Shala Yuan

Mesenchymal Stromal Cells Ameliorate Experimental Autoimmune Encephalomyelitis by Inhibiting CD4 Th17 T Cells in a CC Chemokine Ligand 2-Dependent Manner

The administration of ex vivo culture-expanded mesenchymal stromal cells (MSCs) has been shown to reverse symptomatic neuroinflammation observed in experimental autoimmune encephalomyelitis (EAE). The mechanism by which this therapeutic effect occurs remains unknown. In an effort to decipher MSC mode of action, we found that MSC conditioned medium inhibits EAE-derived CD4 T cell activation by suppressing STAT3 phosphorylation via MSC-derived CCL2. Further analysis demonstrates that the effect is dependent on MSC-driven matrix metalloproteinase proteolytic processing of CCL2 to an antagonistic derivative. We also show that antagonistic CCL2 suppresses phosphorylation of AKT and leads to a reciprocal increased phosphorylation of ERK associated with an up-regulation of B7.H1 in CD4 T cells derived from EAE mice. CD4 T cell infiltration of the spinal cord of MSC-treated group was robustly decreased along with reduced plasma levels of IL-17 and TNF-α levels and in vitro from restimulated splenocytes. The key role of MSC-derived CCL2 was confirmed by the observed loss of function of CCL2−/− MSCs in EAE mice. In summary, this is the first report of MSCs modulating EAE biology via the paracrine conversion of CCL2 from agonist to antagonist of CD4 Th17 cell function.

Mesenchymal stromal cell-derived CCL2 suppresses plasma cell immunoglobulin production via STAT3 inactivation and PAX5 induction

We demonstrate that the secretome of mesenchymal stromal cells (MSCs) suppresses plasma cell (PC) immunoglobulin (Ig) production, induces plasmablast proliferation, and leads to interleukin-10-mediated blockade in vitro. We found that these effects are the result of MSC-derived CC chemokine ligands CCL2 and CCL7. More specifically, MSCs further processed these CC chemokines by the activity of matrix metalloproteinases (MMPs), leading to the generation of proteolytically processed antagonistic CCL2 variant. Neutralizing CCL2 or inhibiting MMP enzymatic activity abolished the PC-suppressive effect of MSCs. We also observed that MMP-processed CCL2 suppresses signal transducer and activator of transcription 3 (STAT3) activation in PC. As a result, the transcription factor PAX5 is induced, thus explaining the inhibition of Ig synthesis. The absence of inhibitory effects by MSC on the humoral response of CCR2(-/-) mice to xenoantigen suggests that MMP-cleaved CCL2/CCR2 interaction as well as downstream phosphatase activity is necessary for antagonistic effect. We tested syngeneic MSCs in hemophilic B6 mice with predeveloped antihuman factor VIII (hFVIII) antibodies and demonstrated a robust decrease in hFVIII-specific IgG levels. Thus, MSCs may play a role in modulating Ig production by PCs via MMP processing of CCL2 and may represent an appealing cell therapy approach for pathologic humoral responses.

Human marrow-derived mesenchymal stromal cells decrease cisplatin renotoxicity in vitro and in vivo and enhance survival of mice post-intraperitoneal injection

Acute kidney injury (AKI) can occur from the toxic side-effects of chemotherapeutic agents such as cisplatin. Bone marrow-derived mesenchymal stromal cells (MSCs) have demonstrated wide therapeutic potential often due to beneficial factors they secrete. The goal of this investigation was to evaluate in vitro the effect of human MSCs (hMSCs) secretome on cisplatin-treated human kidney cells, and in vivo the consequence of hMSCs intraperitoneal (ip) implantation in mice with AKI. Our results revealed that hMSCs-conditioned media improved survival of HK-2 human proximal tubular cells exposed to cisplatin in vitro. This enhanced survival was linked to increased expression of phosphorylated Akt (Ser473) and was reduced by a VEGF-neutralizing antibody. In vivo testing of these hMSCs established that ip administration in NOD-SCID mice decreased cisplatin-induced kidney function impairment, as demonstrated by lower blood urea nitrogen levels and higher survival. In addition, blood phosphorous and amylase levels were also significantly decreased. Moreover, hMSCs reduced the plasma levels of several inflammatory cytokines/chemokines. Immunohistochemical examination of kidneys showed less apoptotic and more proliferating cells. Furthermore, PCR indicated the presence of hMSCs in mouse kidneys, which also showed enhanced expression of phosphorylated Akt. In conclusion, our study reveals that hMSCs can exert prosurvival effects on renal cells in vitro and in vivo, suggests a paracrine contribution for kidney protective abilities of hMSCs delivered ip, and supports their clinical potential in AKI.

A MCP1 fusokine with CCR2-specific tumoricidal activity.

BackgroundThe CCL2 chemokine is involved in promoting cancer angiogenesis, proliferation and metastasis by malignancies that express CCR2 receptor. Thus the CCL2/CCR2 axis is an attractive molecular target for anticancer drug development.MethodsWe have generated a novel fusion protein using GMCSF and an N-terminal truncated version of MCP1/CCL2 (6-76) [hereafter GMME1] and investigated its utility as a CCR2-specific tumoricidal agent.ResultsWe found that distinct to full length CCL2 or its N-truncated derivative (CCL2 5-76), GMME1 bound to CCR2 on mouse lymphoma EG7, human multiple myeloma cell line U266, or murine and human medulloblastoma cell lines, and led to their death by apoptosis. We demonstrated that GMME1 specifically blocked CCR2-associated STAT3 phosphorylation and up-regulated pro-apoptotic BAX. Furthermore, GMME1 significantly inhibited EG7 tumor growth in C57BL/6 mice, and induced apoptosis of primary myeloma cells from patients.ConclusionOur data demonstrate that GMME1 is a fusokine with a potent, CCR2 receptor-mediated pro-apoptotic effect on tumor cells and could be exploited as a novel biological therapy for CCR2+ malignancies including lymphoid and central nervous system malignancies.

A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

Cytokine receptors are randomly distributed on the cell surface membrane and are activated upon binding of their extracellular ligands to mediate downstream cellular activities. We hypothesized that pharmaceutical clustering of ligand-bound, activated receptors may lead to heretofore unrealized gain-of-function with therapeutically desirable properties. We here describe an engineered bifunctional cytokine borne of the fusion of Granulocyte Macrophage Colony Stimulating Factor (GMCSF) and Interleukin-9 (IL9) (hereafter GIFT9 fusokine) and demonstrate that it chaperones co-clustering of the functionally unrelated GMCSF receptor (GMCSFR) and IL9 receptor (IL9R) on cell surface of target cells. We demonstrate that GIFT9 treatment of MC/9 cells leads to transhyperphosphorylation of IL9R-associated STAT1 by GMCSFR-associated JAK2. We also show that IL9R-associated JAK1 and JAK3 augment phosphorylation of GMCSFR-linked STAT5. The functional relevance of these synergistic JAK/STAT transphosphorylation events translates to an increased mitogenic response by GMCSFR/IL9R-expressing primary marrow mast cells. The notion of inducing heterologous receptor clustering by engineered fusokines such as GIFT9 opens the door to a novel type of biopharmaceutical platform where designer fusokines modulate cell physiology through clustering of targeted receptor complexes.

Engineered Fusokine GIFT4 Licenses the Ability of B Cells to Trigger a Tumoricidal T-cell Response

Engineered chimeric cytokines can generate gain-of-function activity in immune cells. Here, we report potent antitumor activity for a novel fusion cytokine generated by N-terminal coupling of GM-CSF to IL4, generating a fusokine termed GIFT4. B cells treated with GIFT4 clustered GM-CSF and IL4 receptors on the cell surface and displayed a pan-STAT hyperphosphorylation associated with acquisition of a distinct phenotype and function described to date. In C57BL/6J mice, administration of GIFT4 expanded endogenous B cells and suppressed the growth of B16F0 melanoma cells. Furthermore, B16F0 melanoma cells engineered to secrete GIFT4 were rejected immunologically in a B-cell-dependent manner. This effect was abolished when GIFT4-expressing B16F0 cells were implanted in B-cell-deficient mice, confirming a B-cell-dependent antitumor effect. Human GIFT4-licensed B cells primed cytotoxic T cells and specifically killed melanoma cells in vitro and in vivo. Taken together, our results demonstrated that GIFT4 could mediate expansion of B cells with potent antigen-specific effector function. GIFT4 may offer a novel immunotherapeutic tool and define a previously unrecognized potential for B cells in melanoma immunotherapy.

Stimulation of Natural Killer Cell-Mediated Tumor Immunity by an IL15/TGFβ-Neutralizing Fusion Protein.

The clinical efficacy of immune cytokines used for cancer therapy is hampered by elements of the immunosuppressive tumor microenvironment such as TGFβ. Here we demonstrate that FIST15, a recombinant chimeric protein composed of the T-cell-stimulatory cytokine IL15, the sushi domain of IL15Rα and a TGFβ ligand trap, can overcome immunosuppressive TGFβ to effectively stimulate the proliferation and activation of natural killer (NK) and CD8+ T cells with potent antitumor properties. FIST15-treated NK and CD8+ T cells produced more IFNγ and TNFα compared with treatment with IL15 and a commercially available TGFβ receptor-Fc fusion protein (sTβRII) in the presence of TGFβ. Murine B16 melanoma cells, which overproduce TGFβ, were lysed by FIST15-treated NK cells in vitro at doses approximately 10-fold lower than NK cells treated with IL15 and sTβRII. Melanoma cells transduced to express FIST15 failed to establish tumors in vivo in immunocompetent murine hosts and could only form tumors in beige mice lacking NK cells. Mice injected with the same cells were also protected from subsequent challenge by unmodified B16 melanoma cells. Finally, mice with pre-established B16 melanoma tumors responded to FIST15 treatment more strongly compared with tumors treated with control cytokines. Taken together, our results offer a preclinical proof of concept for the use of FIST15 as a new class of biological therapeutics that can coordinately neutralize the effects of immunosuppressive TGFβ in the tumor microenvironment while empowering tumor immunity. Cancer Res; 76(19); 5683-95. ©2016 AACR.

Inducible IL10+ Suppressor B Cells Inhibit CNS Inflammation and T Helper 17 Polarization

Granulocyte-macrophage colony-stimulating factor (GMCSF) and MCP3 (aka CCL7) exert complementary, nonoverlapping, proimmune effects on responsive lymphoid and myeloid cells. We hypothesized that a synthetic cytokine linking GMCSF to MCP3 (hereafter GMME3) as part of a single polypeptide would acquire novel, therapeutically desirable immunomodulatory properties. We demonstrate that GMME3 has enhanced CC-chemokine receptor (CCR)-mediated intracellular Ca++ mobilization with selective effects on the CD21hiCD24hi CD1.dhi subset of splenic B cells inducing substantial interleukin 10 (IL10) production. We demonstrate that BGMME3 exert their suppressive effect through an IL10-mediated inhibition of antigen presentation. More importantly, BGMME3 inhibit the reactivation of encephalomyelitis (EAE)-derived or TGFβ/IL6 differentiated Th17 cells by altering their polarization toward a Th1 or Th2 phenotype. The secretion of interferon-γ (IFNγ) and IL4 in turn inhibits IL17 production. The adoptive transfer of BGMME3, but not IL10-/- BGMME3 cells, to mice symptomatic with experimental autoimmune encephalitis significantly improves their disease score and inhibits lymphoid infiltration into the central nervous system (CNS). We propose that designed CCR modulators such as GMME3, allows for conversion of naive B-cells to a novel suppressor phenotype allowing for the personalized cell therapy of autoimmune ailments.Granulocyte-macrophage colony-stimulating factor (GMCSF) and MCP3 (aka CCL7) exert complementary, nonoverlapping, proimmune effects on responsive lymphoid and myeloid cells. We hypothesized that a synthetic cytokine linking GMCSF to MCP3 (hereafter GMME3) as part of a single polypeptide would acquire novel, therapeutically desirable immunomodulatory properties. We demonstrate that GMME3 has enhanced CC-chemokine receptor (CCR)-mediated intracellular Ca(++) mobilization with selective effects on the CD21(hi)CD24(hi) CD1.d(hi) subset of splenic B cells inducing substantial interleukin 10 (IL10) production. We demonstrate that B(GMME3) exert their suppressive effect through an IL10-mediated inhibition of antigen presentation. More importantly, B(GMME3) inhibit the reactivation of encephalomyelitis (EAE)-derived or TGFβ/IL6 differentiated Th17 cells by altering their polarization toward a Th1 or Th2 phenotype. The secretion of interferon-γ (IFNγ) and IL4 in turn inhibits IL17 production. The adoptive transfer of B(GMME3), but not IL10(-/-) B(GMME3) cells, to mice symptomatic with experimental autoimmune encephalitis significantly improves their disease score and inhibits lymphoid infiltration into the central nervous system (CNS). We propose that designed CCR modulators such as GMME3, allows for conversion of naive B-cells to a novel suppressor phenotype allowing for the personalized cell therapy of autoimmune ailments.

A novel parasite-derived suicide gene for cancer gene therapy with specificity for lung cancer cells

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) expressed by the parasite Trypanosoma brucei (Tb) can convert allopurinol, a purine analogue, to corresponding nucleotides with greater efficiency than its human homologue. We have developed a retroviral system that expresses the parasitic enzyme and tested its capacity to activate the prodrug allopurinol to a cytotoxic metabolite. Cytotoxicity assays demonstrated that five non-small cell lung carcinoma cell lines transduced with the construct were sensitized to the prodrug by 2.1- to 7.6-fold compared with control values. This selectivity was not observed in seven other cell lines also expressing the construct, such as breast carcinoma. Assays indicated that enhanced cytotoxicity to allopurinol correlated with induction of apoptosis in lung cancer cells. The selectivity of this suicide gene was not explained either by the TbHGPRT expression or by the allopurinol accumulation. Our study shows that this novel system may represent a therapeutic tool for gene prodrug targeting of lung cancer, considering the fact that allopurinol is well tolerated in humans.

Bone Marrow–Derived Mesenchymal Stromal Cells from Patients with Sickle Cell Disease Display Intact Functionality

Hematopoietic cell transplantation (HCT) is the only cure for sickle cell disease (SCD), but engraftment remains challenging in patients lacking matched donors. Infusion of mesenchymal stromal cells (MSCs) at the time of HCT may promote hematopoiesis and ameliorate graft-versus-host disease. Experimental murine models suggest MSC major histocompatibility complex compatibility with recipient impacts their in vivo function, suggesting autologous MSCs could be superior to third-party MSCs for promoting HCT engraftment. Here we tested whether bone marrow (BM)-derived MSCs from SCD subjects have comparable functionality compared with MSCs from healthy volunteers. SCD MSC doubling time and surface marker phenotype did not differ significantly from non-SCD. Third-party and autologous (SCD) T cell proliferation was suppressed in a dose-dependent manner by all MSCs. SCD MSCs comparably expressed indoleamine-2,3-dioxygenase, which based on transwell and blocking experiments appeared to be the dominant immunomodulatory pathway. The expression of key genes involved in hematopoietic stem cell (HSC)-MSC interactions was minimally altered between SCD and non-SCD MSCs. Expression was, however, altered by IFN-γ stimulation, particularly CXCL14, CXCL26, CX3CL1, CKITL, and JAG1, indicating the potential to augment MSC expression by cytokine stimulation. These data demonstrate the feasibility of expanding BM-derived MSCs from SCD patients that phenotypically and functionally do not differ per International Society of Cell Therapy essential criteria from non-SCD MSCs, supporting initial evaluation (primarily for safety) of autologous MSCs to enhance haploidentical HSC engraftment in SCD.