Clin. exp. Immunol. (1976) 26, 505-510.
Leucocyte migration inhibitory factor (LMIF) profile in primary and secondary immunodeficiency disease A. J. GORSKI, B. DUPONT, J. A. HANSEN, R. O'REILLY, ELISABETH SMITHWICK, RENATA G ORS KA & R. A. GOOD Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, U.S.A.
(Received 26 April 1976) SUMMARY
Lymphocytes from patients with primary and secondary immunodeficiency disease were tested for capacity to produce LMIF after mitogen and antigen stimulation as well as for ability to stimulate and respond in unidirectional MLC-LMIF assay. Different patterns of immune abnormality in vitro were detectable when Con A and Candida albicans antigen were used. In addition, significant abnormalities in LMIF responding and stimulatory capacity were demonstrated in patients with Hodgkin's disease. LMIF production after stimulation with different agents allows for a better characterization of cellular defects in immunodeficiency disease. INTRODUCTION Generation of lymphocyte-effector molecules (lymphokines) in vitro by human lymphocytes stimulated by antigens provides an important tool in the immunological evaluation of patients with immunodeficiency diseases. Migration Inhibitory Factor (MIF) is composed of two different molecules: macrophage MIF (MMIF) and leucocyte MIF (LMIF) which have been shown in vitro to act on different target cells (Rocklin, 1975). It has been demonstrated that lymphokines may be produced independent of DNA synthesis and cellular proliferation (Rocklin et al., 1974; G6rski et al., 1975). It is, therefore, relevant to study the response of lymphocytes in terms of lymphokine production in parallel with the evaluation of the proliferative response induced with mitogens and antigens. The assay of LMIF activity produced by Con A-stimulated lymphocytes has been shown to be a useful parameter in the assessment of immunological abnormalities in patients with Hodgkin's disease and non-Hodgkin's malignant lymphoma (G6rski et al., 1975). In this paper we extend those observations by the study of mitogen-induced LMIF, antigen induced LMIF and the generation of the mediator following allogeneic cell stimulation in the mixed lymphocyte culture assay in patients with primary and secondary immunodeficiency disease. The study demonstrates different patterns of immunological abnormalities in these patients and makes possible a further characterization of the diseases according to the profile of immunological reactivity. MATERIALS AND METHODS In vitro generation of LMIF. This was performed as previously described (G6rski et al., 1975). Briefly, lymphocytes were obtained by Ficoll-Isopaque gradient centrifugation. The cell number was adjusted to 2 x 106 cells/ml in tissue culture medium RPMI 1640 (GIBCO) supplemented with 2 mm of L-glutamine, 25 mm Hepes buffer, 100 i.u. of penicillin and 100 pg/ml of streptomycin. The medium was also supplemented with 17% gammaglobulin-free heat-inactivated foetal calf serum (GIBCO). 100 p1 of cell suspension was placed in sterile microtitre plates (Cooke Laboratory Products Catalogue No 1-221-24-1). Con A (Con A, Pharmacia Fine Chemicals) stimulation of lymphocytes was performed with addition of 25 pl of mitogen solution giving a final Con A concentration of 4 ug/ml. Unstimulated cultures were supplemented with 25 p1 Correspondence: Dr Andrzej G6rski, Institute of Transplantology, Warsaw Medical School, 02-006 Warsaw,
Poland.
505
A. J. Gdrski et a!.
506
of culture medium. All cultures were performed in triplicate. The presence of minute concentration of residual Con A in the supernatants of the cultures does not affect the migration pattern of the purified human granulocytes (G6rski et al., 1975) Furthermore, when purified lymphocytes were treated with puromycin and after three washes in medium assayed for Con A induced LMIF production, it was evident that the puromycin treatment completely abolished the generation of LMIF activity (G6rski et al., 1976). This demonstrates that active protein synthesis is essential in order to generate the migration inhibition measured in this system. Also, alfa-methyl-D-mannoside treatment of the supernatants under circumstances known to bind residual mitogen does not change their inhibitory activity as compared with non-treated LMIF-rich supernatants. Similar observations were made using PHA as stimulant in capillary migration system (Lomnitzer, Rabson & Koornhof, 1975). Candida albicans antigen stimulation was performed with an antigen obtained from organism grown on Sabouraud agar. The extract obtained after sonication was ultracentrifuged and the supernatant was used. The antigen was used in culture in a dilution of 1: 50 with 25 ,1 antigen solution per tissue culture well. Since the concentration of antigen needed to generate LMIF slightly affected the migration of granulocytes the supernatants from unstimulated control cultures were reconstituted with antigen prior to assaying LMIF activity. One-way LMIF production in mixed lymphocyte cultures. Lymphocytes from patients with Hodgkin's disease were used as stimulator and responding cells in unidirectional MLC-LMIF cultures. In this technique puromycin-treated lymphocytes are able to stimulate normal allogeneic lymphocytes being themselves unable to respond with mediator production (Gorski et al., 1976). Briefly, lymphocytes were incubated with 500 pg/ml of puromycin for 90 min at 370C and washed three times with excessive amounts of Hanks's solution. The cell count was adjusted to 2 x 106/ml in medium RPMI 1640 (see above). 50 p1 of stimulator cells were then mixed with 50 p1 of responding cells (untreated) in triplicate cultures and 50 p1 of serum medium to provide a final concentration of 17% of gammaglobulin-free foetal calf serum in a total volume of 150 p1. The lymphocyte cultures were incubated at 37C in a humid atmosphere containing 5% CO2 for 5 days (mitogen and antigen stimulation) or 4 days (allogeneic cells stimulation). Following this, the supernatants from the cultures were collected and tested for LMIF activity. A pool of purified human granulocytes was used as target cells in the LMIF assay performed in agarose dishes. The Migration Index (MI) was calculated after 24 hr of incubation of the dishes according to the ratio: mean migration area in supernatants from lymphocytes stimulated with antigen (mitogen, allogeneic cells) mean migration area in supernatants from lymphocytes cultured in medium alone* Migration index of less than 0-80 was considered to demonstrate in vitro LMIF production. Control and stimulated cultures consisting of lymphocytes from a normal donor were always included in every experiment. Results of replicate tests performed with lymphocytes from normal donors always remained within the range used to define positive LMIF activity and showed 'a day-to-day coefficient of variation of 11% for the same normal donor. Similar data were obtained from a weekly monitoring of a bone marrow transplant recipient following successful marrow engraftment. Repeated testing of two patients with advanced disease (metastatic renal carcinoma, Hodgkin's disease stage III) consistently showed responses in the negative LMIF range (0 84-0 96). Likewise, serial testing of LMIF activity in frozen aliquots of supernatant from the same stimulated culture over a period of one month resulted in a coefficient of variation of 10%. The variability between replicate migration patterns was usually very small with coefficient of variation within the same experiment of less than 5%. In vitro lymphocyte transformation. This was performed using a standardized technique (Dupont & Good, 1975) with 5 x 104 lymphocytes per well in the round-bottomed microtitre plates. Triplicate cultures were performed in a total volume of 200p#1. The cultures were stimulated with Con A (optimal concentration 35 pgfml). Following incubation at 37°C for 72 hr the cultures were labelled with 0 025 mCi of [14C]thymidine (specific activity 50 Ci/mole, New England Nuclear) and after a further 24 hr incubation harvested on GF/C glass fibre filter paper. The filter papers were placed in scintillation vials and
processed for liquid scintillation counting.
RESULTS Mitogen stimulation of lymphocytes This study demonstrates that lymphocytes of patients with primary and secondary immunodeficiency disorders may be defective in secreting LMIF upon Concanavalin stimulation (Fig. 1), as opposed to cells obtained from normal blood donors. Acute lymphatic leukaemia Lymphocytes obtained from patients with ALL in remission usually responded normally to mitogen stimulation. Two patients gave abnormal LMIF response, but those two patients also had a decreased number of E-rosetting cells and poor proliferative response to Con A.
Chronic lymphatic leukaemia Normal LMIF activity could be generated by stimulated lymphocytes from most patients, although *
Also reconstituted with stimulant or obtained from autologous MLC cultures for MLC-LMIF.
LMIF profile in immunodeficiency disease 10
0.8 ~~O*60
.0
.
O__~@LA l°** ° AV *~~~AA* -AA
00 .0
leukoemia Ieukaemia
507
immunodeficiency disease
ff
and other
tumours(A) FIG. 1. Con. A-induced LMIF production in patients with immunodeficiency disease. (*), Bruton's agammaglobulinaemia; (a), Mycosis fungoides; (A\) di George syndrome; (v) chronic mucocutaneous candidiasis.
their proliferative response was always abnormal and did not exceed 6000 ct/mmn. It is interesting to note that lymphocytes of patients treated with cytotoxic therapy (e.g. leukeran, cytoxan) gave a normal response, while some newly diagnosed non-treated patients demonstrated an abnormal LMIF response.
Severe Combined immunodeficiency disease No mediator production could be detected when lymphocytes of children with this profound abnormality of T- and B-cell function (low serum immunoglobulin level, lack of skin-delayed response to bacterial antigens, abolished in vitro proliferative response to antigens, mitogens and allogeneic cells) were stimulated with Con A. However, after a successful bone-marrow transplant this defect may be corrected. Breast cancer and other solid tumours Most patients with solid tumours had lymphocytes that produced a normal LMIF response in vitro. This was frequently observed in a group of newly diagnosed cancer cases. However, patients with advanced disease with metastases as well as recurrent disease often had their lymphocytic reactivity suppressed or gave borderline values. This was in line with observed depression of proliferative response of their lymphocytes to antigens and mitogens.
Various immune abnormalities Two patients with Bruton's aggammaglobulinaemia and apparently normal T-cell functions in vitro had also lymphocytes with normal LMIF secretory capacity. A weak reactivity was demonstrated with cells of two patients with mycosis fungoides. Also, a patient with di George syndrome showed a questionable response in vitro. Lymphocytes of two out of three patients with chronic mucocutaneous candidiasis were able to release mediator in vitro in response to Con A. Mitogen and antigen stimulation of lymphocytes A variety of different patterns of reactivity could be demonstrated in patients with immunological abnormalities as opposed to normal individuals where a typical pattern shows a strong LMIF secretion after Con A stimulation paralleled by slightly lesser response to antigen (Fig. 2). Patients with Hodgkin's disease frequently unable to respond to mitogen showed a depressed response to antigen, although in some instances a positive reaction to Candida albicans was detectable. In most diseases, however, reactivity to antigen seems to be more susceptible to suppressive effiect of an underlying pathological process than a mitogen induced response. This was often observed in patients with solid tumours, where an immunological abnormality might be manifested as a lack of response to antigen while their lymphocytes still could react to a stronger mitogenic stimulus.
508
A. J. Gorski et al x
a)
'O c) 0
-C
Normal Hodgkin's Disease Acute Chronic Severe Solid tumours Chronic lymphatic lymphatic combined mucocutaneous leukoemia leukoemia immunodeficiency candidiasis disease
FIG. 2. Mitogen and antigen stimulation of lymphocytes: LMIF profiles typical for each disease. Solid columns, Con A; open columns, candida albicans. r
J-Q) 0
CD
x
C
c
ES
0
._
0>8 2 12
.
8
0-0
-0
*
4
L FIG. 3. Relationship between Con A-induced LMIF production and lymphocyte transformation in patients with Hodgkin's disease. Lower limit of normal range is indicated.
Stimulator
Responding
Two way
FIG. 4. LMIF production in mixed lymphocyte cultures between allogeneic cells and Hodgkin's lymphocytes assayed in one and two way. Stimulator cells treated with puromycin, responding untreated lymphocytes. (0), Hodgkins disease; (o), normal.
LMIF profile in immunodeficiency disease
509
Interesting observations were made in patients with chronic mucocutaneous candidiasis. A patient after successful Transfer Factor therapy course manifested by regression of skin lesions and positive skin-delayed response to Candida had lymphocytes that produced a strong LMIF activity to both mitogen and Candida antigen. He also demonstrated normal lymphocyte proliferative response to these stimuli. No mediator production to Con A and antigen was detectable during relapse. In one patient with a negative skin-delayed reactivity to Candida antigen no antigen-induced LMIF could be detected, while she still responded to Con A. Although in most cases immunological abnormalities were manifested by both depression of lymphocyte transformation and mediator production, there are situations in which lymphocytes can produce LMIF but transform poorly or vice versa. This was also apparent in a group of non-treated patients with Hodgkin's disease (Fig. 3). When lymphocytes from patients with Hodgkin's disease were cultured with normal allogeneic cells in a two-way mixed lymphocyte culture reaction, several combinations were observed in which no mediator production could be detected in the supernatant (Fig. 4). Further studies using Hodgkin's cells as stimulator and responding lymphocytes revealed that in most cases these lymphocytes were unable to respond to normal allogeneic cells while their stimulatory function was often retained. In preliminary experiments we were also able to demonstrate that Hodgkin's lymphocytes may be able to suppress LMIF generation in mixed lymphocyte cultures between normal allogeneic lymphocytes while normal 'third party' cells do not. DISCUSSION The data presented herewith indicate that testing for Concanavalin-induced LMIF production allows for a detection of significant immune abnormalities in patients with lymphoproliferative diseases, primary immunodeficiency disorders and cancer. A depression or lack of mediator secretion could be demonstrated in patients with Hodgkin's disease, di George syndrome, some cases of chronic mucocutaneous candidiasis as well as some cases of advanced solid tumours. Patients with ALL in remission usually gave normal responses. Most surprisingly, patients with CLL, in spite of abnormal proliferative responses, can frequently give normal LMIF response. Actually, it is possible to isolate a population of T cells that have intact in vitro functions which is diluted by expanding clone of B lymphocytes (Smith, Knowlton & Koons, 1976). Recently, a normal or even increased production of Blastogenic Factor by CLL lymphocytes has been described (Kasakura, 1975). The author also reported correction of impaired mediator production after an immunosuppressive therapy which is in agreement, with our observations with LMIF activity. It has been demonstrated that blastogenic factor is selectively produced by T lymphocytes (Rocklin et al., 1974). A more complex pattern of immune abnormality may be detected by a simultaneous activation of lymphocytes with non-specific mitogen and specific antigen. Generally, subpopulations of lymphocytes responsible for antigen-induced LMIF production seem to be more vulnerable to suppressive effect of disease than mitogen-activable cells, although in a few cases of Hodgkin's disease we observed the reverse situation. If both T and B cells can make LMIF in response to antigen, it may be inferred that the latter population could respond in those cases. It should be underlined that an overall T- and Blymphocyte defect observed in patients with severe combined immunodeficiency disease accounts for lack of reactivity of lymphocytes to either stimuli. The use of specific and non-specific agents to stimulate lymphocytes in patients with chronic mucocutaneous candidiasis shows that there are at least three patterns of immunological reactivity in that disease, from normal response to either stimulant, retained reactivity to mitogen with an abolished one to antigen, to both responses suppressed. T-cell hypofunction in that disease may depend upon presence of T-suppressor cells (Stobo et al., 1976). Thus, a positive response to mitogen with a lack of mediator production after Candida antigen stimulation might indicate that a stronger Con A stimulus is able to overcome this block while specific antigen-sensitive population remains suppressed. It would be of interest to monitor these patients with the proposed system to delineate changes in in vitro responses during their full clinical course and therapy. Finally, our preliminary data obtained with unilateral MLC-LMIF reaction in patients with Hodgkin's
510
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disease demonstrate that this system provides a new and additional methodology for detecting immune abnormalities. Lymphocytes from patients with Hodgkin's disease frequently stimulate subnormal thymidine incorporation by allogeneic lymphocytes and respond poorly in this assay (Twomey et al., 1975). Since it is suggested that different cell subpopulations respond to mitogenic, allogeneic and antigenic stimuli with DNA synthesis (Tourraine et al., 1976), it is also possible that similar situation occurs with mediator production. Furthermore, the data obtained with CLL patients and Hodgkin's disease support the view that different cell subsets respond with DNA synthesis and lymphokine production (Dorkin, Bash & Waksman, 1975). Therefore, our one-way MLC-LMIF system may be useful in further characterization of cellular defects in immunodeficiency disease. The existence of immune abnormalities in lymphoproliferative disorders, immunodeficiency disease and cancer is a well-established fact (Rees et al., 1975; Graze, Perlin & Royston, 1976; Holm et al., 1976; Hansen & Good, 1974; Simo-Camps etal., 1976). Assuming that Con A and allogeneic lymphocytes stimulate primarily T cells in vitro, these findings may be regarded as a further evidence of impaired function of thymus-dependent lymphocytes in these diseases. We introduce herewith a similar methodology to that routinely used for lymphocyte microculture transformation studies (Dupont & Good, 1975) for in vitro detection of disturbances of mediator production with antigen, mitogen and allogeneic stimulation of lymphocytes and call it LMIF immunoprofile. This work was supported by grants from the American Cancer Society, grants from National Cancer Institute CA-087480851, NCI Program Project Grant. NCI-CA 17404-02, National Foundation March of Dimes, The Special Fund for the Advanced Study of Cancer, The J.M. Foundation and Zelda R. Weintraub Foundation. REFERENCES DORKIN, H.G., BASH, J.A. & WmcsmAN, B.H. (1975) Separation of T cell subpopulations capable of DNA synthesis, lymphotoxin release, and regulation of antigen and PHA responses on the basis of density and adherence properties. Proc. nat. Acad. Sci. (Wash), 72, 5090. DUPONT, B. & GOOD, R.A. (1975) Lymphocyte transformation in vitro in patients with immunodeficiency disease: use in diagnosis, histocompatibility testing and monitoring treatment. Immunodeficiency in man and animals (ed. by D. Bergsma), p. 477. Sinauer Associates, Incorporated. G6RSKI, A.J., DUPONT, B., HANSEN, J.A. & GOOD, R.A. (1975) Leukocyte Migration Inhibitory Factor (LMIF) induced by Concanavalin A: standardized microassay for production in vitro. Proc. nat. Acad. Sci. (Wash.), 72, 3197. G6RSKI, A.J., DUPONT, B., HANSEN, J.A. & GOOD, R.A. (1976) LMIF production in unilateral mixed lymphocyte culture reaction. J. Immunol. (In press.) GRAZE, P.R., PERLIN, E. & ROYSTON, I. (1976) In vitro lymphocyte dysfunction in Hodgkin's disease. I. nat. Cancer Inst. 56, 239. HANSEN, J.A. & GOOD, R.A. (1974) Malignant disease of the lymphoid system in immunological perspective. Human Pathol. 5, 567. HOLM, G., MELLSTEDT, H., BJORKHOLM, M., JOHANSSON, B., KILLANDER, D., SUNDBLAD, R. & SODERBERG, G. (1976) Lymphocyte abnormalities in untreated patients with Hodgkin's disease. Cancer, 37, 751. KASAKURA, S. (1975) MLC stimulatory capacity and production of a Blastogenic Factor in patients with chronic lymphatic leukemia and Hodgkin's disease. Blood, 45, 823.
LOMNITZER, R., RABSON, A.R. & KOORNHOF, H.J. (1975) Production of LIF and MIF by PHA-stimulated lymphocytes. Clin. exp. Immunol. 22, 522. REES, J.C., Rossio, J.L., WILSON, H.E., MINTON, J.P. & DODD, M.C. (1975) Cellular immunity in neoplasia. Antigen and mitogen responses in patients with bronchogenic carcinoma. Cancer, 36, 2010. ROCKLIN, R.E., MCDERMOTT, R.P., CHESS, L., ScLossmAN, S.F. & DAVID, J.R. (1974) Studies on mediator production by highly purified human T and B lymphocytes. J. exp. Med. 140, 1303. ROCKLIN, R.E. (1975) Partial characterization of leukocyte inhibitory factor by concanavalin A stimulated human lymphocytes (LIF). J. Immunol. 114, 1161. SIMo CAMPS, E., ANGUERA, A., MA VICH, J., VIDAL-RIBAS, A., SALA, F., SARRIAs, R., GuMMA, J., GRI, E. & MA PUIGDOLLERS J. (1976) Immunologic impairment in patients with non-lymphoid cancer. Cancer, 37, 724. SMITH, J.B., KNOWLTON, R. & KOONS, L.S. (1976) Lymphocyte reactivity in untreated patients with chronic lymphocytic leukemia. Clin. Res. 24, 336 (abstract). STOBO, J.D., PAUL, S., VAN Scoy R.E. & HERMANS, P.E. (1976) Suppressor thymus-derived lymphocytes in fungal infection. J. clin. Invest. 57, 319. TOURAINE, J.L., TOURAINE, F., HADDEN, J.W., HADDEN, E.M. & GOOD, R.A. (1976) 5-Bromodeoxy-uridine-light inactivation of human lymphocytes stimulated by mitogen and allogeneic cells: evidence for distinct T-lymphocyte subsets. Int. Arch. Allergy, (In press.) TWOMEY, J.J., LAUGHTER, A.H., FARROW, S. & DOUGLAS, CH. C. (1975) Hodgkin's disease: an immunodepleting and immunosuppressive disorder. J. cdin. Invest. 56, 467.
Leucocyte migration inhibitory factor (LMIF) profile in primary and secondary immunodeficiency disease A. J. GORSKI, B. DUPONT, J. A. HANSEN, R. O'REILLY, ELISABETH SMITHWICK, RENATA G ORS KA & R. A. GOOD Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, U.S.A.
(Received 26 April 1976) SUMMARY
Lymphocytes from patients with primary and secondary immunodeficiency disease were tested for capacity to produce LMIF after mitogen and antigen stimulation as well as for ability to stimulate and respond in unidirectional MLC-LMIF assay. Different patterns of immune abnormality in vitro were detectable when Con A and Candida albicans antigen were used. In addition, significant abnormalities in LMIF responding and stimulatory capacity were demonstrated in patients with Hodgkin's disease. LMIF production after stimulation with different agents allows for a better characterization of cellular defects in immunodeficiency disease. INTRODUCTION Generation of lymphocyte-effector molecules (lymphokines) in vitro by human lymphocytes stimulated by antigens provides an important tool in the immunological evaluation of patients with immunodeficiency diseases. Migration Inhibitory Factor (MIF) is composed of two different molecules: macrophage MIF (MMIF) and leucocyte MIF (LMIF) which have been shown in vitro to act on different target cells (Rocklin, 1975). It has been demonstrated that lymphokines may be produced independent of DNA synthesis and cellular proliferation (Rocklin et al., 1974; G6rski et al., 1975). It is, therefore, relevant to study the response of lymphocytes in terms of lymphokine production in parallel with the evaluation of the proliferative response induced with mitogens and antigens. The assay of LMIF activity produced by Con A-stimulated lymphocytes has been shown to be a useful parameter in the assessment of immunological abnormalities in patients with Hodgkin's disease and non-Hodgkin's malignant lymphoma (G6rski et al., 1975). In this paper we extend those observations by the study of mitogen-induced LMIF, antigen induced LMIF and the generation of the mediator following allogeneic cell stimulation in the mixed lymphocyte culture assay in patients with primary and secondary immunodeficiency disease. The study demonstrates different patterns of immunological abnormalities in these patients and makes possible a further characterization of the diseases according to the profile of immunological reactivity. MATERIALS AND METHODS In vitro generation of LMIF. This was performed as previously described (G6rski et al., 1975). Briefly, lymphocytes were obtained by Ficoll-Isopaque gradient centrifugation. The cell number was adjusted to 2 x 106 cells/ml in tissue culture medium RPMI 1640 (GIBCO) supplemented with 2 mm of L-glutamine, 25 mm Hepes buffer, 100 i.u. of penicillin and 100 pg/ml of streptomycin. The medium was also supplemented with 17% gammaglobulin-free heat-inactivated foetal calf serum (GIBCO). 100 p1 of cell suspension was placed in sterile microtitre plates (Cooke Laboratory Products Catalogue No 1-221-24-1). Con A (Con A, Pharmacia Fine Chemicals) stimulation of lymphocytes was performed with addition of 25 pl of mitogen solution giving a final Con A concentration of 4 ug/ml. Unstimulated cultures were supplemented with 25 p1 Correspondence: Dr Andrzej G6rski, Institute of Transplantology, Warsaw Medical School, 02-006 Warsaw,
Poland.
505
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506
of culture medium. All cultures were performed in triplicate. The presence of minute concentration of residual Con A in the supernatants of the cultures does not affect the migration pattern of the purified human granulocytes (G6rski et al., 1975) Furthermore, when purified lymphocytes were treated with puromycin and after three washes in medium assayed for Con A induced LMIF production, it was evident that the puromycin treatment completely abolished the generation of LMIF activity (G6rski et al., 1976). This demonstrates that active protein synthesis is essential in order to generate the migration inhibition measured in this system. Also, alfa-methyl-D-mannoside treatment of the supernatants under circumstances known to bind residual mitogen does not change their inhibitory activity as compared with non-treated LMIF-rich supernatants. Similar observations were made using PHA as stimulant in capillary migration system (Lomnitzer, Rabson & Koornhof, 1975). Candida albicans antigen stimulation was performed with an antigen obtained from organism grown on Sabouraud agar. The extract obtained after sonication was ultracentrifuged and the supernatant was used. The antigen was used in culture in a dilution of 1: 50 with 25 ,1 antigen solution per tissue culture well. Since the concentration of antigen needed to generate LMIF slightly affected the migration of granulocytes the supernatants from unstimulated control cultures were reconstituted with antigen prior to assaying LMIF activity. One-way LMIF production in mixed lymphocyte cultures. Lymphocytes from patients with Hodgkin's disease were used as stimulator and responding cells in unidirectional MLC-LMIF cultures. In this technique puromycin-treated lymphocytes are able to stimulate normal allogeneic lymphocytes being themselves unable to respond with mediator production (Gorski et al., 1976). Briefly, lymphocytes were incubated with 500 pg/ml of puromycin for 90 min at 370C and washed three times with excessive amounts of Hanks's solution. The cell count was adjusted to 2 x 106/ml in medium RPMI 1640 (see above). 50 p1 of stimulator cells were then mixed with 50 p1 of responding cells (untreated) in triplicate cultures and 50 p1 of serum medium to provide a final concentration of 17% of gammaglobulin-free foetal calf serum in a total volume of 150 p1. The lymphocyte cultures were incubated at 37C in a humid atmosphere containing 5% CO2 for 5 days (mitogen and antigen stimulation) or 4 days (allogeneic cells stimulation). Following this, the supernatants from the cultures were collected and tested for LMIF activity. A pool of purified human granulocytes was used as target cells in the LMIF assay performed in agarose dishes. The Migration Index (MI) was calculated after 24 hr of incubation of the dishes according to the ratio: mean migration area in supernatants from lymphocytes stimulated with antigen (mitogen, allogeneic cells) mean migration area in supernatants from lymphocytes cultured in medium alone* Migration index of less than 0-80 was considered to demonstrate in vitro LMIF production. Control and stimulated cultures consisting of lymphocytes from a normal donor were always included in every experiment. Results of replicate tests performed with lymphocytes from normal donors always remained within the range used to define positive LMIF activity and showed 'a day-to-day coefficient of variation of 11% for the same normal donor. Similar data were obtained from a weekly monitoring of a bone marrow transplant recipient following successful marrow engraftment. Repeated testing of two patients with advanced disease (metastatic renal carcinoma, Hodgkin's disease stage III) consistently showed responses in the negative LMIF range (0 84-0 96). Likewise, serial testing of LMIF activity in frozen aliquots of supernatant from the same stimulated culture over a period of one month resulted in a coefficient of variation of 10%. The variability between replicate migration patterns was usually very small with coefficient of variation within the same experiment of less than 5%. In vitro lymphocyte transformation. This was performed using a standardized technique (Dupont & Good, 1975) with 5 x 104 lymphocytes per well in the round-bottomed microtitre plates. Triplicate cultures were performed in a total volume of 200p#1. The cultures were stimulated with Con A (optimal concentration 35 pgfml). Following incubation at 37°C for 72 hr the cultures were labelled with 0 025 mCi of [14C]thymidine (specific activity 50 Ci/mole, New England Nuclear) and after a further 24 hr incubation harvested on GF/C glass fibre filter paper. The filter papers were placed in scintillation vials and
processed for liquid scintillation counting.
RESULTS Mitogen stimulation of lymphocytes This study demonstrates that lymphocytes of patients with primary and secondary immunodeficiency disorders may be defective in secreting LMIF upon Concanavalin stimulation (Fig. 1), as opposed to cells obtained from normal blood donors. Acute lymphatic leukaemia Lymphocytes obtained from patients with ALL in remission usually responded normally to mitogen stimulation. Two patients gave abnormal LMIF response, but those two patients also had a decreased number of E-rosetting cells and poor proliferative response to Con A.
Chronic lymphatic leukaemia Normal LMIF activity could be generated by stimulated lymphocytes from most patients, although *
Also reconstituted with stimulant or obtained from autologous MLC cultures for MLC-LMIF.
LMIF profile in immunodeficiency disease 10
0.8 ~~O*60
.0
.
O__~@LA l°** ° AV *~~~AA* -AA
00 .0
leukoemia Ieukaemia
507
immunodeficiency disease
ff
and other
tumours(A) FIG. 1. Con. A-induced LMIF production in patients with immunodeficiency disease. (*), Bruton's agammaglobulinaemia; (a), Mycosis fungoides; (A\) di George syndrome; (v) chronic mucocutaneous candidiasis.
their proliferative response was always abnormal and did not exceed 6000 ct/mmn. It is interesting to note that lymphocytes of patients treated with cytotoxic therapy (e.g. leukeran, cytoxan) gave a normal response, while some newly diagnosed non-treated patients demonstrated an abnormal LMIF response.
Severe Combined immunodeficiency disease No mediator production could be detected when lymphocytes of children with this profound abnormality of T- and B-cell function (low serum immunoglobulin level, lack of skin-delayed response to bacterial antigens, abolished in vitro proliferative response to antigens, mitogens and allogeneic cells) were stimulated with Con A. However, after a successful bone-marrow transplant this defect may be corrected. Breast cancer and other solid tumours Most patients with solid tumours had lymphocytes that produced a normal LMIF response in vitro. This was frequently observed in a group of newly diagnosed cancer cases. However, patients with advanced disease with metastases as well as recurrent disease often had their lymphocytic reactivity suppressed or gave borderline values. This was in line with observed depression of proliferative response of their lymphocytes to antigens and mitogens.
Various immune abnormalities Two patients with Bruton's aggammaglobulinaemia and apparently normal T-cell functions in vitro had also lymphocytes with normal LMIF secretory capacity. A weak reactivity was demonstrated with cells of two patients with mycosis fungoides. Also, a patient with di George syndrome showed a questionable response in vitro. Lymphocytes of two out of three patients with chronic mucocutaneous candidiasis were able to release mediator in vitro in response to Con A. Mitogen and antigen stimulation of lymphocytes A variety of different patterns of reactivity could be demonstrated in patients with immunological abnormalities as opposed to normal individuals where a typical pattern shows a strong LMIF secretion after Con A stimulation paralleled by slightly lesser response to antigen (Fig. 2). Patients with Hodgkin's disease frequently unable to respond to mitogen showed a depressed response to antigen, although in some instances a positive reaction to Candida albicans was detectable. In most diseases, however, reactivity to antigen seems to be more susceptible to suppressive effiect of an underlying pathological process than a mitogen induced response. This was often observed in patients with solid tumours, where an immunological abnormality might be manifested as a lack of response to antigen while their lymphocytes still could react to a stronger mitogenic stimulus.
508
A. J. Gorski et al x
a)
'O c) 0
-C
Normal Hodgkin's Disease Acute Chronic Severe Solid tumours Chronic lymphatic lymphatic combined mucocutaneous leukoemia leukoemia immunodeficiency candidiasis disease
FIG. 2. Mitogen and antigen stimulation of lymphocytes: LMIF profiles typical for each disease. Solid columns, Con A; open columns, candida albicans. r
J-Q) 0
CD
x
C
c
ES
0
._
0>8 2 12
.
8
0-0
-0
*
4
L FIG. 3. Relationship between Con A-induced LMIF production and lymphocyte transformation in patients with Hodgkin's disease. Lower limit of normal range is indicated.
Stimulator
Responding
Two way
FIG. 4. LMIF production in mixed lymphocyte cultures between allogeneic cells and Hodgkin's lymphocytes assayed in one and two way. Stimulator cells treated with puromycin, responding untreated lymphocytes. (0), Hodgkins disease; (o), normal.
LMIF profile in immunodeficiency disease
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Interesting observations were made in patients with chronic mucocutaneous candidiasis. A patient after successful Transfer Factor therapy course manifested by regression of skin lesions and positive skin-delayed response to Candida had lymphocytes that produced a strong LMIF activity to both mitogen and Candida antigen. He also demonstrated normal lymphocyte proliferative response to these stimuli. No mediator production to Con A and antigen was detectable during relapse. In one patient with a negative skin-delayed reactivity to Candida antigen no antigen-induced LMIF could be detected, while she still responded to Con A. Although in most cases immunological abnormalities were manifested by both depression of lymphocyte transformation and mediator production, there are situations in which lymphocytes can produce LMIF but transform poorly or vice versa. This was also apparent in a group of non-treated patients with Hodgkin's disease (Fig. 3). When lymphocytes from patients with Hodgkin's disease were cultured with normal allogeneic cells in a two-way mixed lymphocyte culture reaction, several combinations were observed in which no mediator production could be detected in the supernatant (Fig. 4). Further studies using Hodgkin's cells as stimulator and responding lymphocytes revealed that in most cases these lymphocytes were unable to respond to normal allogeneic cells while their stimulatory function was often retained. In preliminary experiments we were also able to demonstrate that Hodgkin's lymphocytes may be able to suppress LMIF generation in mixed lymphocyte cultures between normal allogeneic lymphocytes while normal 'third party' cells do not. DISCUSSION The data presented herewith indicate that testing for Concanavalin-induced LMIF production allows for a detection of significant immune abnormalities in patients with lymphoproliferative diseases, primary immunodeficiency disorders and cancer. A depression or lack of mediator secretion could be demonstrated in patients with Hodgkin's disease, di George syndrome, some cases of chronic mucocutaneous candidiasis as well as some cases of advanced solid tumours. Patients with ALL in remission usually gave normal responses. Most surprisingly, patients with CLL, in spite of abnormal proliferative responses, can frequently give normal LMIF response. Actually, it is possible to isolate a population of T cells that have intact in vitro functions which is diluted by expanding clone of B lymphocytes (Smith, Knowlton & Koons, 1976). Recently, a normal or even increased production of Blastogenic Factor by CLL lymphocytes has been described (Kasakura, 1975). The author also reported correction of impaired mediator production after an immunosuppressive therapy which is in agreement, with our observations with LMIF activity. It has been demonstrated that blastogenic factor is selectively produced by T lymphocytes (Rocklin et al., 1974). A more complex pattern of immune abnormality may be detected by a simultaneous activation of lymphocytes with non-specific mitogen and specific antigen. Generally, subpopulations of lymphocytes responsible for antigen-induced LMIF production seem to be more vulnerable to suppressive effect of disease than mitogen-activable cells, although in a few cases of Hodgkin's disease we observed the reverse situation. If both T and B cells can make LMIF in response to antigen, it may be inferred that the latter population could respond in those cases. It should be underlined that an overall T- and Blymphocyte defect observed in patients with severe combined immunodeficiency disease accounts for lack of reactivity of lymphocytes to either stimuli. The use of specific and non-specific agents to stimulate lymphocytes in patients with chronic mucocutaneous candidiasis shows that there are at least three patterns of immunological reactivity in that disease, from normal response to either stimulant, retained reactivity to mitogen with an abolished one to antigen, to both responses suppressed. T-cell hypofunction in that disease may depend upon presence of T-suppressor cells (Stobo et al., 1976). Thus, a positive response to mitogen with a lack of mediator production after Candida antigen stimulation might indicate that a stronger Con A stimulus is able to overcome this block while specific antigen-sensitive population remains suppressed. It would be of interest to monitor these patients with the proposed system to delineate changes in in vitro responses during their full clinical course and therapy. Finally, our preliminary data obtained with unilateral MLC-LMIF reaction in patients with Hodgkin's
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disease demonstrate that this system provides a new and additional methodology for detecting immune abnormalities. Lymphocytes from patients with Hodgkin's disease frequently stimulate subnormal thymidine incorporation by allogeneic lymphocytes and respond poorly in this assay (Twomey et al., 1975). Since it is suggested that different cell subpopulations respond to mitogenic, allogeneic and antigenic stimuli with DNA synthesis (Tourraine et al., 1976), it is also possible that similar situation occurs with mediator production. Furthermore, the data obtained with CLL patients and Hodgkin's disease support the view that different cell subsets respond with DNA synthesis and lymphokine production (Dorkin, Bash & Waksman, 1975). Therefore, our one-way MLC-LMIF system may be useful in further characterization of cellular defects in immunodeficiency disease. The existence of immune abnormalities in lymphoproliferative disorders, immunodeficiency disease and cancer is a well-established fact (Rees et al., 1975; Graze, Perlin & Royston, 1976; Holm et al., 1976; Hansen & Good, 1974; Simo-Camps etal., 1976). Assuming that Con A and allogeneic lymphocytes stimulate primarily T cells in vitro, these findings may be regarded as a further evidence of impaired function of thymus-dependent lymphocytes in these diseases. We introduce herewith a similar methodology to that routinely used for lymphocyte microculture transformation studies (Dupont & Good, 1975) for in vitro detection of disturbances of mediator production with antigen, mitogen and allogeneic stimulation of lymphocytes and call it LMIF immunoprofile. This work was supported by grants from the American Cancer Society, grants from National Cancer Institute CA-087480851, NCI Program Project Grant. NCI-CA 17404-02, National Foundation March of Dimes, The Special Fund for the Advanced Study of Cancer, The J.M. Foundation and Zelda R. Weintraub Foundation. REFERENCES DORKIN, H.G., BASH, J.A. & WmcsmAN, B.H. (1975) Separation of T cell subpopulations capable of DNA synthesis, lymphotoxin release, and regulation of antigen and PHA responses on the basis of density and adherence properties. Proc. nat. Acad. Sci. (Wash), 72, 5090. DUPONT, B. & GOOD, R.A. (1975) Lymphocyte transformation in vitro in patients with immunodeficiency disease: use in diagnosis, histocompatibility testing and monitoring treatment. Immunodeficiency in man and animals (ed. by D. Bergsma), p. 477. Sinauer Associates, Incorporated. G6RSKI, A.J., DUPONT, B., HANSEN, J.A. & GOOD, R.A. (1975) Leukocyte Migration Inhibitory Factor (LMIF) induced by Concanavalin A: standardized microassay for production in vitro. Proc. nat. Acad. Sci. (Wash.), 72, 3197. G6RSKI, A.J., DUPONT, B., HANSEN, J.A. & GOOD, R.A. (1976) LMIF production in unilateral mixed lymphocyte culture reaction. J. Immunol. (In press.) GRAZE, P.R., PERLIN, E. & ROYSTON, I. (1976) In vitro lymphocyte dysfunction in Hodgkin's disease. I. nat. Cancer Inst. 56, 239. HANSEN, J.A. & GOOD, R.A. (1974) Malignant disease of the lymphoid system in immunological perspective. Human Pathol. 5, 567. HOLM, G., MELLSTEDT, H., BJORKHOLM, M., JOHANSSON, B., KILLANDER, D., SUNDBLAD, R. & SODERBERG, G. (1976) Lymphocyte abnormalities in untreated patients with Hodgkin's disease. Cancer, 37, 751. KASAKURA, S. (1975) MLC stimulatory capacity and production of a Blastogenic Factor in patients with chronic lymphatic leukemia and Hodgkin's disease. Blood, 45, 823.
LOMNITZER, R., RABSON, A.R. & KOORNHOF, H.J. (1975) Production of LIF and MIF by PHA-stimulated lymphocytes. Clin. exp. Immunol. 22, 522. REES, J.C., Rossio, J.L., WILSON, H.E., MINTON, J.P. & DODD, M.C. (1975) Cellular immunity in neoplasia. Antigen and mitogen responses in patients with bronchogenic carcinoma. Cancer, 36, 2010. ROCKLIN, R.E., MCDERMOTT, R.P., CHESS, L., ScLossmAN, S.F. & DAVID, J.R. (1974) Studies on mediator production by highly purified human T and B lymphocytes. J. exp. Med. 140, 1303. ROCKLIN, R.E. (1975) Partial characterization of leukocyte inhibitory factor by concanavalin A stimulated human lymphocytes (LIF). J. Immunol. 114, 1161. SIMo CAMPS, E., ANGUERA, A., MA VICH, J., VIDAL-RIBAS, A., SALA, F., SARRIAs, R., GuMMA, J., GRI, E. & MA PUIGDOLLERS J. (1976) Immunologic impairment in patients with non-lymphoid cancer. Cancer, 37, 724. SMITH, J.B., KNOWLTON, R. & KOONS, L.S. (1976) Lymphocyte reactivity in untreated patients with chronic lymphocytic leukemia. Clin. Res. 24, 336 (abstract). STOBO, J.D., PAUL, S., VAN Scoy R.E. & HERMANS, P.E. (1976) Suppressor thymus-derived lymphocytes in fungal infection. J. clin. Invest. 57, 319. TOURAINE, J.L., TOURAINE, F., HADDEN, J.W., HADDEN, E.M. & GOOD, R.A. (1976) 5-Bromodeoxy-uridine-light inactivation of human lymphocytes stimulated by mitogen and allogeneic cells: evidence for distinct T-lymphocyte subsets. Int. Arch. Allergy, (In press.) TWOMEY, J.J., LAUGHTER, A.H., FARROW, S. & DOUGLAS, CH. C. (1975) Hodgkin's disease: an immunodepleting and immunosuppressive disorder. J. cdin. Invest. 56, 467.
