New Insights into the Cell Biology of Hematopoietic Progenitors by Studying Prominin-1 (CD133)

Prominin-1 (alias CD133) has received considerable interest because of its expression by several stem and progenitor cells originating from various sources, including the neural and hematopoietic systems. As a cell surface marker, prominin-1 is now used for somatic stem cell isolation. Its expression in cancer stem cells has broadened its clinical value, as it might be useful to outline new prospects for more effective cancer therapies by targeting tumor-initiating cells. Cell biological studies of this molecule have demonstrated that it is specifically concentrated in various membrane structures that protrude from the planar areas of the plasmalemma. Prominin-1 binds to the plasma membrane cholesterol and is associated with a particular membrane microdomain in a cholesterol-dependent manner. Although its physiological function is not yet determined, it is becoming clear that this cell surface protein, as a unique marker of both plasma membrane protrusions and membrane microdomains, might reveal new aspects of the cell biology of rare stem and cancer stem cells. The aim of this review is to outline the recent discoveries regarding the dynamic reorganization of the plasma membrane of rare CD133+ hematopoietic progenitor cells during cell migration and division.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Prominin-1/CD133, a neural and hematopoietic stem cell marker, is expressed in adult human differentiated cells and certain types of kidney cancer.

Human prominin-1/CD133 has been reported to be expressed in neural and hematopoietic stem/progenitor cells and in embryonic, but not adult, epithelia. This lack of detection of human prominin-1, as defined by its glycosylation-dependent AC133 epitope, is surprising given the expression of the murine ortholog in adult epithelia. Here, we demonstrate, by using a novel prominin-1 antiserum (alphahE2), that the decrease of AC133 immunoreactivity observed during differentiation of the colonic adenocarcinoma-derived Caco-2 cells is not paralleled by a down-regulation of prominin-1. We have also shown that alphahE2 immunoreactivity, but not AC133 immunoreactivity, is present in several adult human tissues, such as kidney proximal tubules and the parietal layer of Bowman's capsule of juxtamedullary nephrons, and in lactiferous ducts of the mammary gland. These observations suggest that only the AC133 epitope is down-regulated upon cell differentiation. Furthermore, alphahE2 immunoreactivity has been detected in several kidney carcinomas derived from proximal tubules, independent of their grading. Interestingly, in one particular case, the AC133 epitope, which is restricted to stem cells in normal adult tissue, was up-regulated in the vicinity of the tumor. Our data thus show that (1) in adults, the expression of human prominin-1 is not limited to stem and progenitor cells, and (2) the epitopes of prominin-1 might be useful for investigating solid cancers

Role of lymphocyte activation gene-3 (Lag-3) in conventional and regulatory T cell function in allogeneic transplantation.

Lag-3 has emerged as an important molecule in T cell biology. We investigated the role of Lag-3 in conventional T cell (Tcon) and regulatory T cell (Treg) function in murine GVHD with the hypothesis that Lag-3 engagement diminishes alloreactive T cell responses after bone marrow transplantation. We demonstrate that Lag-3 deficient Tcon (Lag-3(-/-) Tcon) induce significantly more severe GVHD than wild type (WT) Tcon and that the absence of Lag-3 on CD4 but not CD8 T cells is responsible for exacerbating GVHD. Lag-3(-/-) Tcon exhibited increased activation and proliferation as indicated by CFSE and bioluminescence imaging analyses and higher levels of activation markers such as CD69, CD107a, granzyme B, and Ki-67 as well as production of IL-10 and IFN-g early after transplantation. Lag-3(-/-) Tcon were less responsive to suppression by WT Treg as compared to WT Tcon. The absence of Lag-3, however, did not impair Treg function as both Lag-3(-/-) and WT Treg equally suppress the proliferation of Tcon in vitro and in vivo and protect against GVHD. Further, we demonstrate that allogeneic Treg acquire recipient MHC class II molecules through a process termed trogocytosis. As MHC class II is a ligand for Lag-3, we propose a novel suppression mechanism employed by Treg involving the acquisition of host MHC-II followed by the engagement of Lag-3 on T cells. These studies demonstrate for the first time the biologic function of Lag-3 expression on conventional and regulatory T cells in GVHD and identify Lag-3 as an important regulatory molecule involved in alloreactive T cell proliferation and activation after bone marrow transplantation

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies

IL-17 Gene Ablation Does Not Impact Treg-Mediated Suppression of Graft-Versus-Host Disease after Bone Marrow Transplantation

AbstractRegulatory T cell (Treg) immunotherapy is a promising strategy for the treatment of graft rejection responses and autoimmune disorders. Our and other laboratories have shown that the transfer of highly purified CD4+CD25+Foxp3+ natural Treg can prevent lethal graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation across both major and minor histocompatibility barriers. However, recent evidence suggests that the Treg suppressive phenotype can become unstable, a phenomenon that can culminate in Treg conversion into IL-17–producing cells. We hypothesized that the intense proinflammatory signals released during an ongoing alloreaction might redirect a fraction of the transferred Treg to the Th17 cell fate, thereby losing immunosuppressive potential. We therefore sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model. We show that although Il17 gene ablation results in a mildly enhanced Treg immunosuppressive ability in vitro, such improvement is not observed when IL-17–deficient Treg are used for GVHD suppression in vivo. Similarly, when we selectively blocked IL-1 signaling in Treg, that was shown to be necessary for Th17 conversion, we did not detect any improvement on Treg-mediated GVHD suppressive ability in vivo. Furthermore, upon ex vivo reisolation of transferred wild-type Treg, we detected little or no Treg-mediated IL-17 production upon GVHD induction. Our results indicate that blocking Th17 conversion does not affect the GVHD suppressive ability of highly purified natural Treg in vivo, suggesting that IL-17 targeting is not a valuable strategy to improve Treg immunotherapy after hematopoietic cell transplantation

WT Treg and Lag-3^−/− Treg show similar protection against GVHD.

<p>(A) Foxp3 staining (upper panel) and Lag-3 staining (lower panel) of CD4⁺CD25⁺ T cells isolated from WT mice (black histogram) and Lag-3^−/− mice (dashed histogram). For Lag-3 staining, lower panel, regulatory T cells were activated for 2 days with anti-CD3/anti-CD28 beads. Cells were gated on CD4⁺Foxp3⁺. The shaded histogram represents the isotype staining control. The histograms are representative of 2 independent experiments. (B) ³H-thymidine incorporation of WT C57BL/6J responders to Balb/c stimulators in the presence of either WT Treg or Lag-3^−/− Treg at different Treg to responder ratios, R: responders, S: stimulators. (C–D) Lethally irradiated Balb/c recipients were co-transplanted with 5×10⁶ TCD-BM and 5×10⁵ WT or Lag-3^−/− Treg on day 0 followed by infusion of 1×10⁶<i>luc⁺</i> WT Tcon on day 2. (C) Percent survival of mice after transplantation. Graph contains data pooled from 3 independent experiments (n = 15). P = 0.034 for WT Treg+Tcon vs. Tcon and P = 0.033 for Lag-3^−/− Treg+Tcon vs. Tcon. (D) <i>In vivo</i> BLI images of mice receiving WT Tcon alone, WT Tcon+WT Treg and WT Tcon+Lag-3^−/− Treg. Upper panels: BLI images taken at different time points after transplantation; lower panels: the corresponding quantitative analysis of the BLI signal. The BLI images are representative of 3 independent experiments.</p

Modeling Chronic Graft-versus-Host Disease in MHC-Matched Mouse Strains: Genetics, Graft Composition, and Tissue Targets

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation. Acute GVHD (aGVHD) results from direct damage by donor T cells, whereas the biology of chronic GVHD (cGVHD) with its autoimmune-like manifestations remains poorly understood, mainly because of the paucity of representative preclinical models. We examined over an extended time period 7 MHC-matched, minor antigen-mismatched mouse models for development of cGVHD. Development and manifestations of cGVHD were determined by a combination of MHC allele type and recipient strain, with BALB recipients being the most susceptible. The C57BL/6 into BALB.B combination most closely modeled the human syndrome. In this strain combination moderate aGVHD was observed and BALB.B survivors developed overt cGVHD at 6 to 12 months affecting eyes, skin, and liver. Naïve CD4$^{+}$ cells caused this syndrome as no significant pathology was induced by grafts composed of purified hematopoietic stem cells (HSCs) or HSC plus effector memory CD4$^{+}$ or CD8$^{+}$ cells. Furthermore, co-transferred naïve and effector memory CD4$^{+}$ T cells demonstrated differential homing patterns and locations of persistence. No clear association with donor Th17 cells and the phenotype of aGVHD or cGVHD was observed in this model. Donor CD4$^{+}$ cells caused injury to medullary thymic epithelial cells, a key population responsible for negative T cell selection, suggesting that impaired thymic selection was an underlying cause of the cGVHD syndrome. In conclusion, we report for the first time that the C57BL/6 into BALB.B combination is a representative model of cGVHD that evolves from immunologic events during the early post-transplant period

Regulatory T cells acquire MHC class II complexes from host APC.

<p>(A) Lethally irradiated Balb/c (H-2K^d) recipients were co-transplanted with 5×10⁶ TCD-BM, 1×10⁶ Tcon from CD45.1⁺ C57BL/6 (H-2K^b) mice and 1×10⁶ WT or Lag-3^−/− Treg. MHC class II expression on donor Treg was assessed 5 days after transplantation. Cells were gated on CD45.1 negative, H2Kb positive (panel i), then CD4 and Foxp3 positive events (panel ii). Panels iii and iv represent the MHC class II expression level on day 0 and day 5 respectively. (B) MHC class II staining on Treg of donor origin (WT or Lag-3^−/−). Grey histogram represents cells stained with isotype control. Cells were gated on donor CD4 positive, Foxp3 positive events. The histograms are representative of 2 independent experiments each having 4 mice/group.</p

Lag-3^−/− Tcon proliferate faster than WT Tcon.

<p>(A) Quantitative analysis of photon emission <i>in vitro</i> by an equal number of <i>luc⁺</i> WT and <i>luc⁺</i> Lag-3^−/− Tcon. (B) Lethally irradiated Balb/c recipients were transplanted with 5×10⁶ TCD-BM and 5×10⁵<i>luc⁺</i> WT or Lag-3^−/− Tcon. <i>In vivo</i> BLI images at different times after transplantation show more expansion of Lag-3^−/− Tcon. The graph on the right represents the quantitative analysis of the BLI signal. (C) <i>Ex vivo</i> images of the intestinal tract on day 4 displayed increased BLI signal in MLN of recipients receiving <i>luc⁺</i> Lag-3^−/− Tcon. The ex vivo images are representative of 5 mice/group. The bar graph on the right represents the quantitative analysis of the <i>ex vivo</i> BLI signal from spleen, pLN, and MLN. Bar depicts mean plus or minus SD, n = 5 mice/group. (*P<0.05, **P<0.01, ***P<0.001).</p

Surface expression of Lag-3 increases upon T cell activation <i>in vitro</i> and <i>in vivo</i>.

<p>(A) T cells were incubated with anti-CD3/anti-CD28 activation beads in the presence of 1000 U/mL hrIL-2. Lag-3 expression was assessed over a period of 7 days. The grey solid histograms represent cells stained with isotype control Ab. Cells were gated on CD4⁺ and the numbers in the right side corner represent the percentage of Lag-3 positive cells. Data is representative of 3 independent experiments. (B) Balb/c (H-2K^d) recipients were co-transplanted with 5×10⁶ TCD-BM and 1×10⁶ Tcon from C57Bl/6 (H-2K^b) mice. At indicated time points after transplantation, donor T cells were re-isolated from spleen and LN and analyzed for Lag-3 expression. Grey histograms represent cells stained with isotype controls. Cells were gated on donor CD4⁺ cells and the numbers in the right side corner represent the percentage of Lag-3 positive cells. Data is representative of two independent experiments.</p