~
CancerImmunolImmunother(1992) 34:221-227
ancer
mmunology mmunothëPapy
© Springer-Verlag 1992
Tissue distribution of adoptively transferred adherent lymphokine-activated killer cells assessed by different cell labels P. Basse I *, R. B. Herberman 1, M. Hokland 2, and R. H. Goldfarb 1 1PittsburghCancerInstituteand Departmentsof Pathologyand Medicine,Universityof Pittsburgh,Pittsburgh,PA 15 213, USA. 2 Instituteof Medical Microbiology,Universityof Aarhus, DK-8000,Am'husC, Denmark Received 19 August 1991/Accepted25 September1991 Summary. Assessment of the tissue distribution of adoptively transferred adherent lymphokine-activated killer A-LAK) cells by use of 51Cr indicated that these effector cells, after an initial phase in the lungs, distributed in high numbers to liver and spleen (30% and 10% of injected dose, respectively). However, when this experiment was repeated with 125IdUrd as cell label, fewer than 2% and 0.5% of the injected cells distributed into liver and spleen respectively. To analyse this discrepancy, we compared the tissue distribution of 51Cr- and 125IdUrd-labelled A-LAK cells with that indicated by alternative direct visual methods for identification of the injected cells, such as fluorescent dyes (rhodamine and H33342) or immunohistochemical staining of asialo-GMl-positive cells. The number of i.v. injected A-LAK cells found in the liver by all visual methods ranged from 1% to 5% of the injected dose, supporting the data obtained with 125IdUrd, whereas 25%-30% of the 51Cr label was consistently found in this organ. Autoradiography of the liver 24 h after i. v. injection of 51Cr-labelled cells revealed a background activity that was four- to fivefold higher than the control level, indicating substantial non-specific accumulation in the liver of 51Cr released from A-LAK cells. We conclude that 51Cr cannot be reliably used in investigations of cell traffic to the liver because of non-specific accumulation of the 51Cr label, particularly in this organ. In contrast, labelling with 125IdUrd or rhodamine and immunohistochemical staining of asialo-GMl-positive cells appear to be reliable and essentially equivalent methods for investigations of the fate of adoptively transferred A-LAK cells. Using these methods, we found that only few A-LAK cells redistribute to the liver upon i.v., i.e. systemic, injection, whereas 40%-50% of locally (intraportally) injected A-LAK cells remain in the liver for at least 24 h.
Institute of Medical Microbiology,Universityof Aarhus, DK-8000, AarhusC, Denmark Present address:
Offprint requests to:
P. Basse at present address
Key words: LAK migration - Asialo-GM1 antibody Fluorescence - Rhodamine - Hoechst 33342
Introduction Adoptive immunotherapy using lymphokine-activated killer (LAK and A-LAK) cells and interleukin-2 (IL-2) has proven successful in many animal models [7, 14, 16, 17, 23, 24, 26, 28] whereas the results of clinical studies have been less encouraging, with response rates of 20% or more observed only for melanomas and renal cell carcinomas [26, 27]. Many efforts have thus been invested in improving this therapeutic modality and further clarifying the mechanism(s) behind the in vivo anti-cancer effect of LAK and adherent (A)-LAK cells. Among many unresolved questions, it seems very important to analyse the migratory pattern of adoptively transferred effector cells, in order to evaluate their capability of reaching sites of tumour growth. Recently the in vivo tissue distribution of 51Cr or [111In]indium-oxine-labelled LAK and A-LAK cells has been assessed in both normal and tumour-bearing animals [1, 8, 20-22, 25]. On the basis of these results, it is widely accepted that 30%-50% of i. v. injected LAK and A-LAK cells redistribute to liver and spleen after an initial phase in the lungs. Therefore, orte would expect that upon i.v. administration a reasonable number of adoptively transferred LAK and A-LAK cells would be able to reach malignant lesions in organs such as the liver. However, studies of tumour cell traffic and leucocyte migration have indicated a definite risk of overestimating the distribution of cells into liver and spleen when using 51Cr and [11qn]indium oxine, because of non-specific uptake of released label by these organs [2, 31]. This phenomenon might have led also to an overestimation of the migratory capacity of LAK and A-LAK cells. In order to evaluate this potential problem in the context of adoptively transferred A-LAK cells and to elucidate
222 f u r t h e r t h e m i g r a t o r y p o t e n t i a l o f t h e s e cells, w e h a v e i n vestigated their tissue distribution in normal recipients, using different methods for identification of the injected cells. I d e n t i f i c a t i o n o f a s a t i s f a c t o r y l a b e l a m o n g t h o s e previously published should facilitate further studies on the biological potential of LAK and A-LAK cells and possibly facilitate the design of new strategies for immunotherapy w i t h t h e s e cells.
Materials and methods Animals. Male C57BL/6 mice, 1 0 - 1 4 weeks of age, were used throughout the study. Preparation ofA-LAK cells. The preparation of A-LAK cells has been described elsewhere [11, 29]. Briefly, single-cell suspensions from spleens were prepared in RPMI-1640 medium and red blood cells were lysed by incubation with ammonium chloride/potassium buffer. After two washes, cells were transferred into Tl50 plastic flasks with 50 ml complete medium consisting of RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 20 mM HEPES buffer, 0.8 g/1 streptomycin, 1.6 × 10-s U/1 penicillin, 10 ml/1 100 × non-essential amino acids (Gibco), 50 gM 2-mercaptoethanol and 1000 U/tal IL-2 (kindly provided by the Cetus corporation, Emeryville, Calif., and EuroCetus, Amsterdam, The Netherlands). After 2 - 3 days of incubation at 37 ° C in 5% CO2 in air, non-adherent spleen cells were removed and the flasks were gently washed with pre-warmed (37" C) complete medium to remove cells not firmly attached to the plastic; 50 ml fresh complete medium was then added and the cells were cultured for an additional 2 - 3 days. After a total of 5 days in culture, cells were harvested with 0.02% EDTA and washed twice in RPMI-1640 medium before use. In accordance with previous descriptions [11, 12], such cells were phenotypically at least 95% large granular lymphocytes, at least 98% asialo-GM1 +, 60% - 85% NK1.1 t, but only 4% - 11% Lyt 2+ and less than 2% L3T4 +. Labelling of A-LAK cells with 51Cr or 1251dUrd. Samples of ( 2 - 4 ) x 107 A-LAK cells in 50 ml complete medium were incubated for 18-22 h with 1 - 2 g C i 125IdUrd ([l:sI]iododeoxyuridine, specific activity = 5 Ci/mg, obtained from Amersham, Buckinghamshire, England). The cells were washed twice and incubated in 0.5 ml complete medium containing 100 gl 51Cr for 50 min at 37 ° C. After three washes, the cells were resuspended in RPMI-1640 medium to the appropriate concentration. This procedure resulted in 3 0 - 5 0 cpm/103 cells with 125IdUrd and 120-200 cpm/10» cells with 51Cr. with viabilities exceeding 95% as judged by trypan blue exclusion. Labelling of A-LAK cells with rhodamine or H33342. Samples of ( 3 - 6 ) x 1 0 8 A-LAK cells were incubated with 15 gg rhodamine (TRITC, isomer R, Sigma, T2018) in 50 ml RPMI-1640 medium for 30 min, at 37 ° C (this low dose of rhodamine was found not to alter the proliferative properties or the cytotoxic capacity of the cells; data not shown). Samples of 5 × 108 A-LAK cells were labelled with 10 gM Hoechst no. 33342 (Sigma, B2261) for 15 min at 37°C as described by Weston et al. [30]. After labelling, cells were washed twice and resuspended in RPMI-1640 medium to appropriate concentrations. Inoculation ofA-LAK cells. Intravenous injections of 15 × 106 A-LAK cells in a volume of 200 gl were performed in the lateral tail vein. Before intraportal injections, mice were anaesthetized with nembutal and the anterior abdominal wall was opened. A branch of the portal vein was identified and 10 x 106 A-LAK cells in a volume of 200 gl were injected using a 30-gauge needle. Bleeding was terminated by light compression and the abdominal wall was closed with surgical clips. Mice received i. p. injections of RPMI-1640 medium (control) or 50000 U IL-2 in RPMI1640 medium 0, 8, and 16 h after injection of A-LAK cells. Measurement of radioactiviß,. Between 2 h and 24 h after injection, groups of animals were sacrificed and organs were removed and
processed for gamma counting as follows: each organ was placed in vials containing 1 ml 70% ethyl alcohol and counted immediately in a gamma counter adjusted to detect gamma rays from 51Cr (260-400 keV). After counting, the organs were placed in 50 mi vials containing 70% alcohol, which was replaced once a day for 3 days, by which time 99% of the acid/ethanol-soluble 125IdUrd was removed, the remaining organ radioactivity being associated only with whole DNA molecules [3, 15]. The organs were placed in the gamma counter now adjusted to detect 1251 (20-48 keV). Corrections were made for decay and spill-over. Data are presented as the mean percentage recovery of radioactivity per organ of the injected dose.
Fluorescence microscopy. Mice received i.v. injections of 200 gl India ink (Rönnings Perle-Tush, Denmark), diluted to a final concentration of 12 mg/tal, 30 min before removal of organs. Organs were fixed in 4% formalin for 18 h and thereafter placed in 30% sucrose for an additional 18 h. The tissues were frozen in n-hexane at -70°C and 8-gm cryosections were prepared, Rhodamine- or H33342-1abelled A-LAK cells were identified using a Zeiss fluorescence microscope with a HBO 200 w/4 mercury vapour lamp. Estimation of the total number of fluorescent cells per organ. The mean number (n) of fluorescent cells/mm2 liver tissue, sampled from 2 0 - 3 0 different areas of each liver, was determined. Fluorescent material found inside accumulations of India ink was regarded as having been taken up by macrophages and was not counted. Given the section thickness (8 gin) and the volume of the livers, the total number N of fluorescent cells per organ was estimated as: N = n × organ volume/section thickness It should be noted that such an estimation of the number of rhodamine-labelled cells per organ will result in some overestimation of the number of cells. The diameter of most A-LAK cells exceeds the section thickness and many fluorescent cells will, therefore, be visible in more than one section [10]. Analysis of serial sections indicated that approximately 30% of the rhodamine-labelled cells are visible in more than one section. The data given in Tables 3 and 4 can therefore be expected to overestimate the true number of rhodamine-labelled cells per organ by approximately 30%, Since H33342 stains only the cell nucleus [18], fewer cells would be visible in more than one section and the overestimation of the number of H33342-1abelled cells per organ because of this phenomenon would be rauch less pronounced.
Immunohistochemistry. Livers were removed and immediately frozen at -70°C in n-hexane, and 8-gm sections were cut from different areas of the organ. The sections were stained by a two-layer immunoperoxidase technique, using rabbit anti-(asialo-GMa) antibody (Wako Chemical GmbH, Neuss 1, FRG) at a 1 : 250 dilution as the first layer. As a control, sections were incubated with normal rabbit serum in a 1:4 dilution. Peroxidase-conjugated swine anti-(rabbit immunoglobulin) (code P217, Dako-Patts Copenhagen, Denmark) in a 1 : 25 dilution was used as the second layer and diaminobenzidine (D-5637, Sigma Chemical Company) was used as a chromogen in all experiments. Sections were counterstained with trypan blue (0.25%) for 15 s followed by Meyer's haematoxylin for 30 s. Estimation of the total number of asialo-GMl-positive cells per organ. The number, N, of asialo-GM1 + cells in each organ was estimated from the following formula: N = [n × (organ volume/section thickness) x 0.67] - E where n is the mean number asialo-GM1+ cells/mme liver tissue, sampled from 2 0 - 3 0 different areas of each liver, 0.67 is a correction factor adjusting for the bias caused by the visibility of some cells in more than one section (Basse et al. in press) and E is the number of endogenous asialo-GM1 + cells in livers from normal and IL-2-treated animals (0.46 × 10 6 and 1.36 x 106 respectively).
Autoradiography. Cytospin preparations of 125IdUrd-labelled A-LAK cells or sections of liver tissue from animals previously injected with 51Cr-labelled A-LAK cells were dipped in a photographic emulsion
223 Table 1. Percentage recovery of adherent lymphokine-activated killer (A-LAK) cells in lung, liver and spleen 2 h and 24 h after i. v. injection of 2 x 106 double-labelled, murine A-LAK cells into C57BL/6 mice Time after injection
Radiolabel a
Recovery b (%)
(h) Lung
Liver
Spleen
Total
2
5~Cr 125IdUrd
78.3 (8.11) 80.8 (8.22)
13.0 (2.9) 7.2 (1.3) c
2.3 (0.9) 0.9 (0.3) c
106.1 (12.3) 97.8 (9.3)
24
51Cr 125IdUrd
0.9 (0.3) 0.7 (0.4)
27.0 (4.3) 1.4 (0.9) c
5.6 (0.9) 0.1 (0.1) c
42.4 (6.7) 9.7 (4.5) c
a A-LAK cells were labelled with both 51Cr and 125IdUrd b Data are presented as mean percentage recovery ( + SD) of the total radioactivity injected into the animal
o Significantly different from the corresponding 51Cr value by Student's t-test (P
CancerImmunolImmunother(1992) 34:221-227
ancer
mmunology mmunothëPapy
© Springer-Verlag 1992
Tissue distribution of adoptively transferred adherent lymphokine-activated killer cells assessed by different cell labels P. Basse I *, R. B. Herberman 1, M. Hokland 2, and R. H. Goldfarb 1 1PittsburghCancerInstituteand Departmentsof Pathologyand Medicine,Universityof Pittsburgh,Pittsburgh,PA 15 213, USA. 2 Instituteof Medical Microbiology,Universityof Aarhus, DK-8000,Am'husC, Denmark Received 19 August 1991/Accepted25 September1991 Summary. Assessment of the tissue distribution of adoptively transferred adherent lymphokine-activated killer A-LAK) cells by use of 51Cr indicated that these effector cells, after an initial phase in the lungs, distributed in high numbers to liver and spleen (30% and 10% of injected dose, respectively). However, when this experiment was repeated with 125IdUrd as cell label, fewer than 2% and 0.5% of the injected cells distributed into liver and spleen respectively. To analyse this discrepancy, we compared the tissue distribution of 51Cr- and 125IdUrd-labelled A-LAK cells with that indicated by alternative direct visual methods for identification of the injected cells, such as fluorescent dyes (rhodamine and H33342) or immunohistochemical staining of asialo-GMl-positive cells. The number of i.v. injected A-LAK cells found in the liver by all visual methods ranged from 1% to 5% of the injected dose, supporting the data obtained with 125IdUrd, whereas 25%-30% of the 51Cr label was consistently found in this organ. Autoradiography of the liver 24 h after i. v. injection of 51Cr-labelled cells revealed a background activity that was four- to fivefold higher than the control level, indicating substantial non-specific accumulation in the liver of 51Cr released from A-LAK cells. We conclude that 51Cr cannot be reliably used in investigations of cell traffic to the liver because of non-specific accumulation of the 51Cr label, particularly in this organ. In contrast, labelling with 125IdUrd or rhodamine and immunohistochemical staining of asialo-GMl-positive cells appear to be reliable and essentially equivalent methods for investigations of the fate of adoptively transferred A-LAK cells. Using these methods, we found that only few A-LAK cells redistribute to the liver upon i.v., i.e. systemic, injection, whereas 40%-50% of locally (intraportally) injected A-LAK cells remain in the liver for at least 24 h.
Institute of Medical Microbiology,Universityof Aarhus, DK-8000, AarhusC, Denmark Present address:
Offprint requests to:
P. Basse at present address
Key words: LAK migration - Asialo-GM1 antibody Fluorescence - Rhodamine - Hoechst 33342
Introduction Adoptive immunotherapy using lymphokine-activated killer (LAK and A-LAK) cells and interleukin-2 (IL-2) has proven successful in many animal models [7, 14, 16, 17, 23, 24, 26, 28] whereas the results of clinical studies have been less encouraging, with response rates of 20% or more observed only for melanomas and renal cell carcinomas [26, 27]. Many efforts have thus been invested in improving this therapeutic modality and further clarifying the mechanism(s) behind the in vivo anti-cancer effect of LAK and adherent (A)-LAK cells. Among many unresolved questions, it seems very important to analyse the migratory pattern of adoptively transferred effector cells, in order to evaluate their capability of reaching sites of tumour growth. Recently the in vivo tissue distribution of 51Cr or [111In]indium-oxine-labelled LAK and A-LAK cells has been assessed in both normal and tumour-bearing animals [1, 8, 20-22, 25]. On the basis of these results, it is widely accepted that 30%-50% of i. v. injected LAK and A-LAK cells redistribute to liver and spleen after an initial phase in the lungs. Therefore, orte would expect that upon i.v. administration a reasonable number of adoptively transferred LAK and A-LAK cells would be able to reach malignant lesions in organs such as the liver. However, studies of tumour cell traffic and leucocyte migration have indicated a definite risk of overestimating the distribution of cells into liver and spleen when using 51Cr and [11qn]indium oxine, because of non-specific uptake of released label by these organs [2, 31]. This phenomenon might have led also to an overestimation of the migratory capacity of LAK and A-LAK cells. In order to evaluate this potential problem in the context of adoptively transferred A-LAK cells and to elucidate
222 f u r t h e r t h e m i g r a t o r y p o t e n t i a l o f t h e s e cells, w e h a v e i n vestigated their tissue distribution in normal recipients, using different methods for identification of the injected cells. I d e n t i f i c a t i o n o f a s a t i s f a c t o r y l a b e l a m o n g t h o s e previously published should facilitate further studies on the biological potential of LAK and A-LAK cells and possibly facilitate the design of new strategies for immunotherapy w i t h t h e s e cells.
Materials and methods Animals. Male C57BL/6 mice, 1 0 - 1 4 weeks of age, were used throughout the study. Preparation ofA-LAK cells. The preparation of A-LAK cells has been described elsewhere [11, 29]. Briefly, single-cell suspensions from spleens were prepared in RPMI-1640 medium and red blood cells were lysed by incubation with ammonium chloride/potassium buffer. After two washes, cells were transferred into Tl50 plastic flasks with 50 ml complete medium consisting of RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 20 mM HEPES buffer, 0.8 g/1 streptomycin, 1.6 × 10-s U/1 penicillin, 10 ml/1 100 × non-essential amino acids (Gibco), 50 gM 2-mercaptoethanol and 1000 U/tal IL-2 (kindly provided by the Cetus corporation, Emeryville, Calif., and EuroCetus, Amsterdam, The Netherlands). After 2 - 3 days of incubation at 37 ° C in 5% CO2 in air, non-adherent spleen cells were removed and the flasks were gently washed with pre-warmed (37" C) complete medium to remove cells not firmly attached to the plastic; 50 ml fresh complete medium was then added and the cells were cultured for an additional 2 - 3 days. After a total of 5 days in culture, cells were harvested with 0.02% EDTA and washed twice in RPMI-1640 medium before use. In accordance with previous descriptions [11, 12], such cells were phenotypically at least 95% large granular lymphocytes, at least 98% asialo-GM1 +, 60% - 85% NK1.1 t, but only 4% - 11% Lyt 2+ and less than 2% L3T4 +. Labelling of A-LAK cells with 51Cr or 1251dUrd. Samples of ( 2 - 4 ) x 107 A-LAK cells in 50 ml complete medium were incubated for 18-22 h with 1 - 2 g C i 125IdUrd ([l:sI]iododeoxyuridine, specific activity = 5 Ci/mg, obtained from Amersham, Buckinghamshire, England). The cells were washed twice and incubated in 0.5 ml complete medium containing 100 gl 51Cr for 50 min at 37 ° C. After three washes, the cells were resuspended in RPMI-1640 medium to the appropriate concentration. This procedure resulted in 3 0 - 5 0 cpm/103 cells with 125IdUrd and 120-200 cpm/10» cells with 51Cr. with viabilities exceeding 95% as judged by trypan blue exclusion. Labelling of A-LAK cells with rhodamine or H33342. Samples of ( 3 - 6 ) x 1 0 8 A-LAK cells were incubated with 15 gg rhodamine (TRITC, isomer R, Sigma, T2018) in 50 ml RPMI-1640 medium for 30 min, at 37 ° C (this low dose of rhodamine was found not to alter the proliferative properties or the cytotoxic capacity of the cells; data not shown). Samples of 5 × 108 A-LAK cells were labelled with 10 gM Hoechst no. 33342 (Sigma, B2261) for 15 min at 37°C as described by Weston et al. [30]. After labelling, cells were washed twice and resuspended in RPMI-1640 medium to appropriate concentrations. Inoculation ofA-LAK cells. Intravenous injections of 15 × 106 A-LAK cells in a volume of 200 gl were performed in the lateral tail vein. Before intraportal injections, mice were anaesthetized with nembutal and the anterior abdominal wall was opened. A branch of the portal vein was identified and 10 x 106 A-LAK cells in a volume of 200 gl were injected using a 30-gauge needle. Bleeding was terminated by light compression and the abdominal wall was closed with surgical clips. Mice received i. p. injections of RPMI-1640 medium (control) or 50000 U IL-2 in RPMI1640 medium 0, 8, and 16 h after injection of A-LAK cells. Measurement of radioactiviß,. Between 2 h and 24 h after injection, groups of animals were sacrificed and organs were removed and
processed for gamma counting as follows: each organ was placed in vials containing 1 ml 70% ethyl alcohol and counted immediately in a gamma counter adjusted to detect gamma rays from 51Cr (260-400 keV). After counting, the organs were placed in 50 mi vials containing 70% alcohol, which was replaced once a day for 3 days, by which time 99% of the acid/ethanol-soluble 125IdUrd was removed, the remaining organ radioactivity being associated only with whole DNA molecules [3, 15]. The organs were placed in the gamma counter now adjusted to detect 1251 (20-48 keV). Corrections were made for decay and spill-over. Data are presented as the mean percentage recovery of radioactivity per organ of the injected dose.
Fluorescence microscopy. Mice received i.v. injections of 200 gl India ink (Rönnings Perle-Tush, Denmark), diluted to a final concentration of 12 mg/tal, 30 min before removal of organs. Organs were fixed in 4% formalin for 18 h and thereafter placed in 30% sucrose for an additional 18 h. The tissues were frozen in n-hexane at -70°C and 8-gm cryosections were prepared, Rhodamine- or H33342-1abelled A-LAK cells were identified using a Zeiss fluorescence microscope with a HBO 200 w/4 mercury vapour lamp. Estimation of the total number of fluorescent cells per organ. The mean number (n) of fluorescent cells/mm2 liver tissue, sampled from 2 0 - 3 0 different areas of each liver, was determined. Fluorescent material found inside accumulations of India ink was regarded as having been taken up by macrophages and was not counted. Given the section thickness (8 gin) and the volume of the livers, the total number N of fluorescent cells per organ was estimated as: N = n × organ volume/section thickness It should be noted that such an estimation of the number of rhodamine-labelled cells per organ will result in some overestimation of the number of cells. The diameter of most A-LAK cells exceeds the section thickness and many fluorescent cells will, therefore, be visible in more than one section [10]. Analysis of serial sections indicated that approximately 30% of the rhodamine-labelled cells are visible in more than one section. The data given in Tables 3 and 4 can therefore be expected to overestimate the true number of rhodamine-labelled cells per organ by approximately 30%, Since H33342 stains only the cell nucleus [18], fewer cells would be visible in more than one section and the overestimation of the number of H33342-1abelled cells per organ because of this phenomenon would be rauch less pronounced.
Immunohistochemistry. Livers were removed and immediately frozen at -70°C in n-hexane, and 8-gm sections were cut from different areas of the organ. The sections were stained by a two-layer immunoperoxidase technique, using rabbit anti-(asialo-GMa) antibody (Wako Chemical GmbH, Neuss 1, FRG) at a 1 : 250 dilution as the first layer. As a control, sections were incubated with normal rabbit serum in a 1:4 dilution. Peroxidase-conjugated swine anti-(rabbit immunoglobulin) (code P217, Dako-Patts Copenhagen, Denmark) in a 1 : 25 dilution was used as the second layer and diaminobenzidine (D-5637, Sigma Chemical Company) was used as a chromogen in all experiments. Sections were counterstained with trypan blue (0.25%) for 15 s followed by Meyer's haematoxylin for 30 s. Estimation of the total number of asialo-GMl-positive cells per organ. The number, N, of asialo-GM1 + cells in each organ was estimated from the following formula: N = [n × (organ volume/section thickness) x 0.67] - E where n is the mean number asialo-GM1+ cells/mme liver tissue, sampled from 2 0 - 3 0 different areas of each liver, 0.67 is a correction factor adjusting for the bias caused by the visibility of some cells in more than one section (Basse et al. in press) and E is the number of endogenous asialo-GM1 + cells in livers from normal and IL-2-treated animals (0.46 × 10 6 and 1.36 x 106 respectively).
Autoradiography. Cytospin preparations of 125IdUrd-labelled A-LAK cells or sections of liver tissue from animals previously injected with 51Cr-labelled A-LAK cells were dipped in a photographic emulsion
223 Table 1. Percentage recovery of adherent lymphokine-activated killer (A-LAK) cells in lung, liver and spleen 2 h and 24 h after i. v. injection of 2 x 106 double-labelled, murine A-LAK cells into C57BL/6 mice Time after injection
Radiolabel a
Recovery b (%)
(h) Lung
Liver
Spleen
Total
2
5~Cr 125IdUrd
78.3 (8.11) 80.8 (8.22)
13.0 (2.9) 7.2 (1.3) c
2.3 (0.9) 0.9 (0.3) c
106.1 (12.3) 97.8 (9.3)
24
51Cr 125IdUrd
0.9 (0.3) 0.7 (0.4)
27.0 (4.3) 1.4 (0.9) c
5.6 (0.9) 0.1 (0.1) c
42.4 (6.7) 9.7 (4.5) c
a A-LAK cells were labelled with both 51Cr and 125IdUrd b Data are presented as mean percentage recovery ( + SD) of the total radioactivity injected into the animal
o Significantly different from the corresponding 51Cr value by Student's t-test (P
