Clin. exp. Immunol. (1976) 26, 310-313.
Local adoptive transfer to mice of human delayed hypersensitivity REACTIONS BY THE RADIOISOTOPIC FOOTPAD ASSAY NORITOSHI TAKEICHI*, C. W. BOONE* & E. KLEINt * Cell Biology Section, Laboratory of Viral Carcinogenesis, Viral Oncology Program, National Cancer Institute, U.S. Department of Health, Education and Welfare, Bethesda, Maryland and t Department of Dermatology, Rosmell Park Memorial Institute, Buffalo, N.Y., U.S.A.
(Received 26 April 1976) SUMMARY
Human delayed hypersensitivity to living BCG organisms and/or Varidase was adoptively transferred to mice and assayed in mice by a radioisotope footpad assay (FPA). A mixture of the antigen and peripheral blood lymphocytes from patients (positive skin reactions to PPD and/or Varidase) or healthy volunteers was inoculated into the footpads of lethally X-irradiated mice. A positive footpad reaction was accompanied by an increased leakage of the radiolabelled serum protein from the blood stream into the intercellular space at the site of inoculation, and was measured by the 'foot-count ratio', or radioactivity in the test foot divided by radioactivity in the contralateral foot. The intensity of the footpad reaction correlated directly with the skin test response of the human lymphocyte donors.
INTRODUCTION We have previously described a radioisotope footpad assay (FPA) for delayed hypersensitivity in mice both to tuberculin and to tumour-specific antigens of the transplantation type (Paranjpe & Boone, 1972; Takeichi & Boone, 1975). We showed that this assay can be applied between species by adoptive transfer of a mixture of tumour cells and lymphocytes from the tumour-immune or tumour-bearing rats to the footpad of mice (Takeichi & Boone, 1975; Takeichi, Economou & Boone, 1976). This report describes the local adoptive transfer to X-irradiated mice of human delayed hypersensitivity against skin antigens measured by the radioisotope FPA. MATERIALS AND METHODS Animals. An inbred strain of 8-12-week-old male and female BALB/cAnN mice was obtained from the Small Animal Section, Veterinary Resources Branch, National Institutes of Health. Skin test antigens. Tuberculin, purified protein derivative (PPD), 5 TU/0-1 ml was purchased from-Park, Davis & Co., Detroit, Michican. Varidase skin test antigen, 50 u streptokinase/ml was obtained from Roswell Park Memorial Institute, Department of Drug Formation & Development, Buffalo, New York. Living BCG (Phipps strain, TMC No. 1029, Trudeau Institute Saranac Lake, New York) was provided kindly by Dr H. J. Rapp, Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Lymphocyte donors. Lymphocytes from eleven individuals (three with melanoma, one with mycosis fungoides and seven healthy volunteers) were separated from heparinized venous blood by a modified Ficoll-Hypaque technique using Lymphoprep (Nyegaard & Co., As. Oslo, Norway). Fifteen ml whole blood was layered on 20 ml of Lymphoprep fluid in a 50-ml tube and centrifuged for 30 min at 18-200C, at exactly 400 g. The lymphocytes at the interface were transferred to a 50-ml tube, 20-30 ml phosphate buffer solution (PBS) plus 5% foetal calf serum (FCS) was added, and the cells were centrifuged for 10 min at 18-20'C at 350 g. They were resuspended in PBS plus 5% FCS, centrifuged at 18-20'C at 160 g for 10 min to remove most of the platelets, and then resuspended in RPMI1640 medium plus 10% FCS. The total yield of lymphocytes from 80-100 ml of whole blood from each individual donor was 10-20x 107 cells. A smear of the final preparation revealed 80-90% small lymphocytes, 10-20% monocytes, and 2-5% polymorphonuclear leucocytes. Viability counts ranged from 95-100% as determined by trypan blue exclusion. Correspondence: Dr Noritoshi Takeichi, Cell Biology Section, Laboratory of Viral Carcinogenesis, Viral Oncology Program, National Cancer Institute, U.S. Department of Health, Education and Welfare, Bethesda, Maryland 20014, U.S.A.
310
Xenogeneic transfer of human DH reaction
311
Skin test. These were performed with 0 1 ml (5 u) PPD and 0 1 ml (5 u of streptokinase) Varidase, given i.c. at the same time as blood was drawn for lymphocytes. Induration was measured at 24 and 48 hr and recorded as the smallest and largest diameter in mm. Between 0 and 5 mm was considered negative, between 5 and 10 mm weak positive, between 10 and 20 mm positive and over 20 mm strong positive. Radioisotope FPA. Xenogeneic transfer of the DH reaction to skin test antigens between human and mouse was carried out by the method described in previous papers (Paranjpe & Boone, 1972; Takeichi & Boone, 1975). Four million lymphocytes from individual donors were mixed with 1 x 106 living BCG or 5 u streptokinase and injected into the left rear footpad of mice previously X-irradiated with 500 R. Immediately afterwards, 125I-labelled mouse serum albumin or human serum (Mallikrodt Chemical Works, St Louis, Missouri) was injected i.p. (106 ct/min per mouse). Twenty-four hr later the test foot and contralateral foot were excised at the junction of the lower and middle thirds of the tibia and counted in a gamma spectrometer. Results were expressed as the foot-count ratio = ct/min in the test foot divided by ct/min in the contralateral control foot. The significance of the footpad reaction of the group of animals was evaluated by comparing the mean foot-count ratio of the test foot to the control foot by Student's t-test; P-value less than 0 05 was considered significant. X-irradiation. All recipient mice were immunosuppressed with 500 R of X-irradiation (Westinghouse Quadrocondex X-ray Unit, 200 kV, 15 mA at a 25 cm distance from the X-ray source) 24 hr before use.
RESULTS Local adoptive transfer of human delayed hypersensitivity to mice Delayed hypersensitivity to BCG and/or Varidase was adoptively transferred from human to Xirradiated mice with lymphocytes isolated from peripheral blood. Three melanoma patients, who had TABLE 1. Xenogeneic adoptive transfer of delayed hypersensitivity to BCG and/or varidase from human to irradiated mice using a radioisotopic footpad assay
Therapy or Sex immunization with BCG
No. of donors
Diagnosis
1
Melanoma
37
M
Yes
2
Melanoma
44
M
Yes
3
53
M
No
4
Mycosis fungoides Melanoma
60
M
Yes
5
Normal
30
F
No
6
Normal
35
M
No
7
Normal
36
M
No
8
Normal
48
M
No
9
Normal
36
M
No
10
Normal
16
M
No
11
Normal
23
M
No
*
Age
Skin reaction* to
Foot-count ratio+ s.e.t to
PPD
Varidase
BCG
34x 34 (24 hr) 28x 28 (48 hr) 28x 28 35 x 40 30x 30 n.d. 20x 35 20x 35 18 x 20 30x35 16x20 30x 35 18x20 28 x 30 8x 10 lOx 12 Negative Negative Negative Negative Negative
55x 55 55x 55 5x 5 n.d. 20x 20 n.d. n.d. n.d. 5x 5 5x5 7x 12 7x 12 Negative Negative n.d. n.d. n.d. n.d. Negative Negative Negative
2-11+0-12$
1-45+0-04¶1
1P97+0-09+
1-13+0-03
1-89 x 0-08t
1P38 + 0 04¶
1-74+ 0 07$
1-24+ 0-03
1-86+ 0 09t
1 09+ 0 03
1-67+0-05§
1-12+0-03
1-63+0 05§
1 11+003
1-30+0-03
n.d.
1-31+0-04
n.d.
1-41+0 04
1P09+ 003
1-37+0-04
1-06+ 005
Varidase
Size of indurated areas are given by largest x smallest diameter in mm.
t Mean foot-count ratio from patients No. 9, 10 and 11 (skin test negative to PPD) was 1-33 + 0 03. Mean foot-count ratio from patients No. 7, 10 and 11 (skin test negative to varidase) was 1-09+ 0*03, mean foot-count ratio of living BCG (1 x 106 organisms/mouse) alone was 1-20+ 0 05. Mean foot-count ratio of varidase (5 u streptokinase/mouse) alone was 1 09+ 0 03. t Mean foot-count ratio from each donor versus mean foot-count ratio from patients No. 9, 10 and 11, P< 0 001. § Mean foot-count ratio from each donor versus mean foot-count ratio from patients No. 9, 10 and 11, P< 0 01. ¶ Mean foot-count ratio from each donor versus mean foot-count ratio from patients No. 7, 10 and 11, P< 0-05.
Noritoshi Takeichi, C. W. Boone & E. Klein
312
2-00 E
.~i*80+1
.2
1.60
0
'
1-40
0 0
1-20± 100
500 700 900 300 Skin reaction to PPD in humans (product of 2 diameters in mm)
1100
FIG. 1. Local adoptive transfer in mice of human delayed hypersensitivity to PP D.
been treated with BCG for more than 1 year, and one patient with mycosis fungoides had strong positive skin reactions to PPD at both 24 and 48 hr. One melanoma patient (No. 1) and one mycosis fungoides patient (No. 3) showed strong positive skin reactions to the varidase. Four of seven healthy volunteers had positive skin reactions to PPD and two of five tested had weak positive skin reactions to the Varidase. Peripheral lymphocytes from eight tuberculin-positive donors all produced significant reactions when they were mixed with living BCG and injected into the footpads of X-irradiated mice (Table 1). Two Varidase-positive patients (Nos. 1 and 3) also showed positive adoptive footpad reactions to the Varidase in mice. The intensity of the footpad reaction closely paralleled the intensity of the delayed cutaneous reaction in the human donating the test lymphocytes (Fig. 1). The specificity of the radioisotope FPA is shown by the dissociation of the footpad reaction with the lymphocytes from the melanoma patient (No. 2) and from healthy volunteers (Nos. 5 and 6) to BCG and Varidase, as contrasted with the positive footpad reactions of lymphocytes from the melanoma patient (No. 1) and the mycosis fungoides patient (No. 3) to both PPD and Varidase.
DISCUSSION
Delayed hypersensitivity to chemicals or micro-organisms (Asherson & Zembala, 1975; Chase, 1945; Paranjpe & Boone, 1972) or cell-mediated immunity against tumour specific antigens (Bard & Pilch, 1969; Delorme & Alexander, 1964; Fischer & Hammond, 1966; Old et aL., 1962; Takeichi & Boone, 1975; XWepsic, Zbar & Rapp, 1970) can be passively transferred to normal recipient animals with peritoneal exudate, lymph node, or spleen cells from immunized animals. Recent work by Edmund Klein and his associates (Rosenfeld, Bernhard & Klein, 1973) using a guinea-pig-to-mouse system, and by Wallace (1958), in a rat-to-guinea-pig system, have shown the feasibility of the adoptive transfer of cell-mediated immunity to xenogeneic animals. XWe have developed an in vivo assay for antitumour immunity in allogeneic and xenogeneic systems (Takeichi & Boone, 1975; Takeichi, Economou & Boone, 1976). The cellular immunity to Gross virus-induced tumiour cells in rats was adoptively transferred to mice using the radioisotope FPA. The recipient mice were suppressed by X-irradiation of 500R to eliminate a host-versus-graft rejection. A graft-versus-host reaction by the donor rat lymphoid cell was not seen in the time period of the experiments. The decreased footpad reaction in heavily irradiated mice (800 Rbntgens or more) indicates that cells of host origin may be necessary for the manifestation of the antitumiour cell-mediated immunity in the footpad. The host cells are not T-dependent lymphocytes because the footpad reaction could be adoptively transferred to athymic nude mice (Takeichi & Boone, 1975). These cells may be macrophages
Xenogenetic trahsfer ofhuman DH reaction
313
which are relatively resistant to X-irradiation, as reported by other investigators (Tubergen & Feldman, 1971; Zarling & Tevethia, 1973), although a recent report appears to show that histoincompatible macrophages will not support the reaction between antigens and T-cells (Doherty & Zinkernagel, 1975). A mixture of living BCG and peripheral blood lymphocytes from tuberculin-positive donors produced significant footpad reactions whose intensity was proportional to the intensity of the skin reactions to PPD in the humans donating the lymphocytes. The fact that the footpad reaction induced by the lymphocyte-Varidase mixture was inferior to that of the lymphocyte-BCG mixture may be the result of the Varidase being quickly absorbed into the blood stream because of its soluble state. Local adoptive transfer of human cell-mediated immunity to irradiated mice using the radioisotope FPA provides a unique, non-perturbational assay system for studying immunological phenomena in humans. The applicability of this system to the bioassay of antitumour immunity in human cancer patients is under study. The authors wish to thank the following members of the Department of Dermatology, Roswell Park Memorial Institute, Buffalo New York 14203: Drs Seung-Yil Song and David J. Klein, for providing blood samples from patients, and Mr Robert W. Case, for his expert technical and general assistance.
REFERENCES ASHERTON, G.L. & ZEMBALA, M. (1975) Contact sensitivity in the mouse. IV. The role of lymphocytes and macrophages in passive transfer and the mechanism of their interaction. J. exp. Med. 132, 1. BARD, D.S. & PILCH, Y.H. (1969) The role of the spleen in immunity to a chemically induced sarcoma in C3H mice. Cancer Res. 29, 1125. CHASE, M.W. (1945) The cellular transfer of cutaneous hypersensitivity to tuberculin. Exp. biol. Med. 59, 134. DELORME, E.J. & ALEXANDER, P. (1964) Treatment of primary fibrosarcoma in the rat with immune lymphocytes. Lancet, ii, 117. DOHERTY, P.C. & ZINKERNAGEL, R.M. (1975) H-2 compatible is required for T-cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus, _. exp. Med. 14, 502. FISCHER, J.C. & HAMMOND, W.G. (1966) Inhibition of tumor growth by syngeneic spleen cell transfer. Surg. Forum, 18, 102. OLD, L.J., BOYSE, E.A., CLARK, D. & CARSWELL, E.A. (1962) Antigenic properties of chemically-induced tumors. Ann. N.Y. Acad. Sci. 101, 80. PARANJPE, M.S. & BOONE, C.W. (1972) Delayed hypersensitivity to simian virus 40 tumor cells in BALB/c mice demonstrated by a radioisotopic footpad assay. J. nat. Cancer Inst. 48, 563.
ROSENFELD, S.S., BERNHARD, J.D., KLEIN, E. (1973) Passive transfer of cell-mediated immunity in xenogeneic animals. Cell. Immunol. 8, 403. TAKEICHI, N. & BOONE, C.W. (1975) Local adoptive transfer of the antitumor cellular immune response in syngeneic and allogeneic mice studied with a rapid radioisotopic footpad assay. _7. nat. Cancer Inst. 55, 183. TAKEICHI, N., ECONOMOu, G.C. & BOONE, C.W. (1976) Local adcptive transfer of antitumor cellular immunity to xenogeneic animals studied with a rapid radioisotopic footpad assay. 3. nat. Cancer Inst. 57, 131. TUBERGEN, D.G. & FELDMAN, J.D. (1971) The role of thymus and bone marrow cells in delayed hypersensitivity. 3. exp. Med. 134, 1144. WALLACE, J.H. (1958) Leukocytic transfer of delayed sensitivity to normal guinea pigs from rat infected with mycobacterium lepraemuricum. Y. Immunol. 80, 362. WEPSIc, H.T., ZBAR, B. & RAPP, H.J. (1970) Systemic transfer of tumor immunity: Delayed hypersensitivity and suppression of tumor growth. ]. nat. Cancer Inst. 44, 955. ZARLING, J.M. & TEVETHIA, S.S. (1973) Transplantation immunity to simian virus 40-transformed cells in tumorbearing mice. II. Evidence for macrophage participation at the effector level of tumor cell rejection. J7. nat. Cancer Inst. 50, 149.
Local adoptive transfer to mice of human delayed hypersensitivity REACTIONS BY THE RADIOISOTOPIC FOOTPAD ASSAY NORITOSHI TAKEICHI*, C. W. BOONE* & E. KLEINt * Cell Biology Section, Laboratory of Viral Carcinogenesis, Viral Oncology Program, National Cancer Institute, U.S. Department of Health, Education and Welfare, Bethesda, Maryland and t Department of Dermatology, Rosmell Park Memorial Institute, Buffalo, N.Y., U.S.A.
(Received 26 April 1976) SUMMARY
Human delayed hypersensitivity to living BCG organisms and/or Varidase was adoptively transferred to mice and assayed in mice by a radioisotope footpad assay (FPA). A mixture of the antigen and peripheral blood lymphocytes from patients (positive skin reactions to PPD and/or Varidase) or healthy volunteers was inoculated into the footpads of lethally X-irradiated mice. A positive footpad reaction was accompanied by an increased leakage of the radiolabelled serum protein from the blood stream into the intercellular space at the site of inoculation, and was measured by the 'foot-count ratio', or radioactivity in the test foot divided by radioactivity in the contralateral foot. The intensity of the footpad reaction correlated directly with the skin test response of the human lymphocyte donors.
INTRODUCTION We have previously described a radioisotope footpad assay (FPA) for delayed hypersensitivity in mice both to tuberculin and to tumour-specific antigens of the transplantation type (Paranjpe & Boone, 1972; Takeichi & Boone, 1975). We showed that this assay can be applied between species by adoptive transfer of a mixture of tumour cells and lymphocytes from the tumour-immune or tumour-bearing rats to the footpad of mice (Takeichi & Boone, 1975; Takeichi, Economou & Boone, 1976). This report describes the local adoptive transfer to X-irradiated mice of human delayed hypersensitivity against skin antigens measured by the radioisotope FPA. MATERIALS AND METHODS Animals. An inbred strain of 8-12-week-old male and female BALB/cAnN mice was obtained from the Small Animal Section, Veterinary Resources Branch, National Institutes of Health. Skin test antigens. Tuberculin, purified protein derivative (PPD), 5 TU/0-1 ml was purchased from-Park, Davis & Co., Detroit, Michican. Varidase skin test antigen, 50 u streptokinase/ml was obtained from Roswell Park Memorial Institute, Department of Drug Formation & Development, Buffalo, New York. Living BCG (Phipps strain, TMC No. 1029, Trudeau Institute Saranac Lake, New York) was provided kindly by Dr H. J. Rapp, Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Lymphocyte donors. Lymphocytes from eleven individuals (three with melanoma, one with mycosis fungoides and seven healthy volunteers) were separated from heparinized venous blood by a modified Ficoll-Hypaque technique using Lymphoprep (Nyegaard & Co., As. Oslo, Norway). Fifteen ml whole blood was layered on 20 ml of Lymphoprep fluid in a 50-ml tube and centrifuged for 30 min at 18-200C, at exactly 400 g. The lymphocytes at the interface were transferred to a 50-ml tube, 20-30 ml phosphate buffer solution (PBS) plus 5% foetal calf serum (FCS) was added, and the cells were centrifuged for 10 min at 18-20'C at 350 g. They were resuspended in PBS plus 5% FCS, centrifuged at 18-20'C at 160 g for 10 min to remove most of the platelets, and then resuspended in RPMI1640 medium plus 10% FCS. The total yield of lymphocytes from 80-100 ml of whole blood from each individual donor was 10-20x 107 cells. A smear of the final preparation revealed 80-90% small lymphocytes, 10-20% monocytes, and 2-5% polymorphonuclear leucocytes. Viability counts ranged from 95-100% as determined by trypan blue exclusion. Correspondence: Dr Noritoshi Takeichi, Cell Biology Section, Laboratory of Viral Carcinogenesis, Viral Oncology Program, National Cancer Institute, U.S. Department of Health, Education and Welfare, Bethesda, Maryland 20014, U.S.A.
310
Xenogeneic transfer of human DH reaction
311
Skin test. These were performed with 0 1 ml (5 u) PPD and 0 1 ml (5 u of streptokinase) Varidase, given i.c. at the same time as blood was drawn for lymphocytes. Induration was measured at 24 and 48 hr and recorded as the smallest and largest diameter in mm. Between 0 and 5 mm was considered negative, between 5 and 10 mm weak positive, between 10 and 20 mm positive and over 20 mm strong positive. Radioisotope FPA. Xenogeneic transfer of the DH reaction to skin test antigens between human and mouse was carried out by the method described in previous papers (Paranjpe & Boone, 1972; Takeichi & Boone, 1975). Four million lymphocytes from individual donors were mixed with 1 x 106 living BCG or 5 u streptokinase and injected into the left rear footpad of mice previously X-irradiated with 500 R. Immediately afterwards, 125I-labelled mouse serum albumin or human serum (Mallikrodt Chemical Works, St Louis, Missouri) was injected i.p. (106 ct/min per mouse). Twenty-four hr later the test foot and contralateral foot were excised at the junction of the lower and middle thirds of the tibia and counted in a gamma spectrometer. Results were expressed as the foot-count ratio = ct/min in the test foot divided by ct/min in the contralateral control foot. The significance of the footpad reaction of the group of animals was evaluated by comparing the mean foot-count ratio of the test foot to the control foot by Student's t-test; P-value less than 0 05 was considered significant. X-irradiation. All recipient mice were immunosuppressed with 500 R of X-irradiation (Westinghouse Quadrocondex X-ray Unit, 200 kV, 15 mA at a 25 cm distance from the X-ray source) 24 hr before use.
RESULTS Local adoptive transfer of human delayed hypersensitivity to mice Delayed hypersensitivity to BCG and/or Varidase was adoptively transferred from human to Xirradiated mice with lymphocytes isolated from peripheral blood. Three melanoma patients, who had TABLE 1. Xenogeneic adoptive transfer of delayed hypersensitivity to BCG and/or varidase from human to irradiated mice using a radioisotopic footpad assay
Therapy or Sex immunization with BCG
No. of donors
Diagnosis
1
Melanoma
37
M
Yes
2
Melanoma
44
M
Yes
3
53
M
No
4
Mycosis fungoides Melanoma
60
M
Yes
5
Normal
30
F
No
6
Normal
35
M
No
7
Normal
36
M
No
8
Normal
48
M
No
9
Normal
36
M
No
10
Normal
16
M
No
11
Normal
23
M
No
*
Age
Skin reaction* to
Foot-count ratio+ s.e.t to
PPD
Varidase
BCG
34x 34 (24 hr) 28x 28 (48 hr) 28x 28 35 x 40 30x 30 n.d. 20x 35 20x 35 18 x 20 30x35 16x20 30x 35 18x20 28 x 30 8x 10 lOx 12 Negative Negative Negative Negative Negative
55x 55 55x 55 5x 5 n.d. 20x 20 n.d. n.d. n.d. 5x 5 5x5 7x 12 7x 12 Negative Negative n.d. n.d. n.d. n.d. Negative Negative Negative
2-11+0-12$
1-45+0-04¶1
1P97+0-09+
1-13+0-03
1-89 x 0-08t
1P38 + 0 04¶
1-74+ 0 07$
1-24+ 0-03
1-86+ 0 09t
1 09+ 0 03
1-67+0-05§
1-12+0-03
1-63+0 05§
1 11+003
1-30+0-03
n.d.
1-31+0-04
n.d.
1-41+0 04
1P09+ 003
1-37+0-04
1-06+ 005
Varidase
Size of indurated areas are given by largest x smallest diameter in mm.
t Mean foot-count ratio from patients No. 9, 10 and 11 (skin test negative to PPD) was 1-33 + 0 03. Mean foot-count ratio from patients No. 7, 10 and 11 (skin test negative to varidase) was 1-09+ 0*03, mean foot-count ratio of living BCG (1 x 106 organisms/mouse) alone was 1-20+ 0 05. Mean foot-count ratio of varidase (5 u streptokinase/mouse) alone was 1 09+ 0 03. t Mean foot-count ratio from each donor versus mean foot-count ratio from patients No. 9, 10 and 11, P< 0 001. § Mean foot-count ratio from each donor versus mean foot-count ratio from patients No. 9, 10 and 11, P< 0 01. ¶ Mean foot-count ratio from each donor versus mean foot-count ratio from patients No. 7, 10 and 11, P< 0-05.
Noritoshi Takeichi, C. W. Boone & E. Klein
312
2-00 E
.~i*80+1
.2
1.60
0
'
1-40
0 0
1-20± 100
500 700 900 300 Skin reaction to PPD in humans (product of 2 diameters in mm)
1100
FIG. 1. Local adoptive transfer in mice of human delayed hypersensitivity to PP D.
been treated with BCG for more than 1 year, and one patient with mycosis fungoides had strong positive skin reactions to PPD at both 24 and 48 hr. One melanoma patient (No. 1) and one mycosis fungoides patient (No. 3) showed strong positive skin reactions to the varidase. Four of seven healthy volunteers had positive skin reactions to PPD and two of five tested had weak positive skin reactions to the Varidase. Peripheral lymphocytes from eight tuberculin-positive donors all produced significant reactions when they were mixed with living BCG and injected into the footpads of X-irradiated mice (Table 1). Two Varidase-positive patients (Nos. 1 and 3) also showed positive adoptive footpad reactions to the Varidase in mice. The intensity of the footpad reaction closely paralleled the intensity of the delayed cutaneous reaction in the human donating the test lymphocytes (Fig. 1). The specificity of the radioisotope FPA is shown by the dissociation of the footpad reaction with the lymphocytes from the melanoma patient (No. 2) and from healthy volunteers (Nos. 5 and 6) to BCG and Varidase, as contrasted with the positive footpad reactions of lymphocytes from the melanoma patient (No. 1) and the mycosis fungoides patient (No. 3) to both PPD and Varidase.
DISCUSSION
Delayed hypersensitivity to chemicals or micro-organisms (Asherson & Zembala, 1975; Chase, 1945; Paranjpe & Boone, 1972) or cell-mediated immunity against tumour specific antigens (Bard & Pilch, 1969; Delorme & Alexander, 1964; Fischer & Hammond, 1966; Old et aL., 1962; Takeichi & Boone, 1975; XWepsic, Zbar & Rapp, 1970) can be passively transferred to normal recipient animals with peritoneal exudate, lymph node, or spleen cells from immunized animals. Recent work by Edmund Klein and his associates (Rosenfeld, Bernhard & Klein, 1973) using a guinea-pig-to-mouse system, and by Wallace (1958), in a rat-to-guinea-pig system, have shown the feasibility of the adoptive transfer of cell-mediated immunity to xenogeneic animals. XWe have developed an in vivo assay for antitumour immunity in allogeneic and xenogeneic systems (Takeichi & Boone, 1975; Takeichi, Economou & Boone, 1976). The cellular immunity to Gross virus-induced tumiour cells in rats was adoptively transferred to mice using the radioisotope FPA. The recipient mice were suppressed by X-irradiation of 500R to eliminate a host-versus-graft rejection. A graft-versus-host reaction by the donor rat lymphoid cell was not seen in the time period of the experiments. The decreased footpad reaction in heavily irradiated mice (800 Rbntgens or more) indicates that cells of host origin may be necessary for the manifestation of the antitumiour cell-mediated immunity in the footpad. The host cells are not T-dependent lymphocytes because the footpad reaction could be adoptively transferred to athymic nude mice (Takeichi & Boone, 1975). These cells may be macrophages
Xenogenetic trahsfer ofhuman DH reaction
313
which are relatively resistant to X-irradiation, as reported by other investigators (Tubergen & Feldman, 1971; Zarling & Tevethia, 1973), although a recent report appears to show that histoincompatible macrophages will not support the reaction between antigens and T-cells (Doherty & Zinkernagel, 1975). A mixture of living BCG and peripheral blood lymphocytes from tuberculin-positive donors produced significant footpad reactions whose intensity was proportional to the intensity of the skin reactions to PPD in the humans donating the lymphocytes. The fact that the footpad reaction induced by the lymphocyte-Varidase mixture was inferior to that of the lymphocyte-BCG mixture may be the result of the Varidase being quickly absorbed into the blood stream because of its soluble state. Local adoptive transfer of human cell-mediated immunity to irradiated mice using the radioisotope FPA provides a unique, non-perturbational assay system for studying immunological phenomena in humans. The applicability of this system to the bioassay of antitumour immunity in human cancer patients is under study. The authors wish to thank the following members of the Department of Dermatology, Roswell Park Memorial Institute, Buffalo New York 14203: Drs Seung-Yil Song and David J. Klein, for providing blood samples from patients, and Mr Robert W. Case, for his expert technical and general assistance.
REFERENCES ASHERTON, G.L. & ZEMBALA, M. (1975) Contact sensitivity in the mouse. IV. The role of lymphocytes and macrophages in passive transfer and the mechanism of their interaction. J. exp. Med. 132, 1. BARD, D.S. & PILCH, Y.H. (1969) The role of the spleen in immunity to a chemically induced sarcoma in C3H mice. Cancer Res. 29, 1125. CHASE, M.W. (1945) The cellular transfer of cutaneous hypersensitivity to tuberculin. Exp. biol. Med. 59, 134. DELORME, E.J. & ALEXANDER, P. (1964) Treatment of primary fibrosarcoma in the rat with immune lymphocytes. Lancet, ii, 117. DOHERTY, P.C. & ZINKERNAGEL, R.M. (1975) H-2 compatible is required for T-cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus, _. exp. Med. 14, 502. FISCHER, J.C. & HAMMOND, W.G. (1966) Inhibition of tumor growth by syngeneic spleen cell transfer. Surg. Forum, 18, 102. OLD, L.J., BOYSE, E.A., CLARK, D. & CARSWELL, E.A. (1962) Antigenic properties of chemically-induced tumors. Ann. N.Y. Acad. Sci. 101, 80. PARANJPE, M.S. & BOONE, C.W. (1972) Delayed hypersensitivity to simian virus 40 tumor cells in BALB/c mice demonstrated by a radioisotopic footpad assay. J. nat. Cancer Inst. 48, 563.
ROSENFELD, S.S., BERNHARD, J.D., KLEIN, E. (1973) Passive transfer of cell-mediated immunity in xenogeneic animals. Cell. Immunol. 8, 403. TAKEICHI, N. & BOONE, C.W. (1975) Local adoptive transfer of the antitumor cellular immune response in syngeneic and allogeneic mice studied with a rapid radioisotopic footpad assay. _7. nat. Cancer Inst. 55, 183. TAKEICHI, N., ECONOMOu, G.C. & BOONE, C.W. (1976) Local adcptive transfer of antitumor cellular immunity to xenogeneic animals studied with a rapid radioisotopic footpad assay. 3. nat. Cancer Inst. 57, 131. TUBERGEN, D.G. & FELDMAN, J.D. (1971) The role of thymus and bone marrow cells in delayed hypersensitivity. 3. exp. Med. 134, 1144. WALLACE, J.H. (1958) Leukocytic transfer of delayed sensitivity to normal guinea pigs from rat infected with mycobacterium lepraemuricum. Y. Immunol. 80, 362. WEPSIc, H.T., ZBAR, B. & RAPP, H.J. (1970) Systemic transfer of tumor immunity: Delayed hypersensitivity and suppression of tumor growth. ]. nat. Cancer Inst. 44, 955. ZARLING, J.M. & TEVETHIA, S.S. (1973) Transplantation immunity to simian virus 40-transformed cells in tumorbearing mice. II. Evidence for macrophage participation at the effector level of tumor cell rejection. J7. nat. Cancer Inst. 50, 149.
