Lymphocyte Activation: Clinical Aspects

14th International Leucocyte Culture Conference, Heidelberg 21

Lymphocyte Activation: Clinical Aspects

Central Lab. of the Netherl. Rt:d Cross Blood Transf. Service and the Lab. of Exp. and Clin. Immunol. of tht: Univ. of Amsterdam, *Dept . of Pediatrics, University Hospital, Leiden, The Netherlands

T lymphocyte characteristics in bone marrow transplanted patients

A. ASTALDI, R. J. v. D, GRIEND, H. G. [)E BRUIN, C. J. M. LEUPERS, P. TH. A. SCHELLEKENS,

and J. M. VOSSEN""

The relative distribution of T cell subsets, as defined by the monoclonal antibodies OKT. was analyzt:d in peripheral blood lymphocytes of 8 children foUowing bone marrow transplantation for aplastic anemia. The same ce ll s were also studied with biochemical (LDH isoenzyme pattern, 5'-nucleotidase and purine nucleoside phosphorylase enzyme activities, amount of intracellular cyclic AMP) and immunological (binding of peanut lectin, Til and Ty) markers . A close correlation was found during the whole follow up period between the lack of lymphocyte prol iferative response to mitogens and to allogeneic cells on the one hand and a thymocyte-like pattern of the biochemical markers on the other hand. Changes in the binding of monoclonal antibodies were observed with time. The number of lymphocytes binding OKT 3 reached n ormal values shortly after transplantation. In 5 patientS, lymphocytes binding OKT 8 were found in high numbers and lymphocytes binding OKT 4 in low numbers. This resu lted in an invened ratio OKT 4/0KT 8 as compared with lymphocy tes from normal individua ls. In all patients lymphocytes bound abnormally high amounts of OKT lD. In the course of time a trend towards normalization was observed fo r all parameters investigated; however, the kinetics of the recovery showed a marked heterogeneity . From the analysis of th is phenomenon , we conclude that, among other conditions yet unknown, a minimum of 20 % OKT 4-positive lymphocy tes is required fo r a normal proli ferative response to T cell mitogens in vitro. No other co rrelation was found between any lymphocyte phenotype. as defined by the OKT antibodies and proliferative response in vitro. Furthermore, lymphocytes of one patient with chronic Graft-versus-host disease (> 50 % OKT 8-positive) failed to suppress the proliferative response to T cell mitogens of lymphocy tes of the histoidentieal bone marrow donor.

Medizinische Univenitatsklinik Marburg. FRG

Inhibition of the autologous mixed lymphocyte reactivity of normal lymphocytes by serum fractions from patients with Hodgkin's disease

M. B EGEMANN

In previous reports it was demonstrated that the autologous mixed lymphocyte react ivity (MlR) of lymphocytes from patients with Hodgkin's disease is impai red. There is some evidence that fa'tors produced by lymphocy te subpopulati ons are responsible for tbis depressed 'T-cell reactivity and that such factors circulate in the blood of these patients. Therefore we substitu ted serum from patients with Hodgk in 's d isease fo r normal human ABserum in autologous mixed lymphocyte cultures of healthy donors. In these experiments the autologous MLR was su ppressed significantly by 19 of 25 patients' sera as compared to a

22 14th International Leucocyte Culture Conference. Heidelberg

control group of sera from normal persons. Moreover. to characterize the serum fraction inhibiting the T -ceII proliferation we fractionated normal and patients' sera by Amiconmembrane filtration and by Sephadex-gel filtration. In the autologous lymphocyte cultures, fractions with a molecular weight between 50,000 and 300,000 and above 300,000 0 reduced the lymphocyte reactivity more or less. The s trongest suppression in these experiments was seen in fractions with 1I molecular weight of more than 100,000 D, After remov ing IgG from these fractions by a bso rption with protein A, they lost their property to reduce the T-cell proliferation. First experiments suggest that circulating immune complexes in these sera are involved in this inhibition process. These experiments demonstrate that immunoglobulins play an important role in the inhibition by H odgkin's sera of lymphocyte reactivity in vitro, Nevertheless, our data demonstrate that also other factors in different serum fractions take part in these reactions,

Supported by the Deutsche Forsehungsgemeinschaft (SFB 103) and the Kulemann-Stiftung,

Memorial Sloan-Kettering Cancer Center, New York, N ,Y, 10021 , USA

Use of microbial B lymphocyte activators in analysis of primary immunodeficiency

S, CUNNINGHAM-RUNDlES, C. CUt\'NINGHAM-R UNDlES, D. I. MA, F . P . SIEGAL, C. K OSlOFF,

and R. A. GOOD

Lymphocyte activation jn vhro induced by whole cell preparations of E, cofj and S. aureus and an extract of C. aJbt'cans was studied in 39 patients with common variable immunodeficiency having reduced serum immunoglobulins IgG, IgM and IgA and either normal or low numbers of B cells. Thirteen patients with selective IgA deficiency were also studied, No patient had associated malignancy and none were receiving drugs known to affect jn vt'rro lymphocyte cellular response. Lymphocytes were isolated from periphenl blood and cultured jn yj tro in RPMI 1640 with 15 % pooled normal human serum in triplicate. Proliferation was quantified by I~C thymidine uptake and evaluated as mean cpm. The data were obtained over a 4 year period and were compared to results with 39 healthy volunteers matched for each test date. Study of separated normal lymphocytes showed that B lymphocytes were required for nonnal response to t he microbial activators. Study of cord blood lymphocytes indicated that S. aureus and E. coJj are mitagens and that lymphocyte response to C. aJbjcans required presensitiz.ation. The patient group was observed to have marked depression of response to all three activators compared to that of matched controls (p < 0.01) and 70 % o f patients had negative response to at least one B lymphocyte activator; however, negative response to one activator and strongly positive response to anomer were found in patients studied individually. Depressed lymphocyte response did not corrdate with either B cell number or Bff ratio. Examination of patient lymphocyte respanse to S. aureus and E. coHin association with serum IgG levels demonstrated that patients with serum IgG < 100 mg/dl had markedly lower lymphocyte responses than those with serum Ig > 300 mgldl. B cell predominance was clearly observed in these serum-supplemented cultures. No similar correlation with PHA activation was observed. I n contrast only 2 of 13 patients with IgA deficiency had negative responses to the microbial activators and 2 additional patients had responses that were 30 % o f normal. These studies further indicate that preparation technique is important in the development of B lymphocyte activators and that strong reagents are specifically useful in the analysis of primary immunodeficiency. The data demonstrate that defective proliferative response in common variable immunodeficiency may be observed independently of B cell numbers which were normal in these patients. An intrinsic defect with implications for essential host defense against infection was clearly recognized.

14th International Leucocyte Culture Conference, H eidelberg 23

Department of Med icine, Yale University School of Medicine, New Haven, Connecticut 065 10, USA

A quantitative assay for measuring the spontaneous activity of human suppressor cells activated in vivo

JOHN M. DWYER and CHALINE C. JOHNSON

A number of diseases have been causally linked to a deficient functioning of immunoregulatory cells. Qualitative studies of such defects have involved the induction in virro of suppressor cell activity by Concanavalin A . More d ifficu lt to study has been the suppressor activity of regulatory cells thought t o be promoting d isease through inappropriately excessive suppression of effector mechanisms. In some primary and acqu ired humo ral immune deficiency states, neoplasia, parasitic, mycobacterial and viral infections, and granulomatous conditions, inadequate immune responses have been attrib uted to inappropriately excessive supp ression by regulatory cells. If this is true, freshly drawn peripheral blood lymphocytes obtained from pat ients with active disease shou ld possess a greater degree of _spontaneous . suppressor cen (SSC) activity than lymphocytes from healthy controls. We have developed and standardized a reproduc ible assay for SSC activity. Allogeneic responder cells are stimulated with supraoptimal (H ), optimal (M), and sub-optimal (L) mitogenic doses of phytohemagglutin (PHA) in the presence of either mitomycin-C-treated B cells (control cultures) or fresh putative SSe. Spontaneous suppressor cell activity is then calculated by the formula:

!:l cpm from responder cells + suppressor cells 1- X 100

!:l cpm from responder cells + .. B. cells In 50 studies of normal blood, significant spontaneous suppressor cell activity was observed,

the percentage supp ress ion ± SEM of the ind icator system being 28 ± 2.5 (H ), 32 ± 2.9 (M ) and 42 ± 3.6 (L). Significant result.<; werc not obtained by examining the response to PHA in the presence or absence of putative suppressor cell s. The number of cells in culture must be kept constant as crowding and the metabolic activity of the putative suppressor cells produced a passive suppression of the immune response. As an allogeneic culture system was used to measure SSC activity it was necessary to determinc whether a mixed lymphocyte reaction could induce suppressor cell activity in the 72 hrs that the responder cells were exposed to

PHA. While allogeneic culture systems did induce significant suppressor cell activity after 7 days of co·cuhure, they could not induce this act ivity in three days. To test th is assay in a system widely beli eved to feature increased sse activity, we studied ten samples of cord blood lymphocytes. These cells suppressed (% ± SEM) adult lymphocy tes responding to PHA by 41 ± 3.6 (H), 44 ± 3.3 (M). and 50 ± 6.2 (L). Spo ntaneous suppressor cell activity was found in both T-enriched and T-depleted peripheral blood fractions, suggesting that more t han one cell may be capable of cxening th is affect. As would be expected if our assay were detecting biologically significant suppressor cell activity, there was an inverse correlation between the response of cord blood lymphocytes to Con A and the level of their spontaneous activity.

Medical College of Georgia and Veterans Administration Medical Center, Augusta, Georgia, USA

Immunocompetence in Hodgkin's disease: a survival predictor

G UY B. FAGUET and HARRY C. DAVIS

We previously demonstrated (J. e lm. Invest. 56: 951, 1975) various degrees of immunoincompetence in 74 % of 35 previously untreated patients with Hodgkin's disease, as judged by

24 14th International Leucocyte Culture Conference, Heidelberg

their lymp hocyte response profile to a wide spectrum of PHA concentrations. Half of the patients are alive and in relapsc-free complete remission 72 to 114 months (median> 83 months) from diagnosis. The remain ing patients have died with the median survival of 18.5 months. The power of immunocompetence (IC) to discriminate between patients with opposed outcomes was evaluated by multiple linea r regression and discriminant analys is a nd compared to that of conventional risk factors. These included stage, constitutional symptoms (CS), histopathology (HP), delayed hyperse:nsitivity reactions (D H R) to a battery of skin tests, sex, age, and circulating lymphocyte: count (LC). IC demonstrated the highest correlation (R = .537) with survival status, followed by age (r = .522), and by CS, DHR, Le, stage and sex (r:5: .314). In addition, IC was a required common denominator for deriving best sets of two or more variables (R = .671 to .814). An IC-based model ineluding age, CS and HP generated a highly discriminant (R = .784) model not significantly improved by stage (p > .159). sex (p > .448). LC (p > .614) or DHR (p > .713). The utility of IC and oftheICbased model was ascertained by classifying each patient acco rding to expected versus known survival status. Correct classification was achieved in 82.4 % of the cases by IC alone and 91.2 % oE the Ie-based model. The generalizability of the models to Hodgkin 's populations was assessed by predicting their multiple correlation coefficients. Pred icted correlation (R) was AS for Ie alone and .71 for the Ie-based model, and was not meaningfu lly improved by stage, sex, LC or DHR. In contrast, the conventional Hodgkin's evaluation traid of stage, CS and H P showed an actual and predicted correlation with survival status of R = .550 and R = .51, respectively, comparable to IC alone but lower than the IC-based model. In addition, the conventional triad correctly classified fewer patients (70.6 %) than Ie alone and the Ie-based model. We conclude the IC is a powerful discriminant risk factor in Hodgkin's discase which appears to exert a pivotal role on survival. An IC-based model not requiring s taging procedure data generated functions o f much greater discriminant power and general izability than the combination of stage, CS and HP. Ie-based models are expected to provide a more discriminating basis for clinical evaluation, treatment selection and for predicting prognosis of patients with Hodgkin 's disease.

lThe Netherlands Cancer Institute, leentral Laboratory of the N etherlands Red C ross Blood Transfusion Service and Un iversity of Amsterdam, The Netherlands

Studies o n the mechanism of drug sensitizat ion in mice: diphenylhydantoin-induced T-cell-dependent proliferation and activation of B cells

H ELCA GLEICHMANN 1, THADDxus RADASZKIEWICZ1 , and STEVEN S. P ALS2

A wide spectrum of diseases, such as the formation of various autoantibodies and dcvelopment of Iymphoprolifcrative disorders can occur in patients under chronic treatment with the anti-epileptic drug dipheny lhydantoin (DPH) (1). To date, the mechanism of the induction of these adverse effects remained obscure. Interestingly, the majority of the DPH-induced symptoms are similar to those resulting from allorcactive T lymphocytes in graft-versus-host reaction (GVHR) in animal and man ( I). Therefore, it was postulated that GVHR-like cell interactions can occur in autologous systems if T cells were activated by DPH-modified Structures on lymphoid cells. Results obtained by means of thc popl.iceallymph node (PLN) assay suppOrt this concept (2). Injection of the hydrophobic compound DPH, which is not 3.

mitogen, induced T -cell-dependent PLN enlargement, wherca..<; the solvent of DPH or the watersoluble drug phenobarbital fai led to do so. Phenobarbital was chosen fo r comparison because it is chemical ly and pharmacologically rd ated to DPH but is not known to induce: such a broad spectrum of diseases. In MLC reaction, T -cell-dependent proliferative responses


of responder cells cultured together with autologous DPH -treated stimulator cells were found. Recent results obtained by in vitro analyses and histologic examination indicated that proliferating B cells comprised the majority of the cells of the draining PLNs. Furthermore, a marked increa~e of IgM- and IgG-secreting cells (100- to 300-fold, respectively ) were found by means of the staphylococcal protein A plaque assay. It is conceivable that the observed DPHinduced B cell ac6vation in mice conrributes to the pathogenesi.'i of autoimmunization and, together with other factors , development of lymphadenopathy in DPH -treated patients.

1. GLEICHMA l\'N, E., and H . Gl. F:ICHMANN. 1980. Spectrum of diseases caused by aUoreaetive T cells, mode of sensitization to the drug diphenylhydanto in, and possible role of SLEtypical self-antigens in B cell triggering. In: Tmmunoregu lation and Auto immunity , R. S. KRAKAUJ:::R and M. K. C ATHCART, cds. E lsevier, North Holland, p. 73 .

2. GUICHMANN, H. 1981. Studies on the mechanism of drug sensitization: T-cell-dependenr popliteal lymph node react ion to diphenylhydantoin . Clin. Immuno!. Immunopathol., in press.

Dept. of Immunology and University Hospital, Uppsala, Sweden

Autologous and allogeneic MLR reactivity in patients with chronic lymphocytic leukemia (eLL)

D . K ABHITZ, C. ANDRICH ETTO, B. SrMoNSSON, and H . WIGZEI.L

Controversial reports exist as to the stimulatory capacity in mixed lymphocyte react ions (MLR) of lymphocytes from patiems with CLL of B-celJ type. Furthermore, it has been reported that T lymphocytes from these patients fa il to react in the autologous MLR (AMLR) using autologous non-T cell s a.~ stimulator. The AMLR is thought to reflect an important immunoregulatory interaction between T lymphocytes and non-T cells. Accord ingly, the defective AMLR i n eLL has been connected with the uncontroll ed clonal B cell proliferation in this disease (1, 2).

We have studied in more detail the role of various mononuclear cell suhpopulations in autologous and allogeneic MLR. In our experimems, highly purified T ce ll populat ions from CLL patients (95 % E-rosetting cells) responded vigorously in allogeneic MLR but fai led to react against autologous non-T cells (obtained by dep let ion of E-roseuing ce lls). However, in all patient.~ stud ied so far , a positive autologous MLR was observed when a particu lar light d~nsity fraction obtained after Percoll density fractionation of patient's non-T cells was used as stimu lator. The very same fraction (Fr. I, density ~ 1.069 g/ Ol i), containing ~ 5 % of total cells recovered from Percoll grad ients was also highly e nriched for cells stimulating in allogeneic MLR. In contrast, fraction III (density ~ 1.074 g/ml), comprising up to 95 % of cdls recovered from Percoll gradients including the vast majority of leukemic B cells, failed to elici t any stimulation of both autologous and allogeneic T lymphocytes. Fr. III cells, however, expressed la-like antigens as judged by f luorescence using monoclonal anti-DR antibodies. In preliminary experiments the absent stimulator activity of Fr. III cells cou ld not be restored by hapten-modification using TNP or FITC, a procedure known to augment self-reactivi ty in normal donors. Thus, all stimulator activity of C LL mononuclear cells could be attributed to

non-leukemic non-T cells, presumabl y monocytes andlor normal B lymphocytes. The defect of autologous MLR in eLL appears to be due to dilution of stimulating cells with inactive blkemic B c eJls.

t. SMITH,]. B., R. P. K NOWLTON , and L. S. KOONS. 1977. J. Nat!. Cancer Inst. 58: 579. 2. WOLOS, J. A., and F. R. DAVEY. 1980. Cancer 45: 893.

Supported by a fe llowship from DFG (Ka 50211).


Section of Clinical Immunology, Cleveland Clinic, Cleveland, Ohio, USA

Assays of suppressor lymphocyte function in SLE patients and first order relatives

R. S. KRAKAUER, M. MAYES, and J. D. CLOUGH

We investigated suppressor lymphocyte function in several patients with SLE and first order relatives by several different assays. These included allogeneic induced suppression of immunoglobulin synthesis, Con-A-induced suppression of immunoglobulin synthesis, and the natural suppression seen at high allogeneic T : B cell ratios. In addition we investigated Con-A-induced suppression of lymphocyte proliferation. Patients studied, unless otherwise noted, were not on steroids or cytotoxic drugs at the time of study. In three families, as noted, more than one individual had SLE. We find that Con-A-induced suppression of lymphocyte proliferation is defective in both active and inactive SLE and also in first order relatives of patients with SLE. Allogeneic suppression of immunoglobulin synthesis in vitro is defective in SLE patients both active and inactive but not in first order relatives of said patients. This holds true both for families with a single patient and those with two patients. Con-A-induced suppression of immunoglobulin synthesis in vitro is defective in patients with active disease, normal in those with inactive disease, and normal in first order relatives. In fact, the degree of this suppressor cell defect correlates with disease activity as measured by native DNA binding. Natural suppression at high allogeneic T : B cell ratios without T cell mitOgens is reduced in a minority of patients with active SLE and is normal in all patients receiving glucocorticoids. We believe that these findings explain discrepancies in the literature concerning whether the suppressor T cell defect in SLE is related to disease activity and that the various suppressor cell assays employed are measuring different subpopulations of S\lppressor T cells. It therefore appears that some populations may represent a necessary precondition for development of disease (perhaps genetically determined) while others may be involved in disease propagation or flare.

Supported by USPHS grant AI-16621 and a grant from the Kroc Foundation for the Advancement of Medical Science.

Department of Internal Medicine, 2nd Medical Clinic and Immunopathology Division; 1st Medical Clinic, Padua University School of Medicine, Italy

Ia antigens on T cells with different affinity for SRBC

A. PEZZUTrO, G. SEMENZATO. C. AGOSTINI, G. GASI'AROrfO, A. CORSANO, and U. FAGJOLO

Much attention has been focused both in mice and man on the role of Ia antigens in the cellular interactions occurring during the immune response. In humans la-like antigens were first recognized on B lymphocytes, monocytes and non-blood cells, but their presence was recently demonstrated also in a low proportion « 5 %) of normal circulating T cells. The function of these la + T cells is at present not completely defined; it has been demonstrated however, that some T cells express Ia antigens after immunological activation. Moreover, the allogeneic help nonnally provided by l' lymphocytes in the Plaque Forming Cell generation assay is abolished when the cells are pre-treated with an anti-b antiserum and complement. These observations suggest that Ia + T lymphocytes may playa regulatory role in the cellular

14th International Leucocyte Cuhure Conference, Heidelberg 27

cooperations in the course of the immune response. In this study, performed on normal peripheral mononuclear cells, we evaluated whether a correlation exists between the Ia antigens expression and the T cell affinity for sheep red blood cells (SRBC). On the basis of their ability to form E rosettes with unsensitized SRBC, two subpopulations of human T lymphocytes were separated from peripheral blood by using a sedimentation technique. The first subset (Active Rosette Forming Cell fraction which comprises abour 30 °/ ... of the T population, ARFC) binds SRBC with an elevated affinity; the second fraction (Late Rosette Forming Cells, which comprise the remaining 70 % of the T cells, LRFC) possesses low affinity receptors. Ia + T cells were identified in the two populations by indirect immunofluorescencc using a monoclonal antibody to framework determinants of HLA-Ia-like antigens. Ia + T cells were mostly detected in the ARPC fraction (9.2 % ± 1.6 SE) whereas LRFC lacked these antigens (1.7 % ± 0.4 SE) p < 0.001. Two distinct types of fa + T cells have been described: large blastoid T cells obtained after in vitro stimulation which display a strong positivity, and a small percentagc of normal circulating T lymphocytes which show a weak expression of Ia antigens. These latter have been shown to increase after immunization and in various diseases. The significance of ARFC is not yet completely determined. Their increase after in vivo and in vitro stimulations could support the hypothesis that we are dealing with a lymphocyte subset actively involved in immune processes. Following this interpretation, the enrichement of Ia + T cells in ARFC may further substantiate this hypothesis.

Cancer Control Agency of British Columbia, Vancouver, B.C., and University of British Columbia, Vancouver, Canada

Modulation 01 lymphocyte activation by plasmapheresis in advanced malignant melanoma

FERNANDO A. SA UNAS, KlAN H. WH., and H Ul.BERT K. B. SILVER

The purpose of this study was to examine the effect of plasmapheresis in the removal of specific and non-specific factors interfering with lymphocyte activation and to evaluate clinical response in seven selected advanced disease malignant melanoma patients denoted A to G. These patient." were subjected to serial plasmaphere~is with prc- and post-treatment serum and lymphocytes evaluated by the following in vitro assays previously described by us (In: Serological Analysis of Human Cancer Antigens, S. ROSfNBERC, Ed., p. 539, Acad. Press, N.Y., 1980; Cancer Res. , 38: 401-407, 1978; and Cancer Res., 39: 5036-5042. 1979): circulating immune complexes (CIC). anti-xenogeneic oncofetal antibody (anti-XOFA), total serum sialic acid (SA), and mixed lymphocyte reactivity (MiR). Each patient was followed for 4,6,7,9,12,16 and 19 months respectively. On one-way MLR stimulation with allogeneic cells, patients' lymphocytes tended to incorporate less thymidine than controls and this value remained depressed or further decreased after plasmapheresis for all 25 treatments in patients A, B, E, F, and G. The single evaluable pre and post MLR in patient C and the first MLR of patient D demonstrated increased thymidine incorporation following plasmapheresis. Conversely, autOlogou~ thymidine incorporation consistently increased in all patients, but not in patients C and D. Each plasmapheresi~ led to a measurable decrease in CIC, anri-XOFA and SA. CIC and anti-XOFA concentrations were independent and related to tumor burden as reflected by SA. A dynamic interaction of immune reactants was observed during 27 plasmapheresis treatments. Assuming that similar clearance rates for CIC, free antibody and unbound antigen were achieved upon plasmapheresis treatment, and that antigen concentration in these patients remained consistently in excess due to increased tumor burden, we observed that factors which reflected patient MLR capability included both pre-treatment concentrations of CIC and free antibody. Patients A, C, D. E, and G reported symptomatic


improvement following treatments in the absence of objective response in their advanced disease. In view of the relationship of tumor burden, CIC and anti-XOFA to effect of plasmapheresis on MLR, more timely treatment in patients with less extensive disease should be evaluated. This information would provide a better understanding of the observed modulation of lymphocyte activation by plasmapheresis.

Klinikum Steglitz, Dept. Haematology, Free University of Berlin, Berlin

Impaired B lymphocyte function in patients with malignant lymphoma and multiple myeloma

G. SIEBER, B. ENDERS, H. T EICHMANN, F. HERRMANN, and H. ROHL

The generation of immunoglobulin-secreting cells upon stimulation with Pokeweed mitogen (PWM) was studied in patients with malignant lymphoma and multiple myeloma using a reverse hemolytic plaque assay. The experiments revealed that different mechanisms are responsible for the defects of B cell activation and differentiation which are characteristic for these diseases.

In Hodgkin's disease the majority of patients with an active stage of the disease exhibited greatly diminished responses of their peripheral B lymphocytes to PWM. In some of these patients suppressor cells were found to cause the unresponsiveness, in others, however, an intrinsic defect of the circulating B cells - sometimes combined with a defective T helper cell capacity - was found. Interestingly, some patients with completely abolished responses of their peripheral B lymphocytes exhibited normal reactivity of their splenic B cells. It could be further shown that the defect of B cell differentiation could be corrected by therapy.

In patients with Non-Hodgkin Lymphoma an intrinsic B cell defect was found to be the only cause for the unresponsiveness observed in most of them. In contrast to the patients with Hodgkin's disease the B cell defect could not be corrected by therapy. Isolated T cells of these patients were functionally normal helper cells, and nonc of the hitherto studied patients had demonstrable suppres.~or cells in the peripheral blood.

We have further studied B cell function in patients with multiple myeloma which have often severe decreased levels of the non-paraproteinemic immunoglobulins in the serum. The experiments revealed that about half of the patients studied had a markedly decreased in vitro B cell reactivity which was due to an intrinsic defect of the cells. Only one patient showed a cell-mediated suppression, whereas humoral suppressive factors in the patients' serum could not he observed.

Department of Renal Transplantation and Immunogenetics, Medical Institute, Riga, Latvian SSR, USSR

Investigation of T ",-effector function in kidney allotransplantation

A. SOCHN EV, B. SHlf, and E. ARJKOVA

The T -effectors of d~layed hypersensitivity reaction (T dh,) were investigated in 26 recipients before and after kidney transplantation by means of microdroplet leucocyte migration


inhibition test (LMIT), using a modification of the technique and formulae described by WE,ESE et al. 1978. Specific antigens for each transplant recipient were prepared from the donor spleen taken at the time of nephrectomy. 16 of the 26 recipient.~ studied before allotransplantation showed leucocyte migration inhibition to the donor specific antigen. Migration index (MI) in this group was MI = 66.1 ± 3.7%. These patients underwent clinical rejection in early postoperative period (3 months) in 80.5 % of cases. The remaining recipients showed no evidence of leucocyte migration inhibition (M I = 95.2 ± 2.5 %) and demonstrated clinical rejection only in 30 % of cases. Investigation of Tdh< -effector dynamics after allotransplantation showed that 2-4 days before or during rejection in 80 % of cases inhibition of leucocyte migration wa.~ observed in LMIT (MI = 58.7 ± 3.7%). According to the data received, the microdroplet agarose LMIT is onr of the acceptable methods of immunologic monitoring in clinical kidney allotransplantation.

Thc Nethcrland.~ Cancer Institute and Central Laboratory of the Netherland.~ Red Cross Blood Transfusion Service, Amsterdam, The Netherlands

Phorbol-ester induced proliferation in human T -cell leukemia

F. A. VYTH-DREESE, J. E. DE VRIES, H. SPITS, and J. H. VAN DJ::R REIJDEN

The mitogenic reactivity of peripheral blood lymphocytes (PBL) from 5 patients with T-cell chronic lymphocytic leukemia (T-CLL) and 6 patients with acute T-celllcukemia (T-ALL) were investigated. The PBL of only 2 T-CLL patients reacted to phytohemagglutinin (PHA); lH-thymidine e H-TdR) incorporation in the PBL of onc of these patients followed normal dose-response curves, whereas the PBL of the other patient were only found to proliferate after prolonged incubation at suboptimal PHA concentrations. The non-responsiveness of the PBL of the other 9 patients could not be attributed to different dose-response cha racteri.~tics or altered kinetics. Stimulation of these PBL with PHA in the presence of the tumor promotor 12-0-tetradecanoyl phorbol in 13-acetate (TPA) resulted in the induction of proliferation in 3 out of 3 T-CLL and 4 out of 6 T-ALL patients. The extent of 3H-TdR incorporation varied from 2500 to 52,000 cpm per culture in the individual patients and was found to depend on the mitogen dose used (concentration range tested: PHA 0.2-2 f.lg /ml, TPA 10- 3 to 10- 1 f-lg/ml). Furthermore, addition of TPA induced an increase in the PHA response of the PBL of the 2 TCLL patients which showed to respond to PHA. A direct correlation was observed between the induction and enhancement of proliferation and the generation of Interleukin 2 (IL2) in the supernatant of PHA + TPA stimulated cultures of 6 out of 7 leukemic cell populations tested. Il2, which acts as a second signal in normal-T-cell proliferation, was produced only in minor amounts after stimulation with TPA alone. This minimal IL2 production showed to correlate with a 3H-TdR incorporation just above background, observed in both T-ALL and T-Cll patients, although the 3H_TdR incorporation in the latter was slightly higher. The results indicate that 1. the lack of PHA reactivity of leukemic T cell cultures can be attributed to the failure of III production, 2. stimulation with TPA (in the concentration range tested) results in borderline 3H_TdR incorporation as well as IL2 production, 3. TPA strongly induces IL2 production in the presence of PHA, which may act as an activating signal. The data will be discussed in relation to the phenotype of the leukemic T cells detected by reactivity with monoclonal antibodies.


Hotel Dieu Hospital, Kingston, Ontario, Canada

Blood mononuclear cell numbers and B cell function in patients with autoimmune thyroid disorders

]. R. WAtL, P. BANDY, and E. A. RYAN

We have previously described decreased levels of peripheral blood total and «activated,. T cells in patients with Graves' hyperthyroidism (GH), subacute thyroiditis (SAT), and Graves' ophthalmopathy (GO) (1). In this study we have measured numbers of blood B cells, mononuclear cells with suppressor function (low affinity T cells, stable T cells, and Ty cells), and T~ cells, in patients with autoimmune thyroid disorders using rosette tests. B cells were also enumerated using a monoclonal antibody against human Ia antigen. In addition tests for B cell function, assessed as lymphocyte transformation responses to the human B cell mitogen Nocardia water soluble mitogen (NWSM), were carried out. Percentage stable T cells was increased in patients with primary hypothyroidism (PH) (mean 8.5 % ± 3.2 SE, n = 8) compared to normal subjects (2.7 % ± 0.47 SE, n = 30, P < 0.05) but normal in patients with GH, SAT, Hashimoto's thyroiditis (HT) and silent thyroiditis (ST). Percentage low affinity T cells was decreased in patients with PH (8 % ± 3.3 SE, n = 7) compared to normals (14.9 % ± 1.3 SE, n = 34, P < 0.05), but normal in patients with other thyroid disorders. Percentage Ty cells was increased in patients with SAT (33% ± 8.9 5E, n = R) compared to normals (15.60

/0 ± 0.4 SEt n = 30, p < 0.01), but normal in patients with GH, GO, ST, HT, and PH, whilst levels of Tu cells were similar in normals and all groups of patients. Pecentage B cells was increased in patients with GH (18.1 % ± 4.6 SE, n = 15) compared to normals (12.1 % ± LOSE, n = 8, P < 0.01), but normal in patients with SAT, 5T, HT, and PH_ The finding of increased numbers of B cells (Ia +) in GH was confirmed using monoclonal antibodies. B cell function was normal in all groups of patients including those with GH (despite increased levels of B cells).

The mechanism for increase percentage Ty cells in SAT is unclear although likely to reflect either a physiological response to thyroid antigen release or a non-specific response to the underlying viral infection. Abnormalities of low affinity and stable T cells in primary hypothyroidism may reflect a non-specific effect of hypothyroidism on immune function (2). Various blood mononuclear cell populations are presently being enumerated in patients with autoimune thyroid disorders using monoclonal antibodies and levels correlated with helper, suppressor, and killer activity.

1. WALL, J. R., B. GRAY, and D. M. GREENWOOD. 1976. Total and ... activated,. peripheral blood T lymphocytes in patients with thyroid disorders. Acta Endocrinologica (Kbh) 85: 753.

2. WALl., J. R., D. M. JOYNER, E. A. RYAN, and S. F. G. WRF.N. 1979. Effect of experimental hyper- and hypothyroidism on Band T lymphocyte reactivity to bacterial, fungal and tissue antigens. Proc. Symposium, Autoimmune Aspects of Endocrine Disorders, Pisa, Italy, Academic Press 33: 215.


University of Adelaide, Woodville, Australia

Spontaneous plaque forming cells in the peripheral blood of patients with systemic Lupus erythematosus

A. G. WANGf:L, A. MILTON, and B. EGAN

A reverse haemolytic plaque assay using staphylococcal protein A coupled to sheep red blood cells (SRBC) after the method of GRONOWICZ et a1. (1976) was set up in Cunningham cham bers. Optima were established for the source and agc of SRBC and for the dilutions of antisera and complemem. Using this method, the numbers of Picoll-H ypaque isolated peripheral blood lymphocytes (PBL) secreting IgG, IgA or IgM without preceding culture or mitogen stimulation were estimated in patients with systemic lupus erythematosus (SLE) and control subjects. Five patients with clin ically inactive SLE at the time of study had values similar to those of the control subjects. IgA secreting cells were the most numerous. In contrast, five patients who had clinically active SL£ had markedly increased numbers of PBL secreting IgG, IgA and IgM. IgA-secreting cells outnumbered the others in four of the five patients. The results confirm the expected polyclonal B cell activation in patients with SLE. Control experiments confirmed that the plaques were due to Ig secretion by lymphoid cells rather than to immune complexes adsorbed onto Fc receptor bearing cells or to passively adsorbed Ig. Preliminary results in two patients with active SLE using native DNA prepared from Tl bacteriophage a.~ the «developing antigen,. suggest that PBL secreting nDNA antibody can also be demonstrated by this method.

1. GRONOWICZ, E., A. COUTINHO, and F. MEI.CHERS. 1976. A plaque assay for all cells secreting Ig of a given type or class. Eur. J. Immunol. 6: 588.

Instimr G. Roussy, Villejuif, France

A monoclonal antibody against a Burkitt lymphoma associated antigen

J. WmLS, M. FELLOUS, and T. TURSZ

A monoclonal antibody, referred to as 38.13, was obtained by fusing murine myeloma cells with Lewis rat splenocytes sensitized with Daudi cells (human Burkitt lymphoma, containing Epstein-Barr virus genome, but Jacking HLA A, B, C and ill microglobulin molecules at the cell surface). 38.13 antibody was demonstrated to be a rat IgM. By complement-dependent


microcytotoxicity and indirect immunofluorescence assays, 38.13 antibody was shown to react specifically with cells derived from Burkitt tumors, including both Epstein-Barr virus genome carrying and Epstein-Barr virus negativc Burkitt lymphoma. By contrast Epstein-Barr virus containing lymphoblastoid cell lines, derived from normal B lymphocytes, were not recognized by 38.13 antibody. Malignant fresh cells from patients affected with various lymphoproliferative disorders were negative, except 4/ 8 cases of abdominal Burkitt-like lymphomas. Normal lymphocytes from peripheral blood, spleen, lymph node, tonsil and bone-marrow and mitogen (PHA, PWM and Con A) activated blasts were also negative. Thus 38.13 antibody apparently recognized a Burkitt-associated antigen, which is clearly not related to EpsteinBarr virus. The pattern of reactivity of 38.13 antibody with various Burkitt lymphoma cells appeared quite heterogenous, and some Burkitt were constantly negative. 38.13 antibody could thus define a peculiar subgroup of Burkitt lymphoma.

National Institutes of Health, Bethesda, MD, USA

Specific anti-influenza antibody production in vitro by cultures of human peripheral blood mononuclear cells (PBMC)

R. YARCHOAN, B. MURPHY, W. STR.OBER, W. E. BIDDISON, R. M. BlAESE, and D. L. NELSON

Much of our understanding of the immunoregulation of the human antibody response has derived from the study of immunoglobulin production in vitro induced by polyclonal activators. In order to study the regulation of the humoral immune system in response to specific antigens, we have investigated the production of specific anti-influenza virus antibody in vitro by cultures of human PBMC stimulated by influenza virus. 2 X 10~ washed PBMC were cultured in flat-bottomed wclls for 12 days in 2 ml of RPMI 1640 medium with 10% fetal calf serum, antibiotics, and 4 mM L-glutamine. Cumulative antibody secretion was measured by ELISA. Antibody could be detected in cultures stimulated with live A/Hong Kong/68 (H3N2). purified fonnalin-inactivated A/Aichi/68 (H3N2), and live B/Hong Kong/ 73 influenza viruses. Antibody production was specific for the stimulating virus as cultures stimulated with A/Hong Kong made anti-type A virus antibody but not anti-type B antibody and vice versa. Antibody production was first detected between days 3 and 5 and reached a maximal rate between days 5 and 8. Cell separation experiments revealed that antibody production by B cells required the presence of both monocytes and T cells. Helper activity could be provided by irradiated (20CO R) T cells and was found in the OKT4+ but not the OKT4 - subset of T cells; B cells cultured with OKT4 + T cells produced an average of 20 times more specific antibody than B cells cultured with OKT4 - T cells. Irradiation (2000 R) of the OKT4- T cell subset to potentially eliminate suppressor T cells did not


significantly augment the helper activity. We have used this system to study specific in vitro antibody production in patients with several immunodeficiency diseases. 5 of 10 patients studied with common variable hypogammaglobulinemia (CVH) produced measurable antibody compared to 10 of 10 normal adult controls. The class of antibody was examined in 4 of the patients who made antibody and compared to 4 normal controls. While the IgM antiinfluenza virus antibody made by the patients (28.2 ± 15.0 ng/ml; mean ± SEM) was comparable to that made by the controls (15.5 ± 5.0 ng/ml), the IgG antibody produced by the patients was significantly less than made by these same controls (5.4 ± 2.9 vs 54.4 ± 36.1 ng/ml). Thus, a subset of patients with CVH can produce specific antibody in vitro. In contrast, no specific antibody was produced in cultures of cells from 2 patients with x-linked hypogammagJohinemia and growth hormone deficiency or 5 patients with WiskottAldrich syndrome; only 1 of 5 patients with ataxia telangiectasia made measurable antibody, and this one patient made approxim'J.[ely 1.5 ng/ml of total antibody. Further studies of in vitro specific antibody responses in normal individuals and immunodeficient patients should advance our understanding of the maturation and immunoregubtion of the human humoral immune system.

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Lymphocyte Activation: Clinical Aspects