Tumor-associated mesenchymal stromal cells modulate macrophage phagocytosis in stromal-rich colorectal cancer via PD-1 signaling.

CMS4 colorectal cancer (CRC), based on the consensus molecular subtype (CMS), stratifies patients with the poorest disease-free survival rates. It is characterized by a strong mesenchymal stromal cell (MSC) signature, wound healing-like inflammation and therapy resistance. We utilized 2D and 3D in vitro, in vivo, and ex vivo models to assess the impact of inflammation and stromal cells on immunosuppression in CMS4 CRC. RNA sequencing data from untreated stage II/III CRC patients showed enriched TNF-α signatures in CMS1 and CMS4 tumors. Secretome from TNF-α treated cancer cells induced an immunomodulatory and chemotactic phenotype in MSC and cancer-associated fibroblasts (CAFs). Macrophages in CRC tumours migrate and preferentially localise in stromal compartment. Inflammatory CRC secretome enhances expression of PD-L1 and CD47 on both human and murine stromal cells. We demonstrate that TNF-α-induced inflammation in CRC suppresses macrophage phagocytosis via stromal cells. We show that stromal cell-mediated suppression of macrophage phagocytosis is mediated in part through PD-1 signaling. These data suggest that re-stratification of CRC by CMS may reveal patient subsets with microsatellite stable tumors, particularly CMS4-like tumors, that may respond to immunotherapies.

Targeting stromal cell sialylation reverses T cell-mediated immunosuppression in the tumor microenvironment.

Immunosuppressive tumor microenvironments (TMEs) reduce the effectiveness of immune responses in cancer. Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote tumor progression by enhancing immune cell suppression in colorectal cancer (CRC). Hyper-sialylation of glycans promotes immune evasion in cancer through binding of sialic acids to their receptors, Siglecs, expressed on immune cells, which results in inhibition of effector functions. The role of sialylation in shaping MSC/CAF immunosuppression in the TME is not well characterized. In this study, we show that tumor-conditioned stromal cells have increased sialyltransferase expression, α2,3/6-linked sialic acid, and Siglec ligands. Tumor-conditioned stromal cells and CAFs induce exhausted immunomodulatory CD8+ PD1+ and CD8+ Siglec-7+/Siglec-9+ T cell phenotypes. In vivo, targeting stromal cell sialylation reverses stromal cell-mediated immunosuppression, as shown by infiltration of CD25 and granzyme B-expressing CD8+ T cells in the tumor and draining lymph node. Targeting stromal cell sialylation may overcome immunosuppression in the CRC TME.

Precision Medicine and Novel Therapeutic Strategies in Detection and Treatment of Cancer: Highlights from the 58th IACR Annual Conference.

Innovation in both detection and treatment of cancer is necessary for the constant improvement in therapeutic strategies, especially in patients with novel or resistant variants of cancer. Cancer mortality rates have declined by almost 30% since 1991, however, depending on the cancer type, acquired resistance can occur to varying degrees. To combat this, researchers are looking towards advancing our understanding of cancer biology, in order to inform early detection, and guide novel therapeutic approaches. Through combination of these approaches, it is believed that a more complete and thorough intervention on cancer can be achieved. Here, we will discuss the advances and approaches in both detection and treatment of cancer, presented at the 58th Irish Association for Cancer Research (IACR) annual conference.

Subconjunctival administration of low-dose murine allogeneic mesenchymal stromal cells promotes corneal allograft survival in mice.

BACKGROUND: Systemic administration of mesenchymal stromal cells (MSCs) has been efficacious in many inflammatory disease settings; however, little data are available on the potential immunomodulatory effects following local MSC administration in the context of corneal transplantation. The purpose of this study was to assess the potential of subconjunctival injection of MSCs to promote corneal allograft survival. METHODS: MSCs were isolated from female C57BL/6 (H-2k) or Balb/c (H-2d) mice and extensively characterized. An allogeneic mouse corneal transplant model was used with Balb/c mice as recipients of C57BL/6 grafts. A dose-finding study starting with 5 × 105 MSCs injected subconjunctivally at day - 7 was tested first followed by a more clinically translatable low-dose single or dual injection strategy on day - 1 and day + 1 before/after transplantation. Graft transparency served as the primary indicator of transplant rejection while neovascularization was also recorded. Lymphocytes (from draining lymph nodes) and splenocytes were isolated from treatment groups on day 2 post-transplantation and characterized by flow cytometry and qRT-PCR. RESULTS: Both high- and low-dose injection of allogeneic MSCs on day - 7 led to 100% graft survival over the observation period. Moreover, low-dose dual subconjunctival injection of 5 × 104 allogeneic MSCs on day - 1 or day + 1 led to 100% allograft survival in transplant recipients (n = 7). We also demonstrate that single administration of allogeneic MSCs on either day - 1 or day + 1 promotes rejection-free graft survival in 100% (n = 8) and 86% (n = 7) of transplanted mice, respectively. Early time point ex vivo analysis suggests modulation of innate immune responses towards anti-inflammatory, pro-repair responses by local MSC administration. CONCLUSION: This work demonstrates that low-dose subconjunctival injection of allogeneic MSCs successfully promotes corneal allograft survival and may contribute to refining future MSC immunotherapies for prevention of corneal allograft rejection.

Cyclophosphamide alters the tumor cell secretome to potentiate the anti-myeloma activity of daratumumab through augmentation of macrophage-mediated antibody dependent cellular phagocytosis.

Multiple Myeloma (MM) is a malignant disorder of plasma cells which, despite significant advances in treatment, remains incurable. Daratumumab, the first CD38 directed monoclonal antibody, has shown promising activity alone and in combination with other agents for MM treatment. Daratumumab is thought to have pleiotropic mechanisms of activity including natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). With the knowledge that CD38-expressing NK cells are depleted by daratumumab, we sought to investigate a potential mechanism of enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) by combining daratumumab with cyclophosphamide (CTX). Cyclophosphamide's immunomodulatory function was investigated by conditioning macrophages with tumor cell secretome collected from cyclophosphamide treated MM cell lines (CTX-TCS). Flow cytometry analysis revealed that CTX-TCS conditioning augmented the migratory capacity of macrophages and increased CD32 and CD64 Fcγ receptor expression on their cell surface. Daratumumab-specific tumor clearance was increased by conditioning macrophages with CTX-TCS in a dose-dependent manner. This effect was impeded by pre-incubating macrophages with Cytochalasin D (CytoD), an inhibitor of actin polymerization, indicating macrophage-mediated ADCP as the mechanism of clearance. CD64 expression on macrophages directly correlated with MM cell clearance and was essential to the observed synergy between cyclophosphamide and daratumumab, as tumor clearance was attenuated in the presence of a FcγRI/CD64 blocking agent. Cyclophosphamide independently enhances daratumumab-mediated killing of MM cells by altering the tumor microenvironment to promote macrophage recruitment, polarization to a pro-inflammatory phenotype, and directing ADCP. These findings support the addition of cyclophosphamide to existing or novel monoclonal antibody-containing MM regimens.

A 3D View of Colorectal Cancer Models in Predicting Therapeutic Responses and Resistance.

Although there have been many advances in recent years for the treatment of colorectal cancer (CRC), it still remains the third most common cause of cancer-related deaths worldwide. Many patients with late stage CRC display resistance to multiple different therapeutics. An important aspect in developing effective therapeutics for CRC patients is understanding the interactions that take place in the tumor microenvironment (TME), as it has been shown to contribute to drug resistance in vivo. Much research over the past 100 years has focused on 2D monolayer cultures or in vivo studies, however, the efficacy in translating these to the clinic is very low. More recent studies are turning towards developing an effective 3D model of CRC that is clinically relevant, that can recapitulate the TME in vitro and bridge the gap between 2D cultures and in vivo studies, with the aim of reducing the use of animal models in the future. This review summarises the advantages and limitations of different 3D CRC models. It emphasizes how different 3D models may be optimised to study cellular and extracellular interactions that take place in the TME of CRC in an effort to allow the development of more translatable effective treatment options for patients.

Pre-analytical mysteries: A case of severe hypervitaminosis D and mild hypercalcaemia.

We describe a case of severe hypervitaminosis D and mild hypercalcaemia in a 68-year-old woman who presented with fatigue and weight loss. Her 25-hydroxy vitamin D (25OHD) was > 400 nmol/L (50-150) and corrected serum calcium was 2.83 mmol/L (2.1-2.6). Her intact parathyroid hormone (PTH) was 4.9 pmol/L (2.0-9.5). Further investigation revealed an IgM kappa paraprotein, and a bone marrow aspirate confirmed a diagnosis of lymphoplasmacytic lymphoma/Waldenstrom's macroglobulinemia (LPL/WM). As the vitamin D level was discordant with the patient's other results and presentation, the presence of an assay interferent was suspected. A 1-in-2 dilution of the sample returned a 25OHD result of 84 nmol/L in keeping with the presence of an interferent. Testing for rheumatoid factor was negative. The sample was treated with an antibody blocking reagent (Scantibodies) and results were not consistent with heterophile antibody interference. The sample was then analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS), which returned a 25OHD result of 82 nmol/L. Testing on an alternative immunoassay platform produced a 25OHD result of 75 nmol/L. Reapeted testing on the original platform following reduction of the monoclonal paraprotein with chemotherapy, returned a result of 64 nmol/L. The patient's mild hypercalcaemia persisted following resolution of the monoclonal paraprotein, in keeping with a diagnosis of primary hyperparathyroidism. This case highlights the potential for paraproteins to cause assay interference, and the importance of considering interference when results are incongruous with the clinical presentation.

TGF-ß1-Licensed Murine MSCs Show Superior Therapeutic Efficacy in Modulating Corneal Allograft Immune Rejection In Vivo.

Mesenchymal stromal cells (MSCs) are a promising therapeutic option for multiple immune diseases/disorders; however, efficacy of MSC treatments can vary significantly. We present a novel licensing strategy to improve the immunosuppressive capacity of MSCs. Licensing murine MSCs with transforming growth factor-ß1 (TGF-ß MSCs) significantly improved their ability to modulate both the phenotype and secretome of inflammatory bone marrow-derived macrophages and significantly increased the numbers of regulatory T lymphocytes following co-culture assays. These TGF-ß MSC-expanded regulatory T lymphocytes also expressed significantly higher levels of PD-L1 and CD73, indicating enhanced suppressive potential. Detailed analysis of T lymphocyte co-cultures revealed modulation of secreted factors, most notably elevated prostaglandin E2 (PGE2). Furthermore, TGF-ß MSCs could significantly prolong rejection-free survival (69.2% acceptance rate compared to 21.4% for unlicensed MSC-treated recipients) in a murine corneal allograft model. Mechanistic studies revealed that (1) therapeutic efficacy of TGF-ß MSCs is Smad2/3-dependent, (2) the enhanced immunosuppressive capacity of TGF-ß MSCs is contact-dependent, and (3) enhanced secretion of PGE2 (via prostaglandin EP4 [E-type prostanoid 4] receptor) by TGF-ß MSCs is the predominant mediator of Treg expansion and T cell activation and is associated with corneal allograft survival. Collectively, we provide compelling evidence for the use of TGF-ß1 licensing as an unconventional strategy for enhancing MSC immunosuppressive capacity.

Biochemical evaluation of kidney disease.

Different biochemical markers exist in both blood and urine for assessing renal function. Most of these biomarkers have advantages and limitations associated with their use, which is important to consider when ordering and utilising them in the clinical setting. The ideal marker should be able to detect acute kidney injury (AKI) at the onset and be used for the diagnosis and ongoing monitoring and management of kidney disease. The search for such a marker is ongoing, as all potential candidates thus far are associated with certain limitations. This article will attempt to compare and contrast established and emerging kidney disease markers.

TNF-α/IL-1ß-licensed mesenchymal stromal cells promote corneal allograft survival via myeloid cell-mediated induction of Foxp3+ regulatory T cells in the lung.

Mesenchymal stromal cells (MSCs) have shown promise as a therapy for immune-mediated disorders, including transplant rejection. Our group previously demonstrated the efficacy of pretransplant, systemic administration of allogeneic but not syngeneic MSCs in a rat cornea transplant model. The aim of this study was to enhance the immunomodulatory capacity of syngeneic MSCs. In vitro, MSCs licensed with TNF-α/IL-1ß (MSCsTNF-α/IL-1ß) suppress syngeneic lymphocyte proliferation via NO production. In vivo, when administered post-transplantation, nonlicensed syngeneic MSCs improved graft survival from 0 to 50% and MSCsTNF-α/IL-1ß, in an NO-dependent manner, improved survival to 70%. Improved survival was associated with increased CD4+CD25+forkhead box P3+ regulatory T (Treg) cells and decreased proinflammatory cytokine expression in the draining lymph node. MSCsTNF-α/IL-1ß demonstrated a more potent immunomodulatory capacity compared with nonlicensed MSCs, promoting an immune-regulatory CD11b+B220+ monocyte/macrophage population and significantly expanding Treg cells in the lungs and spleen. Ex vivo, we observed that lung-derived myeloid cells act as intermediaries of MSC immunomodulatory function. MSC-conditioned myeloid cells suppressed stimulated lymphocyte proliferation and promoted expansion of Treg cells from naive lymphocytes. This work illustrates how syngeneic MSC therapy can be enhanced by licensing and optimization of timing strategies and further highlights the important role of myeloid cells in mediating MSC immunomodulatory capacity.-Murphy, N., Treacy, O., Lynch, K., Morcos, M., Lohan, P., Howard, L., Fahy, G., Griffin, M. D., Ryan, A. E., Ritter, T. TNF-α/IL-1ß-licensed mesenchymal stromal cells promote corneal allograft survival via myeloid cell-mediated induction of Foxp3+ regulatory T cells in the lung.

Third-Party Allogeneic Mesenchymal Stromal Cells Prevent Rejection in a Pre-sensitized High-Risk Model of Corneal Transplantation.

High-risk cornea transplant recipients represent a patient population with significant un-met medical need for more effective therapies to prevent immunological graft rejection due to heightened anti-donor immune response. In this study, a rat model of pre-existing anti-donor immunity was developed in which corneal allografts were rejected earlier than in non-pre-sensitized recipients. In this model, third-party (non-donor, non-recipient strain) allogeneic mesenchymal stromal cells (allo-MSC) were administered intravenously 7 and 1 days prior to transplantation. Rejection-free graft survival to 30 days post-transplant improved from 0 to 63.6% in MSC-treated compared to vehicle-treated control animals (p = < 0.0001). Pre-sensitized animals that received third-party allo-MSC prior to transplantation had significantly higher proportions of CD45+CD11b+ B220+ monocytes in the lungs 24 h after the second MSC injection and significantly higher proportions of CD4+ FoxP3+ regulatory T cells in the graft-draining lymph nodes at the average day of rejection of control animals. In in vitro experiments, third-party allo-MSC polarized primary lung-derived CD11b/c+ myeloid cells to a more anti-inflammatory phenotype, as determined by cytokine profile and conferred them with the capacity to suppress T cell activation via prostaglandin E2 and TGFß1. In experiments designed to further validate the clinical potential of the protocol, thawed cryopreserved, third-party allo-MSC were shown to be similarly potent at prolonging rejection-free corneal allograft survival as their freshly-cultured counterparts in the pre-sensitized high-risk model. Furthermore, thawed cryopreserved third-party allo-MSC could be co-administered with mycophenolate mofetil without adversely affecting their immunomodulatory function. In conclusion, a clinically-relevant protocol consisting of two intravenous infusions of third-party allo-MSC during the week prior to transplantation, exerts a potent anti-rejection effect in a pre-sensitized rat model of high-risk corneal allo-transplantation. This immune regulatory effect is likely to be mediated in the immediate post-transplant period through the promotion, by allo-MSC, of alternatively-activated macrophages in the lung and, later, by enhanced regulatory T-cell numbers.

Stromal Cell PD-L1 Inhibits CD8+ T-cell Antitumor Immune Responses and Promotes Colon Cancer.

Stromal cells of mesenchymal origin reside below the epithelial compartment and provide structural support in the intestine. These intestinal stromal cells interact with both the epithelial cell compartments, as well as infiltrating hematopoietic immune cells. The importance of these cells in regulating immune homeostasis during inflammation is well recognized. However, little is known about their function and phenotype in the inflammatory tumor microenvironment. Using a syngeneic, immunogenic model of colorectal cancer, we showed that TNFα-initiated inflammatory signaling in CT26 colorectal cancer cells selectively induced PD-L1 expression in stromal cells. Using CD274 shRNA and antibody-mediated approaches, we showed that stromal cell PD-L1 potentiated enhanced immunosuppression, characterized by inhibition of activated CD8+ granzyme B-secreting T cells in vitro, and the inhibition of CD8+ effector cells was associated with enhanced tumor progression. Stromal cell immunosuppressive and tumor-promoting effects could be reversed with administration of anti-PD-1 in vivo We validated our findings of stromal cell CD274 expression in two cohorts of clinical samples and also observed PD-L1 induction on human stromal cells in response to exposure to the inflammatory secretome from human colon cancer cells, irrespective of microsatellite instability. Collectively, our data showed that tumor-associated stromal cells support T-cell suppression by PD-L1 induction, which is dependent on colon cancer inflammatory signaling. Our findings reveal a key role of mesenchymal stromal cells PD-L1 in suppression of CD8+ antitumor immune responses and potentiation of colorectal cancer progression. Cancer Immunol Res; 6(11); 1426-41. ©2018 AACR.

Interspecies Incompatibilities Limit the Immunomodulatory Effect of Human Mesenchymal Stromal Cells in the Rat.

Mesenchymal stem/stromal cells (MSC) are an immunomodulatory cell population which are under preclinical and clinical investigation for a number of inflammatory conditions including transplantation. In this study, a well-established rat corneal transplantation model was used to test the ability of human MSC to prolong corneal allograft rejection-free survival using a pre-transplant intravenous infusion protocol previously shown to be efficacious with allogeneic rat MSC. Surprisingly, pre-transplant administration of human MSC had no effect on corneal allograft survival. In vitro, human MSC failed to produce nitric oxide and upregulate IDO and, as a consequence, could not suppress rat T-cell proliferation. Furthermore, human MSC were not activated by rat pro-inflammatory cytokines. Thus, interspecies incompatibility in cytokine signaling leading to failure of MSC licensing may explain the lack of in vivo efficacy of human MSC in a rat tissue allotransplant model. Interspecies incompatibilities should be taken into consideration when interpreting preclinical data efficacy data in the context of translation to clinical trial. Stem Cells 2018;36:1210-1215.

Anti-Donor Immune Responses Elicited by Allogeneic Mesenchymal Stem Cells and Their Extracellular Vesicles: Are We Still Learning?

Mesenchymal stromal cells (MSC) have been used to treat a broad range of disease indications such as acute and chronic inflammatory disorders, autoimmune diseases, and transplant rejection due to their potent immunosuppressive/anti-inflammatory properties. The breadth of their usage is due in no small part to the vast quantity of published studies showing their ability to modulate multiple immune cell types of both the innate and adaptive immune response. While patient-derived (autologous) MSC may be the safer choice in terms of avoiding unwanted immune responses, factors including donor comorbidities may preclude these cells from use. In these situations, allogeneic MSC derived from genetically unrelated individuals must be used. While allogeneic MSC were initially believed to be immune-privileged, substantial evidence now exists to prove otherwise with multiple studies documenting specific cellular and humoral immune responses against donor antigens following administration of these cells. In this article, we will review recent published studies using non-manipulated, inflammatory molecule-activated (licensed) and differentiated allogeneic MSC, as well as MSC extracellular vesicles focusing on the immune responses to these cells and whether or not such responses have an impact on allogeneic MSC-mediated safety and efficacy.

Regulating Immunogenicity and Tolerogenicity of Bone Marrow-Derived Dendritic Cells through Modulation of Cell Surface Glycosylation by Dexamethasone Treatment.

Dendritic cellular therapies and dendritic cell vaccines show promise for the treatment of autoimmune diseases, the prolongation of graft survival in transplantation, and in educating the immune system to fight cancers. Cell surface glycosylation plays a crucial role in the cell-cell interaction, uptake of antigens, migration, and homing of DCs. Glycosylation is known to change with environment and the functional state of DCs. Tolerogenic DCs (tDCs) are commonly generated using corticosteroids including dexamethasone, however, to date, little is known on how corticosteroid treatment alters glycosylation and what functional consequences this may have. Here, we present a comprehensive profile of rat bone marrow-derived dendritic cells, examining their cell surface glycosylation profile before and after Dexa treatment as resolved by both lectin microarrays and lectin-coupled flow cytometry. We further examine the functional consequences of altering cell surface glycosylation on immunogenicity and tolerogenicity of DCs. Dexa treatment of rat DCs leads to profoundly reduced expression of markers of immunogenicity (MHC I/II, CD80, CD86) and pro-inflammatory molecules (IL-6, IL-12p40, inducible nitric oxide synthase) indicating a tolerogenic phenotype. Moreover, by comprehensive lectin microarray profiling and flow cytometry analysis, we show that sialic acid (Sia) is significantly upregulated on tDCs after Dexa treatment, and that this may play a vital role in the therapeutic attributes of these cells. Interestingly, removal of Sia by neuraminidase treatment increases the immunogenicity of immature DCs and also leads to increased expression of pro-inflammatory cytokines while tDCs are moderately protected from this increase in immunogenicity. These findings may have important implications in strategies aimed at increasing tolerogenicity where it is advantageous to reduce immune activation over prolonged periods. These findings are also relevant in therapeutic strategies aimed at increasing the immunogenicity of cells, for example, in the context of tumor specific immunotherapies.

Mesenchymal stem cell therapy to promote corneal allograft survival: current status and pathway to clinical translation.

PURPOSE OF REVIEW: This article reviews the literature on the therapeutic potential of mesenchymal stem cells (MSCs) to prolong corneal allograft survival. RECENT FINDINGS: To date, only small numbers studies have investigated the MSC ability to modulate corneal allograft survival. Most reports have shown positive results, which is encouraging, however as different MSC-application strategies (time point of injection, cell number/number of injections, route of injection, MSC source, MSC licensing) have been employed in various animal models it is difficult to compare and validate the results. The MSC ability to promote graft survival has been attributed to their modulation of the recipient immune system, altering the Th1/Th2 balance, expanding Foxp3 regulatory T cells, polarizing macrophages and inhibiting intra-graft infiltration of antigen presenting cells. More in depth analysis is required to elucidate the mechanism of MSC-immunomodulation in vivo. SUMMARY: MSCs have shown the potential to modulate corneal allograft rejection in various models using MSCs from different species. In particular for high-risk patients with poor prognosis MSC therapy might be a promising approach to promote corneal allograft survival. First-in-man clinical trials with MSC will hopefully shed new light on MSC-mediated immunomodulation in vivo and contribute to the restoration of vision in patients receiving corneal allografts.

Corneal Immunosuppressive Mechanisms, Anterior Chamber-Associated Immune Deviation (ACAID) and Their Role in Allograft Rejection.

Corneal transplantation is the most frequently performed transplant procedure in humans. Human leukocyte antigen matching, while imperative for other types of organ transplants, is usually not performed before cornea transplantation. With the use of topical steroid immunosuppressants, which are subsequently tailed off to almost zero, most corneal transplants will not be rejected in recipients with low risk of graft rejection. This phenomenon has been described as immune privilege by Medawar many years ago. However, this immune privilege is relative and can be easily eroded, e.g. by postoperative nonspecific inflammation or other causes of corneal or ocular inflammation. Interestingly, corneas that are at high risk of rejection have a higher failure rate than other organs. Considerable progress has been made in recent years to provide a better understanding of corneal immune privilege. This chapter will review current knowledge on ocular immunosuppressive mechanisms including anterior chamber-associated immune deviation and discuss their role(s) in corneal allograft rejection. Ultimately, this evolving information will be of benefit in developing therapeutic strategies to prevent corneal transplant rejection.

Chondrogenic differentiation increases antidonor immune response to allogeneic mesenchymal stem cell transplantation.

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3(+) and CD68(+) infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use.