Differential requirement of GRP94 and GRP78 in mammary gland development.

Glucose Regulated Protein (GRP) 94 and GRP78 are critical molecular chaperones and regulators of signaling. Conditional knockout mouse models have revealed tissue specific requirements for GRP94 and GRP78, including selection for allele retention in specific cell types. Here we report the consequences of mammary-targeted knockout of these GRPs. Our studies revealed that MMTV-Cre, Grp94(f/f) mammary glands, despite GRP94 deficiency, exhibited normal proliferation and ductal morphogenesis. Interestingly, MMTV-Cre, Grp78(f/f) mammary glands displayed only slightly reduced GRP78 protein levels, associating with the retention of the non-recombined Grp78 floxed alleles in isolated mammary epithelial cells and displayed phenotypes comparable to wild-type glands. In contrast, transduction of isolated Grp78(f/f) mammary epithelial stem/progenitor cells with adenovirus expressing GFP and Cre-recombinase was successful in GRP78 ablation, and the GFP sorted cells failed to give rise to repopulated mammary glands in de-epithelialized recipient mice. These studies imply GRP78, but not GRP94, is required for mammary gland development.

Suppression of rheumatoid arthritis B cells by XmAb5871, an anti-CD19 antibody that coengages B cell antigen receptor complex and Fcγ receptor IIb inhibitory receptor.

OBJECTIVE: Engagement of Fcγ receptor IIb (FcγRIIb) suppresses B cell activation and represents a promising target for therapy in autoimmunity. The aim of this study was to characterize B cell immunosuppression mediated by the Fc-engineered antibody, XmAb5871, which coengages FcγRIIb with the B cell antigen receptor (BCR) complex and that is currently in clinical development for the treatment of rheumatoid arthritis (RA). Because rheumatoid factor (RF) might interfere with the binding of XmAb5871 to FcγRIIb, we correlated RF titers with the potency of XmAb5871. METHODS: We analyzed the expression of CD19, FcγRIIb, and CD86 on naive and memory B cells from 50 patients with RA and 66 healthy donors, quantified XmAb5871-induced promotion of FcγRIIb phosphorylation and suppression of calcium flux in activated B cells, measured CD86 inhibition in whole blood, and correlated RF and anti-citrullinated protein antibody (ACPA) levels with drug potency. We engrafted RA peripheral blood mononuclear cells (PBMCs) into SCID mice, treated them with XmAb5871, and quantified human total IgG, total IgM, and anti-tetanus IgG antibody levels in vivo. RESULTS: B cells from all donors expressed CD19 and FcγRIIb, and the expression of FcγRIIb was higher on naive, but not memory, B cells from donors with RA compared with healthy donors. BCR-mediated calcium flux was suppressed by XmAb5871 and was associated with FcγRIIb phosphorylation. XmAb5871 inhibited CD86 induction, and the levels of RF and ACPAs did not affect efficacy. XmAb5871 suppressed B cell activation regardless of disease severity. In SCID mice engrafted with PBMCs from a patient with RA, XmAb5871 suppressed humoral responses. CONCLUSION: Coengagement of the BCR complex and FcγRIIb by XmAb5871 inhibits B cell activation and function. The similar potency in patients with RA and healthy donors and the absence of autoantibody interference suggest that XmAb5871 may represent a new therapeutic strategy to suppress autoreactive B cells in RA.

Immune suppression in cynomolgus monkeys by XPro9523: an improved CTLA4-Ig fusion with enhanced binding to CD80, CD86 and neonatal Fc receptor FcRn.

The CTLA4-Ig fusion proteins abatacept and belatacept are clinically proven immunosuppressants used for rheumatoid arthritis and renal transplant, respectively. Given that both biologics are typically administered chronically by infusion, a need exists for a next-generation CTLA4-Ig with more convenient dosing. We used structure-based protein engineering to optimize the affinity of existing CTLA4-Ig therapeutics for the ligands CD80 and CD86, and for the neonatal Fc receptor, FcRn. From a rationally designed library, we identified four substitutions that enhanced binding to human CD80 and CD86. Coupled with two IgG1 Fc substitutions that enhanced binding to human FcRn, these changes comprise the novel CTLA4-Ig fusion protein, XPro9523. Compared with abatacept, XPro9523 demonstrated 5.9-fold, 23-fold, and 12-fold increased binding to CD80, CD86, and FcRn, respectively; compared with belatacept, CD80, CD86, and FcRn binding increased 1.5-fold, 7.7-fold, and 11-fold, respectively. XPro9523 and belatacept suppressed human T cell proliferation and IL-2 production more potently than abatacept. XPro9523 also suppressed inflammation in the mouse collagen-induced arthritis model. In cynomolgus monkeys, XPro9523 saturated CD80 and CD86 more effectively than abatacept and belatacept, potently inhibited IgM and IgG immunization responses, and demonstrated longer half-life. Pharmacokinetic modeling of its increased potency and persistence suggests that, in humans, XPro9523 may demonstrate superior efficacy and dosing convenience compared with abatacept and belatacept.

A super TLR agonist to improve efficacy of dendritic cell vaccine in induction of anti-HCV immunity.

Persistent infections caused by pathogens such as hepatitis C virus are major human diseases with limited or suboptimal prophylactic and therapeutic options. Given the critical role of dendritic cell (DC) in inducing immune responses, DC vaccination is an attractive means to prevent and control the occurrence and persistence of the infections. However, DCs are built-in with inherent negative regulation mechanisms which attenuate their immune stimulatory activity and lead to their ineffectiveness in clinical application. In this study, we developed a super DC stimulant that consists of a modified, secretory Toll-like Receptor (TLR)-5 ligand and an inhibitor of the negative regulator, suppressor of cytokine sinaling-1 (SOCS1). We found that expressing the super stimulant in DCs is drastically more potent and persistent than using the commonly used DC stimuli to enhance the level and duration of inflammatory cytokine production by both murine and human DCs. Moreover, the DCs expressing the super stimulant are more potent to provoke both cellular and humoral immune responses against hepatitis C virus (HCV) antigen in vivo. Thus, the strategy capable of triggering and sustaining proinflammatory status of DCs may be used to boost efficiency of DC vaccine in preventing and combating the persistent infection of HCV or other chronic viruses.

The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche.

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology.

Enhanced generation of myeloid lineages in hematopoietic differentiation from embryonic stem cells by silencing transcriptional repressor Twist-2.

The self-renewal and multilineage differentiation of embryonic stem cells (ESC) is largely governed by transcription factors or repressors. Extensive efforts have focused on elucidating critical factors that control the differentiation of specific cell lineages, for instance, myeloid lineages in hematopoietic development. In this study, we found that Twist-2, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in inhibiting the differentiation of ESC. Murine ES cells, in which Twist-2 expression is silenced by lentivirally delivered shRNA, exhibit an enhanced formation of primary embryoid bodies (EB) and enhanced differentiation into mesodermally derived hematopoietic colonies. Furthermore, Twist-2 silenced (LV-siTwist-2) ESC display significantly increased generation of myeloid lineages (Gr-1(+) and F4/80(+) cells) during in vitro hematopoietic differentiation. Treatment with the Toll-like receptor (TLR) 4 ligand synergistically stimulates the generation of primary EB formation as well as of hematopoietic progenitors differentiated from LV-siTwist-2 ES cells. Thus, this study reveals the critical role of the transcriptional repressor Twist-2 in regulating the development of myeloid lineage in hematopoietic differentiation from ESC. This study also suggests a potential strategy for directional differentiation of ESC by inhibiting a transcriptional repressor.

Embryonic stem cells and mammary luminal progenitors directly sense and respond to microbial products.

Stem cells are normally maintained in a quiescent state and proliferate only under certain conditions; however, little is known about the biological stimuli that initiate the proliferation and differentiation of stem cells. In this study, we found that functional Toll-like receptors (TLRs) are expressed on mouse embryonic stem (ES) cells and that TLR ligands stimulate ES cell proliferation and promote their hematopoietic differentiation. TLR ligands activate TLR-mediated signaling pathways, leading to the altered expression of numerous genes in ES cells. Moreover, TLR ligands efficiently stimulate the proliferation and expansion of adult stem cells and progenitors of nonhematopoietic tissues, such as mammary glands and intestine as well. We further found that mammary luminal progenitor cells (Lin(-)CD29(+)CD61(+)) express TLR4-MD2 complex and actively proliferate, resulting in the enhanced growth of mammospheres in response to TLR ligands. Thus, mouse ES cells and adult tissue-specific stem cells/progenitors directly sense and respond to microbial products, which function as a class of foreign, but biological stimuli for stem cell/progenitor proliferation. This finding expands the biological role of TLRs and has implications in understanding stem cell biology, tissue repair/homeostasis, and the role of infection and inflammation in malignant transformation.

Twist-2 controls myeloid lineage development and function.

Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2-deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPalpha. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-gamma (IFNgamma) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.

Targeting tumor-associated macrophages as a novel strategy against breast cancer.

Tumor-associated macrophages (TAMs) are associated with tumor progression and metastasis. Here, we demonstrate for the first time that legumain, a member of the asparaginyl endopeptidase family functioning as a stress protein, overexpressed by TAMs, provides an ideal target molecule. In fact, a legumain-based DNA vaccine served as a tool to prove this point, as it induced a robust CD8+ T cell response against TAMs, which dramatically reduced their density in tumor tissues and resulted in a marked decrease in proangiogenic factors released by TAMs such as TGF-beta, TNF-alpha, MMP-9, and VEGF. This, in turn, led to a suppression of both tumor angiogenesis and tumor growth and metastasis. Importantly, the success of this strategy was demonstrated in murine models of metastatic breast, colon, and non-small cell lung cancers, where 75% of vaccinated mice survived lethal tumor cell challenges and 62% were completely free of metastases. In conclusion, decreasing the number of TAMs in the tumor stroma effectively altered the tumor microenvironment involved in tumor angiogenesis and progression to markedly suppress tumor growth and metastasis. Gaining better insights into the mechanisms required for an effective intervention in tumor growth and metastasis may ultimately lead to new therapeutic targets and better anticancer strategies.

Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer.

Endoglin (CD105), a co-receptor in the TGF-beta receptor complex, is over-expressed on proliferating endothelial cells in the breast tumor neovasculature and thus offers an attractive target for anti-angiogenic therapy. Here we report the anti-angiogenic/anti-tumor effects achieved in a prophylactic setting with an oral DNA vaccine encoding murine endoglin, carried by double attenuated Salmonella typhimurium (dam-, AroA-) to a secondary lymphoid organ, i.e., Peyer's patches . We demonstrate that an endoglin vaccine elicited activation of antigen-presenting dendritic cells, coupled with immune responses mediated by CD8+ T cells against endoglin-positive target cells. Moreover, we observed suppression of angiogenesis only in mice administered with the endoglin vaccine as compared to controls. These data suggest that a CD8+ T cell-mediated immune response induced by this vaccine effectively suppressed dissemination of pulmonary metastases of D2F2 breast carcinoma cells presumably by eliminating proliferating endothelial cells in the tumor vasculature. It is anticipated that vaccine strategies such as this may contribute to future therapies for breast cancer.

A DNA-based cancer vaccine enhances lymphocyte cross talk by engaging the NKG2D receptor.

The NKG2D receptor is a stimulatory receptor expressed on NK cells and activated CD8 T cells. We previously demonstrated that engaging the NKG2D receptor markedly improved the efficacy of a survivin-based DNA vaccine. The combination vaccine, encoding both the NKG2D ligand H60 and survivin, activates innate and adaptive antitumor immunity and results in better protection against tumors of different origin and NKG2D expression levels. Here we demonstrate that the enhanced vaccine efficacy is in part attributable to increased cross talk between lymphocytes. Depletion of CD8 T cells during priming reduces the vaccine-induced activation of dendritic cells (DCs) and NK cell activity. Depletion of NK cells during priming leads to reduced DC activation and CTL activity. However, depletion of CD4 T cells results in the activation of DCs, NK cells, and CD8 T cells and enhances NK cell activity. The pH60/Survivin vaccine also increases DCs and NK cells but decreases CD4 T cell homing to Peyer patches, presumably as a result of changes in the homing receptor profile. Thus, by preferentially activating and attracting positive regulators and reducing negative regulators in Peyer patches, this dual-function DNA vaccine induces a microenvironment more suitable for NK cell activation and T cell priming.

Combined dendritic cell cryotherapy of tumor induces systemic antimetastatic immunity.

PURPOSE: Cryotherapy of localized prostate, renal, and hepatic primary tumors and metastases is considered a minimally invasive treatment demonstrating a low complication rate in comparison with conventional surgery. The main drawback of cryotherapy is that it has no systemic effect on distant metastases. We investigated whether intratumoral injections of dendritic cells following cryotherapy of local tumors (cryoimmunotherapy) provides an improved approach to cancer treatment, combining local tumor destruction and systemic anticancer immunity. EXPERIMENTAL DESIGNS: The 3LL murine Lewis lung carcinoma clone D122 and the ovalbumin-transfected B16 melanoma clone MO5 served as models for spontaneous metastasis. The antimetastatic effect of cryoimmunotherapy was assessed in the lung carcinoma model by monitoring mouse survival, lung weight, and induction of tumor-specific CTLs. The mechanism of cryoimmunotherapy was elucidated in the melanoma model using adoptive transfer of T cell receptor transgenic OT-I CTLs into the tumor-bearing mice, and analysis of Th1/Th2 responses by intracellular cytokine staining in CD4 and CD8 cells. RESULTS: Cryoimmunotherapy caused robust and tumor-specific CTL responses, increased Th1 responses, significantly prolonged survival and dramatically reduced lung metastasis. Although intratumor administration of dendritic cells alone increased the proliferation rate of CD8 cells, only cryoimmunotherapy resulted in the generation of effector memory cells. Furthermore, cryoimmunotherapyprotected mice that had survived primary MO5 tumors from rechallenge with parental tumors. CONCLUSIONS: These results present cryoimmunotherapy as a novel approach for systemic treatment of cancer. We envisage that cryotherapy of tumors combined with subsequent in situ immunotherapy by autologous unmodified immature dendritic cells can be applied in practice.

Expression of FasL by tumor cells does not abrogate anti-tumor CTL function.

The effects of Fas-ligand (FasL) expression by tumor cells on their tumorigenicity and immunogenicity have been reported as opposite, contradictory results. In some systems the killing of Fas positive cytotoxic T-cells (CTL) by FasL expressing tumors resulted in increased tumorigenicity while in other systems tumors expressing FasL were eliminated by neutrophil mediated inflammation. In the present study, we investigated how FasL expression influences the low immunogenic Lewis lung carcinoma clone D122 and its highly immunogenic MHC I (H-2Kb) and B7-1 (CD80) transfectant 39.5-B7, by transfecting the human FasL (FasL) gene into these cells. Despite the fact that FasL-expressing cells kill effectively appropriate target cells (L1210-fas) compared to parental cells (D122) and low expressors (DFasL-33), these tumor cells were completely rejected in syngeneic mice (C57BL/6), but not in Fas mutant B6-MRL mice, suggesting that functional Fas receptor expression in the host was required to induce an anti-tumor mechanism. In addition, although FasL-expressing immunogenic tumor cells (39.5-B7-FasL 7) kill effectively target cells in vitro, both the transfectant and the mock transfectant (39.5-B7-pBabe) were rejected in syngenic mice. The sensitivity of FasL expressing tumor cells to lysis by CTLs was similar to that of FasL non-expressors. Therefore, these results indicate that FasL expression on immunogenic tumor cells does not affect their immunogenicity in vivo, as well as CTL functions in vitro.

In vivo rejection of tumor cells dependent on CD8 cells that kill independently of perforin and FasL.

Perforin/granzyme B- and Fas/FasL-mediated killing pathways are the main effector mechanisms of CTL and NK cells in antitumor immune responses. In this study, we investigated the relative role of these two lytic mechanisms in protection of the host from tumor progression, as well as spontaneous metastasis, using the D122 Lewis lung carcinoma and its gene-modified cells. Utilizing perforin knockout mice (B6-PKO) and Fas and FasL mutant (B6-MRL and B6-Smn) mice, we found that perforin expression in the host plays a crucial function in the prevention of metastasis. However, local tumor rejection of an H-2K(b) and B7-1 transfectant, 39.5-B7 cells, was not dependent either on perforin or Fas/FasL expression in vivo. In addition, CTL lysis of 39.5-B7 cells was independent of perforin and Fas/FasL interactions in 18-hour in vitro assays. We also confirmed that CD8 T-cells were responsible for rejecting 39.5-B7 local tumors, yet cytokines, TNF-alpha and gammaIFN were not involved in tumor rejection in vivo. Furthermore, blocking assays using caspase inhibitors (zVAD-fmk, zLETD-fmk and zLEHD-fmk) showed that, whereas caspase activation was partially required to induce 39.5-B7 lysis mediated by the perforin-dependent pathway, 39.5-B7 lysis by CTLs through the perforin-independent mechanism required caspase activation. Thus, these results suggested that perforin, Fas/FasL, gammaIFN and TNF-alpha independent lytic mechanisms, mediated by CD8 T cells, have a crucial role in rejection of 39.5-B7 cells in vivo. Caspase activation is a pre requisite for apoptosis of targets by CTLs.