The fate of transplanted xenogeneic bone marrow‐derived stem cells in rat intervertebral discs

Intervertebral disc degeneration is a major cause and a risk factor for chronic low back pain. The potential of using stem cells to treat disc degeneration has been raised. The aims of our study were to assess whether xenogeneic bone‐marrow derived stem cells could survive in a rat disc degeneration model and to determine which cell types, if any, survived and differentiated into disc‐like cells. Human bone‐marrow derived CD34⁺ (hematopoietic progenitor cells) and CD34^? (nonhematopoietic progenitor cells, including mesenchymal stem cells) cells were isolated, fluorescent‐labeled, and injected into rat coccygeal discs. The rats were sacrificed at day 1, 10, 21, and 42. Treated discs were examined by histological and immunostaining techniques and compared to control discs. The survival of transplanted cells was further confirmed with a human nuclear specific marker. Fluorescent labeled CD34^? cells were detected until day 42 in the nucleus pulposus of the injected discs. After 3 weeks these cells had differentiated into cells expressing chondrocytic phenotype (Collagen II and Sox‐9). In contrast, the fluorescent labeled CD34⁺ cells could not be detected after day 21. No fluorescence‐positive cells were detected in the noninjected control discs. Further, no inflammatory cells infiltrated the nucleus pulposus, even though these animals had not received immunosuppressive treatment. Our data provide evidence that transplanted human BM CD34^? cells survived and differentiated within the relative immune privileged nucleus pulposus of intervertebral disc degeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:374–379, 2009     

CD146 as a new marker for an increased chondroprogenitor cell sub‐population in the later stages of osteoarthritis

Cartilage‐derived mesenchymal stem cells (MSCs) have been isolated with different methods. In this study lateral and medial femoral condyles were respectively collected from patients with late‐stage osteoarthritis during the total knee arthroplasty. After digestion of the cartilage tissues with type II collagenase and analysis by fluorescence‐activated cell sorting (FACS) with CD146, a chondroprogenitor cell sub‐population were isolated and purified. The expression of other MSC‐associated markers in the CD146⁺ chondroprogenitors was analyzed by flow cytometry. Multi‐lineage differentiation capacity of CD146⁺ chondroprogenitors was compared with that of unsorted chondrocytes and adipose‐derived MSCs (ADMSCs). Higher percentage of CD146⁺ chondroprogenitors isolated from the medial femoral condyles was observed than that from the lateral. CD146⁺ chondroprogenitors expressed high levels of MSC‐specific surface antigens, and showed higher chondrogenesis capacity than ADMSCs and unsorted chondrocytes in a 3D cell pellet culture model. Thus CD146 might be a new cell surface marker for cartilage progenitor cell population in the late‐stage osteoarthritis. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:84–91, 2015.     

Both CD45RA+ and CD45RO+ human CD4+ T cells drive direct xenogeneic T‐cell responses against porcine aortic endothelial cells

Kim CH, Oh K, Kim D‐E, Lee SB, Yang JH, Lee G, Cho J, Lee D‐S. Both CD45RA⁺ and CD45RO⁺ human CD4⁺ T cells drive direct xenogeneic T‐cell responses against porcine aortic endothelial cells. Xenotransplantation 2010; 17: 224–232. © 2010 John Wiley & Sons A/S. Abstract: Background: Xenogeneic cellular immune responses are mediated by either direct or indirect pathways depending on the participation of donor or host antigen presenting cells, respectively. The contribution of direct response of human T cells, especially memory T cells, to porcine antigen presenting cells is currently unknown. Here, we sought to determine whether human peripheral blood memory/activated phenotype T cells are directly responsive to porcine endothelial cells. Methods: Porcine aortic endothelial cells (PAECs) were prepared from Yorkshire or miniature pigs. Highly purified human T cells, including naïve and memory/activated phenotype cells, were incubated with PAECs with or without the addition of exogenous cytokines. T‐cell proliferation and T‐cell receptor (TCR) Vβ usage in response to PAECs were analyzed. Results: Both CD8⁺ and CD4⁺ T cells responded directly to PAECs and exhibited exclusive responsiveness to SLA class I and class II molecules, respectively. Naïve and memory/activated phenotype CD4⁺ T cells responded against PAECs, whereas only naïve phenotype CD8⁺ T cells contributed to such a response. In addition, both populations of xenogeneic human CD4⁺ T cells exhibited similar and diverse Vβ usage. Conclusion: Due to the considerable contribution of human CD45RO⁺CD4⁺ T cells to the xenoreactivity against PAECs, effective control of xenogeneic memory/activated T‐cell responses would significantly affect long‐term survival of transplanted grafts.     

Differential Requirement of CD27 Costimulatory Signaling for Naïve Versus Alloantigen‐Primed Effector/Memory CD8+ T Cells

CD8⁺ memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T‐cell costimulatory pathway in alloreactive CD8⁺/CD4⁺ T‐cell activation. CD27‐deficient (CD27^?/?) and wild‐type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4⁺ or CD8⁺ T cells, suggesting that CD27 is not essential during primary T‐cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27^?/? or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor‐type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40‐ but not day 10‐sensitized CD27^?/? recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8⁺ T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27^?/? recipients of class I‐ but not class II‐mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8⁺ memory T cells in acute and chronic allograft rejection.     

Prolongation of Cardiac Allograft Survival by a Novel Population of Autologous CD117+ Bone Marrow‐Derived Progenitor Cells

Autologous CD117⁺ progenitor cells (PC) have been successfully utilized in myocardial infarction and ischemic injury, potentially through the replacement/repair of damaged vascular endothelium. To date, such cells have not been used to enhance solid organ transplant outcome. In this study, we determined whether autologous bone marrow‐derived CD117⁺PC could benefit cardiac allograft survival, possibly by replacing donor vascular cells. Autologous, positively selected CD117⁺PC were administered posttransplantation and allografts were assessed for acute rejection. Although significant generation of recipient vascular cell chimerism was not observed, transferred PC disseminated both to the allograft and to peripheral lymphoid tissues and facilitated a significant, dose‐dependent prolongation of allograft survival. While CD117⁺PC dramatically inhibited alloreactive T cell proliferation in vitro, this property did not differ from nonprotective CD117^? bone marrow populations. In vivo, CD117⁺ PC did not significantly inhibit T cell alloreactivity or increase peripheral regulatory T cell numbers. Thus, rather than inhibiting adaptive immunity to the allograft, CD117⁺ PC may play a cytoprotective role in prolonging graft survival. Importantly, autologous CD117⁺PC appear to be distinct from bone marrow‐derived mesenchymal stem cells (MSC) previously used to prolong allograft survival. As such, autologous CD117⁺PC represent a novel cellular therapy for promoting allograft survival.     

Parathyroid hormone regulates the distribution and osteoclastogenic potential of hematopoietic progenitors in the bone marrow

Parathyroid hormone (PTH) increases both the number of osteoclast in bone and the number of early hematopoietic stem cells (HSCs) in bone marrow. We previously characterized the phenotype of multiple populations of bone marrow cells with in vitro osteoclastogenic potential in mice. Here we examined whether intermittent administration of PTH influences these osteoclast progenitor (OCP) populations. C57BL/6 mice were treated with daily injections of bPTH(1–34) (80 µg/kg/day) for 7 or 14 days. We found that PTH caused a significant increase in the percentage of TN/CD115⁺CD117^high and TN/CD115⁺CD117^int cells (p < .05) in bone marrow on day 7. In contrast, PTH decreased the absolute number of TN/CD115⁺CD117^low cells by 39% on day 7 (p < .05). On day 14, there was no effect of PTH on osteoclast progenitor distribution in vivo. However, PTH treatment for 7 and 14 days did increase receptor activator of NF‐κB ligand (RANKL)– and macrophage colony‐stimulating factor (M‐CSF)–stimulated in vitro osteoclastogenesis and bone resorption in TN/CD115⁺ cells. In the periphery, 14 days of treatment increased the percentage and absolute numbers of HSCs (Lin^?CD117⁺Sca‐1⁺) in the spleen (p < .05). These data correlated with an increase in the percent and absolute numbers of HSCs in bone marrow on day 14 (p < .05). Interestingly, the effects on hematopoietic progenitors do not depend on osteoclast resorption activity. These results suggest that in vivo PTH treatment increased in vitro osteoclastogenesis and resorption without altering the number of osteoclast precursors. This implies that in vivo PTH induces sustained changes, possibly through an epigenetic mechanism, in the in vitro responsiveness of the cells to M‐CSF and RANKL. © 2011 American Society for Bone and Mineral Research.     

Sox‐9 facilitates differentiation of adipose tissue‐derived stem cells into a chondrocyte‐like phenotype in vitro

The purpose of this study is to test whether ectopic expression of Sox‐9 can induce adipose tissue‐derived stem cells (ASCs) to function as real nucleus pulposus (NP) cells in vitro. Adenoviral vectors expressing Sox‐9 were reported to infect the chondroblastic and human disc cells, which resulted in increased Sox‐9 and type II collagen production. ASCs were isolated from rat inguinal adipose pad, characterized, and transduced in vitro with a retroviral vector encoding the Sox‐9 gene. Sox‐9‐engineered ASCs (ASCs/Sox‐9) were induced for the chondrocyte‐like cell differentiation by 3D cultured in alginate beads and TGF‐β3 for 2 weeks. Expression of exogenous Sox‐9 protein was detected. Type II collagen and Aggrecan gene expressions of induced ASCs/Sox‐9 were measured using real‐time PCR; proteoglycans expressions were measured by checking the glycosaminoglycan content and type II collagen production by enzyme‐linked immunosorbent assay. Isolated ASCs were CD 29⁺/CD44⁺/C‐Kit^?/Lin^?/CD34^?/CD45^?. ASCs/Sox‐9 expressed marked increase in exogenous Sox‐9 protein. After induction, type II collagen gene expression was sevenfold higher in mRNA levels, with an approximately twofold increase in protein levels of ASCs/Sox‐9 compared to ASCs. Type II collagen and proteoglycan productions were significantly increased in the ASCs/Sox‐9 compared to the ASCs. In addition, co‐culture of induced ASCs/Sox‐9 with matured NP cells resulted in enhanced increase in proteoglycan and type II collagen production. Constitutive retroviral expression of Sox‐9 could efficiently induce ASCs differentiation into chondrocyte‐like cells. This novel approach may provide a practicable system for a simple and rapid differentiation of ASCs into chondrocyte‐like cells which may be potentially used as a stem cell‐based therapeutic tool for the treatment of degenerative disc diseases. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1291–1297, 2011     

Parathyroid hormone induces differentiation of mesenchymal stromal/stem cells by enhancing bone morphogenetic protein signaling

Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with parathyroid hormone 1 receptor (PTH1R) and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1⁺CD45^–CD11b^–MSCs, showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone led to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMP receptor type II (BMPRII) significantly in C2C12 cells, and PTH treatment significantly enhanced cell-surface binding of ¹²⁵I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted green fluorescent protein (GFP)-labeled Sca-1⁺CD45^–CD11b^–MSCs into the bone marrow cavity of Rag2^−/− immunodeficient mice showed that PTH enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH enhancement of MSC differentiation to the osteoblast lineage occurs through a PTH- and LRP6-dependent pathway by endocytosis of the PTH1R/LRp6 complex, allowing enhancement of BMP signaling. © 2012 American Society for Bone and Mineral Research.     

PD‐1 Analysis on CD28−CD27− CD4 T Cells Allows Stimulation‐Independent Assessment of CMV Viremic Episodes in Transplant Recipients

Expression of the inhibitory receptor programmed death 1 (PD‐1) on cytomegalovirus (CMV)‐specific CD4 T cells defines a phenotype associated with CMV viremia in transplant recipients. Moreover, CD28^?CD27^? double negativity is known as a typical phenotype of CMV‐specific CD4 T cells. Therefore, the co‐expression of inhibitory receptors on CD28^?CD27^? CD4 T cells was assessed as a rapid, stimulation‐independent parameter for monitoring CMV complications after transplantation. Ninety‐three controls, 67 hemodialysis patients and 81 renal transplant recipients were recruited in a cross‐sectional and longitudinal manner. CMV‐specific CD4 T cell levels quantified after stimulation were compared to levels of CD28^?CD27^? CD4 T cells. PD‐1 and cytotoxic T lymphocyte–associated antigen 4 (CTLA‐4) expression on CD28^?CD27^? CD4 T cells were related to viremia. A percentage of ≥0.44% CD28^?CD27^? CD4 T cells defined CMV seropositivity (93.3% sensitivity, 97.1% specificity), and their frequencies correlated strongly with CMV‐specific CD4 T cell levels after stimulation (r = 0.73, p < 0.0001). Highest PD‐1 expression levels on CD28^?CD27^? CD4 T cells were observed in patients with primary CMV viremia and reactivation (p < 0.0001), whereas CTLA‐4 expression was only elevated during primary CMV viremia (p < 0.05). Longitudinal analysis showed a significant increase in PD‐1 expression in relation to viremia (p < 0.001), whereas changes in nonviremic patients were nonsignificant. In conclusion, increased PD‐1 expression on CD28^?CD27^? CD4 T cells correlates with CMV viremia in transplant recipients and may serve as a specific, stimulation‐independent parameter to guide duration of antiviral therapy.

     

Myeloid‐derived suppressor cells increase and inhibit donor‐reactive T cell responses to graft intestinal epithelium in intestinal transplant patients

Recent advances in immunosuppressive regimens have decreased acute cellular rejection (ACR) rates and improved intestinal and multivisceral transplant (ITx) recipient survival. We investigated the role of myeloid‐derived suppressor cells (MDSCs) in ITx. We identified MDSCs as CD33⁺CD11b⁺ lineage(CD3/CD56/CD19)^?HLA‐DR^?/low cells with 3 subsets, CD14^?CD15^? (e‐MDSCs), CD14⁺CD15^? (M‐MDSCs), and CD14^?CD15⁺ (PMN‐MDSCs), in peripheral blood mononuclear cells (PBMCs) and mononuclear cells in the grafted intestinal mucosa. Total MDSC numbers increased in PBMCs after ITx; among MDSC subsets, M‐MDSC numbers were maintained at a high level after 2 months post ITx. The MDSC numbers decreased in ITx recipients with ACR. MDSC numbers were positively correlated with serum interleukin (IL)‐6 levels and the glucocorticoid administration index. IL‐6 and methylprednisolone enhanced the differentiation of bone marrow cells to MDSCs in vitro. M‐MDSCs and e‐MDSCs expressed CCR1, ‐2, and ‐3; e‐MDSCs and PMN‐MDSCs expressed CXCR2; and intestinal grafts expressed the corresponding chemokine ligands after ITx. Of note, the percentage of MDSCs among intestinal mucosal CD45⁺ cells increased after ITx. A novel in vitro assay demonstrated that MDSCs suppressed donor‐reactive T cell–mediated destruction of donor intestinal epithelial organoids. Taken together, our results suggest that MDSCs accumulate in the recipient PBMCs and the grafted intestinal mucosa in ITx, and may regulate ACR.     

Recombinant Human Erythropoietin Treatment of Chronic Renal Failure Patients Normalizes Altered Phenotype and Proliferation of CD4‐positive T Lymphocytes

Patients with chronic renal failure (CRF) receive recombinant human erythropoietin (rhEPO) for the correction of anemia. However, rhEPO also has an immunomodulatory effect. Detailed changes of phenotype and function of CD4⁺ T lymphocytes in CRF patients receiving rhEPO have not been reported yet; their study may bring insight into understanding of this immunomodulatory action of rhEPO. Two groups of CRF patients were included into the study: those treated; and those not receiving rhEPO. The expression of activation markers on CD4⁺ lymphocytes was measured with flow cytometry, both ex vivo and in vitro. The kinetics of CD4⁺ T lymphocytes proliferation was calculated using a dividing cells tracing method and numerical approach. Significantly higher percentages of CD4⁺CD95⁺, CD4⁺HLA‐DR⁺ cells, and lower percentages of CD4⁺CD69⁺ and CD4⁺CD28⁺ cells were observed in both rhEPO‐treated and untreated patients when compared with healthy controls. Changes in the proportions of CD4⁺CD28⁺ and CD4⁺HLA‐DR⁺ subpopulations were dependent on the type of rhEPO, being more pronounced for rhEPOβ. CD4⁺ lymphocytes from untreated patients exhibited decreased expression of CD28 and CD69 after stimulation in vitro, whereas the expression of these antigens on lymphocytes of rhEPO‐treated patients was similar to that observed in healthy controls. Fewer CD4⁺CD28⁺ T lymphocytes of untreated patients proliferated in vitro; these cells had longer G0→G1 time, which negatively correlated with surface expression of CD28. Our study confirms that rhEPO treatment normalizes activation parameters of CD4⁺ T lymphocytes and their proliferative capacity, which could explain earlier described immunomodulatory effects of rhEPO in patients suffering from CRF.     

Alloreactive natural killer cells promote haploidentical hematopoietic stem cell transplantation by expansion of recipient‐derived CD4+CD25+ regulatory T cells

Alloreactive NK cells (Allo‐NKs) have been shown to exert advantageous effects on the outcomes of haploidentical hematopoietic stem cell transplantation (Haplo‐HSCT) for cancer treatment. However, the mechanisms of action of Allo‐NKs remain unclear. We established a novel Haplo‐HSCT conditioning regimen composed of Allo‐NKs and a low dose of immunosuppressive drugs (Allo‐NKs + Chemo) to investigate alternative mechanisms besides direct cytotoxicity. The inhibitory effects of different cell subsets on the donor–recipient mixed lymphocyte reactions (MLRs) were evaluated after Haplo‐HSCT. The quantities and functions of CD4⁺CD25⁺ regulatory T cells (Tregs) and dendritic cells (DCs) in the spleen and the thymus were examined. Our results showed that the Allo‐NKs + Chemo regimen induced systemic tolerance, and that CD4⁺CD25⁺ Tregs played a significant role in inducing and maintaining systemic tolerance after Haplo‐HSCT. Alloreactive NK cells promoted the expansion of recipient‐derived CD4⁺CD25⁺CD127^? Tregs in the thymus and the spleen which could be amplified in vitro by the immature donor‐derived DC subset isolated from the thymus of Allo‐NKs + Chemo‐treated mice. Our findings suggested that Allo‐NKs are capable of inducing systemic tolerance after Haplo‐HSCT by assembling donor‐derived immature DCs to expand recipient‐derived Treg cells in the thymus.     

Rapamycin‐Conditioned Donor Dendritic Cells Differentiate CD4+CD25+Foxp3+ T Cells In Vitro with TGF‐β1 for Islet Transplantation

Dendritic cells (DCs) conditioned with the mammalian target of rapamycin (mTOR) inhibitor rapamycin have been previously shown to expand naturally existing regulatory T cells (nTregs). This work addresses whether rapamycin‐conditioned donor DCs could effectively induce CD4⁺CD25⁺Foxp3⁺ Tregs (iTregs) in cell cultures with alloantigen specificities, and whether such in vitro‐differentiated CD4⁺CD25⁺Foxp3⁺ iTregs could effectively control acute rejection in allogeneic islet transplantation. We found that donor BALB/c bone marrow‐derived DCs (BMDCs) pharmacologically modified by the mTOR inhibitor rapamycin had significantly enhanced ability to induce CD4⁺CD25⁺Foxp3⁺ iTregs of recipient origin (C57BL/6 (B6)) in vitro under Treg driving conditions compared to unmodified BMDCs. These in vitro‐induced CD4⁺CD25⁺Foxp3⁺ iTregs exerted donor‐specific suppression in vitro, and prolonged allogeneic islet graft survival in vivo in RAG^?/‐ hosts upon coadoptive transfer with T‐effector cells. The CD4⁺CD25⁺Foxp3⁺ iTregs expanded and preferentially maintained Foxp3 expression in the graft draining lymph nodes. Finally, the CD4⁺CD25⁺Foxp3⁺ iTregs were further able to induce endogenous naïve T cells to convert to CD4⁺CD25⁺Foxp3⁺ T cells. We conclude that rapamycin‐conditioned donor BMDCs can be exploited for efficient in vitro differentiation of donor antigen‐specific CD4⁺CD25⁺Foxp3⁺ iTregs. Such in vitro‐generated donor‐specific CD4⁺CD25⁺Foxp3⁺ iTregs are able to effectively control allogeneic islet graft rejection.     

CD4+ CD25+ regulatory T cells suppressed the indirect xenogeneic immune response mediated by porcine epithelial cell pulsed dendritic cells

Nishimura T, Onda M, Takao S. CD4⁺ CD25⁺ regulatory T cells suppressed the indirect xenogeneic immune response mediated by porcine epithelial cell pulsed dendritic cells. Xenotransplantation 2010; 17: 313–323. © 2010 John Wiley & Sons A/S. Abstract: Background: CD4⁺ CD25⁺ regulatory T cells have been reported to suppress T cell‐mediated xenogeneic immune responses. Although the direct T cell response to xenogeneic cells is important, the indirect xenogeneic immune response mediated by dendritic cells (DCs) is also likely involved in rejection. We have generated an in vitro indirect immune reaction model and evaluated the effect of CD4⁺ CD25⁺ regulatory T cells on this system. Methods: Human DCs were generated from peripheral blood and cultured with X‐ray‐irradiated porcine kidney epithelial cells. Porcine cell‐pulsed DCs were mixed with autologous CD4⁺ T cells, CD4⁺ CD25^? T cells and/or CD4⁺ CD25⁺ T cells. After 7 days of culture, T cell proliferation was measured. Results: The co‐culture of human DCs and X‐ray‐irradiated porcine epithelial cells resulted in observable DC phagocytic activity within 2 days. These porcine cell–pulsed DCs stimulated CD4⁺ T cell proliferation much more potently than unpulsed DCs or porcine cells. This proliferation was blocked by CTLA4‐Ig or an anti‐HLA‐DR antibody. CD4⁺ CD25⁺ regulatory T cells also suppressed CD4⁺ CD25^? T cell proliferation in response to porcine cell‐pulsed DCs. Conclusions: An in vitro model of the indirect xenogeneic immune response was established. Porcine cell‐pulsed DCs stimulated CD4⁺ T cells, and CD4⁺ CD25⁺ regulatory T cells suppressed this response.     

Characterization of Human CD8+TCR− Facilitating Cells In Vitro and In Vivo in a NOD/SCID/IL2rγnull Mouse Model

CD8⁺/TCR^? facilitating cells (FCs) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPCs). Human FC phenotype and mechanism of action remain to be defined. We report, for the first time, the phenotypic characterization of human FCs and correlation of phenotype with function. Approximately half of human FCs are CD8⁺/TCR^?/CD56 negative (CD56^neg); the remainder are CD8⁺/TCR^?/CD56 bright (CD56^bright). The CD56^neg FC subpopulation significantly promotes homing of HSPCs to BM in nonobese diabetic/severe combined immunodeficiency/IL‐2 receptor γ‐chain knockout mouse recipients and enhances hematopoietic colony formation in vitro. The CD56^neg FC subpopulation promotes rapid reconstitution of donor HSPCs without graft‐versus‐host disease (GVHD); recipients of CD56^bright FCs plus HSPCs exhibit low donor chimerism early after transplantation, but the level of chimerism significantly increases with time. Recipients of HSPCs plus CD56^neg or CD56^bright FCs showed durable donor chimerism at significantly higher levels in BM. The majority of both FC subpopulations express CXCR4. Coculture of CD56^bright FCs with HSPCs upregulates cathelicidin and β‐defensin 2, factors that prime responsiveness of HSPCs to stromal cell–derived factor 1. Both FC subpopulations significantly upregulated mRNA expression of the HSPC growth factors and Flt3 ligand. These results indicate that human FCs exert a direct effect on HSPCs to enhance engraftment. Human FCs offer a potential regulatory cell‐based therapy for enhancement of engraftment and prevention of GVHD.

     

Gr‐1+CD11b+ myeloid‐derived suppressor cells: Formidable partners in tumor metastasis

The growth and metastasis of solid tumors not only depends on their ability to escape from immune surveillance but also hinges on their ability to invade the vasculature system as well as to induce the formation of new blood vessels. Gr‐1⁺CD11b⁺ myeloid‐derived suppressor cells (MDSCs), overproduced in tumor‐bearing hosts, contribute significantly to all these aspects. They also have a potential role in the osteolysis associated with bone metastases. They are formidable partners in tumor metastasis. © 2010 American Society for Bone and Mineral Research     

Higher dose of CD34+ peripheral blood stem cells is associated with better survival after haploidentical stem cell transplantation in pediatric patients

Haploidentical stem cell transplantation (SCT) is increasingly used to treat pediatric patients with malignant or nonmalignant hematological disorders. The CD34+ dose of bone marrow or peripheral blood stem cells (PBSCs) has been shown to be an important determinant of the transplant outcome in adults under various preparative regimens. However, knowledge of the effect of the CD34+ dose in pediatric haploidentical SCT is limited. We analyzed the data of 348 pediatric patients (aged 2‐18 years) with acute or chronic leukemia, myelodysplastic syndrome (MDS), and other hematological disorders that received a transplant between 2002 and 2012. The results of multivariate analysis showed that PBSC CD34+ counts greater than 1.01 × 10⁶ kg^?1 improved platelet engraftment, improved overall survival, and reduced nonrelapse mortality. In contrast, a higher PBSC CD34+ dose did not affect the incidence of acute or chronic graft‐versus‐host disease, including engraftment syndrome. These data suggest that a PBSC CD34+ dose greater than 1.01 × 10⁶ kg^?1 is optimal for pediatric haploidentical SCT.     

Hypoxic culture conditions induce increased metabolic rate and collagen gene expression in ACL‐derived cells

There has been substantial effort directed toward the application of bone marrow and adipose‐derived mesenchymal stromal cells (MSCs) in the regeneration of musculoskeletal tissue. Recently, resident tissue‐specific stem cells have been described in a variety of mesenchymal structures including ligament, tendon, muscle, cartilage, and bone. In the current study, we systematically characterize three novel anterior cruciate ligament (ACL)‐derived cell populations with the potential for ligament regeneration: ligament‐forming fibroblasts (LFF: CD146^neg, CD34^negCD44^pos, CD31^neg, CD45^neg), ligament perivascular cells (LPC: CD146^posCD34^negCD44^pos, CD31^neg, CD45^neg) and ligament interstitial cells (LIC: CD34^posCD146^neg, CD44^pos, CD31^neg, CD45^neg)—and describe their proliferative and differentiation potential, collagen gene expression and metabolism in both normoxic and hypoxic environments, and their trophic potential in vitro. All three groups of cells (LIC, LPC, and LFF) isolated from adult human ACL exhibited progenitor cell characteristics with regard to proliferation and differentiation potential in vitro. Culture in low oxygen tension enhanced the collagen I and III gene expression in LICs (by 2.8‐ and 3.3‐fold, respectively) and LFFs (by 3‐ and 3.5‐fold, respectively) and increased oxygen consumption rate and extracellular acidification rate in LICs (by 4‐ and 3.5‐fold, respectively), LFFs (by 5.5‐ and 3‐fold, respectively), LPCs (by 10‐ and 4.5‐fold, respectively) as compared to normal oxygen concentration. In summary, this study demonstrates for the first time the presence of three novel progenitor cell populations in the adult ACL that demonstrate robust proliferative and matrix synthetic capacity; these cells may play a role in local ligament regeneration, and consequently represent a potential cell source for ligament engineering applications. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:985–994, 2016.     

Donor‐Specific CD8+Foxp3+ T Cells Protect Skin Allografts and Facilitate Induction of Conventional CD4+Foxp3+ Regulatory T Cells

CD4⁺ regulatory T cells play a critical role in tolerance induction in transplantation. CD8⁺ suppressor T cells have also been shown to control alloimmune responses in preclinical and clinical models. However, the exact nature of the CD8⁺ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen‐specific CD8⁺Foxp3⁺ T cells can be induced and significantly expanded by stimulating naïve CD8⁺ T cells with donor dendritic cells in the presence of IL‐2, TGF‐β1 and retinoic acid. These CD8⁺Foxp3⁺ T cells express enhanced levels of CTLA‐4, CCR4 and CD103, inhibit the up‐regulation of costimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor‐specific and contact‐dependent manner. Importantly, upon adoptive transfer, the induced CD8⁺Foxp3⁺ T cells protect full MHC‐mismatched skin allografts. In vivo, the CD8⁺Foxp3⁺ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4⁺Foxp3⁺ T cells and concurrently suppress effector T cell expansion. We conclude that donor‐specific CD8⁺Foxp3⁺ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.     

Flt3L‐mobilized dendritic cells bearing H2‐Kbm1 apoptotic cells do not induce cross‐tolerance to CD8+ T cells across a class I MHC mismatched barrier

Tolerization of allogeneic CD8⁺ T cells is still a pending issue in the field of transplantation research to achieve long‐term survival. To test whether dendritic cells (DC) bearing allogeneic major histocompatibility complex (MHC) class I mismatched apoptotic cells could induce cross‐tolerance to alloreactive CD8⁺ T cells, the following experimental strategy was devised. Rag2/γ_c KO B6 mice were treated with Fms‐like tyrosine kinase 3 ligand (Flt3L)‐transduced B16 melanoma cells to drive a rapid expansion and mobilization of DC in vivo. Of all DC populations expanded, splenic CD11c⁺CD103⁺CD8α⁺ DC were selectively involved in the process of antigen clearance of X‐ray irradiated apoptotic thymocytes in vivo. Considering that CD11c⁺CD103⁺CD8α⁺ DC selectively take up apoptotic cells and that they are highly specialized in cross‐presenting antigen to CD8⁺ T cells, we investigated whether B6 mice adoptively transferred with Flt3L‐derived DC loaded with donor‐derived apoptotic thymocytes could induce tolerance to bm1 skin allografts. Our findings on host anti‐donor alloresponse, as revealed by skin allograft survival and cytotoxic T lymphocyte assays, indicated that the administration of syngeneic DC presenting K^bm1 donor‐derived allopeptides through the indirect pathway of antigen presentation was not sufficient to induce cross‐tolerance to alloreactive CD8⁺ T cells responding to bm1 alloantigens in a murine model of skin allograft transplantation across an MHC class I mismatched barrier.