Immunological Studies in Mycosis Fungoides*

THE JOTJRN.L OP INVESTIGATIVE DERMAToLoGY

Copyright

Vol. 46, No. 1

1966 by The Williams & Wilkins Co.

Printed in U.S.A

IMMUNOLOGICAL STUDIES IN MYCOSIS FUNGOIDES* W. KENNETH BLAYLOCK, M.D.t, JOOST OPPENHEIM, M.D4 AND

RICHARD A. MALMGREN, MD.

Fluorescent antibody technic :—Horse antiIn recent years it has been demonstrated that animals can be immunized against certain serum prepared against M.F. tissue and serum an unimmunized horse were conjugated with chemically induced and viral induced tumors from fluorescein isothiocyanate by the technic of Riggs

which originated in the same inbred strain (8). Tissues to be tested were sectioned at 5

of animals (1, 2, 3, 4, 5). This finding has re- microns with a Spencer rotary microtome in a awakened interest in the concept of tumor an- cryostat. The following tissues were exposed to horse anti-M.F. serum and to contigens. Although the immunity invoked by conjugated jugated normal horse serum: M.F. skin tumors, these tumor or viral antigens seems to be car- uninvolved skin of patients with M.F., M.F. ried in the lymphoid cells of the immune ani- lymph nodes, normal lymph nodes, dermatopathic mal rather than in the serum, the existence of lymph nodes, psoriatic skin, skin tumors of reticu-

such antigens makes the likelihood of their lum cell sarcoma, and carcinoma metastatic to the demonstration using heterologous immune serum seem reasonable. Toward this end, heterologous and autologous anti-serum against mycosis fun-

skin. Whole cell preparations of Hela cells,1 and epithelial cells2 grown in tissue culture, bone cells from human fetus grown in tissue culture media,8

variety of immunologic technics.

prints) from patients having M.F., acute lymphocytic leukemia, uremia, multiple myeloma and

lymphocytes from M.F. patients obtained by goides (M.F.) tissue has been studied with a thoracic duct cannulation, bone marrow cells (im-

congenital heart disease were also reacted on

MATERIALS AND METHODS

slides with conjugated serum.

Immunization procedure :—A previously unimAbsorption studies :—Four 2 ml aliquots of horse munized ten-year-old horse, Joe II, was immunized anti-M.F. serum were absorbed with small with homogenates of M. F. tumor tissue as pre- amounts (0.1 ml) of 0.85% saline homogenates of viously described (6). normal human lymph nodes, M.F. tumors, norComplement fixation tests:—Complement fixa- mal skin and normal human serum. These antition tests to characterize the antigens in the M.F. gens were added twice daily until no further pre-

tumor homogenates were done according to the method of Kent and Fife (7). Tumor homogenates were prepared in 0.85% saline with a Virtis "45" tissue homogenizer as previously described (6). These homogenates were fractionated with the Spinco ultracentrifuge as follows: the original homogenate was centrifuged at 30,000 G for 15

cipitate was observed. This serum was then

centrifuged and the supernatant used as antibody in the complement fixation reaction. In a similar manner the serum labelled with fluorescein isothiocyanate was absorbed with the antigens listed above.

Short term lymphocyte cultures:—Patients in

minutes and a pellet (Pi) was obtained. This this study included normal young volunteers as pellet (P1) was re-suspended in 0.85% saline and well as patients with M.F. Sixty ml of heparinized this homogenate was again centrifuged at 30,000 blood from each patient (10 units of heparin/ml)

G for 15 minutes and a second pellet (P2) was were allowed to sediment at room temperature obtained. In like manner this pellet (P2) was re- for 1 to 2 hours. The buffy coat was withdrawn suspended in saline and centrifuged. This proce- and sufficient cells were mixed with 8 ml of 199 dure was repeated a total of 4 times. A portion of tissue culture medium (containing penicillin and the supernatants from the 4 procedures was pooled streptomyein) to obtain a cell suspension with a and centrifuged at 100,000 G for 2 hours. Each pel-

white blood count of 1000—2000/cu mm and a con-

let (Pi-P5) and a small aliquot from each super- centration of about 30% autologous plasma. To a natant was used as the antigen in a complement 1 fixation reaction with horse anti-M.F. serum, and Hela cell line obtained from Dr. Stanley Burthe titers were recorded. ban: Division of Biological Standards, National * From the National Cancer Institute, Bethesda, Institutes of Health. These cells were cultured in Maryland Public Health Service, U.S. Department of Health, Education, and Welfare. t Dermatology Branch, N.C.I.; Present address: Dermatology Division, Department of Medicine, Medical College of Virginia, Richmond, Virginia. t Medicine Branch, N.C.I. § Pathological Anatomy Branch, N.C.I. Received for publication December 5, 1964.

28

calf serum.

2 NCTC strain 3075—obtained from Dr. Virginia J. Evans: National Institutes of Health. Epithelial cells from skin grown on chemically defined medium devoid of any protein (9).

8NCTC strain 3354—obtained from Dr. Virginia

J. Evans: National Institutes of Health. Established from explants of femoral epiphysis of a human fetus (9).

ThIMIJNOLOGY STUDIES IN MYCOSIS FTJNGOIDES

29

series of 12 ml aliquots of the cell suspension the marked anti-complementary effect in the comfollowing were added: nothing, 02 ml phytohe- plement fixing system. This system was theremagglutinin M,4 normal horse serum and horse

anti-M.F. serum in various dilutions. The cells were cultured at 37° for 5 days. The cultures were

then centrifuged at 500 rpm for 5 minutes, and the supernatant was discarded. The cells were fixed

in modified Carnoy's fixative (3:1 absolute ethyl alcohol:glacial acetic acid) for 10 minutes. The fixed cells were placed on slides with a pipette, air dried, and stained with Giemsa (Harleco).

Immunization of patients with autologous mycosis fun goides tumor tissue :—Five patients with M.F. lymphoma tumors and plaques were injected

fore unsatisfactory in evaluating the antibody absorbing qualities of various tissue antigens.

The major portion of the antigen from the M.F. tumor was saline soluble as measured by the complement fixation technic. The antigen was easily recovered in the supernatant fraction (S1) following 30,000 G centrifugation of this homogenate while it was completely removed from this fraction (S1) by centrifugation at 100,000 G for 2 hours. (Table I)

intracutaneously with autologous tumor homogPrevious fluorescent antibody studies (6) enized in saline plus Freund's complete adjuvant. have demonstrated that the horse anti-M.F. The material for injection was prepared as follows: a 6 mm punch biopsy specimen was taken serum is directed toward the peripheral rim

from a M.F. plaque and homogenized under of cytoplasm of cells in the tumor infiltrate. sterile conditions with a Virtis "45" tissue homog- Cross-reactivity was noted in the complement enizer. Three ml of complete Freund's adjuvant were added to 3 ml of tumor homogenate con- fixation reaction between horse anti-M.F. taining 200 mgm of wet weight tumor tissue. Each serum and normal lymph node homogenates. patient received 0.1 ml of autologous tumor plus It is possible that there are common antigens Freund's adjuvant intracutaneously once each in the reticulum cells in the M.F. tumors and week for 6—7 weeks. This material was stored at the reticulum cells or lymphocytes in normal 4° C during the 6 weeks period. Serum samples were taken from the antecubital vein from 2 of lymph nodes. An alternate explanation would the patients near the end of the active immuni- be that the antigen in the M.F. tumor homogzation period while the other 2 samples were ob- enate is really the cytoplasm from the normal tained 6 months and 11 months following the last injection of tumor homogenate. The globulin was removed with ammonium sulfate and labelled with fluorescein isothiocyanate according to the technique of Riggs (8). Fluorescein labelled globulins were then applied to autologous and heterologous lymphocytes obtained from thoracic dut cannulations of patients with M.F. The tissues were cx-

anined microslopically with ultraviolet micro-

scope. RESULTS AND DISCUSSION

lymphocytes contained in this infiltrate. Fluorescent antibody studies :—Fluorescence

was observed in the stroma of all tissue exposed to conjugated horse anti-M.F. serum. This was not observed in tissue treated in the same fashion with conjugated normal horse serum globulin. In addition to stromal fluorescence, the peripheral rim of cytoplasm of the

cells in the dermal infiltrate in M.F. tumors fluoresced when horse anti-M.F. serum was added to these sections. Conjugated normal horse serum globulin did not produce this result. (Table II) Cells from the buffy coat of whole blood in patients having M.F. showed cytoplasmic fluorescence when tested with

Complement fixation tests :—As previously reported (6) horse anti-M.F. serum fixed complement in the presence of M.F. tumor tissue and normal human lymph nodes while it did not fix complement in the presence of normal skin or human serum. This indicates that the horse anti-M.F. serum. This was reduced when antigen involved in this reaction is found in a blocking procedure was done by adding nfllymphoid or reticulum cells both of which are conjugated horse anti-M??. serum before the found in M.F. tumor infiltrates as well as in conjugated anti-M.F. serum was added to the normal lymph nodes, spleen, liver, lungs, etc.

Absorption of horse anti-M.F. serum with human red blood cells from all major human blood types failed to reduce the complement fixing titer. Horse anti-M.F. serum absorbed with normal skin homogenates produced a from Difco Labs, Detroit, Michigan.

cells. Thoracic duct lymphocytes from 2 patients having M.F. showed cytoplasmic fluorescence when exposed to conjugated horse anti-

M.F. serum. Fluorescence was reduced in a blocking test when unconjugated horse antiM.F. serum was added prior to the conjugated horse serum.

Several bone marrow imprints from autopsy

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

30

TABLE I to conjugated horse anti-M.F. serum revealed Complement fixing activity of ultracentrifuge cytoplasmic fluorescence in all these cells. After conjugated horse anti-M.F. serum was fractions of mycosis fun goides tumor homogenates CF activity of supernatant with horse anti-mycosis fungoides serum

CF activity of pellet* with horse anti-mycosis fungoides serum

1:16 (S1)**

1:4 (Ps)t

1:8 (S2) 1:2 (S5) 1:2 (54)

neg (P2) neg (P3) neg (P4)

negY (S5)

1:8 (P5)

* Each pellet was resuspended in 0.85% saline.

negative refers to no complement fixing ability

of the serum. ** S refers to the supernatant fraction from cen-

trifugation of the M. F. tumor homogenate at 30,000 X G for 15 minutes. 52 refers to the supernatant fraction from centrifugation of the resuspended P1 at 30,000 X G for 15 minutes.

absorbed with M.F. tumor homogenates, it failed to fluoresce the stroma and peripheral rim of cytoplasm in M.F. tumor cells. When anti-M.F. serum was absorbed with normal fresh lymph node homogenates, there was a reduction of cytoplasmic fluorescence while normal skin homogenates and normal human serum did not significantly alter the fluorescent properties of this serum. These studies indicate that the cells in the M.F. tumor infiltrate share a common antigen with normal lymph node cells and lymphocytes from peripheral blood.

There was no reaction between horse antiM.F. serum and reticulum cell sarcoma (6), TABLE II

Fluorescent antibody studies S refers to the supernatant fraction from centrifugation of the resuspended P2 at 30,000 X G Reaction between fluorescent labelled horse for 15 minutes. anti-mycosis fungoides serum and various tissues. S4 refers to the supernatant fraction from cenCyto. Neg. cytotrifugation of the resuspended P3 at 30,000 X G plasmic plasmic No. of for 15 minutes. fluoresSuoresTissue sections different cence with cence with S5 refers to the supernatant fraction from cenpatients rh. B.S.A.1 rh. E.S.A. plus Joe plus Joe trifugation of the combined S1, S2 , S3, S4 at II H.S.2 II H.S.

100,000 X G for 120 minutes. f P1 refers to the pellet fraction from centrifugation of the M. F. tumor homogenate at 30,000 X G for 15 minutes.

P2 refers to the pellet fraction from centrifugation of the resuspended P1 at 30,000 X G for 15 minutes. P5 refers to the pellet fraction from centrifugation of the resuspended P2 at 30,000 X G for 15 minutes. P4 refers to the pellet fraction from centrifugation of the resuspended P3 at 30,000 X G for 15 minutes. P5 refers to the pellet fraction from centrifugation of the combined S1 , S2 , Ss , S4 , at 100,000 X G for 120 minutes.

fungoides

9

9

0

Thoracic duct lym-

2

2

0

3

2

1

1

1

0

2

1

1

1

0

1

1

0

1

1

1

0

Mycosis

skin tumors

phocytes from myco-

sis fungoides pa-

tients Buffy coats from patients with mycosis fungoides Bone marrow imprints

from mycosis fun-

goides patients Normal bone marrow imprints Acute lymphocytic leukemic bone marspecimens were tested with conjugated horse row imprint anti-M.F. serum. Cytoplasmic fluorescence in Multiple myeloma the white cell precursors was noted in one case bone marrow imprint

of M.F. and 1 out of 2 bone marrow im-

Hela cells

1 0 1 prints from normal subjects. Imprints from Epithelial cells f 3075 1 1 0 acute lymphocytic leukemia and multiple Bone cells f 3354 myeloma did not show cytoplasmic fluores1 rh. B. S. A. refers to rhodarnine bovine serum cence.

Hela cells, epitheial cells 3O75, and bone

albumin.

Joe II H. S. is horse anti-mycosis fungoides cells 3354 grown in tissue culture media exposed serum.

IMMUNOLOGY STUDIES IN MYCOSIS flJNGOIDES

31

nor plasma cells from multiple myeloma or 56° did not block its cytotoxic and agglutinatacute lymphocytic leukemia cells, indicating ing effects. The method of short term culture of lymthat these cells probably do not share the common antigen in question with M.F. tumors and phocytes for cytogenetic studies has been normal lymph node. In these conditions there shown to be an excellent means of evaluating may well be a loss of antigens. In order to be lymphocytic immunologic competence (10). certain about a gain or loss of antigens in my- Lymphocytes transform to large immature cosis fungoides, individual abnormal reticulum pyroninophilic cells. These cells produce proteins cells and normal lymphocytes must be isolated (11), RNA (12), DNA (13) and undergo mitosis from the tumor and exposed to the conjugated when they are stimulated in vitro by various antihorse sera. gens to which they have been previously exposed Short term peripherai WBC cultures:— in vivo (14, 15). Phytohemagglutinin and staphyAfter 5 days of incubation, the in vitro cultures lococcal filtrates (16) initiate this transformaof WBC from 4 M.F. patients and 4 normal tion in approximately 75% of the lymphocytes subjects responded equally well to phytohemag- and produce mitotic indices ranging from 1— glutinin (PHA) with the usual 75—85% lym- 10%. Purified protein derivative (14) some enphocyte transformation and 0.5—1.5% mitotic dotoxins (17), polio vaccine (15), tetanus toxoid response. In contrast, the unstimulated WEC (15), diphtheria toxoid (15), and other antigens cultures contained 10—25% monocytic cells and usually transform less than 30% of the lymphono transformed lymphocytes nor mitoses. cytes cultured for 5 days from subjects with a The addition of normal horse serum had history of exposure to these antigens. It was recently reported that a rabbit antivariable effects on WBC growth. Small amounts (0.025-0.1 nil) had no effect on the cultures, while human WBC serum was as effective a lympho-

a higher concentration (0.375 ml. per 12 ml) cyte stimulant as phytohemagglutinin (18). caused death of all cells in the cultures of 2 of Although fluorescent antibody techn&cs demthe M.F. patients and 2 normal subjects and onstrated antibodies in the horse anti-Mi'. was mildly cytotoxic in the others. One of the serum to human lymphocytes and reticulum patients with a history of horse serum immuni- cells in M.F. tumors, the response of our cell zation had a mild transformation response cultures is more complex. Recent studies using with 5—10% typical transformed lympho- rabbit anti-human skin sera and rabbit antisquamous cell tumor sera have also demoncytes. Horse anti-M.F. serum was more cytotoxic strated that these sera have cytotoxic, agglutinatthan the control horse serum and caused cell ing and occasional lymphocyte transforming death in 75% of the cultures when the higher effects when these sera are exposed to human

concentration (0.375 ml/12 ml) was used. lymphocytes in tissue culture.

Horse anti-M.F. serum was moderately toxic at the lower concentration (0.025—0.1 ml/12 ml), but cell clumps consisting of mononuclear cells, predominantly macrophages, survived in all of the cultures. Only a few of the cells appeared morphologically similar to the PHA transformed lymphocytes. The other cells were large mononuclear cells with very basophilic

There was no difference in the effect of horse

anti-M.F. serum on lymphocytes from M.F. patients and lymphocytes from normal volunteers. This might be expected since peripheral lymphocytes usually appear normal in patients

with M.F. until late in the illness. The M.F. patients' response to the PHA-M also indicates that they function normally in this respect.

The marked cytotoxic effect of horse anticytoplasm, rounded nuclei and Mi'. serum may have obscured lymphocyte prominent nucleoli. Unlike the PHA "blasts", granular

stimulation in these cultures, and the minimal however, their cytoplasm was more basophilic, lymphocyte transformation present may have multicolored and frequently contained eosino- been due to a response to heterologous antigens philic granular particles. The mitogenic effect present in the horse serum which triggered a

of the horse anti-M.F. serum was minimal response similar to that elicited by antigens. with only 2 patients showing a mitotic index in Horse anti-M.F. serum probably contains the range of 1 per 1000 mononuclear cells. heterologous antibodies to various parts of the Heating horse anti-M.F. serum for 1 hour at lymphocyte and reticulum cells, and this ac-

32

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

counts for its varied effects on the cultured bodies by fluorescent antibody and complement WBC's.

fixation technics.

Auto-antibody studies in patients not previously treated with

Autologous injections of mycosis fungoides tumor tissue

chemotherapeutic agents

Four patients with active M.F. received

The serum globulins of 4 patients with mycosis fungoides in early phases of disease development were conjugated with fluorescein isothiocyanate and applied to sections of their own tumor tissue. There was no evidence for circulating antibodies against M.F. tumor tissue in these patients. Serum from 10 patients with M.F. did not fix complement in the presence of autologous tumor tissue.

small intraeutaneous injections of autologous M.F. tumor tissue homogenized with complete Freund's adjuvant. These injections produced mild systemic responses including 1—2 C ele-

vations of body temperature. Local effects included edema, erythema, and ulceration. The granuloma produced by these injections healed with scar formation over a 6 weeks period. No definite clinical improvement of the primary

The concept of immunity to tumor cells disease was noted during and following the is not a new one, and several investigators in the past 10 years have described circulating antibodies to autologous malignant tissue (19, 20). Early in M.F. there is an abundance of lymphocytes, plasma cells and histiocytes surrounding only a small number of abnormal

treatment period. No fluctuations in total WBC

"M.F." cells. As the disease progresses, there is

tion of M.F. patients, cytoplasmic fluorescence

or in blood lymphocytes were noted. The serum globulins from these patients were conjugated with fluorescein isothiocyanate. With

addition of this conjugated globulin to lymphocytes obtained by thoracic duct cannula-

an increase in the number of abnormal cells of either autologous or homologous lymphoand a gradual decline in the number of normal cytes was noted with 3 out of 4 sera. (Table cells. If this represents a failure of the host III) One serum fluoresced autologous lymphodefense mechanism, it might be expected that cytes from the patient with M.F. This fluorescirculating antibodies to tumor cells which might cence was blocked in a blocking procedure. be present early in the disease would be difficult Since 3 of these patients definitely showed

to demonstrate in the late stages of the illness. evidence of anti-lymphocyte antibodies in their Such an event might hamper demonstrating anti- serum following injections of autologous M.F.

TABLE III Immune fluorescence of lymphocytes with serum from patients injected with autologous M.F. tumor tissue Patients whose serum was labelled with fluorescein isothiocyanate

after injections of M.F. tumors

E.W. H.B. C.T. A.A.

Time serum was labelled after last injection of autologous M.F. tissue

Fluorescent studies with autologous lymphocytes'

Pre' block

One week before Cytoplasmic last injection fluorescence

Post' block

Fluorescent studies with heterologous lymphocytes Pre-block

Post-block

No fluorescence No fluorescence No fluorescence

of cytoplasm 11 months after last No fluorescence No fluorescence N.D. injection Six months after N.D. N.D. Cytoplasmic

last injection fluorescence At time of last in- No fluorescence No fluorescence Cytoplasmic jection fluorescence

N.D. No fluorescence

N.D.

'Lymphocytes obtained from thoracic duct. Rh. B.S.A. + Fluorescein conjugated autologous serum globulin + autologous lymphocytes. IJnconjugated autologous serum + rh. B.S.A. + Fluorescein conjugated autologous serum globulin + autologous lymphocytes. N.D.—not done.

33

IMMUNOLOGY STUDIES IN MYCOSIS FUNGOIDES

tissue, a drop in total blood lymphocytes might be expected. This was not observed. One of the 4 patients injected showed slight clinical improvement, but this may have been related to

2. Foley, E. J.: Antigenic properties of methyl

an increase in circulating steroids or to the

4. Revesz, L.: Detection of antigenic difference

natural course of the illness rather than to any effect of the injections. SUMMARY AND CONCLUSIONS

Horse anti-M.F. serum fixed complement in the presence of M.F. tumor homogenates and normal lymph node homogenates. Tlltracentrifuge fractionation of these tumor homogenates indicated that the antigen is saline soluble and can be completely removed by centrifugation for 2 hours at 100,000 G.

Fluorescein isothiocyanate conjugated horse

anti-M.F. serum labelled the peripheral rim of cytoplasm in the cells of the infiltrate in M.F. tumors. Similarly conjugated normal horse globulin failed to do so. Cytoplasm of the following cells was labelled by horse antiM.F. serum: Hela cells, human bone cells and epithelial cells in tissue culture media, buffy coat cells, and pure lymphocyte mixtures. The

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