IMPROVEMENT IN DELAYED HYPERSENSITIVITY IN HODGKIN'S DISEASE WITH TRANSFER FACTOR:

LYMPHAPHERESIS AND CELLULAR IMMUNE REACTIONS OF NORMAL DONORS

AMANULLAH KHAN, MD, PHD,* JOSEPH M. HILL, MD, ' AYTEN MACLELLAN, MD,' ELLEN LOEB, MD,* NORWOOD 0. HILL, MD,Il AND SIMONE THAXTON, BS**

Passive transfer of delayed hypersensitivity was achieved, with normal transfer factor, in patients with Hodgkin's disease in remission. The cellular immune responses of the recipients improved. It is suggested that, in addition to specific effect the transfer factor (or factors) has a nonspecific effect causing improve- ment in the state of delayed hypersensitivity of the recipient in general. The average number of E-rosette T lymphocytes was 46.3% after the transfer factor treatment in Hodgkin's disease. The control patients with Hodgkin's disease, not receiving transfer factor, had a value of 37.8%. Removal of 4.9 X lo8 to 1.08 X 10" lymphocytes did not diminish the delayed hypersensitivity of the donor. Side effects attributable to transfer factor were not seen.

Cancer 36:86-89, 1975.

HE IMPAIRMENT OF DELAYED HYPERSENSI- T tivity was shown to be related to the prog- nosis of the patient with Hodgkin's Passive transfer of delayed hypersensitivity to tuberculin was attempted with leukocytes in Hodgkin's disease, but the results had been dis- app~in t ing .~~ '~ ' ' Improvement in cellular im- munity was shown with the administration of transfer factor in diseases such as candidiasis, Wiscott-Aldrich syndrome, Chediak-Higashi

Presented at the 10th Annual hleetinn of the American Society of Clinical Oncology, Houston, Texas, blarch 27-28. 1974.

From the Wadley Institutes of Molecular Medicine, Dal-

Supported by the Wadley Guild Research Fund. Chairman, Department of Immunotherapy, Leland

Fikes Research Institute, Division of Wadley Institutes of Molecular h.ledicine. ' Executive Director, Wadley Institutes of Molecular

hledicine. Chief of Pediatric Service, Granville C. Morton Cancer

& Research Hospital, Division of Wadley Institutes of Molecular Medicine.

I Chief of Medical Services, Granville C. Morton Cancer & Research Hospital.

" Director, Wadley Central Blood Bank, Division of Wadley Institutes of Molecular Medicine.

* * Department of Immunotherapy, Leland Fikes Research Institute.

Address for reprints: Amanullah Khan, MD, PhD, Chairman, Department of Irnmunotherapy, Wadley In- stitutes of Molecular Medicine, 9000 Harry Hines, Dallas, TX. 75235.

las, TX.

Received for publication .July 31, 1974.

syndrome, tuberculosis, and l e p r o ~ y . ~ ~ ~ ~ ' ~ ~ ' ~ ~ ~ ' ~ ~ ~ I t has become possible to obtain large numbers of lymphocytes with the availability of continu- ous flow cell separators, and thus obtain larger amounts of transfer factor. The present study was undertaken to see if delayed hypersensitivity can be transferred passively in Hodgkin's dis- ease in remission, with large doses of transfer factor. It was also given to patients with dis- seminated malignancies. The effect on the delayed hypersensitivity of the donor following the donation of large amounts of lymphocytes was also studied. Preliminary results of part of this study have been reported earlier.'

MATERIALS AND METHODS

Eight patients with Hodgkin's disease received transfer factor in different doses. Transfer factor was given after remission had been achieved with chemotherapy or radiotherapy or a combination of the two. Seven other patients were observed as controls. The distribution of patients in the two groups regarding the stage of the disease and the histologic type is given in Table 1.

T h e delayed hypersensitivity of all the patients and the would-be donors was tested with a panel of skin-test antigens by intradermal injection of 0.1 ml volume. This included four to six of the following antigens: A) mumps skin- test antigen (Eli Lilly and Company); B)Der-

86

No. 1 TRANSFER FACTOR IN HODCKIN'S DISEASE Khan et af. 87

matophytin (Holister Stier Laboratory); C) and D) Histoplasmin and purified protein derivative (PPD), 5 tuberculin units/O.l ml (Parke Davis & Co.); and E) Varidase (streptokinase 100 units/ml, streptodorinase 10 units/ml) (Lederle Labs). Skin test reactions were graded by measuring the average diameter of erythema and induration at 24 and 48 hours (6-10 mm 1+ , 11-15 mm = 2+, 16-20 mm = 3+, and 21 mm or more = 4+). Dinitrochlorobenzene (DNCB) sensitization was carried out by apply- ing 1 mg of this substance (dissolved in 0.1 ml of acetone) to the skin of the forearm; the challenge was performed after a period of 2 weeks or more. Skin tests were performed 3 weeks to 27 months after the cessation of chemotherapy or radio- therapy. The median interval between the cessa- tion of chemotherapy or radiotherapy and the skin tests was 3 Yi months in the control group and 2 months in the transfer factor group. The median lapsed time between the completion of chemotherapy or radiotherapy and the ad- ministration of transfer factor was 3 '/4 months.

Only the contacts of the patient were screened to be the donors for transfer factor. The donor was chosen on the basis of normal lymphocyte blastogenic response, the presence of the highest number of positive delayed hypersensitivity reactions, and a good health record. The lymphocyte-rich fraction of leukocytes was col- lected with the aid of continuous flow centrifuge (Celltrifuge-Aminco) during a period of 3-4 hours. The cells were disrupted by 10 cycles of freezing and thawing, and the dialyzable transfer factor was prepared as described previously.' In an attempt to standardize our preparation of transfer factor, it was prepared in such a manner that the final product contained transfer factor from 10' leukocytes/ml (mean lymphocyte concentration 85%). The transfer factor was given intramuscularly. The max- imum volume administered to a patient was 49 ml, and the minimum volume was 30 ml, given in divided doses at varying intervals. The fre-

TABLE 1. Distribution of Patients in the Control and Irnmunotherapy Groups

No. of patients Control Imrnunotherapy

Stage of disease I I 3 4 111 2 2 IV 2 2

Histologic type Lymphocytic

Nodular

hlixed

predominance 1 -

sclerosing 3 3

cellularity 3 5

quency varied from daily to every 4 months. The maximum single dose was 20 ml (representing 2 X 10" leukocytes).

RESULTS

Tables 2 and 3 show the skin reactions in both the control and the transfer factor groups. The reactions varied from 1+ to 4+. There were some positive skin tests in both groups initially. Table 3 shows the skin test reactions of the donors, along with the initial and post-transfer- factor reactions of the recipients. Passive transfer of delayed hypersensitivity, to one or more antigens, was achieved from the donors to the recipients in five patients. Three patients in the immunotherapy group also converted from negative to positive DNCB hypersensitivity, sug- gesting improvement in the responses in general. DNCB reactivity was transferred passively to one patient in the immunotherapy group. Two patients in the control group also converted to positive reactions (table 2). Two patients belonging to the control group died too soon to permit followup studies. Patient MC died from disseminated varicella zoster infection, and H B had septicemia. Both patients were free of Hodgkin's disease at autopsy.

Peripheral T lymphocytes were determined

TABLE 2. Delayed Hypersensitivity 0 1 the Controls

Patient Mumps PPD Histo Derrn Varidase IINCH

h1.C.' H.B.. A.hl . C.M. J .H. S.L. L.K.

* Died. ' Results in parentheses indicate positive reactions developed during the period of observation.

88 CANCER July 1975 Vol. 36

T A B L E 3. The Reactivity of the Donors and the Recipients 01 Transfer Factor to \.'arious Antiqens

Test subject hiumps PPD Histo Derrn \'aridase DNCB

+ f - - Donor* + Patient (R.E.) ' - -

(converted)l + + + + + - - -

Donor + + Patient (J.K.) - -

(converted) + + Donor + + Patient (L.C.) + (converted)

Donor + Patient (T.W.) - -

(converted)

-

-

Donor Pa!ient (JT.) (converted)

- Donor + Patient (C.J.) - - (converted) + Donor + Patient (T.F.) - - (converted)

Donor + Patient (\V.hl.) + (converted)

-

- -

none

none

+ + + + - -

+ - - +

+ -

+

+

- +

+

* Skin test reactions of the donor. ' Skin reactivity of patients before the administration of transfer factor

Skin tests converted to positive after giving transfer factor.

by spontaneous rosette formation with sheep erythrocytes (E-rosettes),2 in patients treated with transfer factor and the control group. The percentage of T cells in the transfer group was 46.3%, compared to a value of 37.8% in the con- trol group of Hodgkin's disease in remission (the difference was not statistically significant). The mean normal value of E-rosette T lymphocytes is 65% in our hands.

Table 4 shows the number of lymphocytes removed from one of the donors during a single run of lymphapheresis. There was no significant difference in his absolute lymphocyte count and his percentage of T lymphocytes in peripheral blood before and after lympha- pheresis. Similarly, Table 5 shows that his

TABLE 4. Effect of Lymphapheresis on the Peripheral Lymphocytes (1.08 X 10" Lymphocytes Removed)

Count of the donor Before After

Lymphocytes/ 2295 3264

T lymphocytes 57% 62% h.1 rn

24-hour and 48-hour skin reactions were un- affected in intensity following lymphapheresis. The lymphocyte blastogenesis to PHA remained unchanged following the removal of lympho- cytes. Two other donors were tested following the removal of 4.9 X 10' and 7.09 X 10' lymphocytes. Their skin tests also remained un- changed.

DISCUSSION Delayed hypersensitivity was transferred pas-

sively from healthy donors to five patients with Hodgkin's disease in remission. In addition to the passive transfer of delayed hypersensitivity, DNCB hypersensitivity developed in three patients who were initially negative. Similarly some patients developed positive reactions which were not present in the donor of the transfer factor. This suggests that transfer factor not only conferred the delayed hypersensitivity, but also improved the immune reactivity of the patients. This raises the question of specificity of transfer factor. The dialyzable transfer factor is a crude preparation. Therefore, the improve- ment may be due either to a nonspecific effect of

No. 1 TRANSFER FACTOR IN HODCKIN’S DISEASE Khan et al. 89

transfer factor (or factors), or another factor in the preparation capable of doing so. An alter- nate explanation could be that repeated testing was able to bring out dormant reactivity.

The donors for transfer factor were immediate contacts of the patient. It was thought that the contacts would be superior donors if the etiologic agent of this disease happened to be a virus. The healthy contacts would have the max- imum likelihood of exposure and possible resistance to the etiologic agent. Moreover, in an earlier study, csing macrophage migration in- hibition test, we had found a high degree of cel- lular immunity against Hodgkin’s tissue among the contacts of the patient (80% positive MIF).

The percentage of T lymphocytes (E-rosette) in all the Hodgkin’s patients was low as com- pared to normal. Recently, similar results have also been reported by others.’’ Although the average value was higher in the transfer factor group, the difference was not statistically signifi- cant. It has been suggested that the decrease in E-rosette forming cells may reflect altered cell surface.’ Correction of such a defect will ob- viously be important. It needs to be seen if prolonged treatment with transfer factor can change the E-rosette values significantly.

There has been concern about depleting transfer factor of the donor. We were taking large numbers of lymphocytes from the donors, therefore, we were concerned about altering the cellular immunity or the delayed hypersen-

TABLE 5. Skin Reactions of the Donor Pre- and Post- Lymphapheresis

Skin reactions 24 hours 48 hours

Antigen Pre Post Pre Post ~~

Mumps 4 + 4+ 4+ 4+ Histoplasmin 2+ 4+ 4+ 4+ Dermatophytin 1 + 3 + 2+ 2+ P.P.D. - - - - Varidase 4+ 4+ 4+ 4+

sitivity of the donor following lymphapheresis. T o determine the effect of removal of lymphocytes on delayed hypersensitivity, the donors were tested before and after lympha- pheresis. It was seen that removal of lymphocytes up to 1.8 X 10” did not alter the delayed hyper- sensitivity of the donors. The effect of repeated lymphapheresis still remains to be seen.

Transfer factor in 5- to 95-ml doses failed to give objective responses in nine patients with disseminated malignancies (acute lymphocytic leukemia 1, lymphosarcoma 1, carcinoma of breast 2, synovial sarcoma 1, malignant mela- noma 3, and carcinoma of lung 1). We, there- fore, feel that the most opportune occasion to give transfer factor may be the time when the tumor load is minimum, such as following chemo- therapy, radiotherapy, or surgery. Improvement of cellular immunity at this stage may help prolong the disease-free period and prevent varicella zoster infection in Hodgkin’s disease.

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I . Aisenberg, A. C.: Studies on delayed hypersensitivity in Hodgkin’s disease. J. C h . Inuest. 41 :1964-1970, 1962.

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3. Bulloci, W. E. , Fields, J. P., and Brandriss, M. W.: An evaluation of transfer factor as immunotherapy for patients with lepromatous leprosy. N . Engl. J . Med. 287: 1053-1059, 1972.

4. Fazio, hl., Calciati, A,, and DePaoli, M.: Dermatite al- lergica de contatto nel linfogranuloma maligno. Minerua Dcr- matol. 37:259-261, 1962.

5. Fazio, M., and Calciati, A,: An attempt to transfer tuberculin hypersensitivity in Hodgkin’s disease. Panmrnerua Med. 4:164-167, 1962.

6. Kaplan, H. S., Bobrove, A. M., Fuks, Z., and Strober, S.: T and B cells in Hodgkin’s disease. N. Engl. J. Med. 290:971, 1974.

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8. Khan, A,, Hill, J. M., Loeb, E., MacLellan, A., and Hill, N. 0.: Management of Chediak-Higashi syndrome with transfer factor. Am. J. Dis. Child. 126:797-799, 1973.

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Hill, N. 0.: Reconstitution of cellular immunity in Hodgkin’s disease with transfer factor. Recent Results Concn Rer. 49:74-80, 1974.

10. Levin, A. S., Spitler, L. E., Stites, D. P., et al.: Wiskott-Aldrich syndrome, a genetically determined cellular immunologic deficiency-Clinical and laboratory responses to therapy with transfer factor. Proc. Natl. Acad. Scr. USA 67:821-828, 1970.

11. Levy, R., and Kaplan, H. S.: Impaired lymphocyte function in untreated Hodgkin’s disease. N . Engl. J. Med. 290:181-186, 1974.

12. Muftuoglo, A. U., and Balkuv, S.: Passive transfer of tuberculin sensitivity to patients with Hodgkin’s disease. N . Engl. 3. Med. 277:126-129, 1967.

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14. Schulkind, M . L., Adler, W. H., 111, Alterneier, M’. A,, 111, et al.: Transfer factor in the treatment of a case of chronic mucocutaneous candidiasis. Cell. Immunol. 3:606-615, 1972.

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16. Spitler, L. E., Levin, A. S., Stites, D. P., et al.: The Wiskott-Aldrich syndrome-Results of transfer factor therapy. J. Clin. Inuest. 51 :3216-3224, 1972.