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The lack of understanding of the interplay between hematopoietic stem cells (HSCs) and the immune system has severely hampered the stem cell research and practice of transplantation. Major problems for allogeneic transplantation include low levels of donor engraftment and high risks of graft-versus-host disease (GVHD). Transplantation of purified allogeneic HSCs diminishes the risk of GVHD, but results in decreased engraftment. Here we show that ex vivo expanded mouse HSCs efficiently overcame the major histocompatibility complex barrier and repopulated allogeneic recipient mice. An 8-day expansion culture led to a 40-fold increase of the allograft ability of HSCs. Both increased numbers of HSCs and culture-induced elevation of expression of the immune inhibitor CD274 (B7-H1 or PD-L1) on the surface of HSCs contributed to the enhancement. Our study indicates the great potential of utilizing ex vivo expanded HSCs for allogeneic transplantation, and suggests that the immune privilege of HSCs can be modulated.
Hematopoietic stem cells (HSCs) have been used in transplantation to treat patients with leukemia, lymphoma, some solid cancers, and autoimmune diseases (Bryder et al., 2006). In particular, allogeneic bone marrow (BM) transplantation is potentially curative for both inherited and acquired hematopoietic diseases (Gyurkocza et al.). Two major problems, failure of engraftment and graft-versus-host disease (GVHD), have severely limited the progress in the field, however. Although the inclusion of donor T cells in transplantation enhances donor engraftment and has graft-versus-leukemia effects, it causes life-threatening GVHD. Transplantation of purified allogeneic HSCs diminishes the risk of GVHD, but also results in decreased engraftment (Shizuru et al., 1996; Wang et al., 1997). It is not clear why most allogeneic HSCs cannot escape immune rejection and whether the allograft efficiency of HSCs can be improved. The resolution of these questions will promote the understanding of the immunology of HSCs and other stem cells and greatly improve the practice of allogeneic transplantation.
We recently developed an efficient culture system for ex vivo expansion of HSCs (Zhang and Lodish, 2008). This system is based on the use of serum-free culture medium supplemented with several growth factors including SCF, TPO, FGF-1/Flt3-L, IGFBP2, and angiopoietin-like proteins (Angptls) (Huynh et al., 2008; Zhang et al., 2006; Zhang et al., 2008). In vivo studies suggested that Angptls are new molecular components of the microenvironment of fetal liver and adult HSCs (Chou and Lodish, 2010; Zheng et al., 2011), and Angptl1 and 2 are essential to HSC development in zebrafish (Lin and Zon, 2008). We and others have used this culture system to expand mouse and human HSCs for transplantation or genetic modification purposes (Akala et al., 2008; Carter et al.; Chen et al., 2009; Drake et al., 2011; Heckl et al., 2011; Huynh et al., 2008; Khoury et al., 2011; Kiel et al., 2007; Stern et al., 2008; Zhang et al., 2006; Zhang et al., 2008; Zhao et al.). There are two important features of this HSC culture system: the increased number of repopulating HSCs (Huynh et al., 2008; Zhang et al., 2006; Zhang et al., 2008), and the change of surface expression of many surface proteins (Zhang and Lodish, 2005). While the expansion of repopulating HSCs were validated by transplanting cultured HSCs into congeneic or immune deficient mice in these previous studies, we hypothesized that ex vivo expansion of HSCs may also modulate the immunological properties of HSCs so that they possess an altered ability to cross the immune barrier upon allogeneic transplantation. To test this hypothesis, we started to compare the allograft abilities of freshly isolated HSCs and ex vivo expanded HSCs in allogeneic transplantation models.
Using a well-established mouse model for fully allogeneic transplantation (sFig. 1), we compared the allograft abilities of freshly isolated and ex vivo expanded HSCs from CD45.1 C57BL/6 donors transplanted into lethally irradiated BALB/c (CD45.2) recipients. The culture was performed in our optimized STFIA medium (Huynh et al., 2008; Zhang et al., 2006) for 8 days that allows ex vivo expansion of HSCs. Consistent with previously reported results (Shizuru et al., 1996; Wang et al., 1997), a relatively large number (1,000 or more) freshly isolated BM Lin−Sca-1+Kit+CD34−Flk2− HSCs were needed for successful allograft (Fig. 1A–C). By striking contrast, the cultured progeny of 50 or more input equivalent HSCs were capable of achieving the same level of allograft (Fig. 1D–E). Similar to freshly isolated HSCs, cultured HSCs were capable of multilineage differentiation in allogeneic mice (Fig. 1B, 1C, and 1E) and no sign of GVHD was observed. This suggests that ex vivo expanded HSCs have enhanced allograft abilities compared with freshly isolated cells.
The above strategy may result in the death of mice when donor HSCs are not capable of engrafting recipients. To ensure recipient mice survive after transplantation and to better quantitate the allograft abilities of different donor cells, we performed allogeneic transplantation by including competitors (Fig. S1). These competitors are total BM cells freshly isolated from the same type of mice as the recipients; these cells provide short-term radio-protection and serve as internal controls but also significantly enhance the host immune rejection and increase the difficulty of donor engraftment. Figure 2 shows the result of a representative competitive allogeneic transplantation from donor C57BL/6 (CD45.1) to BALB/c (CD45.2) recipients. Although 10,000 freshly isolated CD45.1 C57BL/6 BM Lin−Sca-1+Kit+CD34−Flk2− HSCs failed to engraft into the BALB/c recipients in the presence of competitors (0%, left, Fig. 2A), their cultured progenies had dramatically increased engraftment (55%, right, Fig. 2A). Similar results were obtained from the measurement of major histocompatibility complex (MHC) markers of donors and recipients (Fig. 2B). This allogeneic reconstitution sustained over time (Fig. 2C) and the donor cells repopulated the lymphoid and myeloid lineages (Fig. 2D–E), attesting to the engraftment of the donor long-term HSCs. Again no sign of GVHD was observed in the transplanted mice. To test whether allogenic donor HSCs were tolerated in the host, we performed secondary transplantation by isolating BM cells from the primary recipients and transplanting them into secondary BALB/c recipients. We found that the original CD45.1 donor cells successfully repopulated secondary recipients (Fig. 2F–G). The successful secondary transplantation indicates that the allogeneic donor HSCs were already tolerated after the primary transplantation. This result was further confirmed by the Mixed Lymphocyte Reaction (MLR) experiment showing that BALB/c T cells were not stimulated by the original donor derived cells in primary transplanted mice, but reacted to the counterpart cells isolated from CD45.1 C57BL/6 mice (Fig. 2H). Therefore the competitive allogeneic transplantation (Fig. 2) gave similar results as the non-competitive allograft (Fig. 1).
We further employed a third transplantation model to compare the abilities of donor HSCs before and after ex vivo expansion to engraft the allogeneic recipient mice with sublethal irradiation. Again the ex vivo expanded HSCs achieved markedly increased allograft compared to their uncultured counterparts (Fig. S2a). All these results indicate a dramatic enhancement of allograft ability of HSCs after ex vivo expansion.
Moreover, to directly compare the allograft capacities of HSCs before and after ex vivo expansion, we co-transplanted freshly isolated CD45.2 C57BL/6 HSCs and ex vivo expanded progenies of CD45.1 C57BL/6 HSCs into the same BALB/c recipient mice (Fig. S2b). Ex vivo expanded HSCs demonstrated a clear advantage over freshly isolated HSCs in this direct competitive allograft setting (Fig. S2b).
This ability of cultured HSCs to overcome the allogeneic barrier was not restricted to the use of particular allogeneic transplantation models. In addition to using C57BL/6 mice and BALB/c mice as the donor and recipient respectively, we tested a number of other donor/recipient combinations and reached the same conclusion. For example, ex vivo expanded HSCs isolated from FVB (CD45.1) mice had much greater ability to repopulate CD45.2 C57BL/6 recipients than their freshly isolated counterparts (Fig. S2c).
Because we used a culture system that expands HSCs, we sought to determine the contribution of the increase of HSC numbers during ex vivo expansion to the increased allograft ability by conducting limiting dilution analyses (Huynh et al., 2008; Zhang et al., 2006; Zheng et al., 2011). First, we used competitive syngeneic transplantation to calculate the numbers of repopulating CD45.1 C57BL/6 BM HSCs before and after ex vivo expansion. When we cultured HSCs in the optimized STFIA medium for 8 days, we obtained 11-fold (= 69/6, Table S1a, Fig. S2d) expansion in the number of HSCs as determined by limiting dilution analysis in syngeneic transplantation. Next, we quantitated the allograft abilities of these HSCs before and after culture by competitive allogeneic transplantation into BALB/c mice. For freshly isolated donor HSCs, the frequency of allograftable cells was 1/43,818, whereas the frequency in those cells cultured in the STFIA medium was 1/945 of input equivalent cells, determined by the competitive allogeneic transplantation (Fig. 2I, Tables S1b–c). This represents a ~40-fold (= 43,818/945) increase of allograft ability when cells were ex vivo expanded. Hence, in this experiment, ex vivo expansion led to 11-fold increase of HSC numbers and 40-fold increase of allograft ability. This result is concordant with previous reports that an increased number of HSCs enhances reconstitution of the hematopoietic compartment across the MHC barrier (Shizuru et al., 1996; Wang et al., 1997). Nevertheless, since the ex vivo expansion of HSCs had 40-fold increase of allograft ability, and the net increase of HSC number was 11-fold, another ~4-fold increase (= 40/11) should be contributed by culture independent of expansion of HSCs.
To further determine whether culture enhances allograft ability independent of expansion, we cultured HSCs in conditions that do not support HSC expansion and used these cells for transplantation. To this end, we cultured HSCs in serum-free medium supplemented with only SCF and TPO (as ST medium, Fig. 2I, Tables S1a–c, Fig. S2d); based on previous results (Huynh et al., 2008; Zhang et al., 2006; Zhang and Lodish, 2004, 2005) and our syngeneic transplantation (Table S1a), this condition does not support HSC expansion. We determined that the allograft frequency for these cultured but unexpanded HSCs was 1/12,332 input equivalent cells (Fig. 2I). This represented a ~4-fold increase (= 43,818/12,332) of allograft ability compared to freshly isolated HSCs. This number is in perfect agreement with the above estimate of a ~4-fold of increase of allograft ability by expansion-independent mechanism(s) based on comparison of results in syngeneic transplantation and allogeneic transplantation. Therefore increase of allograft ability of HSCs does not necessarily need HSC expansion. In summary, our results indicate that both the increase of HSC numbers and expansion-independent characteristics acquired during ex vivo culture contribute to the improved allograft efficiency.
To identify the expansion-independent mechanism for cultured HSCs to cross the MHC barrier, we explored two possibilities: the presence of certain accessory hematopoietic or mesenchymal cells, and a change of HSC immunogenicity during culture. To test the first possibility, we examined whether facilitating cells (Gandy et al., 1999; Kaufman et al., 1994), regulatory T cells (Taylor et al., 2008), or other cells produced during culture supported allograft. It has been established that unique differentiated BM populations as facilitating cells improve allogeneic reconstitution and result in donor-specific transplantation tolerance across MHC disparities (Gandy et al., 1999; Kaufman et al., 1994). The reported facilitating cells express conventional T cell components such as CD8 but are not T cells as they do not express TCR (Kaufman et al., 1994),(Bridenbaugh et al., 2008). Interestingly, facilitating cells induce an increase in numbers of donor regulatory T cells (Treg) (Taylor et al., 2008), which directly facilitate allograft. While freshly isolated HSCs do not contain CD3+ cells (Fig. S3a), after HSCs were cultured for 8 days in STFIA medium, approximately 0.3% of cells possessed the surface phenotype of CD8+CD45R+TCR− (Fig. S3b), the same phenotype as the previously characterized facilitating cells (Kaufman et al., 1994). To test whether the phenotypic “facilitating cells” produced in culture supported allograft, we collected these culture produced CD8+CD45R+TCR− cells by FACS and co-transplanted them with freshly isolated HSCs (1:1 as reported (Kaufman et al., 1994)) for allogeneic transplantation. We did not observe improved transplantation efficiency by including these cultured phenotypic “facilitating cells”, suggesting that they were not functional facilitating cells. In parallel, we were unable to detect phenotypic Treg (FoxP3+CD4+CD25+) cells in the cultures we examined, suggesting the increased allograft of cultured cells was unlikely contributed by production of Treg cells. To further test whether differentiated hematopoietic cells affected allograft, we isolated Lin+ cells from the HSC culture and co-transplanted them with freshly isolated HSCs. These Lin+ cells did not alter allogeneic transplantation efficiency (Fig. S3c). In addition, there were no apparent adherent cells during our 8-day culture, and a CFU-F assay showed no meshenchymal stem cells were produced from the cultured HSCs (Fig. S3d). These results indicate there is no engraftment-enhancing effect from mesenchymal stem cells. Taken together, we concluded that the accessory cells produced during the culture did not significantly contribute to increased allograft ability of ex vivo expanded HSCs.
Next we tested the possibility that the immunogenicity of HSCs changes during culture by examining the expression of surface immune proteins, including MHC-I, MHC-II, CD274 (B7-H1 or PD-L1), CD275 (B7-H2), CD47, CD80, and CD86. The expression of these surface proteins on freshly isolated and cultured cells, as determined by flow cytometry, are summarized in Figs. 3A–B. Almost all the freshly isolated HSCs and cultured cells expressed MHC-I and CD47, whereas very few of either population expressed MHC-II, CD275, CD80, or CD86. By contrast, there was a significant increase of surface expression of CD274 upon culture, as evidenced by an increase of CD274+ cells from 61% to 88% (Fig. 3A). Importantly, cultured cells contained a new population with more than 10-fold increase of CD274 expression (Fig. 3C–D, and fold increase of CD274 staining intensity = 10270/752 in Fig. 3C). There was a greater portion of CD274 positive cells in the phenotypic cultured HSCs as Lin−Sca-1+Kit+CD48− cells (Noda et al., 2008) than in differentiated cultured cells (Fig. 3E), although the expression intensities of CD274 were similar in all fractions of cultured cells (Fig. 3F).
B7 immune proteins belong to the immunoglobulin (Ig) superfamily, with two Ig-like extracellular domains and short cytoplasmic domains. CD274 is a member of the B7 family that is expressed or induced on dendritic cells or non-antigen presenting cells and inhibits T cell or innate activation (Francisco et al.; Zou and Chen, 2008). While Fig. 3D shows that CD274 might be upregulated on cultured HSCs based on phenotypic analysis, because the exact surface phenotype of cultured HSCs is not defined (Zhang and Lodish, 2005), we used the “gold standard” BM reconstitution analysis to test whether CD274 was expressed on functional HSCs and whether its level was altered upon culture. We first sorted freshly isolated BM cells into fractions negative and positive for immunostaining with antibodies against CD274. The repopulation activities of these fractions were then analyzed in the competitive syngeneic transplantation model. All the repopulating activity was within the CD274 positive fraction (Fig. 4A–C), indicating that all freshly isolated HSCs express CD274 on their surface. Since CD274 level was elevated more than 10-fold on some cultured cells (Fig. 3C–D), we sought to determine whether surface expression of CD274 was increased on functional repopulating HSCs after culture. To this end, we fractioned the low positive and high positive cultured cells (as CD274low and CD274high respectively) followed by competitive syngeneic transplantation. The repopulating activity was found in both CD274low and CD274high fractions (Fig. 4D–E, Fig. S4). This reveals that indeed a fraction of HSCs increased their surface expression of CD274 more than 10-fold under our culture conditions. Interestingly, different culture conditions did not change the CD274 upregulation (Fig. 4F), suggesting that the increase of CD274 expression was induced by general proliferation signals in culture, and was independent of HSC expansion.
To determine the role of CD274 in transplantation of HSCs, we utilized mice that are deficient in CD274 (Dong et al., 2004). We showed that CD274 null mice had higher frequency of phenotypic HSCs than wild-type (WT) mice (Fig. S5a), and the same number of freshly isolated CD274 null HSCs or ex vivo expanded null HSCs had slightly higher or similar long-term repopulation as WT HSCs in competitive syngeneic transplantation (Fig. 5A–D). These results suggest that CD274 per se does not significantly support the HSC activity in homeostatic and cultured conditions, concordant with the general normal phenotype of the CD274 null mice in homeostasis (Zou and Chen, 2008). By contrast, cultured CD274 null HSCs showed significantly decreased long-term repopulation in the competitive allogeneic repopulation compared to WT HSCs at 16 weeks post-transplant (Fig. 5E–F). The deficiency of B7-H4, another B7 family immune inhibitor, did not decrease allograft efficiency at 8–16 weeks post-transplant compared to WT HSCs (Fig. 5E–F). To further confirm that the surface CD274 on cultured HSCs facilitates allograft, we performed non-competitive allogeneic transplantation and compared the allograft of 1,000 input equivalent WT HSCs, anti-CD274 neutralizing antibody treated WT HSCs, and CD274 null HSCs after culture (Fig. 5G). Here we used ST medium (that does not support expansion of HSCs) to culture HSCs and specifically evaluate the expansion-independent effect of CD274 on HSC allograft. The 1,000 input equivalent WT HSCs engrafted 5 out of 17 recipients, whereas the anti-CD274 neutralizing antibody treated WT HSCs or the cultured CD274 null HSCs lost donor allograft activity (Fig. 5G). Therefore the deletion of CD274 or treatment with a CD274 neutralizing antibody abrogated the ability of cultured but unexpanded HSCs to cross the MHC barrier. A MLR experiment confirmed that, while cultured WT HSCs significantly inhibited allogeneic T cell activation, cultured CD274 null HSCs did not exhibit this inhibitory effect (Fig. 5H). Anti-PD-1 was capable of decreasing the late apoptosis of activated T cells co-cultured with pre-cultured HSCs (Fig. S5b). These results lead us to conclude that CD274, a ligand known to inhibit T cell responses, is induced on cultured HSCs and possibly some differentiated cells; PD-1 mediated apoptosis of host T cells is one mechanism by which cultured HSCs overcome the MHC barrier in allograft.
While many studies demonstrated direct evidence that CD274 impedes T cell functions, it was reported that CD274 can also suppress the activation of innate immune cells (Yao et al., 2009). We performed a further experiment to distinguish the possible involvement of T cell mediated immune response and innate immunity in the cultured HSC-enabled allograft. To this end, we cultured WT and CD274 null HSCs in the STFIA medium, followed by transplantation into sublethally irradiated SCID BALB/c mice (2.5 Gy). These recipient mice do not have functional T cells or B cells but have normal NK cells. If WT and CD274 null HSCs do not have difference in repopulation in these mice, it would indicate that CD274 mainly work through suppressing allogeneic T cell activation but not innate immunity. Indeed we did not observe difference in allograft abilities of cultured WT and null HSCs in these mice (Fig. 5I). Therefore, consistent with previous studies showing CD274 suppresses T cell-mediated allo-rejection (Francisco et al.; Zou and Chen, 2008), our result suggests that upregulation of CD274 on cultured cells including HSCs inhibited allogeneic T cell response.
To test whether ex vivo expanded HSCs can be used to cure genetic diseases, we ex vivo expanded allogeneic HSCs and transplanted these cells into homozygotic DNA-PK 3A/3A knock-in mice, in which three phosphorylation sites Thr2605, Thr2634, and Thr2643 of DNA-PK were eliminated (Zhang et al., 2011). These mice have defective HSC self-renewal during development and normally die around one month after birth (Zhang et al., 2011). Fig. 6A shows the result of transplantation of WT FVB (CD45.1) donor into DNA-PK knock-in mice of the CD45.2 C57BL/6/129 background. Whereas freshly isolated Lin−Sca-1+Kit+CD34−Flk2− allogeneic HSCs transplanted with 2–4 × 106 Sca-1− helper cells (which made the total number of transplanted cells the same as or more than the number of cultured cells transplanted) engrafted only 1 out of 9 recipients, their cultured progeny successfully engrafted and rescued all recipients. The rescued mice had almost 100% donor reconstitution and lymphoid, myeloid, and erythroid lineages were repopulated at 4 months post-transplantation (Fig. 6B). Our result demonstrated that ex vivo expanded HSCs can be successfully used in fully non-matched allogeneic transplantation to rescue the lethal phenotype of genetically mutated mice.
It is important to know whether a similar alteration of CD274 occurs on human HSCs upon culture. To this end, we determined the expression of CD274 on freshly isolated and cultured human cord blood HSCs. While only ~10% of freshly isolated human Lin−CD34+CD38−CD90+ cells express CD274 on their surface, the CD274+ population increased to more than 50% after culture (Fig. 7A–E). MLR analysis showed that the elevated CD274 expression on cultured human cord blood HSCs indeed suppressed the proliferation of allogeneic T cells, and this ability was abrogated by the anti-CD274 neutralizing antibody treatment (Fig. 7F). When we cultured human cord blood HSCs followed by transplantation into immune deficient NOD/SCID/gamma(c)(null) (NSG) mice, we observed a stimulating effect of Angptl5 on HSC expansion as previously reported (Drake et al., 2011; Khoury et al., 2011; Zhang et al., 2008) (Fig. S6). Nevertheless, this enhanced ability to engraft NSG mice was not affected by anti-CD274 neutralizing antibody (Fig. S6). This result is similar to what we observed in allograft in SCID BALB/c mice (Fig. 5I), suggesting that human CD274 suppresses allogeneic T cell activation but not innate immunity. The upregulation of CD274 on cultured human HSCs may enable these stem cells to possess an enhanced allograft ability.
In this study, we demonstrated that ex vivo expanded HSCs more efficiently overcome MHC barriers and repopulated allogeneic recipient mice than freshly isolated HSCs. As measured by limiting dilution analysis, there was a 40-fold increase in the allograft ability of HSCs cultured for only 8 days compared to that of the freshly isolated HSCs. To identify the underlying mechanisms, we found that both increased numbers of HSCs and cultured-induced elevation of expression of the immune inhibitor CD274 on the surface of HSCs contributed to the enhanced allograft efficiency. As a proof-of-principle that ex vivo expanded HSCs can be used to cure genetic diseases in allogeneic recipients, we used ex vivo expanded allogeneic HSCs for transplantation and successfully rescued the lethal phenotype of DNA-PK knock-in mice.
We used three models of allogeneic transplantation: non-competitive transplantation into lethally irradiated recipients, competitive transplantation into lethally irradiated recipients, and non-competitive transplantation into sublethally irradiated recipients. While the first model was well-established and allows fewer numbers of donor cells for engraftment, it may result in the mouse death if donor HSCs cannot repopulate recipients. The second and third models ensure the survival of all recipients and better mimic the human transplantation scenario in which reduced intensity conditioning is often applied. Nevertheless, due to the enhanced host immune rejection, more than 10-fold of freshly isolated allogeneic donor HSCs are needed for successful engraftment in these models. This also underscores the importance of the increased number and MHC matching of donor HSCs in the clinical setting.
Our findings may shed new light on allogeneic transplantation of human HSCs into patients, which cannot be appropriately modeled by xenograft into immune-deficient mouse recipients. Two major problems, failure of engraftment and GVHD, have limited the progress in allogeneic transplantation. A strategy that significantly improves donor engraftment and reduces the risk of GVHD compared to current practice is needed. Transplantation of freshly isolated allogeneic HSCs indeed decreases the risk of GVHD, but results in much lower engraftment (Shizuru et al., 1996; Wang et al., 1997). Here we show that ex vivo expanded mouse HSCs possess two advantages: increased HSC numbers and the enhanced immune feature to evade host rejection, therefore having dramatically enhanced allogeneic engraftment. Importantly, similar to freshly isolated HSCs (Shizuru et al., 1996; Wang et al., 1997), no sign of GVHD was observed after allogeneic transplantation of ex vivo expanded HSCs. This is expected because the condition of our (or other) HSC culture supports expansion of HSCs, along with production of differentiated myeloid but not much lymphoid cells. The culture thus does not seem to produce the source cells including T cells that may cause GVHD. Therefore, ex vivo expanded mouse HSCs appear to be an appropriate cell source to solve the problems of allogeneic transplantation in the mouse model. Based on these results of mouse HSCs and the elevation of CD274 on cultured human HSCs, we propose that ex vivo expansion of human HSCs may benefit the practice of allogeneic transplantation for patients. This would apply to non-matched or low-matched donor human cord blood, BM, or mobilized peripheral blood HSCs. If donor human HSCs can be expanded in culture and engraft non-matched or low-matched patients without GVHD, this strategy will possibly lead to an ultimate solution to problems in allogeneic transplantation.
It is known that some adult stem cells, such as mesenchymal and amnion stem cells, but not embryonic stem cells, are capable of avoiding rejection through production of immunosuppressive molecules and can be used in intra- and even interspecies transplantation (Salem and Thiemermann; Swijnenburg et al., 2008). Here we demonstrated that the immune inhibitor CD274 is expressed on freshly isolated HSCs and its expression dramatically increased upon culture. Interestingly, CD274 does not appear to significantly affect the repopulation of long-term HSCs before and after culture as determined by syngeneic transplantation, suggesting its main role is not regulation of the regular activity of HSCs, but modulation of immunological properties of these cells. This was confirmed by the result that the deletion of CD274 or treatment with a CD274 neutralizing antibody abrogated the ability of cultured but unexpanded HSCs to cross the MHC barrier. CD274 was shown in previous studies to be expressed on activated immune cells and parenchymal cells and in immune-privileged sites such as eyes and placenta (Francisco et al.; Zou and Chen, 2008). CD274 is also selectively expressed by various cellular components in the tumor microenvironment, where it inhibits tumor-specific T-cell immunity by inducing T cell apoptosis and delay rejection (Zou and Chen, 2008). Here we provided an example suggesting that HSCs possess the ability to evade the rejection of the acquired immune system by regulating the expression of their own surface immune inhibitor such as CD274. Besides HSCs, the elevation of CD274 on hematopoietic progenitors produced during culture also might have contributed to the enhanced allograft. However, it is interesting to note that more differentiated Lin+ cells elicit no effect, although they also express CD274. This may be contributed by the different cellular locations of HSCs/progenitors and more differentiated hematopoietic cells home after transplantation. It therefore will be interesting to study where the T cell-mediated immune response occurs for allogeneic transplanted HSCs in the future. In addition, it is noteworthy that CD274 may not be the only immune suppressor acts on the HSC allograft. This is because that, although CD274-null HSCs behave much worse in allogeneic transplantation than their WT counterparts, they still possess a certain ability for allogeneic engraftment. Consistent with the elevation of the expression of immune inhibitor CD274 upon culture, co-stimulatory molecules such as CD80 and CD86 lost their expression on some cells after culture. All these observations clearly indicate that ex vivo culture significantly modulates the immunogenicity of stem cells. The identification of additional immune molecules whose alterations can regulate allograft will enable the complete resolution of the issue of immune rejection in allogeneic transplantation.
While our study suggests that the upregulation of CD274 on cultured cells including HSCs inhibited allogeneic T cell response, a related example is surface expression of CD47, which enables HSCs and leukemia cells to evade innate macrophage phagocytosis (Jaiswal et al., 2009). Based on these results, we hypothesize that all homeostatic HSCs express low levels of surface immune suppressors, and the levels of these suppressors can be induced by stress or immune signals. These immune suppressors may thus modulate HSC immunogenicity and, therefore, contribute to the “immune privilege” of HSCs. This regulatable “immune privilege” should be advantageous to HSCs, as it may allow these important stem cells to rapidly adjust to altered environment or to protect them from the excessive immune activation and even potential autoimmune disorder. Whether the expression of CD274 on HSCs or cancer cells can be regulated in vivo and its biological significance warrants further investigation.
Furthermore, we speculate that a common mechanism exists for regulation of expression of immune inhibitory signals in some other types of stem cells – similar to that in tumor cells. The expression and regulation of immune inhibitors on stem cells per se may allow these cells to survive an unexpected immune attack. It will be interesting to study the immunology of stem cells by investigating the roles of surface immune molecules on embryonic stem cells, induced pluripotent stem cells, other adult stem cells, and cancer stem cells.
In summary, our study demonstrated the great benefits of ex vivo expansion of HSCs for overcoming problems in allogeneic transplantation, and revealed the importance of an immune inhibitor on the surface of HSCs. This work should shed new light on understanding the immunology of HSCs and other stem cells and may lead to development of novel strategies for successful allogeneic transplantation of human patients.
Indicated numbers of BM Lin−Sca-1+Kit+ CD34−Flk-2− cells were isolated from 8–12 week old mice and 150–200 of them were plated into each well of a U-bottom 96-well plate (3799; Corning) with 200 μl of the indicated medium. STFIA medium was defined as Stemspan serum-free medium (StemCell Technologies) supplemented with 10 μg/ml heparin, 10 ng/ml mouse SCF, 20 ng/ml mouse TPO, 10 ng/ml human FGF-1, 100 ng/ml IGFBP2, and 500 ng/ml Angptl3 as described (Huynh et al., 2008) which was used in Fig. 2 experiments. In repeated experiments and experiments described in other figures, we refer STFIA medium as the same above medium except using serum-free conditioned medium collected from Angptl2 transfected 293T cells as described (Zhang et al., 2006) (that contains both IGFBP2 (Huynh et al., 2008) and Angptl2) to replace recombinant IGFBP2 and Angptl3. This Angptl2 supplemented medium worked equivalently and reproducibly supported HSC expansion in all experiments. ST medium was defined as Stemspan supplemented with 10 μg/ml heparin, 10 ng/ml mouse SCF, and 20 ng/ml mouse TPO. STF medium was ST medium supplemented with 10 ng/ml human FGF-1. STFA medium was STF medium supplemented with 500 ng/ml Angptl3. STFI medium was STF medium supplemented with 100 ng/ml IGFBP2. Unless otherwise described, cells were cultured for 8 days at 37°C in 5% CO2 and the normal level of O2. The culture duration of 8 days was shorter than that we described in previous studies (Huynh et al., 2008; Zhang et al., 2006). Because a substantially more number of cells were needed for allogeneic transplantation, we plated 150–200 HSCs per well in our experiments, instead of 20 HSCs described previously (Huynh et al., 2008; Zhang et al., 2006) for congeneic transplantation. We typically observed a ~200-fold increase of total number of cells after 8 days of culture. Therefore a 100 input cells produced 2.32 ± 0.28 × 104 total cells after 8 days of culture. This 8-day culture thus allowed us to harvest cells from the culture wells before the expanded cells exhausted the medium. For the purpose of transplantation, we pooled cells from at least 10 culture wells before the indicated numbers of cells were transplanted into each mouse. Flow cytometry analysis was performed to confirm multilineage reconstitution as we described (Simsek et al., 2010; Zheng et al., 2011). Calculation of CRUs in limiting dilution experiments was conducted using L-Calc software (StemCell Technologies) (Huynh et al., 2008; Simsek et al., 2010; Zheng et al., 2011).
For allogeneic transplantation without competitors, the indicated numbers of mouse donor cells before or after culture were injected intravenously via the retro-orbital route into each of a group of 6–9 week old recipient mice immediately after irradiation with a lethal dose of 9 or 9.5 Gy for BALB/c or 10 Gy for C57BL/6 mice. Sublethal irradiation of BALB/c mice in Fig. S2a and of SCID-BALB/c mice in Fig. 5I were performed at a dose of 7.5 Gy and 2.5 Gy respectively. For competitive allogeneic transplantation, the indicated mouse donor cells before or after culture were mixed with 1–2 × 105 (as indicated) freshly isolated competitor bone marrow cells before transplantation. When indicated, one million bone marrow cells collected from primary recipients were used for the secondary transplantation into lethally irradiated BALB/c mice. The antibody blocking treatment was conducted by incubating cultured HSCs with 50 μg/ml anti-CD274 neutralizing antibody (Cat# 16-5982-81, eBioscience) for 2 h followed by washing before transplantation.
MLR was performed similarly as we described (Curiel et al., 2003). Briefly, for mouse MLR in Fig. 5H, BALB/c splenocyte CD90.2+ T cells were plated in 96-well plate (flat-bottom) pre-coated with 1 μg/ml anti-CD3, followed by co-culture with 8-d pre-cultured irradiated C57BL/6 HSCs. For human MLR in Fig. 7F, peripheral blood CD3+ cells were plated in the presence of 2.5 μg/ml anti-CD3 and co-cultured with 8-d pre-cultured irradiated allogeneic cord blood HSCs. Proliferation was measured at day 3 of incubation at 37°C and 5% CO2 following pulsing with [3H]TdR using a liquid scintillation counter. When indicated, 50 μg/ml anti-CD274 neutralizing antibody (Cat# 16-5983-82) was used to treat the cultured cells for 2 h.
More protocol information is available in the Supplemental Experimental Procedures.
Support to C. C. Z. was from NIH grant K01 CA 120099, American Society of Hematology Junior Faculty Award, AHA 09BGIA2230372, DOD PR093256, CPRIT RP100402, and the Gabrielle’s Angel Foundation. Support to B. C. was from NIH grant R01 CA50519.
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