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From the Deutsches Krebsforschungszentrum Heidelberg, Abteilung Zelluläre Immunologie, FSP Tumorimmunologie, Heidelberg, Germany; the Deutsches Krebsforschungszentrum Heidelberg, Abteilung Experimentelle Pathologie, FSP Krebsrisikofaktoren und Krebsprävention, Heidelberg, Germany; and the Deutsches Krebsforschungszentrum Heidelberg, Abteilung Biostatistik, FSP Bioinformatik, Heidelberg, Germany.
Graft-versus-leukemia (GVL) and Graft-versus-host (GVH) reactions were compared after systemic transfer of allogeneic antitumor immune T lymphocytes from B10.D2 (H-2d; MIsb) into DBA/2 (H-2d; MIsa) mice. Before immune cell transfer, recipient DBA/2 mice were sublethally irradiated with 5 Gy to prevent host-versus-graft reactivity. Recipients were either bearing syngeneic metastatic ESb lymphomas (GVL system) or were normal, non-tumor-bearing mice (GVH system). We previously reported that this adoptive immunotherapy protocol (ADI) had pronounced GVL activity and led to immune rejection of even advanced metastasized cancer. In this study, monoclonal antibodies were used for immunohistochemical analysis of native frozen tissue sections from either spleen or liver to distinguish donor from host cells, to differentiate between CD4 and CD8 T lymphocytes, and to stain sialoadhesin-positive macrophages at different time points after cell transfer. The kinetics of donor cell infiltration in spleen and liver differed in that the lymphoid organ was infiltrated earlier (days 1 to 5 after transfer) than the nonlymphoid organ (days 5 to 20). After reaching a peak, donor cell infiltration decreased gradually and was not detectable in the spleen after day 20 and in the liver after day 30. The organ-infiltrating donor immune cells were mostly T lymphocytes and stained positive for CD4 or CD8 T-cell markers. A remarkable GVL-associated observation was made with regard to a subset of macrophages bearing the adhesion molecule sialoadhesin (SER+ macrophages). In the livers of tumor-bearing mice, their numbers increased between days 1 and 12 after ADI by a factor greater than 30. Double-staining for donor cell marker and SER showed that the sialoadhesin-expressing macrophages were of host origin. The SER+ host macrophages from GVL livers were isolated by enzyme perfusion and rosetting 12 days after ADI, when they reached peak values of about 60 cells per liver lobule, and were tested, without further antigen addition, for their capacity to stimulate an antitumor CD8 T-cell response. The results of this immunologic analysis suggest that these cells in the liver function as scavengers of the destroyed metastases and as antigen-processing and -presenting cells for antitumor immune T cells.
VARIOUS BIOLOGIC STRATEGIES for treatment of cancer are based on the transfer of cells with particular functions. Two examples of such cell-based therapies are (1) transfer of hematopoietic stem cells for bone marrow reconstitution (bone marrow transplantation [BMT]) after high-dose chemotherapy and (2) transfer of activated tumor-reactive lymphocytes for adoptive cellular immunotherapy (ADI) of primary tumor and metastases.1-3 A combined effect of stem cell and immune cell reconstitution may explain the beneficial effects of allogeneic versus autologous BMT.4 Such graft-versus-leukemia (GVL) effects can be observed for instance in a proportion of chronic myelogenous leukemia (CML) patients that receive allogeneic HLA-matched BMT, provided that the bone marrow cells contain allogeneic T cells.5 Allogeneic GVL reactivity can be transfered with donor buffy coat lymphocytes,6-8 but it is usually associated with a risk for development of graft-versus-host (GVH) disease with significant morbidity and mortality. The difficulty in dissociating GVL from GVH reactivity prevents exploitation of BMT for its full potential. To improve this situation, a better understanding of mechanisms of GVL and GVH reactivity is urgently needed.
We recently established an interesting animal model system for the investigation of GVL and GVH reactivity of immune lymphocytes. Using freshly in situ-activated antitumor immune cells from major histocompatability complex (MHC)-congenic mice, we obtained a strong GVL effect in the absence of severe GVH disease.9 The murine leukemia system consists of various well-defined metastasizing sublines (ESb, ESb-MP, and ESbL-lacZ) that are derived from the chemically induced DBA/2 lymphoma L 5178 Y.9 The ADI effects are achieved by the transfer of in situ-activated tumor-reactive lymphocytes from the tumor-resistant strain B10.D2 (H2d, Mlsb) into susceptible syngeneic tumor-bearing DBA/2 (H2d, Mlsa) mice. A single transfer of 2 × 107 allogeneic antitumor immune spleen cells into 5 Gy pretreated tumor-bearing mice resulted in regression, encapsulation, and rejection of primary cancer and eradication of metastases as well as in significant improvements of overall survival. This was true even when the therapy was administered very late, such as 4 weeks after tumor transplantation.9 The therapy effect could be demonstrated and quantified by (1) counting tumor cells and tumor-infiltrating lymphocytes in frozen tissue sections, (2) FACscan analysis of ESbL-lacZ cells and host cells reisolated from the livers of untreated and treated mice, and (3) high-field nuclear magnetic resonance (NMR)-spectroscopy of phosphor metabolites of primary tumors in individual live animals as a noninvasive method.9
The reasons for the predominantly protective effects of this ADI protocol and a possibly downregulated GVH reactivity are not yet entirely clear. The B10.D2 anti-ESb immune spleen cells used contain ESb tumor antigen-specific as well as DBA/2 host antigen-specific T cells.10 Because we observed in some experiments a late chronic GVH reaction,11,12 a contribution of minor histocompatibility antigen-reactive T cells and thus of a GVH reactivity in this antitumor effect cannot be excluded. B10.D2 immune cells are likely to react against a minor lymphocyte stimulatory antigen from DBA/2 mice (Mlsa) that was discovered more than 20 years ago13 and that is now known to be a superantigen (SAg) encoded by endogenous mouse mammary tumor virus (MMTV) proviruses that have randomly integrated into germ cells.14,15 Mls-1a16-18 represents a viral SAg (vSAG7) that binds to MHC class II molecules. Preliminary findings indicate that vSAG7, which behaves as a self-tolerogen in DBA/2 mice by deleting T cells with certain v The present study was undertaken to compare the kinetics of organ infiltration by donor immune cells in tumor-bearing (GVL system) or non-tumor-bearing (GVH system) mice. For this purpose, we used standard histologic and newly established immunohistologic stainings of frozen tissue sections from livers and spleens. The ability to distinguish donor from host cells by antibodies against an allelic form of Animals
Tumor Lines
ADI Protocol On day 0 (d0), DBA/2 recipients were inoculated with 2 × 105 ESb-MP tumor cells intradermally. To generate allogeneic immune effector cells, ESb-MP lymphoma cells were injected intravenously into B10.D2 donor mice at a dose of 105 cells.11 Seven days later, these effector cells were isolated from the spleen and transferred intravenously (3 × 107 cells/200 µL RPMI-1640 medium) into 5 Gy (60Co source; Gammatron F 80S; Siemens München, Germany) sublethally irradiated recipient DBA/2 mice. In the GVH system, the recipients were normal DBA/2 mice, whereas in the GVL system, they were mice bearing ESb-MP tumors and metastases 23 days after tumor transplantation. Tumor-bearing DBA/2 mice of the control group remained untreated.Antibodies and Other Reagents The following rat MoAbs were used as culture supernatants: antimouse CD4 (clone GK 1.5),24 antimouse CD8 (clone 53-6-72),25 and antimouse sialoadhesin (SER-4).26 The rat MoAbs were visualized by using a biotin-conjugated donkey antirat Ig reagent followed by streptavidin-AP (both from Dianova, Hamburg, Germany). The mouse MoAb Lym 11,27 a hybridoma supernatant, directed allele-specific against 2Mb of B10.D2 mice, was kindly provided by P. Robinson (London, UK). The following antibodies were obtained commercially: rat antimouse CD4 or CD8, biotin-conjugated (Life Technologies, Eggenstein, Germany), polyclonal donkey antimouse IgG (H+L) antibodies, horseradish peroxidase (PO)-linked or alkaline phosphatase (AP) or fluorescein isothiocyanate (FITC)-linked F(ab)'2 fragments (Dianova).
Anti- Flow Cytometric Analysis Cells (1 × 106) were washed with FACS buffer (5% fetal calf serum, 0.1% NaN3 in PBS) and incubated on ice for 20 minutes with preformed immune complexes from MoAb Lym 11 and FITC-linked polyclonal donkey antimouse Ig antibody. The cells were then extensively washed and stained with 1.5 µmol/L propidium iodide to exclude dead cells. Flow cytometric analysis was performed using a FACScan (Becton Dickinson, Heidelberg, Germany). Excitation laser frequency was 488 nm and fluorescence emission was detected at 575 nm. Typically, 10,000 events were collected and data were expressed as histograms.Separation of Macrophages from Spleen Cells Single-cell suspensions from spleens were prepared by mechanical dissociation. After lysis of erythrocytes by a short NH4Cl hypotonic solution treatment, residual cells were washed twice and resuspended in RPMI-1640 medium with antibiotics, 10% heat-inactivated fetal calf serum, and 50 µmol/L 2-mercaptoethanol (Sigma) followed by incubation on tissue culture petri dishes for 2 hours at 37°C in 5% CO2. The nonadherent cells were collected.Immunohistochemistry Tissue preparation. Livers and spleens were removed and snap-frozen by immersing in liquid nitrogen. Five-micrometer-thick consecutive cryostat sections were mounted on uncovered glass slides. After drying overnight at room temperature, the sections were fixed in aceton for 10 minutes at room temperature and air dried.
Control and Data Evaluation The distribution of the reaction products of the one-step method was compared with the results of conventionally performed indirect immunostainings at the same antibody dilutions as those found to give optimal staining for the complexes. Sections from liver and spleen of B10.D2 mice served as a positive test and those from DBA/2 served as negative controls. To determine nonspecific background staining, further negative controls were required involving the following modifications of the above-described immunoenzymatic staining. (1) The specific primary Igs were substituted with Ig from normal serum used to dilute the immunoreagents or with nonimmune serum of the same animal species that provided the primary antibodies in the two-step or the one-step method. The protein concentration of these solutions was chosen to be that found optimal in the complexes. (2) The primary antibody was interchanged by another mouse antibody (anti--galactosidase; Boehringer Mannheim, Mannheim, Germany) in both the one-step and two-step stainings. (3) The immune complexes in all combinations were also applied without the saturation step with mouse serum. Only after a clear discrimination between donor and host tissue staining was achieved was donor cell infiltration in host organs quantified.
Immunization of DBA/2 Mice and Isolation of Immune Spleen Cells DBA/2 mice were immunized three times by subcutaneous injection with 2 × 107 irradiated (200 Gy) ESb cells (batch 289) in 100 µL RPMI-1640 medium. Inactivation of tumor cells was achieved by X-irradiation with 100 Gy from a 137Cs source (Gammacell 1000; Atomic Energy of Canada Ltd, Ottawa, Canada). One week after the last immunization, single-cell suspensions from spleens were prepared by mechanic dissociation, treated with NH4Cl hypotonic solution, washed, and incubated in plastic culture dishes for 2 hours at 37°C in 5% CO2 to remove adherent cells. Spleens from normal animals (nonprimed) were prepared following the same protocol. Only nonadherent cells (lymphocytes) were used for the functional assays.
Isolation of SER+ Macrophages From the Liver Liver sinusoidal cells from tumor-bearing mice treated with ADI and from normal mice were isolated by perfusion through the portal vein with pronase and collagenase followed by metrizamide gradient, as previously described.29 Cells were incubated in plastic culture dishes for 2 hours at 37°C in 5% CO2. After the removal of nonadherent cells by washing, Kupffer cells (adherent cells) were scraped with a rubber spatula, washed twice, and resuspended in PBS without divalent cations (Biochrom KG, Berlin, Germany). The SER+ macrophage population was isolated by means of a rosetting technique using unopsonized sheep erythrocytes, as described previously.30 In the case of normal liver, which contains only few SER+ macrophages, we used the whole sinusoidal cell population for the functional assays.
Functional Assays to Determine T-Cell Stimulatory Capacity of Liver-Derived Cells Spleen lymphocytes isolated from normal (nonprimed) and immunized animals were double-stained with antibodies against activation markers and analyzed by flow cytometry (day 0). Another part of lymphocytes (responders) were incubated for 6 days in 24-well plates (Renner, Darmstadt, Germany) with the following stimulator cells (responder:stimulatory cell ratio = 10:1): (1) SER+ liver macrophages, isolated from tumor-bearing DBA-2 mice 12 days after ADI; (2) 200-Gy irradiated ESb289 cells; and (3) liver sinusoidal cells isolated from normal (non-tumor-bearing) animals. The cultured lymphocytes were stained with the same antibodies as at day 0 followed by FACS analysis. The following antimouse antibodies were used: FITC-conjugated anti-interleukin-2 (IL-2) receptor -chain (CD25) and phycoerythrin (PE)-conjugated CD8a (Ly-29; Pharmingen, San Diego, CA). Cells were incubated at 4°C for 10 minutes with the antibodies, washed, and treated with 1.5 µmol/L propidium iodide (final concentration). FACS analysis was performed using a FACScan (Becton Dickinson). Thirty thousand cells per sample were simultaneously measured for FSC and integrated side scatter as well as green (FL1) and red (FL2 and FL3) fluorescences (expressed as logarithm of the integrated fluorescence light). Recordings were made only on popidium iodide-negative (viable) cells of the red (FL3) fluorescence, excluding aggregates whose FSCs were out of range. Expression of cell surface molecules was analyzed by dot blots of the red fluorescence (FL2) versus green fluorescence (FL1) and data were expressed as the percentage of CD25+ cells (FL1) among the CD8+ T-cell population (FL2).
Detection of Donor Cells in Host Tissue by Anti- 2M (anti-2Mb, Lym 11) that is expressed in donor but not host cells and a polyclonal donkey antimouse Ig F(ab)'2 reagent linked to either PO or to AP. To reduce possible interactions of this second antibody with mouse Ig on cells, we first established a new technique of immune complex preformation and determined the optimal molecular proportions between the Lym 11 hybridoma supernatant and the second antibody, as described in the Materials and Methods. FACS flow cytometric analysis was performed to determine the specificity of immune complex binding in a quantitative way and to exclude a possible exchange between donor and host cells. As can be seen from Fig 1, 100% of donor cells were stained by the immune complexes, whereas there was no significant staining of host DBA/2 cells. When donor cells were mixed with host cells, they could be clearly distinguished even when the majority were of host origin. The percentage of positive cells obtained was close to the theoretical expectation, thus excluding significant exchanges of immune complex-bound donor 2Mb with host cell 2Ma chains.
Kinetics of Donor Cell Infiltration in Host Organs Immune spleen cells from B10.D2 mice that had been immunized against the DBA/2 lymphoma variant ESb-MP were transfered into 5 Gy irradiated ESb-MP tumor-bearing DBA/2 mice, in which they exerted strong GVL effects associated with only mild GVH reactivity. To distinguish between GVL and GVH reactivity, we performed comparative studies in either tumor-bearing mice (GVL situation) or non-tumor-bearing mice (GVH situation). At different times after intravenous immune cell transfer, spleens and livers of the recipients were removed and frozen to be later analyzed by immuno-histochemistry. The amount of detected donor cells was related to the structural units of the respective organs, the liver lobuli, and the white pulp of the spleen.
Distinction Between CD4 and CD8 T Lymphocytes In addition to the total number of infiltrating donor cells in spleen and liver, we determined the amounts of CD4 and CD8 staining T lymphocytes. As can be seen from Fig 4B, 1 day after cell transfer into the 5 Gy irradiated recipients there were about 50 CD4 T lymphocytes per white pulp, whereas there were less than 5 CD8 T cells detectable. Five days after transfer, the CD4 T cells had increased to 75 and the CD8 T cells to 30 cells per unit. From days 8 through 20, a steady state was reached that leveled off at about 55 CD4 and 30 CD8 T cells. Because donor cell infiltration in the spleen steadily decreased after day 8 (Fig 2), it appears that a large proportion of the CD4 and CD8 T cells in the spleen after day 8 were of host origin.Sialoadhesin-Positive Macrophages With the help of the MoAb SER-4,24 we determined the localization and quantity of sialoadhesin-positive macrophages in the spleens and livers of tumor-bearing mice undergoing ADI. The number of these cells in the spleen (Fig 4A) remained rather constant around 50 per unit throughout the detection period. In contrast, in the liver (Fig 4B), we saw a dramatic increase from day 1 to 12 by a factor greater than 30. Thereafter, they slowly decreased to baseline level until day 30. The area under the curve of SER+ cells in the liver (days 1 through 30; 1,211 units) was 1.32-fold larger than the area of donor cells (days 5 through 30; 916 units).Localization of SER+ Macrophages and T Lymphocytes in the Spleen We first investigated the localization of CD4 or CD8 T lymphocytes in the spleens in relation to the position of SER+ macrophages. Figure 5A shows the typical structure in normal control mice of the white pulp with the T lymphocytes in the center and the stained blue SER+ macrophages surrounding. A double staining of CD4 and CD8 T lymphocytes (Fig 5C) showed a pattern of random mixture within the center of the white pulp. In contrast, tumor-bearing mice that were irradiated with 5 Gy on day 16 and then inoculated with immune spleen cells showed 5 days later a different structural organization in the spleen (Fig 5B and D). CD4 T lymphocytes and SER+ macrophages (Fig 5B) were not as nicely separated as in the white pulp from normal mice, and CD4 and CD8 T lymphocytes (Fig 5D) were more separated than in normal spleens. It seems as if the white pulp is in a process of reorganization after irradiation and cell transfer. Spleens of irradiated tumor-bearing control animals not treated by cell transfer showed SER staining such as in normal mice but with decreased intensity.30Increase in SER+ Liver Macrophages Representing a Host Response Figure 6 shows immunohistochemical double stainings from the liver. Figure 6A and B show the positioning between CD4 T lymphocytes and SER+ macrophages in areas of the periportal vein (A) or deep in the parenchyma (B). A close association between the CD4 T lymphocytes and the macrophages can be seen. Figure 6C and D show the positioning of total donor cells (brown) and SER+ macrophages. Whereas clusters of donor cells can be seen around portal veins (Fig 6C) and in the parenchyma (Fig 6D), there were also single donor cells with or without direct contact to SER+ macrophages.
Quantitative Aspects and Statistical Analysis For quantitative comparison of donor cell infiltration in the GVL versus the GVH system, we determined the areas under the curves shown in Fig 2. The data were obtained on the basis of the mean response curves. Further analysis via bootstrapping (1,000 samples) showed the 95% confidence interval (CI; 0.95) that is indicated in parentheses. The following values were obtained: GVL/GVH area (spleen) = 1.54 (1.50; 1.60); GVL/GVH area (liver, days 5 through 30) = 3.00 (2.75; 3.25).
SER Macrophages From GVL Livers Stimulate CD8 Antitumor Memory T Cells
We previously described an animal model for GVL in which the transfer of allogeneic immune T lymphocytes led to strong GVL effects associated with only mild GVH reactivity.9 This situation may be related to the clinical setting of allogeneic BMT in leukemia patients in whom both GVL and GVH reactivity can be seen and in whom a distinction between both activities has been difficult so far.33,34 In this study, we performed a systematic immuno-histochemical analysis of immune donor cell infiltration of lymphoid organs (spleen) and nonlymphoid organs (liver) and compared experimental situations of a pure GVH reaction (in non-tumor-bearing hosts) with reactivity in a GVL situation (in tumor-bearing hosts). A distinction between donor and host cells was made possible by making use of the fact that donor and host cells express a different allelic form of Submitted March 1, 1996;
accepted October 24, 1996.
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Abstract
Introduction
Abstract
receptors in the mouse tissue were blocked with different concentrations of normal mouse serum. The sections were incubated for 20 minutes with 5% to 20% normal mouse serum at 37°C. Removal of the serum was followed by incubation with the preformed immunocomplex that was found to provide the best quality immunostaining for 1 hour at room temperature. At the end of the antibody treatment, the sections were washed in PBS three times for 5 minutes and immersed in the same buffer as used in the developer solution for another 5 minutes.
) or GVH (
) system. Frozen tissue sections were stained for donor cells with anti-
), and SER-4+ macrophages (
). Immunohistochemically stained frozen tissue sections were analyzed under the microscope. The data indicate the means and standard deviations from two experiments with 2 to 3 animals per time point.
) Donor cells; (- - -) CD4 + CD8 cells.
