TY - JOUR
AU - Rogers, Ian M.
AB - Abstract Human hematopoietic stem cell engraftment has been studied extensively using xenograft transplant models with immunocompromised mice. It is standard practice to incorporate mouse models, such as the limiting dilution assay, to accurately assess the number of repopulating stem cells in bone marrow or umbilical cord blood collections or to confirm the long-term repopulating ability of cultured hematopoietic stem cells. In a previous study using a standard NOD/SCID mouse model to assess human hematopoietic stem cell engraftment we observed that all human cells had mouse MHC class I protein on their surface, suggesting that this is a mechanism adopted by the cells to evade host immune surveillance. To determine whether this was a xenograft phenomenon we studied host MHC transfer in an intraspecies mouse model and observed similar results. The transfer of MHC class I proteins has implications for antigen presentation and immune modulation. In this report, we used a standard mouse model of bone marrow transplantation to demonstrate that surface protein transfer between cells plays an important role in protecting donor hematopoietic cells from NK cell and macrophage-mediated rejection. The transfer of intact MHC class I antigens from host cells to transplanted donor cells confers a self identity on these otherwise foreign cells. This gives them the ability to evade detection by the host NK cells and macrophages. Once full donor chimerism is established, transplanted cells no longer require host MHC class I protein transfer to survive. Transplantation, Stem cell-microenvironment interactions, NOD/SCID chimeras, NK cells, Marrow stromal cells Introduction Mouse models of human disease play a key role in advancing therapies toward clinical trials. Mice have been used for four decades to model hematopoietic stem cell transplants (HSCT) and were key in the characterization of the hematopoietic stem cell [[1]]. The HSCT mouse model was adapted to allow for xenotransplants using human cells, and this was important for identifying the human hematopoietic stem cell. Over the years, new mouse models have been developed to improve the engraftment of human cells. NOD/SCID mice are used extensively as they lack T cells, B cells, and have reduced NK cell activity and macrophages. Originally it was thought that the NK cell activity was low or absent in these mice, but it was later demonstrated that pretreating the mice with a neutralizing antibody to NK cells (anti-CD122) greatly enhanced the engraftment level of these mice. NOD/SCID/gamma mice are lacking a functional IL-2 gene so NK-cell activity is nonexistent. These mice produce high levels of engraftment similar to using NOD/SCID mice with the anti-CD122 antibody [[2]]. The NOD/SCID mouse model for human cell engraftment, although well used and characterized, possess an interesting immunological question due to the fact that engraftment of human cells occurs in the presence of active NK cells and macrophages [[3, 4]]. It would be expected that even a small but active population of NK cells should, over time, be capable of destroying all donor cells. We proposed that human donor cells transplanted into NOD/SCID mice are under constant immune surveillance by NK cells and macrophages but have adapted to evade detection. The hybrid resistance model argues that the presence of inhibitory and activating receptors on NK cells allows, under certain circumstances, for NK cell inhibitory receptors to override the activation of NK cells. MHC transgene and F1 hybrid bone marrow transplant experiments suggest that the matched MHC class I protein activated the inhibitory receptor on host NK cells thus stopping NK cell-mediated killing [[5]]. In a previous paper [[14]], we were able to demonstrate that human donor cells were protected from NK cell-mediated killing due to the fact that they obtained the mouse (host) MHC class I molecule that then triggered the recipient NK cell inhibitory receptor. This explains how, even in the continued presence of recipient NK cells, human donor cells are able to evade lysis. Furthermore, we confirmed using fluorescent in situ hybridization that the donor cells only received host protein and not the host MHC class I gene. The transfer of surface proteins between cells, termed trogocytosis, is believed to occur through the movement of large membrane sections or lipid rafts that contain intact functional surface proteins [[6]]. Trogocytosis is a common process that occurs between immune cells and more recently, it has been shown to occur between nonimmune cells including cells of the lung, liver, intestine, and kidney [[7-11]]. In this report, we confirm that trogocytosis occurs for intraspecies HSCT, but once full donor chimerism is established the advantage of host MHC class I acquisition is lost and donor cells that are negative for host MHC class I become evident in the bone marrow. This suggests that not all donor cells undergo protein transfer and in the early stages of engraftment these cells are quickly removed by the host NK cells leaving behind only the donor cells that have host MHC class I on their surface. We also demonstrate here that the transfer is limited to cell surface MHC class I protein and does not include the transfer of genetic material. Materials and Methods Ethics Statement Patient consent was received before umbilical cord blood samples were collected. Samples were collected from anonymous donors with approval from the University of Toronto and Mount Sinai Hospital Research Ethics Boards. All animal work was approved by the Animal Care Committee at the Toronto Centre for Phenogenomics. hUCB samples were collected at the time of delivery by trained personnel following protocols approved by the Mount Sinai Hospital and University of Toronto Human Ethics Committee. hUCB Collection, Processing, and Cryopreservation When human umbilical cord blood (hUCB) samples were received, samples were processed and red blood cells were removed using Pentaspan (Bristol-Meyers Squibb, Montreal, URL: http://www.bmscanada.ca/en). After processing, samples were cryopreserved and stored in liquid nitrogen until use as previously described [[3]]. Stromal Cell Isolation Blood cells were flushed from femurs and tibias with 10 ml of phosphate-buffered saline/2% fetal bovine serum, the bones were then cut into small pieces and cultured in Dulbecco's modified Eagle's medium (DMEM)/10% FBS/antibiotics/1 mg collagenase per ml of media for 1–2 hours at 37°C. After 1–2 hours, the bone was washed with DMEM/5% FBS/antibiotics and then cultured in DMEM/10% FBS/antibiotics for 3 days without any media change. Once adherent cells appear and reach 60%–70% confluence, they were passaged 1:2. Passage two stromal cells were used in the experiment [[12]]. Mice NOD/SCID and NOD/SCID/gamma mice were used as hosts and C57BL/6 and Friend Virus B NIH Jackson (FVB/NJ) mice were used as donors. BALB/c mice were used in the in vitro assay. Mice were housed under pathogen-free conditions at the Toronto Centre for Phenogenomics. All mice, aged 7–9 weeks, were purchased from the Toronto Center for Phenogenomics or the Jackson Laboratory. NOD/SCID and NOD/SCID/gamma mice were irradiated 320 cGy and 200 cGy, respectively, using a GammaCell40 irradiator 4 hours prior to transplantation. For GdCl3-treated mice, GdCl3 (Sigma-Aldrich, St. Louis, URL: http://www.sigmaaldrich.com/canada-english.html) was dissolved in sterile saline and administered intravenously at a dose of 20–40 mg/kg 24 hours prior to irradiation. Preconditioned recipients were transplanted 3.5 million (mouse bone marrow cell [BMC]) or 10 million (hUCB) donor cells intravenously. Mice were euthanized at different time points using CO2. Flow Cytometry Hosts were sacrificed from 1 week to 8 weeks post-transplantation. Peripheral blood was collected by heart puncture and femurs and tibias were excised and flushed for BMCs using 1 ml of PBS/2% FBS. Retrieved cells were centrifuged at 400g, 10°C for 5 minutes. The cell pellet was resuspended in 1 ml RBC Lysis Buffer and centrifuged at 400g, 10°C for 5 minutes. RBC Lysis Buffer was removed after centrifugation and the cell pellet was resuspended at a concentration of 1 × 106 cells in 50 μl PBS/2% FBS. Fifty microliters of cells was aliquoted for each staining unit. Before staining, cells were incubated with anti-CD16/32 antibody (BD Pharmingen, California, URL: http://www.bdbiosciences.com/ca/index.jsp) to block nonspecific binding of IgG, for 10 minutes in ice. 7-AAD and antibodies against CD45, H-2Kd, H-2Kb, H-2Kq, and HLA-ABC were purchased from BD Pharmingen. All antibodies were titrated and cells were stained using the optimal concentrations for 30 minutes on ice. Corresponding isotope controls were used at the same concentration for gating purposes. After incubation, cells were washed with 1 ml PBS/2% FBS and centrifuged 400g, 10°C for 5 minutes. Cells were resuspended in 200 μl PBS/0.2% FBS. Cells were analyzed using a Gallios Flow Cytometer (Beckman Coulter, California, URL: https://www.beckmancoulter.com/wsrportal/wsr/index.htm). Ex Vivo Culture Double MHC class I-positive cells were sorted using a BD FACSAria and collected in DMEM/50% FBS by a qualified personnel. Cells were centrifuged and cultured in DMEM/10% FBS/antibiotics at 37°C, 5% CO2. After culture, cells were collected and centrifuged at 400g, 10°C for 5 minutes. Cells were stained in the same procedure as described above and analyzed using a Gallios Flow Cytometer (Beckman Coulter, California, URL: https://www.beckmancoulter.com/wsrportal/wsr/index.htm). Confocal Microscopy C57BL/6 stromal cells were cocultured with BALB/c blood cells on four-well chamber slides for 3 days at 37°C, 5% CO2. Prior to staining, cells were washed once with PBS and fixed with formalin for 20 minutes at room temperature. After fixation, cells were blocked with anti-CD16/32 antibody (BD Pharmingen, California, URL: http://www.bdbiosciences.com/ca/index.jsp) for 10 minutes at room temperature followed by 30 minutes of blocking buffer (0.1% triton/10% FBS/PBS). Cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-H-2Kb and phycoerythrin (PE)-conjugated anti-H-2Kd antibodies (both at 1/100 dilution) overnight at 4°C. Cells were then washed four times in 0.1% triton/PBS and stained with 4',6-diamidino-2-phenylindole (DAPI). Polymerase Chain Reaction DNA was isolated from BMCs of C57BL/6, NOD/SCID/gamma, and fluorescence-activated cell sorting (FACS)-sorted trogocytosis-positive cells. Primer sequences [[13]]: H-2Kb fwd 5-GGCTCTCACACTATTCAGGT-3, H-2Kb rev 5-GCGTCGCGTTCCCGTTCTT-3, H-2Kd fwd 5-GTTCCAGCGGATGTTCGGC-3, and H-2Kd rev 5-CGTCTCATTCCCGAGCTCCA-3. Polymerase chain reaction (PCR) program for H-2Kb was: 94°C for 2 minutes, 94°C for 2 seconds, 56°C for 10 seconds, 72°C for 8 seconds, and 72°C for 1 minute for 38 cycles. PCR program for H-2Kd was: 94°C for 2 minutes, 94°C for 2 seconds, 60°C for 10 seconds, 72°C for 8 seconds, and 72°C for 1 minute for 38 cycles. PCR was performed using the REDTaq ReadyMix PCR Reaction Mix with MgCl2 (Sigma-Aldrich, St. Louis, URL: http://www.sigmaaldrich.com/canada-english.html) and the PCR programs were ran on the MJ Research PTC-200 Peltier Thermal Cycler. Statistics To determine whether donor cell engraftment was different depending on the mouse strain used for donor cells (FVB/NJ donor cells vs. C57BL/6 donor cells), a two-way ANOVA followed by a Bonferroni post-test was used to compare the two strains. Significance was accepted as *, p < .05. To determine whether there was a significant loss of trogocytosis-positive cells after culture, a Student's t test was used. The same test was used to compare the difference in trogocytosis-positive cells between irradiated and nonirradiated NOD/SCID/gamma mice, and between GdCl3-treated and nontreated NOD/SCID/gamma mice. All tests were completed using GraphPad-Prism software (La Jolla, CA). Results Trogocytosis Protects Donor Cells from Rejection in NOD/SCID Mice A mouse intraspecies transplantation model was established to study trogocytosis. Allogeneic C57BL/6 BMCs (3.5 × 106) (H-2Kb) were transplanted intravenously into sublethally irradiated (320 cGy) NOD/SCID mice (H-2Kd). Recipients were sacrificed weekly for up to 5 weeks post-transplantation. At 1 week post-transplantation, approximately 55% of the BMCs was of donor origin (H-2Kb+) with 91% (range: 88%–97%) of them expressed both host and donor MHC class I protein (H-2Kd+/H-2Kb+), referred to as trogocytosis-positive cells (Fig. 1A). These results were similar to what was observed in our previous xenotransplantations where the majority of human donor cells (95%+) were trogocytosis positive [[14]]. However, unlike hUCB cells used in the xenotransplantation study, allogeneic mouse donor cells can mature and become functional in NOD/SCID recipient mice. Figure 1. Open in new tabDownload slide Trogocytosis protects donor cell from rejection during early post-transplantation in NOD/SCID mice. Sublethally irradiated (320 cGy) NOD/SCID mice were transplanted with 3.5 × 106 C57BL/6 bone marrow cells (BMCs) and sacrificed at (A) 1 week (n = 2), (B) 2 weeks (n = 3), (C) 3 weeks (n = 3), (D) 4 weeks (n = 5), and (E) 5 weeks (n = 2) post-transplantation. Host BMCs were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kb. Percentage of all donor cells that are H-2Kd+/H-2Kb+ or single H-2Kb+ is written on the graph and underlined. (F): The relationship between trogocytosis-positive cells and total donor cells throughout the 5 weeks post-transplantation. Error bars represent SD. Fluorescence-activated cell sorting events count: (A) 116,373, (B) 92,202, (C) 109,956, (D) 70,935, and (E) 72,442. Abbreviation: PE, phycoerythrin. Figure 1. Open in new tabDownload slide Trogocytosis protects donor cell from rejection during early post-transplantation in NOD/SCID mice. Sublethally irradiated (320 cGy) NOD/SCID mice were transplanted with 3.5 × 106 C57BL/6 bone marrow cells (BMCs) and sacrificed at (A) 1 week (n = 2), (B) 2 weeks (n = 3), (C) 3 weeks (n = 3), (D) 4 weeks (n = 5), and (E) 5 weeks (n = 2) post-transplantation. Host BMCs were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kb. Percentage of all donor cells that are H-2Kd+/H-2Kb+ or single H-2Kb+ is written on the graph and underlined. (F): The relationship between trogocytosis-positive cells and total donor cells throughout the 5 weeks post-transplantation. Error bars represent SD. Fluorescence-activated cell sorting events count: (A) 116,373, (B) 92,202, (C) 109,956, (D) 70,935, and (E) 72,442. Abbreviation: PE, phycoerythrin. Full donor chimerism was achieved at 2 weeks post-transplantation and 71% (range: 65%–75%) of the donor cells was trogocytosis-positive (Fig. 1B). As the recipient BMCs were removed during the establishment of full donor chimerism, the single H-2Kb+ donor cells that have not undergone trogocytosis were no longer targeted for destruction, which explains the increase in this donor cell population (Fig. 1B–1E). At 5 weeks post-transplantation, almost all the donor cells were single H-2Kb+ (Fig. 1E). The negative relationship between the percentage of trogocytosis-positive cells and total donor cells as donor chimerism was established is depicted in Figure 1F. This result confirms that cells that have undergone trogocytosis are protected from host NK cells and macrophages allowing them to fully engraft and establish a donor-derived immune system. Once the host immune cells are replaced by the donor cells there is no longer a survival advantage for trogocytosis-positive donor cells over single H-2Kb+ donor cells. All antibody control flow cytometry graphs are in Supporting Information Figures S1–S6. The Transfer of Host MHC I Proteins Is Not Strain Specific To confirm that the transfer of host MHC class I proteins is not limited to donor cells from one mouse strain, 3.5 × 106 allogeneic FVB/NJ BMCs (H-2Kq) were transplanted intravenously into sublethally irradiated (320 cGy) NOD/SCID mice. At 1 week post-transplantation, 85% (range: 84%–86%) of the donor cells was trogocytosis-positive (Fig. 2A). After donor chimerism was established around 2 weeks post-transplantation, the ratio of trogocytosis-positive to single H-2Kq+ donor cell population decreased (Fig. 2B). We then compared the two strain groups (individual mice: FVB/NJ donor cells vs. C57BL/6 donor cells) using a two-way ANOVA followed by a Bonferroni post-test. There were no significant difference (p = .9) between the percentage of trogocytosis-positive C57BL/6 and FVB/NJ donor cells at any given time point, and there were no observable differences in the kinetics of engraftment and the establishment of full donor chimerism (Fig. 2C). Figure 2. Open in new tabDownload slide Host MHC class I protein transfers onto donor cells of different mouse strains. Sublethally irradiated (320 cGy) NOD/SCID mice were transplanted with 3.5 × 106 FVB/NJ bone marrow cells (BMCs) and sacrificed at (A) 1 week (n = 2), and (B) 2 weeks (n = 4) post-transplantation. Individual mouse BMCs were gated on IgG (data not shown). Host BMCs were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kq. Percentage of all donor cells that are H-2Kd+/H-2Kq+ or single H-2Kq+ is written on the graph and underlined. (C): The percentage of trogocytosis-positive cells in NOD/SCID mice transplanted with FVB/NJ or C57BL/6 donor cells during the first 3 weeks post-transplantation. There was no significant difference between the percentage of trogocytosis-positive cells in NOD/SCID mice transplanted with FVB/NJ or C57BL/6 donor cells at all time points (p = .9). Fluorescence-activated cell sorting events count: (A) 179,669 and (B) 5,997. Figure 2. Open in new tabDownload slide Host MHC class I protein transfers onto donor cells of different mouse strains. Sublethally irradiated (320 cGy) NOD/SCID mice were transplanted with 3.5 × 106 FVB/NJ bone marrow cells (BMCs) and sacrificed at (A) 1 week (n = 2), and (B) 2 weeks (n = 4) post-transplantation. Individual mouse BMCs were gated on IgG (data not shown). Host BMCs were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kq. Percentage of all donor cells that are H-2Kd+/H-2Kq+ or single H-2Kq+ is written on the graph and underlined. (C): The percentage of trogocytosis-positive cells in NOD/SCID mice transplanted with FVB/NJ or C57BL/6 donor cells during the first 3 weeks post-transplantation. There was no significant difference between the percentage of trogocytosis-positive cells in NOD/SCID mice transplanted with FVB/NJ or C57BL/6 donor cells at all time points (p = .9). Fluorescence-activated cell sorting events count: (A) 179,669 and (B) 5,997. Trogocytosis-Positive Cells Are of Donor Origin and Result from Protein Transfer In our xenotransplantation study [[14]], we confirmed that all double positive cells were of donor origin by cell culture, immunocytochemistry, and fluorescent in situ hybridization. In this study, we confirm that trogocytosis-positive (H-2Kb+/H-2Kd+) cells from intraspecies transplants also result from intercellular protein transfer and not DNA transfer. First, we used confocal microscopy to confirm the presence of H-2Kd and H-2Kb proteins on trogocytosis-positive cells. H-2Kd and H-2Kb can be seen in large patches on the cell surface confirming the flow cytometry results that trogocytosis-positive cells contain similar amounts of both MHC class I proteins (Fig. 3A). Second, we FACS-sorted trogocytosis-positive cells from NOD/SCID/gamma mice transplanted with C57BL/6 donor cells and cultured them overnight, and reanalyzed the next day for H-2Kd and H-2Kb expression. A t = 0 hour postsort analysis showed that 86.5% of the sorted cells was trogocytosis-positive. After 18 hours in culture, there was a significant decrease in trogocytosis-positive cells (p < .01) with only 36% of the sorted cells remained H-2Kd+/H-2Kb+ (Supporting Information Fig. S7). Trogocytosis-positive cells lost the host MHC protein because there is no gene to produce new protein. To demonstrate that the host MHC gene is not transferred to trogocytosis-positive cells, we performed PCR on DNA obtained from FACS-sorted trogocytosis-positive cells isolated from NOD/SCID/gamma mice transplanted with C57BL/6 donor cells. Primers were targeted to an exon region of H-2Kb and H-2Kd genes. Trogocytosis-positive cells only have the H-2Kb gene (Fig. 3B), which confirms that trogocytosis-positive cells result from protein transfer without the parallel transfer of any genetic material. Figure 3. Open in new tabDownload slide Trogocytosis-positive cells contain only donor H-2Kb gene but express both H-2Kb and host H-2Kd proteins. (A): C57BL/6 stromal cells were cocultured with BALB/c blood cells for 3 days and cells were stained for H-2Kb (green) and H-2Kd (red). Cells expressing both H-2Kb and H-2Kd proteins were observed in the culture (magnification = ×63). (B): Genomic DNA was isolated from C57BL/6, NOD/SCID/gamma, and trogocytosis-positive cells from NOD/SCID/gamma transplanted with C57BL/6 donor cells, and PCR was performed using primers targeted to an exon region of its respective H-2Kb and H-2Kd genes. As controls, DNA from C57BL/6 cells was positive for H-2Kb but negative for the H-2Kd primer, and DNA from NOD/SCID/gamma cells was positive for H-2Kd but negative for H-2Kb primers. Trogocytosis-positive cells only have the donor H-2Kb gene. Figure 3. Open in new tabDownload slide Trogocytosis-positive cells contain only donor H-2Kb gene but express both H-2Kb and host H-2Kd proteins. (A): C57BL/6 stromal cells were cocultured with BALB/c blood cells for 3 days and cells were stained for H-2Kb (green) and H-2Kd (red). Cells expressing both H-2Kb and H-2Kd proteins were observed in the culture (magnification = ×63). (B): Genomic DNA was isolated from C57BL/6, NOD/SCID/gamma, and trogocytosis-positive cells from NOD/SCID/gamma transplanted with C57BL/6 donor cells, and PCR was performed using primers targeted to an exon region of its respective H-2Kb and H-2Kd genes. As controls, DNA from C57BL/6 cells was positive for H-2Kb but negative for the H-2Kd primer, and DNA from NOD/SCID/gamma cells was positive for H-2Kd but negative for H-2Kb primers. Trogocytosis-positive cells only have the donor H-2Kb gene. In line with the results from our previous xenotransplantation study, the inability of trogocytosis-positive cells to maintain expression of host MHC class I protein expression after culture confirms that these cells are of donor origin and they arise from the transfer of host MHC class I proteins to donor cells and not as a result of DNA transfer. Taken together, we can conclude that trogocytosis-positive cells are due to protein transfer and that no genetic material, whole chromosomes or single genes, were transferred in the process. Trogocytosis-Positive Cells Also Have a Survival Advantage in Irradiated NOD/SCID/Gamma Studies with NOD/SCID mice as recipients demonstrated that NK cells and macrophages are prevented from lysing donor cells that have undergone trogocytosis. In order to separate the role of NK cells from that of the macrophages we used NOD/SCID/gamma mice as recipients. These mice allowed us to study the role of trogocytosis in transplantation and engraftment in the absence of T, B, and NK cells and allowed us to focus on the impact of macrophages. Allogeneic C57BL/6 (H-2Kb) donor BMCs (3.5 × 106) were transplanted intravenously into sublethally irradiated (200 cGy) NOD/SCID/gamma mice (H-2Kd). Hosts were sacrificed weekly for up to 4 weeks post-transplantation. At 1 week post-transplantation 76.5% (range: 73%–80%) of the donor cells was trogocytosis-positive (Fig. 4A). Full donor chimerism was achieved at 2 weeks post-transplantation, and we saw the gradual decrease in trogocytosis-positive (H-2Kd+/H-2Kb+) donor cells and an increase in single H-2Kb+ donor cells (Fig. 4B-4D). By 4 weeks post-transplantation, the majority of donor cells were single H-2Kb+ (Fig. 4D). These results are similar to those with NOD/SCID mice transplanted with C57BL/6 BMCs (Fig. 1). We also observed a negative relationship between the percentage of trogocytosis-positive cells and total donor cells that was similar to NOD/SCID mice transplanted with C57BL/6 BMCs (Fig. 4E). This experiment clearly demonstrates that macrophages, like NK cells, can discriminate between cells expressing self and nonself MHC class I proteins and therefore do not attack donor cells that have acquired host MHC class I proteins. These results are in line with past studies that show that macrophages alone are sufficient at rejecting allogeneic donor cells [[15, 16]]. Figure 4. Open in new tabDownload slide Trogocytosis protects donor cell from rejection during early post-transplantation in NOD/SCID/gamma mice. Sublethally irradiated (200 cGy) NOD/SCID/gamma mice were transplanted with 3.5 × 106 C57BL/6 BMCs and sacrificed at (A) 1 week (n = 2), (B) 2 weeks (n = 3), (C) 3 weeks (n = 3), and (D) 4 weeks (n = 4) post-transplantation. Host bone marrow cells were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kb. Percentage of all donor cells that are H-2Kd+/H-2Kb+ or single H-2Kb+ is written on the graph and underlined. (E): The relationship between trogocytosis-positive cells and total donor cells throughout the 4 weeks post-transplantation. Error bars represent SD. Fluorescence-activated cell sorting events counted: (A) 150,867, (B) 145,920, (C) 194,824, and (D) 172,775. Abbreviation: PE, phycoerythrin. Figure 4. Open in new tabDownload slide Trogocytosis protects donor cell from rejection during early post-transplantation in NOD/SCID/gamma mice. Sublethally irradiated (200 cGy) NOD/SCID/gamma mice were transplanted with 3.5 × 106 C57BL/6 BMCs and sacrificed at (A) 1 week (n = 2), (B) 2 weeks (n = 3), (C) 3 weeks (n = 3), and (D) 4 weeks (n = 4) post-transplantation. Host bone marrow cells were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kb. Percentage of all donor cells that are H-2Kd+/H-2Kb+ or single H-2Kb+ is written on the graph and underlined. (E): The relationship between trogocytosis-positive cells and total donor cells throughout the 4 weeks post-transplantation. Error bars represent SD. Fluorescence-activated cell sorting events counted: (A) 150,867, (B) 145,920, (C) 194,824, and (D) 172,775. Abbreviation: PE, phycoerythrin. Host Radiation Influences Donor Cell Survival in NOD/SCID/Gamma Through Macrophage Activation and the Effect Can be Reversed by Macrophage Depletion Macrophages can be activated following exposure to ionizing radiation [[17, 18]]. To determine the effect of radiation-activated macrophages on trogocytosis-mediated engraftment, we used nonirradiated NOD/SCID/gamma hosts and characterized the trogocytosis status of the engrafted cells. As in the previous experiments, 3.5 × 106 allogeneic C57BL/6 BMCs were transplanted intravenously into nonirradiated NOD/SCID/gamma mice. The donor cell engraftment pattern observed in nonirradiated NOD/SCID/gamma mice (Fig. 5A, 5B) differs from what we observed in irradiated hosts (Fig. 4). In nonirradiated hosts, at 2 weeks post-transplantation, over half of the donor cells were lacking host MHC class I proteins (H-2Kd) (56.5%, range: 55%–58%) (Fig. 5A). This single H-2Kb+ donor cell population persisted for 8 weeks post-transplantation (61.5%, range: 61%–62%) (Fig. 5B). Without irradiation, macrophages in NOD/SCID/gamma mice were not activated and were unable to eliminate single H-2Kb+ donor cells. Therefore, the population of cells that has not undergone trogocytosis would survive and proliferate, resulting in a larger portion of single H-2Kb+ donor cells compared to irradiated hosts during the early stages of engraftment. Moreover, these results reinforce that trogocytosis is a natural cellular event and not a by-product of irradiation. Figure 5. Open in new tabDownload slide Acquisition of host MHC class I protein is not a requirement for survival in nonirradiated or GdCl3-treated NOD/SCID/gamma mice. Nonirradiated NOD/SCID/gamma mice were transplanted with 3.5 × 106 C57BL/6 bone marrow cells (BMCs) and sacrificed at (A) 2 weeks (n = 2), and (B) 8 weeks (n = 2) post-transplantation. Host BMCs were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kb. (C): GdCl3-treated and sublethally irradiated (200 cGy) NOD/SCID/gamma mice were transplanted with 3.5 × 106 C57BL/6 BMCs and sacrificed at 1 week post-transplantation (n = 5). Host BMCs were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kb. Fluorescence-activated cell sorting events counted: (A) 183,781, (B) 166,602, and (C) 141,313. Abbreviation: PE, phycoerythrin. Figure 5. Open in new tabDownload slide Acquisition of host MHC class I protein is not a requirement for survival in nonirradiated or GdCl3-treated NOD/SCID/gamma mice. Nonirradiated NOD/SCID/gamma mice were transplanted with 3.5 × 106 C57BL/6 bone marrow cells (BMCs) and sacrificed at (A) 2 weeks (n = 2), and (B) 8 weeks (n = 2) post-transplantation. Host BMCs were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kb. (C): GdCl3-treated and sublethally irradiated (200 cGy) NOD/SCID/gamma mice were transplanted with 3.5 × 106 C57BL/6 BMCs and sacrificed at 1 week post-transplantation (n = 5). Host BMCs were flushed from the femurs and tibias and stained for host H-2Kd and donor H-2Kb. Fluorescence-activated cell sorting events counted: (A) 183,781, (B) 166,602, and (C) 141,313. Abbreviation: PE, phycoerythrin. To confirm that macrophages are responsible for the rejection of single H-2Kb+ donor cells in irradiated NOD/SCID/gamma hosts, the hosts were pretreated with gadolinium chloride (GdCl3) before irradiation and transplantation. GdCl3 inhibits phagocytosis and eliminates macrophages [[19]]. Irradiated NOD/SCID/gamma hosts pretreated with GdCl3 had a different donor cell composition than irradiated but nontreated hosts (Fig. 4: untreated vs. Fig. 5C: GdCl3-treated). GdCl3-treated hosts were more tolerant of single H-2Kb+ donor cells with 42% (range: 34%–50%) of the donor cells being single positive at 1 week post-transplantation, while the irradiated but nontreated hosts had 24% (range: 20%–27%) single H-2Kb+ donor cells. This difference was statistically significant (p < .05). Furthermore, the GdCl3-treated mice had a similar flow cytometry profile to nonirradiated NOD/SCID/gamma hosts (Fig. 5A) thus confirming that activated host macrophages target single H-2Kb+ donor cells and not trogocytosis-positive donor cells. Engrafted Human Donor Cells Are also Trogocytosis-Positive in NOD/SCID/Gamma Hosts Xenotransplantations with immunocompromised mice are an invaluable tool to evaluate how human donor cells will behave in the human transplant setting. We have previously demonstrated that hUCB and hBM cells undergo trogocytosis and are protected from rejection in NOD/SCID mice [[14]]. Here, we extended our xenograft studies to NOD/SCID/gamma mice to determine whether macrophage-mediated rejection of single HLA class I-positive human donor cells occurs. hUCB cells (1 × 107) were transplanted intravenously into sublethally irradiated (200 cGy) NOD/SCID/gamma mice, and as expected trogocytosis-positive human donor cells were protected from destruction during engraftment. Human cells colonized the recipient mice at a slower rate than mouse donor cells did, but we observed at 1 week post-transplantation that donor cells contributed approximately 3% (range: 2.5%–3.7%) of the total cells and approximately 88% (range: 81%–93%) of these cells was trogocytosis-positive (range: 83%–94%) (Fig. 6A). By the second week, donor cell contribution increased to approximately 12% (range: 1.6%–20%) and trogocytosis-positive donor cells comprised 77% (range: 67%–84%) of these cells (Fig. 6B). At 3 weeks post-transplantation, the donor cell contribution increased to 23% (range: 21%–26%) which is similar to the overall donor contribution we observed in xenografts using NOD/SCID as hosts [[14]] but the frequency of trogocytosis was lower, just 34% (range: 21%–47%) of the donor cells (Fig. 6C). It is possible that after the initial irradiation of the hosts and subsequent activation of the macrophages, any newly produced host macrophages are not activated and are unable to attack the donor cells. We do not observe this when mouse donor cells are used as they engraft and achieve donor chimerism rapidly. Figure 6. Open in new tabDownload slide The transfer of host MHC class I proteins also protects hUCB cells from rejection in NOD/SCID/gamma mice. Sublethally irradiated (200 cGy) NOD/SCID/gamma mice were transplanted with 1 × 107 hUCB cells and sacrificed at (A) 1 week (n = 3), (B) 2 weeks (n = 3) and (C) 3 weeks (n = 3) post-transplantation. Host bone marrow cells were flushed from the femurs and tibias and stained for host H-2Kd and donor HLA-ABC. Fluorescence-activated cell sorting events counted: (A) 140,252, (B) 65,647, and (C) 167,408. Figure 6. Open in new tabDownload slide The transfer of host MHC class I proteins also protects hUCB cells from rejection in NOD/SCID/gamma mice. Sublethally irradiated (200 cGy) NOD/SCID/gamma mice were transplanted with 1 × 107 hUCB cells and sacrificed at (A) 1 week (n = 3), (B) 2 weeks (n = 3) and (C) 3 weeks (n = 3) post-transplantation. Host bone marrow cells were flushed from the femurs and tibias and stained for host H-2Kd and donor HLA-ABC. Fluorescence-activated cell sorting events counted: (A) 140,252, (B) 65,647, and (C) 167,408. Stromal Cells Are a Source of MHC Class I Protein for Trogocytosis But They Do Not Acquire MHC Class I Proteins Blood cells are in close contact and constant communication with stromal cells in the bone marrow niche. The intimate communication between blood cells and stromal cells led us to reason that donor blood cells acquire most of the host MHC class I proteins from host stromal cells. To investigate whether MHC class I protein transfers between blood cells and stromal cells, we performed in vivo and in vitro experiments. First, MHC class I expression on CD45+ blood cells and CD45− stromal cells in the bone marrow niche was assessed for trogocytosis (Fig. 7A, 7B). In this experiment, host NOD/SCID/gamma mice were irradiated and treated with GdCl3. This preconditioning regimen would prevent the possible rejection of donor stromal cells and allow us to look at both host and donor stromal populations. BMCs were harvested before full donor chimerism was established. This allowed us to determine whether host and donor stromal cells undergo trogocytosis. Cells were first sorted for CD45+ and CD45− populations then each population was interrogated for host and donor MHC class I proteins. Less than 5% of the stromal cells (CD45−) was trogocytosis-positive although the lack of a clear clustering of data points argues this is background (Fig. 7A). In contrast, 59% (58%–60%) of donor blood cells (CD45+) was trogocytosis-positive confirming that trogocytosis had occurred (Fig. 7B). Our results demonstrate that stromal cells do not receive MHC class I proteins. There could be an intrinsic property of stromal cells that prevents them from receiving proteins through trogocytosis but the reason for this is undetermined. Figure 7. Open in new tabDownload slide Stromal cells can donate MHC class I proteins but they do not acquire foreign MHC class I proteins in vivo and in vitro. In vivo study: GdCl3-treated and sublethally irradiated (200 cGy) NOD/SCID/gamma mice were transplanted with 3.5 × 106 C57BL/6 BMCs and sacrificed at 1 week post-transplantation (n = 2). Host bone marrow cells were flushed from the femurs and tibias and stained for CD45, host H-2Kd, and donor H-2Kb. (A): MHC expression on CD45− (stromal) cells. (B): MHC expression on CD45+ (blood) cells. In vitro study: BALB/c blood cells (H-2Kd) were cultured with C57BL/6 stromal cells (H-2Kb) (n = 2). All cells were collected and stained for CD45, H-2Kd, and H-2Kb. (C): MHC expression on CD45− (stromal) cells. (D): MHC expression on CD45+ (blood) cells. Fluorescence-activated cell sorting events counted: (A) 922, (B) 132,843, (C) 1,296, and (D) 54,896. Abbreviation: PE, phycoerythrin. Figure 7. Open in new tabDownload slide Stromal cells can donate MHC class I proteins but they do not acquire foreign MHC class I proteins in vivo and in vitro. In vivo study: GdCl3-treated and sublethally irradiated (200 cGy) NOD/SCID/gamma mice were transplanted with 3.5 × 106 C57BL/6 BMCs and sacrificed at 1 week post-transplantation (n = 2). Host bone marrow cells were flushed from the femurs and tibias and stained for CD45, host H-2Kd, and donor H-2Kb. (A): MHC expression on CD45− (stromal) cells. (B): MHC expression on CD45+ (blood) cells. In vitro study: BALB/c blood cells (H-2Kd) were cultured with C57BL/6 stromal cells (H-2Kb) (n = 2). All cells were collected and stained for CD45, H-2Kd, and H-2Kb. (C): MHC expression on CD45− (stromal) cells. (D): MHC expression on CD45+ (blood) cells. Fluorescence-activated cell sorting events counted: (A) 922, (B) 132,843, (C) 1,296, and (D) 54,896. Abbreviation: PE, phycoerythrin. Although stromal cells do not acquire foreign MHC class I proteins it does not exclude the possibility of them acting as MHC class I protein donors. To show whether bone marrow stromal cells are a key source of MHC class I protein for trogocytosis, we cocultured C57BL/6 (H-2Kb) bone marrow stromal cells with mononuclear blood cells from BALB/c mice (H-2Kd). After 3 days in culture, the cells were harvested, stained for H-2Kb and H-2Kd, and analyzed by flow cytometry (Fig. 7C, 7D). Cells were gated for blood cell (CD45+) and stromal cell (CD45−) populations and each of these populations was then analyzed for H-2Kd and H-2Kb expression. In line with our above experiment, stromal cells did not receive MHC class I proteins from the blood cells as all stromal cells were positive only for H-2Kb but not for the BALB/c blood cell H-2Kd protein (Fig. 7C). In contrast, 27.5% the CD45+ blood cells was trogocytosis-positive (H-2Kb+/H-2Kd+) (Fig. 7D). These results confirm that trogocytosis involves the unidirectional transfer of host MHC class I protein from stromal cells to blood cells. Discussion In order to improve graft tolerance after nonmyeloablative or reduced intensity conditioning regimens, a firm understanding of host-donor cell interactions and the criteria that make donor cells resistant to host immune attack is required. Our study has focused on mouse intraspecies transplantations using NOD/SCID and NOD/SCID/gamma mice, which enabled us to study the influence of NK-cells and/or macrophages on graft rejection [[15, 16, 20]]. Specifically, we have demonstrated that successful engraftment in NOD/SCID and NOD/SCID/gamma mice is dependent on the donor cells acquiring host MHC class I proteins by trogocytosis, which protects them from NK cell and macrophage-mediated rejection. NK cells are members of the innate immune system and express activating and inhibitory receptors that recognize various ligands and self MHC class I proteins, respectively. Upon interaction with another cell, the NK cell's decision to lyse a target cell depends on the balance of incoming activating and inhibitory signals [[21]]. The “missing self” hypothesis is used to describe the ability of NK cells to recognize “self” MHC proteins by binding through the inhibitory receptor which prevents the NK cell from lysing the target [[22]]. According to the missing self hypothesis, the presence of self MHC class I proteins protects cells from NK cell-mediated lysis through inhibition of the “kill” signal. For instance, a study by Ohlen et al. [[5]] showed that H-2Db cells can be protected from rejection in a H-2Dd recipient by the transgenic expression of H-2Dd. As demonstrated here, in the early stages of engraftment almost all the donor cells in the bone marrow were double positive for both host and donor MHC class I proteins. The presence of host MHC class I proteins on donor cells disguised them as self and allowed them to escape immunological surveillance in NOD/SCID and NOD/SCID/gamma mice. However, when full donor chimerism was achieved, or when host immune cells were inactivated or depleted, having host MHC class I proteins was no longer a requirement for donor cell survival. Hence, we saw a decrease in the frequency of trogocytosis-positive cells. In bone marrow transplantation, the absence of self MHC class I proteins on donor cells would normally activate host NK cells which results in donor cell death. But in the case of trogocytosis where the donor cells display functional host MHC class I proteins, signaling through the inhibitory receptors was initiated and NK cell and macrophage-mediated lysis was terminated. In this study, we demonstrated that macrophages, when activated by radiation, were able to eliminate trogocytosis-negative donor cells but were ineffective toward the trogocytosis-positive donor cells. In addition to NK cells, macrophages also express inhibitory receptors for self MHC class I proteins. In mice, these inhibitory receptors belong to the paired Ig-like receptor family [[23]]. The role of these macrophage inhibitory receptors is still unclear but their structural similarity to NK cell's inhibitory receptors suggests that they may share a similar function [[24, 25]]. Macrophages play a substantial role in graft rejection. Depleting macrophages using dichloromethylene diphosphate liposomes in SCID mice improved engraftment of hUCB [[26]]. Similarly, Liu et al. [[27]] used gadolinium chloride (GdCl3) to inhibit macrophages in Rag−/−γc−/− mice and found improved allogeneic donor cell engraftment compared to untreated mice. The activation of macrophages by radiation also argues that low-dose radiation used during conditioning protocols may be detrimental to the patient by inadvertently activating host macrophages [[28]]. Since trogocytosis involves the transfer of protein and not DNA, it is expected that trogocytosis-positive donor cells will lose expression of host MHC class I proteins over time. When cultured ex vivo, we confirmed that trogocytosis-positive cells are of donor origin and resulted from the transfer of host MHC class I proteins onto donor cells. Using confocal microscopy to analyze trogocytosis-positive cells, we confirmed the presence of both H-2Kb and H-2Kd proteins on the cell surface. Trogocytosis-positive cells were also tested by PCR using probes unique to each of the donor and host MHC class I genes. The PCR results confirmed that the MHC gene is not transferred. Although cell fusion of a few cells or other mechanisms of protein transfer may have occurred and went undetected in our tests, such rare events cannot be responsible for our observations. Collectively, our data suggest that MHC class I proteins were transferred by trogocytosis. Donor HSCs migrate to the bone marrow within hours of transplantation and after engraftment their progeny migrate from the bone marrow back into the peripheral circulation. Therefore, we hypothesized that donor cells acquired a majority of the host MHC class I proteins from stromal cells in the bone marrow niche due to the close contact between these cells. Our in vitro coculture experiment confirmed that stromal cells are a source of MHC class I protein for trogocytosis and that the transfer only occurs in the stromal-to-blood cell direction. The xenograft model of trogocytosis allowed us to look at trogocytosis over a prolonged period of time because donor chimerism is not established. In this study and our previous study using human CD34+ cells as the donor source, trogocytosis-positive cells were found in the bone marrow and peripheral blood for up to 7 months post-transplantation, while lineage negative cells did not result in any observable trogocytosis [[14]]. Coupled with the fact that MHC class I proteins are constantly internalized [[29]], there must be a steady supply of host MHC class I proteins from stromal and possibly nonstromal cells. It is possible that endothelial cells are another source of protein as Herrera et al. [[30]] demonstrated that endothelial cells can donate MHC class I proteins to immune cells. Our previous study suggested that only approximately 10%–15% of the transplanted donor cells initially undergo trogocytosis [[14]], escape immune surveillance, and give rise to all of the donor cells observed during the initial stages of engraftment. However, when donor chimerism is established, trogocytosis-positive donor cells no longer have a survival advantage over trogocytosis-negative donor cells and their numbers decrease to baseline level. Our intraspecies HSCT study demonstrated that even when a small subset of donor cells is able to evade immune surveillance they can engraft and full donor chimerism can be established. Xenotransplants using NOD/SCID mice resulted in an equilibrium being established between trogocytosis-positive donor cells and host NK cells and macrophages. The human donor cells were never able to establish full donor chimerism due to limitations of the mouse bone marrow niche, thus they were constantly attacked but were able to maintain long-term engraftment at moderate levels due to the ability of the donor cells to maintain trogocytosis. The speed of donor cell engraftment was reduced in xenotransplants but we predict that in the clinical setting full donor chimerism would be achievable in an acceptable time frame as a human HSCT would demonstrate similar engraftment kinetics as observed with our intraspecies model and not the xenograft model. We expect that trogocytosis is an important facilitator of engraftment in human HSCTs. For some current conditioning regimens, immune suppression reduces the effect of macrophages and NK cells on donor cell rejection but we hypothesis that trogocytosis can be observed when nonmyeloablative and reduced intensity conditioning regimens are used. Furthermore, we predict that in a clinical setting, NK cell and macrophage suppression are unnecessary due to trogocytosis and maintaining active host macrophages during the initial stage of engraftment can potentially protect the patient from infection, which can be a major factor associated transplant-related mortality [[31]]. Trogocytosis has been primarily described to occur between immune cells [[32-36]] but the transfer between nonimmune cells or between blood and non-blood tissues has been observed [[7-10, 14, 37]]. The mechanism that marks some surface proteins for trogocytosis and not others has yet to be determined. It is not clear what mechanism would allow the transfer of specific proteins in one direction but the current literature suggests that PI3-kinase activation may be the underlying mechanism of trogocytosis, although this does not account for the directionality or specificity of trogocytosis [[38]]. This study provides evidence that upon transplantation, a subset of donor cells acquire “natural” protection from host NK-cells and macrophages through the acquisition of host MHC class I proteins. This protected subset of donor cells is sufficient to form a fully functional immune system as observed by the establishment of full donor chimerism. Although mouse models have been used extensively to determine the engraftment properties of hematopoietic stem cells, our results suggest that only a subset of trogocytosis-positive donor cells engraft. Therefore, it is possible that current animal models underestimate the number of cells with engrafting potential. We have demonstrated that by taking advantage of trogocytosis and the different animal models available we can gain further insight into the role of macrophages and NK cells in graft rejection. Conclusion In this study, we focused on establishing a mouse intraspecies transplantation model to understand the role of MHC class I protein transfer between host and donor cells during bone marrow transplantation. Trogocytosis is a mechanism whereby large amounts of surface protein can be transferred between cells. Trogocytosis-positive donor cells were prevalent in sublethally irradiated NOD/SCID and NOD/SCID/gamma hosts during the early stages of engraftment. However, when donor chimerism was established around 2 weeks post transplantation, the percentage of trogocytosis-positive donor cells decreased. Moreover, the percentage of trogocytosis-positive cells was also reduced in nonirradiated and GdCl3-treated NOD/SCID/gamma mice. Altogether, this suggests that the transfer of host MHC class I protein was a requirement for donor cells survival it is no longer the case when the host NK cells and/or macrophages are absent or removed (either through donor chimerism establishment or immune cell depletion). We also showed that trogocytosis-positive donor cells arise from the transfer of host MHC class I protein without the parallel transfer of genetic material, and that host stromal cells are a source of host MHC class I protein for transfer. Using xenotransplants, we confirmed that MHC protein transfer also occurs on human donor cells which strongly suggest that MHC protein transfer also play a role in human transplants. Acknowledgments Umbilical Cord Blood Collections: Research Centre for Women's and Infants' Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada, http://biobank.lunenfeld.ca. Flow Cytometry: Annie Bang, Lunenfeld Research Institute, Toronto, Canada. 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T.C.: performed experiments, analyzed data, designed experiments, and wrote manuscript; J.W. and M.L.: performed experiments; I.M.R.: designed experiments, analyzed data, and wrote manuscript. © AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - The transfer of host MHC class I protein protects donor cells from NK cell and macrophage-mediated rejection during hematopoietic stem cell transplantation and engraftment in mice
JF - Stem Cells
DO - 10.1002/stem.1458
DA - 2013-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-transfer-of-host-mhc-class-i-protein-protects-donor-cells-from-nk-r9qHVgLQP4
SP - 2242
EP - 2252
VL - 31
IS - 10
DP - DeepDyve
ER -