B cells which spontane­ously secrete immunoglobulin (Ig). Even though these abnormal cells may be detected in a reverse plaque assay, they constitute a very small percentage (usually less than 1 %) of the total number of lymphocytes, making identification of surface antigens on these cells very difficult. To identify surface antigens on Ig secreting cells isolated from lymphadenopathy patients, ficoll-hypaque separated cells were first reacted at 4 °C with monoclonal antibody, followed by a FITC-conjugated goat anti-mouse Ig reagent. Secretion of Ig was then assessed in a reverse plaque assay using a modified Cunningham chamber in which the individual plaque forming cell (PFC) could be viewed with an FA microscope. Using this technique, surface antigens on Ig secreting cells could be identified. In some instance, T cells were depleted by a sheep red blood cell rosetting procedure. In both cases, cells which were identified as Ig secreting PFC were found to be reactive with monoclonal antibodies 4F2 (kindly donated by A. Fauci, NIH, Bethesda, MD) and OKTlO, both of which detect antigens on activated T and B cells. PFC were unreactive with T3, T11, TQ1, B1 and M02. In preliminary experiments, some, but not all PFC, were found to be reactive with B4 and anti-Ia. Depletion of the T cells greatly enriched for both Ig secreting cells and 4F2+ cells. None of the monoclonal antibodies or fluorescein reagents used in this study inhibited the PFC response compared to similarly processed controls. However, the 4°C preincubation did reduce the total PFC detected. The reactivity of Ig secreting cells with monoclonal antibodies specific for mature plasma cells is pending the availability of these antibodies. Analysis of pokeweed mitogen stimulated normal lymphocytes is now in progress.

Program in Infectious Diseases and Clinical Microbiology, The University of Texas Medical School at Houston, PO Box 20708, Houston, TX 77025, U.S.A.

305. T cell recognition of antigen acquired by in vitro priming to Cryptococcus neoformans

GERALDINE P. G. MILLER

A significant number of adult patients have transient or permanent deficiencies of cellular immunity secondary to steroid or cytotoxic therapy for allografts, tumor therapy, and autoimmunity as well as secondary to AIDS. Cryptococcus neoformans, a fungus, is one of several opportunistic pathogens which may cause fatal infection in these individuals. The immune functions which protect normals from this disease and are apparently lacking in immunocompromised patients are not well characterized. To study these immune responses, we have developed an assay measuring in vitro proliferative response to this organism. Lymphocytes from normal, non-sensitized individuals proliferated during primary culture with this organism with late peak proliferation (day nine of culture). During this primary response in vitro, a T8+ subpopulation expanded and little or no IL2 was found in superna­tants of these stimulated cultures. When previously stimulated normal cells were rechallenged

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with the organism in vitro, in the presence of fresh irradiated autologous antigen presenting cells, the peak response was shifted four to five days earlier, the T8+ population was not increased, and IL2 was detected in the supernatants of restimulated cultures. On initial culture, lymphocytes from patients recovering from infection demonstrated early proliferative responses and IL2 production. Two patients were studied before and during therapy while clinically recovering: initial studies revealed their cells behaved like those of non-sensitized individuals. On repeat study, several weeks later, their responses were similar to those of recovered patients and secondary responses of non-sensitized cells. Two patients failed to develop these responses and subsequently died. The studies demonstrate acquisition of T cell recognition of antigen during primary in vitro stimulation of non-sensitized cells and in vivo acquisition of T cell responses in recovering patients. Recently derived cryptococcus-specific T cell lines will allow us to characterize the cellular basis of in vitro acquisition of T cell recognition of this opportunistic pathogen.

Departments of Microbiology and Immunology, The John Curtin School of Medical Research, Australian National University, Canberra, Australia

306. Immunosuppression by a fungal metabolite and as possible aetiology in AIDS

A. MULLBACHER and R. EICHNER

Aspergillus fumigatus, a natural human pathogen, and closely related fungi can produce a metabolite, gliotoxin (GT) or related compounds, all possessing the distinct epidithiadioxo­piperazine ring structure. «GT» at concentrations of 5-50 ng/ml is a potent inhibitor of phagocytosis by macrophages. The induction of alloreactive and MHC-restricted cytotoxic T cells in vitro does not occur with GT-treated stimulator cells. This inhibition is prevented by addition of supernatants of Con-A activated spleen cells. Addition of «GT» to MLC after 48 h has no effect on the generation of 5 day in vitro alloreactive cytotoxic T cells. «GT» also inhibits cell proliferation of spleen cells by either LPS or Con-A. Peripheral blood leucocytes of AIDS patients exhibit similar response patterns as GT-treated rodent lymphoid cells. Thus, we propose that an opportunistic fungal infection in immunocompromised individuals leads to chronic immune dysfunction as observed in AIDS, and is the result of the effects of «GT», or a «GT» like fungal metabolite, on cells of the lymphomyloid lineage.

Immunology Branch, Centers for Disease Control, Atlanta, GA 30333, U.S.A.

307. Alterations in functional subpopulations of the T helper and T suppressor cell populations in AIDS and chronic, unexplained lymphadenopathy

J. K. A. NICHOLSON, J. S. McDOUGAL, T. J. SPIRA, and B. M. JONES

A common finding in AIDS patients is a decrease in the percentage and number of helper T cells (T 4) in the peripheral blood. To determine whether this decrease in T 4 cells was due to a decrease in a particular subpopulation of T4 T cells, we double labeled lymphocytes from AIDS patients or patients with chronic, unexplained lymphadenopathy (a prodrome to AIDS)

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for T4 or TS (suppressor/cytotoxic cells) and Ia or LeuS. (Ia on T cells is a marker of activation; LeuS identifies helper [LeuS-] and feedback suppressor [LeuS+] subpopulations on T4 T cells.) We found a decrease in T4:LeuS+ T cells was responsible for the low numbers of T4 cells in the lymphadenopathy patients. In AIDS patients, there was a decrease in both T4:LeuS+ and T4:LeuS- cells. In both groups of patients, there was a normal representation of Ia on the T 4 + T cells. The increase in TS cells in both patient groups was due to increases in both Ia+ and Ia- T8 cells and LeuS- TS cells.

North Shore University Hospital- Cornell University Medical College, St. Luke's Roosevelt Hospital Center, Nassau County Medical Center and Downstate Medical Center, New York, U.S.A.

308. Abnormalities of B lymphocyte functions in patients with the acquired immunodeficency syndrome (AIDS) and in certain populations at risk

S. PAHWA, M. LANGE, T. QUI LOP, M. GRIECO, A. WARREN, S. FIKRIG, and R. PAHWA

The present study was aimed at investigating B lymphocyte functions in adults and children with established or suspected AIDS and in homosexual (HS) males who were either asymp­tomatic (AS) or had minimal symptoms (S) but did not fit the CDC criteria for AIDS. Functions of B lymphocytes were assessed in polyclonal and antigen-specific assay systems. Polyclonal stimuli included pokeweed mitogen (PWM, T-dependent), Epstein Barr virus (EBV, T-independent) and Staph. aureus of Cowan strain (SAC) with which proliferation of B cells is T -independent but their differentiation into immunoglobulin secreting cells (ISC) is T­dependent. Specific antibody secreting cells (ASC) were generated in culture in response to primary in-vitro sensitization with sheep erythrocytes. ISC and ASC were quantified in reverse and direct hemolytic plaque assays respectively. Delayed addition of the T-cell mitogen concanavalin A (Con A) to lymphocyte cultures was used to amplify ASC responses. As shown below, ISC and ASC responses were blunted in all test groups, and were most severely affected in the symptomatic HS and in patients with AIDS:

n PWM HS:AS (1S) 54.1 ± 14.6 S (17) 6.1 ± 2.2 AIDS: Adult (IS) 17.9 ± 3.S Children ( 5) 7.3 ± 4.1 Normals (20) 90.3 ± 7.6

ISC x 103/10. cells SAC EBV 59.2 ± 13.6 21.4 ± 3.S 17.7 ± 6.7 9.9 ± 2.0

22.0± 5.1 13.2 ± 3.2 22.2 ± 11.6 2.2 ± 0.7 92.4 ± 10.5 3S.S ± 4.1

ASC/1 D· cells SRBC SRBC+Con A

S7.7 ± 29.S 277.4 ± 59.4 29.5 ± 9.2 164.0 ± 42.4

26.5 ± 6.0 66.S ± 9.6 21.0 ± 7.4

159.2 ± 16.9 46.5 ± 14.2

693.6 ± 109.3

T cell subset abnormalities and marked deficiency in T helper function were noted in symptomatic HS and patients with AIDS. Intrinsic B cell dysfunction was suggested by decreased proliferative responses to SAC and increased spontaneous ISC in addition to the poor ISC and ASC responses. Clinical follow-up suggested a relationship between morbidity and mortality and severity of B cell dysfunction. These findings suggest that a combined Band T cell immunodeficiency prevails in AIDS amd may also be present in AS and S individuals in certain risk groups. Antibody replacement might thus be of potential benefit as adjunctive therapy in individuals with defective B cell function.

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Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205, U.S.A.

309. Transformation of human T -cells by HTL V

M. POPOVIC and R. C. GALLO

Human T-cell Iymphotropic retroviruses (HTLV) comprise three major subgroups of human retroviruses. Subgroup I and II (HTL V-I and HTL V-II), also called human T-cell leukemia/lymphoma viruses, are characterized by their ability to transform in vitro human mature T-cells (OKT4+). The third subgroup of human T-cell Iymphotropic retroviruses (HTLV-III) exhibits mainly cytopathic effect upon infection of mature T-cells (OKT4+). Seroepidemiological, in vitro biological and nucleic acid hybridization studies indicate that the most common HTL V-I isolate is etiologically associated with mature T-cell malignancies of adults which clusters in the south of Japan, the Caribbean and Africa. HTL V of subgroup II (HTL V-II) was first isolated from a patient with aT-cell variant of hairy cell leukemia and, to date, this is the only isolate obtained from a patient with a neoplastic disease. Although there are distinct immunological and nucleic acid differences between HTL V-I and HTL V-II, both viruses effectively immortalize human T -cells in vitro. The infected T -cells become trans­formed and differ from normal uninfected T -cells in several well defined in vitro characteris­tics: exhibit permanent growth, altered growth pattern and morphology; demonstrate a decreased requirement or complete independence of exogenous TCGF; possess constitutive production of severallymphokines and expression of TCGF receptors; and express new HLA antigen(s) detected with alloantisera and monoclonal antibodies. In addition, HTLV infection of human helper or cytotoxic T -cells leads to stable alterations of specific T-cell functions. Some possible mechanisms of T-cell transformation induced by HTL V will be discussed.

Beilinson Medical Center, Ichilov Hospital and the Weizman Institute of Science, Israel

310. Effects of a thymic hormone (THF) in vitro on cell-mediated immune functions in patients with acquired immunodeficiency syndrome (AIDS) as tested by theophylline sensitivity and monoclonal antibodies

B. SHOHAT, A. GELLER, I. BLUM, E. MARILUS, H. JOSHUA G. DELPRE, E. THEODOR, and N. TRAININ

T cell subset number and function were studied in 3 Israeli males suffering from AIDS, 2 healthy homosexuals and 3 normal donors, using 3 different methods: 1) monoclonal antibodies OKT4+ and OKT8+; 2) theophylline sensitivity; 3) xenogeneic local graft-versus­host reaction. Sera from the above were tested for suppressive activity on control lymphocytes. The effect of a thymic hormone, THF, in vitro on the cell subsets was also investigated. Serial monitoring ofT cell subsets during a period of 3-12 months demonstrated a reversed T helper: T suppressor ratio in the 3 AIDS patients and in one of the healthy homosexuals who had been a partner of one of the AIDS patients for two years. Incubation of the patients' lymphocytes with THF did not abolish the T suppressor activity nor was the percentage of theophylline suppressor cells diminished. However, THF was found to induce helper cell activity. A suppressor factor which could not be related to acid-labile alpha interferon was demonstrated in the sera of the AIDS patients but was not found in the sera of the healthy homosexuals. THF neutralized the suppressor effect of this serum factor when tested on normallympho­cytes. Addition of AIDS sera to normal T helper cells abolished the helper activity whereas its addition to normal isolated T suppressor cells enhanced the suppressor activity.

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Cyclosporin

Department of Pathology, University of Southern California School of Medicine, Los Angeles, CA 90033, U.S.A.

311. Cyclosporin A inhibits mitogen- and antigen-induced lymphocyte proliferation by different mechanisms

J. F. P. DIXON, K. UYEMURA, and J. W. PARKER

Cyclosporin A (CsA) is a potent immunosuppressant that is effective in preventing allograft rejection. It has been found to inhibit in vitro mitogen- and antigen-induced lymphocyte transformation. This inhibition has been widely assumed to be an effect of CsA on lympho­cytes, particularly T lymphocytes, but we have recently found that the inhibition of mitogen­induced lymphocyte transformation by CsA is due to its effect on adherent accessory cells. In contrast, we now show that for antigen-induced lymphocyte transformation, the principal effect of CsA is on lymphocytes. Using human peripheral blood, we prepared highly purified lymphocyte populations (PL) which did not respond to mitogens without the addition of adherent cell populations (AC) highly enriched for macrophages. The PL were treated with neuraminidase/galactose oxidase (NAGO), phytohaemagglutinin (PHA), or concanavalin A (con A). AC were treated with NAGO (indirect oxidation) or purified protein derivative (PPD). During the first 6 hr of incubation, either PL or AC were treated with 0.1 ug CsA/ml. Following this the cultures were washed extensively and the PL and AC were combined in fresh medium. After 60 hr (120 hr for PPD) the cultures were labelled with 3H-Tdr and harvested. We found that combining CsA-treated PL with untreated AC produced little inhibition of the proliferative response to PHA, con A, or direct or indirect NAGO oxidation, but a major inhibition (80 %) of PPD-induced proliferation. When untreated PL were combined with CsA-treated AC, the situation was reversed with the proliferation induced by PHA, con A, and direct and indirect NAGO oxidation being greatly inhibited (86, 90, 92, and 84 % respectively) and the response of the PPD cultures being unaffected or actually improved. Thus mitogen-induced lymphocyte transformation is blocked by the effect of CsA on accessory cells, whereas the CsA blocking effect in PPD-stimulated cultures is due to a direct lymphocyte effect. These results suggest not only a differential CsA effect, but that different populations of accessory cells operate in the two systems.

lThe Weizmann Institute of Science, Rehovot, 76100, Israel, 2The National Cancer Institute, Bethesda, MD 20205, U.S.A., and 3DuPont de Nemours & Co., Glenolden Laboratory, Glenolden, PA, 191036, U.S.A.

312. Cyclosporin A blocks antigen-induced maturation and lymphokine secretion by memory-like cytotoxic T lymphocyte hybridomas

Y. KAUFMANNl, A. E. CHANG2, R. J. ROBB3, and S. A. ROSENBERd

Cyclosporin A (CsA) is being used as a primary drug to suppress the rejection of non­matched organ transplants. The effect of CsA on immune function has been attributed to its

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ability to abrogate proliferation of T lymphocytes by suppressing synthesis of Interleukin-2 (IL-2) and by blocking expression of IL-2 receptors. The mechanism by which CsA suppresses T cell responses is not yet known. We have employed bifunctional cytotoxic T lymphocyte (CTL) hybridomas, reminiscent of memory CTL, to analyze the effect of CsA on T cell helper and effector functions. These monoclonal hybridomas grow independent of exogenous stimuli, do not express receptors for IL-2 and are induced by T mitogens or cells presenting H-2Db antigens to secrete IL-2 and lyse H-2Db target cells (1, 2). Thus they provide a unique system to study the direct effects of CsA on activation and expression of these T cell activities. We have found that CsA at 20 ng/ml blocks antigen and lectin-driven maturation of the cytolytic activity and IL-2 secretion without affecting cell proliferation. In addition, one monoclonal hybridoma, which is induced by Con A to secrete colony-stimulating factors (CSF) as well as IL-2, is concomitantly blocked by CsA for production of both IL-2 and CSF. We have also found that not only stimulation of lymphokine secretion is blocked by CsA but also ongoing IL-2 production is inhibited. Thus, when CsA is added to preactivated hybrid­omas, IL-2 secretion proceeds uninterrupted for 60 min after CsA addition and then stops, suggesting that an inhibition occurs at a post transcriptional step. These results are consistent with a mechanism of action of CsA on T cell clones, involving a direct block of the induction of multiple lymphokines and the interference of ongoing lymphokine secretion by preactivated T cells. In addition the results suggest that CsA directly interferes with the antigen responsive­ness of helper-independent CTL, as represented by these hybridomas, and prevents their maturation.

1. KAUFMANN, Y., G. BERKE, and Z. ESHHAR. 1981. Proc. Nat!' Acad. Sci. USA 78: 2502. 2. KAUFMANN, Y., and G. BERKE. 1983. J. Immuno!. 131: 50.

School of Biological Sciences and Centre for Medical Research, University of Sussex, Brighton BN1 9QG, U.K.

313. Inhibition of lymphokine production by cyclosporin A is due to inhibition of an early Ca2+ -calmodulin-mediated event

]. E. KAY, C. R. BENZIE, and MARIANNE GUDGEON

The immunosuppressive drug cyclosporin A (CS-A) inhibits the induction of proliferation of T lymphocytes by mitogenic lectins. It has been reported that the production of Inter­leukin-2 (IL-2) and other lymphokines is strongly inhibited by CS-A, but that the responsive­ness of primed lymphocytes to IL-2 is not affected. However, several lines of evidence suggest that lymphokine synthesis and secretion are not the primary target of CS-A action in this system. Firstly, supplementation of cultures with lymphokine-rich supernatants or commer­cial IL-2 preparations has only marginal effects on CS-A sensitivity. Secondly, lymphocytes preincubated with mitogens become completely resistant to CS-A within 6 hr, long before the period of maximum lymphokine secretion, and the development of resistance to CS-A is not delayed by culture medium replacement. Thirdly, phorbol esters induce lymphokine secretion by a CS-A resistant process, but do not reduce the sensitivity of activation by lectins to inhibition by CS-A. Our results suggest that the CS-A-sensitive event occurs in the period 1-6 hr after mitogen addition and is required for many later events, including lymphokine secretion. The development of CS-A-resistance after activation is dependent on the continued presence of Ca2+ in the culture medium. It is abolished by inhibitors of calmodulin function, such as chlorpromazine, but not by cycloheximide (which inhibits protein synthesis), monen­sin (which inhibits protein secretion), ouabain (which inhibits N a + - K + ATPase) or colchicine (which inhibits microtubule formation). The sensitivity of activation to inhibition by CS-A

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and chlorpromazine declines with similar kinetics. We conclude that the primary target of CS-A action is not the synthesis or secretion of lymphokines, but a CaH -calmodulin-dependent process occurring in the first few hours after activation that is essential for the propagation of the signal inducing lymphocyte proliferation.

Supported by the Medical Research Council.

Division of Molecular Pharmacology, Department of Pharmacology and Toxicology, Medical School Hannover, D-3000 Hannover 61, FRG

314. Cyclosporin A inhibits lymphocyte activation by interfering with the early activation of plasma membrane phospholipid metabolism

M. SZAMEL, P. BERGER, and K. RESCH

The cyclic peptide, cyclosporin A, a new immunosuppressive agent, is increasingly used in transplantations. Although its cellular target the T lymphocyte has been defined, the molecular mechanism of its action is still unclear. Rabbit lymph node and thymus lymphocytes were stimulated with concanavalin A (conA). Cyclosporin A inhibited dose-dependently the induction of RNA and DNA synthesis, complete inhibition was observed at a concentration of 200 nmol/ml. Kinetic studies suggested that the immunosuppressive drug interfered with an early step occuring in activated lymphocytes. Amongst the earliest changes detectable in stimulated lymphocytes the turnover of plasma membrane phospholipids is increased, predo­minantly of their fatty acid moieties, catalized by the membrane bound lysolecithin acyltrans­ferase. Cyclosporin A at concentrations similar to those inhibiting macromolecular synthesis also inhibited the conA -stimulated increase in the incorporation of labelled fatty acids into cellular phospholipids. When lymphocytes were stimulated with conA for 1 hour incorpora­tion of labelled oleic acid and arachidonic acid approximately doubled in plasma membrane phospholipids. Cyclosporin A at a concentration of 200 nmol!ml prevented the elevated incorporation of labelled fatty acids into plasma membrane phospholipids of conA-stimulated cells. Concomitantly the activation of lysolecithin acyltransferase, the key enzyme for the incorporation of long chain fatty acids into phospholipids, was strongly inhibited in plasma membranes of lymphocytes stimulated in the presence of cyclosporin A. The results suggest that cyclosporin A inhibits the activation of T lymphocytes by interfering with the early changes in the phospholipid metabolism of the lymphocyte plasma membrane.

This work was supported by the Deutsche Forschungsgemeinschaft.

University of Southern California School of Medicine, Los Angeles, CA, 90033, U.S.A.

315. Cyclosporin A-treated lymphocytes stimulated with both 12-0-tetradecanoyl-phorbol-13-acetate and phytohaemagglutinin require macrophages for transformation

K. UYEMURA, J. F. P. DIXON, and J. W. PARKER

Lymphocyte activation is thought to involve two distinct stimulatory signals. The first signal is delivered by a mitogen or antigen, and soluble macrophage and lymphocyte factors supply the second necessary stimulatory signal for lymphocyte transformation. Numerous

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studies of a number of mammalian species have shown that the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) may induce lymphocyte proliferation by replacement of the macrophage signal. We have previously demonstrated that cyclosporin A (CsA), a potent immunosuppressant which is widely used in organ transplantation, inhibits lymphocyte transformation of a number of mitogens by a direct effect on macrophages. However, TPA­treated macrophages and lymphocytes are not affected by CsA. In view of these findings, we have continued to use TP A and CsA as probes to further investigate the signal requirements of lymphocyte activation. Populations of highly purified lymphocytes (PL) and macrophage enriched accessory cells (AC) were prepared from human peripheral blood. PL failed to respond to TP A and also to phytohaemagglutinin (PHA). Proliferation was restored with the addition of AC to these PL. We confirmed that TPA is a potent co-mitogen with PHA. PL treated with both TPA and PHA proliferated with or without the addition of AC or AC treated with CsA. However, PL treated with TPA, PHA and a 6 hour pulse of CsA did not proliferate when placed into culture in the absence of AC. The addition of CsA-treated or untreated AC to these PL restored the response. Our results indicate that TPA/PHA stimulation of PL occurs in the absence of AC. This is in agreement with other published findings. However, the AC requirement of CsA-treated PL suggests that some macrophage­mediated help is required. Further investigations of TP A and CsA effects may facilitate the dissection of the lymphocyte activation process.