Tissue distribution of adoptively transferred adherent lymphokine-activated killer cells assessed by different cell labels

Summary Assessment of the tissue distribution of adoptively transferred adherent lymphokine-activated killer A-LAK) cells by use of⁵¹Cr 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 with¹²⁵IdUrd 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 of⁵¹Cr- and¹²⁵IdUrd-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-GM₁-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 with¹²⁵IdUrd, whereas 25%–30% of the⁵¹Cr label was consistently found in this organ. Autoradiography of the liver 24 h after i. v. injection of⁵¹Cr-labelled cells revealed a background activity that was four- to fivefold higher than the control level, indicating substantial non-specific accumulation in the liver of⁵¹Cr released from A-LAK cells. We conclude that⁵¹Cr cannot be reliably used in investigations of cell traffic to the liver because of non-specific accumulation of the⁵¹Cr label, particularly in this organ. In contrast, labelling with¹²⁵IdUrd or rhodamine and immunohistochemical staining of asialo-GM₁-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.     

A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (l) and light-chain () constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with¹²⁵I,¹¹¹In or^99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the¹²⁵I-labeled or^99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with¹³¹I,¹¹¹In or^99mTc is promising for the radioimmunoimaging of ovarian cancer.     

Natural killer activity in the rat. IV. Distribution of large granular lymphocytes (LGL) following intravenous and intraperitoneal transfer

Highly enriched populations of rat large granular lymphocytes (LGL) and T lymphocytes were prepared on discontinuous density gradients of Percoll, labeled with either 111In-oxine or 51Cr and injected either intravenously (iv) or intraperitoneally (ip) into normal syngeneic recipients. Following iv inoculation of labeled LGL or T cells into normal recipients, a large proportion of radioactivity (18 to 33%) was recovered within minutes in the lungs. By 2 to 4 hr following transfer, significantly more LGL (13.5%) than T cells (6.4%) remained in the lungs. This difference persisted through 48 hr (5.4 vs 0.8%). Decreasing levels of radioactivity in the lungs were accompanied by corresponding increases in counts in the spleen and liver. At early time points, a significantly higher proportion of T cells was found to distribute to the spleen, while labeled LGL persisted for longer periods in the blood as well as in the lungs. Following ip inoculation into normal recipients, there was a slow clearance of radiolabeled LGL and T cells from the peritoneal cavity, with less than 20% of the radiolabel found in peripheral organs by 24 hr. These results demonstrate a distribution pattern for LGL and T cells that resembles the previously reported proportions of these cells in various organs. In addition, these studies provide a firm basis for the formulation of further experiments to examine the usefulness of adoptive immunotherapy with LGL or immune T cells.     

The in vivo distribution of murine lymphokine activated killer cells in splenectomized host

The in vivo distribution of intravenously injected lymphokine activated killer (LAK) cells, generated in vitro with rIL-2 from normal murine splenocytes, was studied in BALB/c mice and compared with that of normal splenocytes. Both normal splenocytes and LAK cells were labeled with 51Cr, and the results were analyzed at 6, 24, and 48 hours after injection by localization index as the parameter. After injection through tail veins of mice, LAK cells were found to migrate to the spleen, lungs, liver, lymph nodes, bones and the kidneys. The apparent increased distribution pattern of LAK cells to the lung at 6 and 24 hours after injection was not detected when normal splenocytes were injected. Since almost one third of the injected LAK cells were found to localize in the spleen, it was postulated that splenectomy would affect the in vivo organ distribution of LAK cells. Accordingly, the in vivo distribution of LAK cells in splenectomized mice was further investigated. Results indicated that splenectomy enhanced the convergence of LAK cells to the lungs, liver, lymph nodes and bones. Therefore, splenectomy may augment the therapeutic effect of the adoptive transfer of LAK cells in pulmonary, hepatic, lymph node and bony metastases.     

Assessment of in vivo natural antitumor resistance and lymphocyte

Clearance of IV-injected tumor cells has been correlated with levels of natural killer (NK) cell activity in recipient animals. Studies of in vivo tumor cell clearance strongly suggest a relationship between levels of NK cell activity and antitumor or antimetastatic effector function. This study outlines the applicability of three radiolabels, [125I]iododeoxyuridine, ( [125I]dUrd), indium-111-oxine chelate ( [111In]Ox), and chromium-51 (51Cr), to studies of tumor cell clearance in vivo. The suitability of these labels for analysis of the in vivo migration patterns of normal lymphocytes or thymus-derived T cells cultivated in vitro (CTC) is also discussed. The results indicate that [111In]Ox and 51Cr compare favorably with the more widely used [125]dUrd as radiolabels for the assessment of IV-injected tumor cell clearance from the lungs of mice. The rates of clearance of both [111In]Ox and 51Cr, like that for [125I]dUrd, correlate closely with levels of NK-cell activity of the host. Further studies with [111In]Ox reveal that treatment of recipients with anti-asialo GM1 serum, a regimen known to suppress NK-cell activity, demonstrates the appropriate reduction in isotope clearance from the lungs after NK suppression. However, clearance data obtained by monitoring levels of radioactivity in the liver after IV injection must be viewed cautiously, since the same cells labeled with [111In]Ox and [125I]dUrd had a different pattern of clearance from the liver. The same inconsistencies in clearance were observed when [111In]Ox and [125I]dUrd were injected intrafootpad (i.f.p.). Similar effects were observed when [111In]Ox or 51Cr was applied to studies of CTC migration. Levels of [111In]Ox and 51Cr remained high in the liver after IV injection, while [125I]dUrd was rapidly cleared. Normal spleen or thymic lymphocytes exhibited the expected homing to the spleen after labeling with [111In]Ox, indicating a suitability of this label for migration studies, except possibly in the liver. These results with CTC and normal lymphocytes should be considered during the formulation of immunotherapy protocols based on cell migration data, since the choice of radiolabel can result in widely divergent levels of radioactivity accumulated in some organs, and may not provide an accurate representation of the presence of viable, intact, or functional cells.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

Comparison between 125IUdR and 51Cr as cell labels in investigations of tumor cell migration

YAC-1 tumor cells double-labeled with Na₂[⁵¹Cr]O₄ [⁵¹Cr] and [¹²⁵I]iododeoxyuridine [¹²⁵IUdR] were injected intravenously into Balb/c mice in order to investigate their migration and fate 0–4 h after the injection. Whereas the clearance of tumor cells from the lung tissue was similar as judged with both labels, the kinetics of isotope uptake in the liver were strikingly different. Thus, retention of ⁵¹Cr in the liver was very high compared to a much lower and only transient retention of ¹²⁵I. A higher retention of non-tumor cell-associated ⁵¹Cr was also observed in most other organs, resulting in overestimation of the number of viable tumor cells in these organs. Moreover, a marked spontaneous release (> 10% after 12 h) makes ⁵¹Cr less suitable as a cell label than ¹²⁵IUdR. On the other hand, we found that the release of ¹²⁵I from dead cells in vivo depends at least partially on host factors such as macrophages. Consequently, caution must be exerted when tumor cell migration is investigated in animals treated with drugs that might affect the reticuloendothelial system. We conclude that ¹²⁵IUdR is superior to ⁵¹Cr as a cell label for investigation of tumor cell migration in vivo, even though some doubt about the reliability of the number of tumor cells in liver and carcass, predicted by this radiolabel, still remains.     

In vivo distribution and cytokine gene expression by enriched mouse LAK effector cells

Lymphokine activated killer (LAK) cells administered in combination with interleukin 2 (IL2) can mediate antitumor activity in tumor-bearing mice and advanced cancer patients. Relatively little is known about the mechanism by which adoptively transferred LAK cells plus IL2 mediate these antitumor effects in vivo, and it remains unclear to what extent the actual LAK effector cells can accumulate in tumors. In the present study, enriched cytolytic LAK effector cells were obtained by fractionation of bulk LAK cell cultures on Percoll density gradients. About 95% of the total lytic activity was recovered from the 55% of cells isolated in fraction 2 (Fr2). The cells recovered in Fr2 are mostly large, proliferating lymphoblasts that express either the NK-associated surface markers NK1.1 (38%) or LGL-1 (31%), or the cytotoxic T cell phenotype, Lyt2 (39%). The cytolytic lymphoblasts obtained from Fr2 were radiolabelled with either 111Indium-Oxine (111InOx) which labels all cells in the population, or with 125Iododeoxyuridine (125IUdR) which labels only proliferating cells, and injected iv into mice bearing murine renal cancer (Renca). 111InOx-labeled Fr2 cells migrated mostly to spleen (28%) and liver (35%), with approximately 5% of the injected label detectable in the Renca-bearing kidney by 24 hrs. In contrast, Fr2 cells labeled with 125IUdR, which labels only the proliferating blasts thought to include the actual effector cells, exhibited a very different localization pattern. 125IUdR-Fr2 cells were retained in the lungs at higher levels than were 111InOx-Fr2 cells and very little label was detectable in liver (6%), spleen (3%), or tumor bearing kidney (2%) at 24 hrs. These results suggest that most of the large, proliferating lymphoblasts are cleared from the body by 24 hrs and very few localize into even large tumors. Subsequently, Northern blot analyses performed on bulk LAK cells revealed a potent induction of mRNA for TNF alpha by 6 hrs and for IFN gamma by 48 hrs. The intensity of gene expression for both cytokines was increased in Fr2 as compared to the unfractionated bulk LAK cells or to non-cytolytic cells obtained from Fr3. Overall, these results suggest that at least some of the antitumor effects mediated by LAK cells occur by the release of cytokines that synergize with exogenous IL2 for the activation of host effector cells.     

Influence of the donors' clinical status on in vitro and in vivo tumor-cytotoxic activation of interleukin-2-exposed lymphocytes and their circulation in different organs

Summary In-vitro-generated lymphokine-activated killer (LAK) cells of BALB/c mice, bearing the syngeneic colon carcinoma C-26 for 7 days, were as efficient as those from normal mice in lysing C-26 cells whereas LAK cells from 14-day tumor-bearing and 5- and 14-day tumor-resected animals had a lower C-26 cytotoxicity. The level of C-26 lysis returned to normal values 30 days after surgery. To identify the best source of LAK cells in vivo, groups of normal mice were treated with 10⁴, 3×10⁴ or 10⁵ U/day of interleukin 2 (IL-2) for 7 days intraperitoneally (i. p.) or intravenously (i. v.) (3×10⁴ dose only). The highest lysis on C-26 was obtained from peritoneal exudate cells of mice given 3×10⁴ and 10⁵ U whereas spleen cells were lytic only when taken from mice treated with 10⁵ U IL-2. Peripheral blood lymphocytes lacked any cytotoxicity except for the group of mice which received IL-2 i. v. The kinetics of in vivo LAK activation in different organs showed a peak of anti-(C-26) lytic activity at day 5 in peritoneal exudate cells and spleen cells of mice given IL-2 for 5 days whereas administration of LAK cells alone had no effect; IL-2 plus LAK cells gave a lower peak of LAK activity as compared with IL-2 alone. A lower level of in vivo LAK activation was found in mice whose tumor was resected 5 days before; such impairment was evident even 14 days after surgery. Homing experiments were carried out with i. v. injected ⁵¹Cr-labelled LAK cells in normal or tumor-resected mice. In normal mice the highest radioactivity at 30 min was found in the lungs; liver and spleen also showed high radioactivity whereas blood had a negligible amount of radioactivity. Radioactivity declined rapidly in lungs (less than 10% after 24 h) while remaining at appreciable levels in the liver after 24 h and 48 h; spleen showed constant levels of 12%–15%. Homing of LAK cells was altered in mice receiving IL-2 i. p. for 5 days with slower and lower radioactivity peaks in the lung and higher levels in liver. In tumor-excised mice lower levels of radioactivity were found in lungs. These results show that: (a) alterations in LAK activity occur in early-tumor-resected and large-tumor-bearing animals; (b) the route of IL-2 administration is critical in LAK activation in vivo; (c) treatment with IL-2 modifies LAK homing.This study was in part supported by grant no. 87.01565.44 of the Finalized Project Oncology of CNR (Rome, Italy)     

Differential 51Cr uptake of human peripheral lymphocytes separated by density gradient electrophoresis

Human peripheral lymphocytes were labeled with ⁵¹Cr before or after separation by preparative density gradient electrophoresis. In both cases, wide variations in the distribution of ⁵¹Cr in the electrophoresed cells was observed. In general, there was significantly more ⁵¹Cr per cell in the high mobility fractions. These results suggest caution in the interpretation of cytotoxic assays where ⁵¹Cr-labeled lymphocytes are used as target cells and prompt further studies by different separation methods.     

Modification of lymphokine-activated killer cell accumulation into tumor sites by chemotherapy, local irradiation, or splenectomy

The effects of chemotherapy or local irradiation on lymphokine-activated killer (LAK) cell accumulation into tumor sites were investigated. Lymphokine-activated killer cells labeled with 111In-oxine were injected into the caudal vein of C57BL/6 mice that had been previously transplanted with 3LL cancer. An adoptive transfer of LAK cells was carried out 4 days after treatments. Twenty-four hours after the transfer, tumor tissues were excised, and the accumulation of labeled LAK cells in the tumor was measured. In two different experiments, LAK cell accumulation in tumor in the nontreated group was 2.15% and 1.58% of the administered dose per gram of tissue. The accumulation in the groups of mice treated with cyclophosphamide, nimustine hydrochloride, or Adriamycin increased fourfold (7.38% dose/g, 6.61% dose/g), threefold (6.47% dose/g) and twofold (4.46% dose/g), respectively, as compared with the nontreated group. These agents induced significant tumor regression. In the group treated with bleomycin, which showed no significant effect on tumor growth, LAK cell accumulation in tumor remained unaltered (1.57% dose/g). However, the group treated with local irradiation, which induced significant tumor reduction, showed no increase in LAK cell accumulation into tumors. These results suggest that some antitumor drugs enhance LAK cell accumulation into tumor sites and that this increase is due to tumor modification by antitumor drugs.     

New 111In labeling of IgG: 111In-oxine mediated chelation

Current methods of ¹¹¹In chelate conjugation labeling of antibodies expose the protein to pH 5–6 during ¹¹¹In chelation. These conditions could be detrimental if the antibody is acid labile. We have successfully labeled human IgG via the cyclic anhydride of DPTA and ¹¹¹In-oxyquinoline(oxine). Chelation was achieved at pH 6.9–8.4 and was complete within 1 min at room temperature. The chelation was sensitive to trace metal contamination on labware and in some reagents (including commercial ¹¹¹In-oxine).     

Antimetastatic effect of recombinant human macrophage-colony-stimulating factor against lung and liver metastatic B16 melanoma

 We studied the effect of recombinant human macrophage-colony-stimulating factor (rhM-CSF) on the formation of lung and liver metastases following the i.v. injection of the B16 melanoma subline (B16 LiLu) into mice. When rhM-CSF was administered before the B16 inoculation, the number of tumor metastases decreased in the lung and liver. However, the administration of rhM-CSF after B16 inoculation did not produce an antimetastatic effect in the lung, but did in the liver. B16 cells labeled with 5-[¹²⁵I]-iodo-2′-deoxyuridine (¹²⁵I-dUrd) were injected and the arrest of tumor cell emboli was examined in the capillary beds of the lung and liver of mice treated with either vehicle or rhM-CSF. In both groups, there were the same numbers of B16 cells in both the lung and the liver 3 minutes after the B16 injection, and almost all tumor cells died within 24 h. However, the number of cells surviving in the lung was decreased in mice injected with rhM-CSF (37%). There was no difference in the number of cells in the livers of mice treated either with vehicle or rhM-CSF in the first 24 h after tumor cell injection. The administration of rhM-CSF increased NK 1.1⁺ cells in the mouse spleen and facilitated NK activity in vivo. At the same time, the administration of an anti-NK 1.1 antibody blocked the antimetastatic effect of rhM-CSF in the lung but not in the liver. The antibody was effective only when it was injected before the B16 inoculation. These results suggest that the antimetastatic effect of rhM-CSF in the lung was mediated by NK 1.1⁺ cells within 24 h of B16 injection. In contrast, the antimetastatic effect of rhM-CSF in the liver was mediated not only by NK 1.1⁺ cells but also by other antimetastatic systems such as macrophages. Received: 8 April 1996 / Accepted: 26 November 1996     

The in vivo distribution of Cr-labeled Trypanosoma cruzi in mice

The optimal conditions for labeling Trypanosoma cruzi culture forms with ⁵¹CrO₄²⁻ were determined. Labeled trypanosomes or labeled human red blood cells (RBCs) were injected intravenously into normal C3H(He) female mice and the rate of clearance and organ distribution of the isotope were observed over a 30 h period. It was found that trypanosomes and xenogeneic RBCs were cleared rapidly from the peripheral blood and accumulated primarily in the liver, spleen, lungs and kidneys. A difference was noted in accumulation of trypanosomes and RBCs in these mice.     

Induction of lymphokine-activated killer cells from rat thymocytes using recombinant human interleukin-2

Summary Using a 4-h ⁵¹Cr release assay, we observed that thymocytes from Fischer strain rats incubated with recombinant human interleukin-2 (rhIL-2) developed cytotoxicity to YAC-1 lymphoma, 9L-glioma, and B-16 melanoma cells (effector/target ratio =25/1). Induction of the lymphokine-activated killer (LAK) cells was as follows: (1) when 5×10⁶/ml thymocytes were cultured with various concentrations of rhIL-2 (50, 125, 250, 500, or 1,000 units/ml) for 4 days, no cell proliferation was observed at any concentration. However, the LAK cells showed significant cytotoxicity toward all tumor cells at more than 50 units/ml. (2) When 5×10⁶/ml thymocytes were cultured for 1 to 6 days with 250 units/ml of rhIL-2, the harvested cell count decreased markedly after the 2nd day. The cytotoxicity of all the tumor cells became significant after the 2nd day, with peak activity on the 4th day. In rat splenocytes, on the other hand, the LAK cells could not be identified because rat splenocytes developed nonspecific cytotoxicity in medium containing fetal calf serum without adding rhIL-2.     

Efficient radiolabeling of mammalian cells using 111In-tagged liposomes

When indium-111 oxine labeled neutral liposomes were incubated with Chinese hamster V79 cells in the presence of 100 mM calcium, the cell-associated radioactivity increased approximately 75-fold over that observed in the absence of calcium. This is considerably higher (~20 times) than the cellular uptake obtained when these cells are incubated in the presence of ¹¹¹In-oxine alone. The highest uptake of radioactivity occurred when no bovine serum albumin was present in the medium, while as little as 0.001% of the protein greatly reduced the cell-liposome association. These efficient cell labeling conditions were not found to affect the survival of the cells.     

Direct preparation of radioactive fullerenes as a tracer for applications

The C₆₀ and C₇₀ fullerenes were irradiated by high-energy γ-rays and charged particles. The irradiated samples were dissolved in CS₂ and/or toluene and filtered to remove insoluble by-products. Finally, radioactive fullerenes and products labeled with¹¹C or¹³N were isolated and detected in the liquid phase by radiochromatography. It was found that (1) not only¹¹C or¹³N radioactive monomer fullerenes but also their dimers (trimers and, possibly, tetramers) were produced by recoil implantation process following nuclear reaction and (2) the radioactive fullerene labeled with¹¹C yields has led to high yields.     

Preparation of 111in leukocytes after hemolytic removal of erythrocytes

An optimized procedure is described for isolation and highefficiency radiolabeling of leukocytes using ¹¹¹In-oxine. The chief advantages over conventional methods include virtually no loss of leukocytes during washing and separation steps; a significant reduction in the time required to prepare leukocytes for radiolabeling compared to non-hemolytic preparations; a 28% increase in the average labeling efficiency obtained using ¹¹¹In-oxine; >95% cell viability as measured by the trypan blue exclusion test; elimination of contaminating red blood cells from the leukocyte pellet prior to labeling; and 80% survivability at 15 min post injection (measured as per cent of blood activity on leukocyte fraction).     

Comparative studies using 125I- and 111In-labeled monoclonal antibodies

HDP-1 monoclonal antibody was labeled with ¹¹¹In using deferoxamine, diethylenetriaminepentaacetic acid or 1-(para-bromoacetamidobenzyl)-EDTA as chelating agents or with ¹²⁵I. The in vitro binding capacity and stability of the labeled molecules were evaluated using affinity chromatography. The biodistribution and imaging capabilities were compared using an animal model system that does not involve the use of tumors. Similar studies were done using the corresponding labeled F(ab′)₂ and Fab′ fragments. All labeled molecules, except those treated with deferoxamine, were stable in vitro. When tested in vivo, all retained their capacity to localize in the target tissue (lung). The lung %ID/g levels for the ¹¹¹In-labeled molecules were, however, slightly lower than those observed for the corresponding ¹²⁵I-labeled molecules. High uptake was also observed in the liver or kidneys when the ¹¹¹In-labeled molecules were used; no such results were obtained with the ¹²⁵I-labeled molecules. More work appears to be necessary before the use of bifunctional chelates becomes the optimal method for radiolabeling monoclonal antibodies for use in tumor imaging.     

Blood volume inMacaca fascicularis

The plasma and erythrocyte volumes ofMacaca fascicularis were determined using blood labelled with¹²⁵I-serum albumin and⁵¹Cr. It was found that the erythrocyte, plasma and packed cell volumes were 108±6 ml (Mean±S.D.), 210±10 ml and 37±2%, respectively. Total blood volume of macaque was 8% of body weight.