Overexpression of an Engineered SerpinB9 Enhances Allogeneic T-Cell Persistence and Efficacy.

Allogeneic chimeric antigen receptor (CAR)-expressing T cells offer many advantages over autologous therapies, but their benefits are curtailed by graft-versus-host disease (GvHD) and elimination by recipient immune cells. Moreover, just as with autologous therapies, allogeneic CAR T cells are susceptible to activation-induced cell death (AICD) caused by chronic antigen exposure (CAE). Granzyme B (GzmB) and Fas/FasL-initiated, caspase-mediated apoptosis are key mechanisms of T-cell death caused by T/NK cell-mediated allorejection or CAE. We explored a protective strategy of engineering CAR T cells to overexpress variants of the GzmB-specific serine protease inhibitor, SerpinB9 (SB9), to improve allogeneic T-cell persistence and antitumor efficacy. We showed that the overexpression of an SB9 variant with broadened caspase specificity, SB9(CAS), not only significantly reduced rejection of allogeneic CAR T cells, but also increased their resistance to AICD and enabled them to thrive better under CAE, thus improving allogeneic T-cell persistence and antitumor activity in vitro and in vivo. In addition, while SB9(CAS)-overexpression improved the efficacy of allogeneic CAR T-cell therapy by conferring protection to cell death, we did not observe any autonomous growth and the engineered CAR T cells were still susceptible to an inducible suicide switch. Hence, SB9(CAS)-overexpression is a promising strategy that can strengthen current development of cell therapies, broadening their applications to address unmet medical needs.

B7-H3-Targeting Chimeric Antigen Receptors Epstein-Barr Virus-specific T Cells Provides a Tumor Agnostic Off-The-Shelf Therapy Against B7-H3-positive Solid Tumors.

Encouraged by the observations of significant B7-H3 protein overexpression in many human solid tumors compared to healthy tissues, we directed our focus towards targeting B7-H3 using chimeric antigen receptor (CAR) T cells. We utilized a nanobody as the B7-H3-targeting domain in our CAR construct to circumvent the stability issues associated with single-chain variable fragment-based domains. In efforts to expand patient access to CAR T-cell therapy, we engineered our nanobody-based CAR into human Epstein-Barr virus-specific T cells (EBVST), offering a readily available off-the-shelf treatment. B7H3.CAR-armored EBVSTs demonstrated potent in vitro and in vivo activities against multiple B7-H3-positive human tumor cell lines and patient-derived xenograft models. Murine T cells expressing a murine equivalent of our B7H3.CAR exhibited no life-threatening toxicities in immunocompetent mice bearing syngeneic tumors. Further in vitro evaluation revealed that while human T, B, and natural killer cells were unaffected by B7H3.CAR EBVSTs, monocytes were targeted because of upregulation of B7-H3. Such targeting of myeloid cells, which are key mediators of cytokine release syndrome (CRS), contributed to a low incidence of CRS in humanized mice after B7H3.CAR EBVST treatment. Notably, we showed that B7H3.CAR EBVSTs can target B7-H3-expressing myeloid-derived suppressor cells (MDSC), thereby mitigating MDSC-driven immune suppression. In summary, our data demonstrate that our nanobody-based B7H3.CAR EBVSTs are effective as an off-the-shelf therapy for B7-H3-positive solid tumors. These cells also offer an avenue to modulate the immunosuppressive tumor microenvironment, highlighting their promising clinical potential in targeting solid tumors. SIGNIFICANCE: Clinical application of EBVSTs armored with B7-H3-targeting CARs offer an attractive solution to translate off-the-shelf CAR T cells as therapy for solid tumors.

Cord blood-derived Vδ2+ and Vδ2- T cells acquire differential cell state compositions upon in vitro expansion.

Human cord blood-derived γδ T cells (CBγδ) display a highly diverse TCRγδ repertoire and have a unique subtype composition different from fetal or adult peripheral blood counterparts. We expanded CBγδ in vitro using an irradiated Epstein-Barr virus-transformed feeder cell-based modified rapid expansion protocol (REP). Single-cell RNA sequencing tracked progressive differentiation of naïve CBγδ into cells expressing neoantigen-reactive tumor-infiltrating lymphocyte as well as tissue-resident memory precursor-like and antigen-presenting cell-like gene signatures. TCRγδ clonal tracing revealed a bias toward cytotoxic effector differentiation in a much larger proportion of Vδ2- clones compared to Vδ2+ clones, resulting in the former being more cytotoxic at the population level. These clonotype-specific differentiation dynamics were not restricted to REP and were recapitulated upon secondary nonviral antigen stimulations. Thus, our data showed intrinsic cellular differences between major subtypes of human γδ T cells already in operation at early postnatal stage and highlighted key areas of consideration in optimizing cell manufacturing processes.

Determining the Development of Persisters in Extensively Drug-Resistant Acinetobacter baumannii upon Exposure to Polymyxin B-Based Antibiotic Combinations Using Flow Cytometry.

Polymyxin B-based combinations are increasingly prescribed as a last-line option against extensively drug-resistant (XDR) Acinetobacter baumannii It is unknown if such combinations can result in the development of nondividing persister cells in XDR A. baumannii We investigated persister development upon exposure of XDR A. baumannii to polymyxin B-based antibiotic combinations using flow cytometry. Time-kill studies (TKSs) were conducted in three nonclonal XDR A. baumannii strains with 5 log10 CFU/ml bacteria against polymyxin B alone and polymyxin B-based two-drug combinations over 24 h. At different time points, samples were obtained and enumerated by viable plating and flow cytometry. Propidium iodide and carboxyfluorescein succinimidyl ester dyes were used to differentiate between live and dead cells and between dividing and nondividing cells, respectively, at the single-cell level, and nondividing live cells were resuscitated and characterized phenotypically. Our results from viable plating showed that polymyxin B plus meropenem and polymyxin B plus rifampin were each bactericidal (>99.9% kill compared to the initial inoculum) against 2/3 XDR A. baumannii strains at 24 h. By flow cytometry, however, none of the combinations were bactericidal against XDR A. baumannii at 24 h. Further analysis using cellular dyes in flow cytometry revealed that upon exposure to polymyxin B-based combinations, XDR A. baumannii entered a viable but nondividing persister state. These bacterial cells reinitiated division upon the removal of antibiotic pressure and did not have a growth deficit compared to the parent strain. We conclude that persister cells develop in XDR A. baumannii upon exposure to polymyxin B-based combinations and that nonplating methods appear to complement viable-plating methods in describing the killing activity of polymyxin B-based combinations against XDR A. baumannii.

Halted Lymphocyte Egress via Efferent Lymph Contributes to Lymph Node Hypertrophy During Hypercholesterolemia.

Dyslipidemia is a central component of atherosclerosis and metabolic syndrome linked to chronic inflammation and immune dysfunction. Previously, we showed that hypercholesterolemic apolipoprotein E knock out (apoE-/-) mice exhibit systemic effects including skin inflammation and hypertrophic lymph nodes (LNs). However, the mechanisms accounting for LN hypertrophy in these mice remain unknown. Here, we show that hypercholesterolemia led to the accumulation of lymphocytes in LNs. We excluded that the increased number of lymphocytes in expanded LNs resulted from increased lymphocyte proliferation or entry into those LNs. Instead, we demonstrated that the egress of lymphocytes from the enlarged LN of apoE-/- mice was markedly decreased. Impairment in efferent lymphatic emigration of lymphocytes from LNs resulted from an aberrant expansion of cortical and medullary sinuses that became hyperplastic. Moreover, CCL21 was more abundant on these enlarged sinuses whereas lymph levels of sphingosine 1 phosphate (S1P) were decreased in apoE-/- mice. Normal LN size, lymphatic density and S1P levels were restored by reversing hypercholesterolemia. Thus, systemic changes in cholesterol can sequester lymphocytes in tissue draining LNs through the extensive remodeling of lymphatic sinuses and alteration of the balance between retention/egress signals leading to LN hypertrophy which subsequently may contribute to poor immunity. This study further illustrates the role of lymphatic vessels in immunity through the regulation of immune cell trafficking.

Tumor stroma and chemokines control T-cell migration into melanoma following Temozolomide treatment.

The infiltration of T lymphocytes within tumors is associated with better outcomes in cancer patients, yet current understanding of factors that influence T-lymphocyte infiltration into tumors remains incomplete. In our study, Temozolomide (TMZ), a chemotherapeutic drug used to treat metastatic melanoma, induced T-cell infiltration into transplanted melanoma and into genitourinary (GU) tumors in mice developing spontaneous melanoma. In contrast, TMZ treatment did not increase T-cell infiltration into cutaneous tumors, despite similar increases in the expression of the (C-X-C) chemokines CXCL9 and CXCL10 in all sites after TMZ exposure. Our findings reveal that the matrix architecture of the GU tumor stroma, and its ability to present CXCL9 and CXCL10 after TMZ treatment played a key role in favouring T-cell infiltration. We subsequently demonstrate that modifications of these key elements by combined collagenase and TMZ treatment induced T-cell infiltration into skin tumors. T cells accumulating within GU tumors after TMZ treatment exhibited T helper type-1 effector and cytolytic functional phenotypes, which are important for control of tumor growth. Our findings highlight the importance of the interaction between tumor stroma and chemokines in influencing T-cell migration into tumors, thereby impacting immune control of tumor growth. This knowledge will aid the development of strategies to promote T-cell infiltration into cancerous lesions and has the potential to markedly improve treatment outcomes.

Melanoma-initiating cells exploit M2 macrophage TGFß and arginase pathway for survival and proliferation.

M2 macrophages promote tumor growth and metastasis, but their interactions with specific tumor cell populations are poorly characterized. Using a mouse model of spontaneous melanoma, we showed that CD34- but not CD34+ tumor-initiating cells (TICs) depend on M2 macrophages for survival and proliferation. Tumor-associated macrophages (TAMs) and macrophage-conditioned media protected CD34- TICs from chemotherapy in vitro. In vivo, while inhibition of CD115 suppressed the macrophage-dependent CD34- TIC population, chemotherapy accelerated its development. The ability of TICs to respond to TAMs was acquired during melanoma progression and immediately preceded a surge in metastatic outgrowth. TAM-derived transforming growth factor-ß (TGFß) and polyamines produced via the Arginase pathway were critical for stimulation of TICs and synergized to promote their growth.

Inflammatory lymphangiogenesis: cellular mediators and functional implications.

In adult mammals, lymphatic vessels have been shown to respond to their environment by undergoing lymphangiogenesis, the formation of new lymphatic vessels from preexisting ones. Accumulating experimental and preclinical studies demonstrate that lymphangiogenesis is associated with many inflammatory diseases and may represent an attractive therapeutic target for inflammatory diseases. Thus, a better understanding of how lymphangiogenesis is regulated and contribution to inflammation is critical and may benefit clinical research targeting chronic inflammatory diseases. This review discusses the biological functions of lymphangiogenesis during inflammation and our current understanding of the key cellular players that can either support or limit lymphangiogenesis. Current data suggest that the context and time frame in which lymphangiogenesis occurs will determine its impact on the course of inflammation.

CD8 T cells regulate allergic contact dermatitis by modulating CCR2-dependent TNF/iNOS-expressing Ly6C+ CD11b+ monocytic cells.

Monocytes and their derived cells have critical roles in inflammation and immune defense. However, their function in skin diseases such as allergic contact dermatitis remains poorly defined. Using a model of contact hypersensitivity (CHS) toward 2,4-dinitrochlorobenzene, we show that Ly6C+ CD11b+ monocytic cells participate in the pathophysiology of CHS and their accumulation is regulated by effector CD8 T cells. These Ly6C+ CD11b+ monocytic cells are the primary contributors of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) and derive from Ly6C(hi)CCR2+ monocytes, as they were absent in non-inflamed skin and accumulate as a consequence of inflammation in a C-C chemokine receptor type 2 (CCR2)-dependent manner. Importantly, CCR2(-/-) mice, or wild-type mice depleted of monocytes via clodronate liposomes, display a marked decrease in TNF-α and iNOS expression accompanied by attenuated skin inflammation. Using transgenic mice and antibody depletion, we show that effector CD8 T cells regulate the accumulation of Ly6C+ CD11b+ monocytic cells through IL-17 and activate them for TNF-α and iNOS through IFN-γ. CD8 T cell-derived IFN-γ was also critical for the accumulation of the major histocompatibility complex II-expressing Ly6C+ CD11b+ subset, which expressed intermediate levels of CD11c and costimulatory molecules. Taken together, our findings provide further insight into the pathophysiology of allergic contact dermatitis by showing that CD8 T cells regulate the inflammatory cascade through TNF/iNOS-expressing Ly6C+ CD11b+ monocytic cells.

The DNA damage response induces antigen presenting cell-like functions in fibroblasts.

The DNA damage response (DDR) alerts the immune system to the danger posed by DNA damage through the induction of damage-associated molecular pattern molecules, chemokines, and ligands for activating immune receptors such as lymphocyte function-associated antigen 1 (LFA-1), NKG2D, and DNAX accessory molecule 1 (DNAM-1). Here we provide evidence that OVA(257-264) -pulsed fibroblasts gain the ability to activate naïve OT-I CD8(+) T cells in response to DNA damage. The ability of fibroblasts to activate OT-I CD8(+) T cells depended on the upregulation of ICAM-1 on fibroblasts and DNAM-1 expression of CD8(+) T cells. OVA(257-264) -pulsed fibroblasts were able to induce a protective T-cell response against B16-OVA cells in a DDR-dependent manner. Hence, the DDR may alert the immune system to the presence of potentially dangerous cells by upregulating the expression of ligands that can induce the activation of innate and adaptive immune cells.

Neutrophils contribute to inflammatory lymphangiogenesis by increasing VEGF-A bioavailability and secreting VEGF-D.

Lymphangiogenesis is an important physiological response to inflammatory insult, acting to limit inflammation. Macrophages, dendritic cells, and lymphocytes are known to drive lymphangiogenesis. In this study, we show that neutrophils recruited to sites of inflammation can also coordinate lymphangiogenesis. In the absence of B cells, intranodal lymphangiogenesis induced during prolonged inflammation as a consequence of immunization is dependent on the accumulation of neutrophils. When neutrophils are depleted in wild-type mice developing skin inflammation in response to immunization or contact hypersensitization, lymphangiogenesis is decreased and local inflammation is increased. We demonstrate that neutrophils contribute to lymphangiogenesis primarily by modulating vascular endothelial growth factor (VEGF)-A bioavailability and bioactivity and, to a lesser extent, secreting VEGF-D. We further show that neutrophils increased VEGF-A bioavailability and bioactivity via the secretion of matrix metalloproteinases 9 and heparanase. Together, these findings uncover a novel function for neutrophils as organizers of lymphangiogenesis during inflammation.

Hypertonic saline reduces vascular leakage in a mouse model of severe dengue.

Dengue (DEN) is a mosquito-borne viral disease and represents a serious public health threat and an economical burden throughout the tropics. Dengue clinical manifestations range from mild acute febrile illness to severe DEN hemorrhagic fever/DEN shock syndrome (DHF/DSS). Currently, resuscitation with large volumes of isotonic fluid remains the gold standard of care for DEN patients who develop vascular leakage and shock. Here, we investigated the ability of small volume of hypertonic saline (HTS) suspensions to control vascular permeability in a mouse model of severe DEN associated with vascular leakage. Several HTS treatment regimens were considered and our results indicated that a single bolus of 7.5% NaCl at 4 mL per kg of body weight administered at the onset of detectable vascular leakage rapidly and significantly reduced vascular leak for several days after injection. This transient reduction of vascular leakage correlated with reduced intestine and liver damage with restoration of the hepatic functions, and resulted in delayed death of the infected animals. Mechanistically, we showed that HTS did not directly impact on the viral titers but resulted in lower immune cells counts and decreased systemic levels of soluble mediators involved in vascular permeability. In addition, we demonstrated that neutrophils do not play a critical role in DEN-associated vascular leakage and that the therapeutic effect of HTS is not mediated by its impact on the neutrophil counts. Together our data indicate that HTS treatment can transiently but rapidly reduce dengue-associated vascular leakage, and support the findings of a recent clinical trial which evaluated the efficacy of a hypertonic suspension to impact on vascular permeability in DSS children.

Lymphatic vessels are essential for the removal of cholesterol from peripheral tissues by SR-BI-mediated transport of HDL.

Removal of cholesterol from peripheral tissues to the bloodstream via reverse cholesterol transport (RCT) is a process of major biological importance. Here we demonstrate that lymphatic drainage is required for RCT. We have previously shown that hypercholesterolemia in mice is associated with impaired lymphatic drainage and increased lipid accumulation in peripheral tissues. We now show that restoration of lymphatic drainage in these mice significantly improves cholesterol clearance. Conversely, obstruction of lymphatic vessels in wild-type mice significantly impairs RCT. Finally, we demonstrate using silencing RNA interference, neutralizing antibody, and transgenic mice that removal of cholesterol by lymphatic vessels is dependent on the uptake and transcytosis of HDL by scavenger receptor class B type I expressed on lymphatic endothelium. Collectively, this study challenges the current view that lymphatic endothelium is a passive exchange barrier for cholesterol transport and provides further evidence for its interplay with lipid biology in health and disease.

Expansion of cortical and medullary sinuses restrains lymph node hypertrophy during prolonged inflammation.

During inflammation, accumulation of immune cells in activated lymph nodes (LNs), coupled with a transient shutdown in lymphocyte exit, results in dramatic cellular expansion. Counter-regulatory measures to restrain LN expansion must exist and may include re-establishment of lymphocyte egress to steady-state levels. Indeed, we show in a murine model that egress of lymphocytes from LNs was returned to steady-state levels during prolonged inflammation following initial retention. This restoration in lymphocyte egress was supported by a preferential expansion of cortical and medullary sinuses during late inflammation. Cortical and medullary sinus remodeling during late inflammation was dependent on temporal and spatial changes in vascular endothelial growth factor-A distribution. Specifically, its expression was restricted to the subcapsular space of the LN during early inflammation, whereas its expression was concentrated in the paracortical and medullary regions of the LN at later stages. We next showed that this process was mostly driven by the synergistic cross-talk between fibroblastic reticular cells and interstitial flow. Our data shed new light on the biological significance of LN lymphangiogenesis during prolonged inflammation and further underscore the collaborative roles of stromal cells, immune cells, and interstitial flow in modulating LN plasticity and function.

Impact of postgrafting immunosuppressive regimens on nonrelapse mortality and survival after nonmyeloablative allogeneic hematopoietic stem cell transplant using the fludarabine and low-dose total-body irradiation 200-cGy.

The development of nonmyeloablative (NM) hematopoietic cell transplantation (HCT) has extended the potential curative treatment option of allografting to patients in whom it was previously contraindicated because of advanced age or comorbidity. Acute and chronic graft versus host disease (GVHD) and its consequent nonrelapse mortality (NRM), remains the major limitation of NM HCT. In this report, we analyzed the outcome of 67 patients (median age, 45 years) with hematologic diseases receiving NM conditioning with fludarabine 90 mg/m(2) and total body irradiation (TBI) 200-cGy, followed by filgrastim-mobilized peripheral blood stem cell transplant from HLA identical (n = 61), 5/6 antigen-matched related (n = 1), 6/6 antigen-matched unrelated (n = 3), and 5/6 antigen-matched unrelated (n = 2) donors. The first cohort of 21 patients were given cyclosporine (CSP) and mycophenolate mofetil (MMF) as postgrafting immunosuppression, whereas the subsequent cohort was given additional methotrexate (MTX) and extended duration of CSP/MMF prophylaxis in an attempt to reduce graft-versus-host disease (GVHD). Sixty-four (95%) patients engrafted and 3 (5%) had secondary graft failure. Myelosuppression was moderate with neutrophil counts not declining below 500/microL in approximately 25% of patients, and with more than half of the patients not requiring any blood or platelet transfusion. The 2-year cumulative interval (CI) of grade II-IV, grade III-IV acute GVHD and chronic GVHD were 49%, 30%, and 34%, respectively. The 2-year probability of NRM, overall (OS), and progression-free (PFS) survival were 27%, 43%, and 28%, respectively. GVHD-related death accounted for 85% of NRM. Compared with patients receiving CSP/MMF, patients receiving extended duration of CSP/MMF with additional MTX in postgrafting immunosuppression had a significantly lower risk of grade III-IV acute GVHD (CI 20% versus 52%; P = .009) and NRM (CI at 2 years: 11% versus 62%; P < .001), without any significant adverse impact on the risk of relapse (CI at 2 years: 59% versus 33%; P = .174) Subgroup analysis of a cohort of patients given MTX/CSP/MMF showed that patients with "standard risk" diseases (n = 21) had a 3-year OS and PFS of 85% and 65%, respectively. This compares favorably to the 41% (P = .02) and 23% (P = .03) OS and PFS, respectively, in patients with "high-risk" diseases (n = 25). In conclusion, the addition of MTX onto the current postgrafting immunosuppression regimen with extended CSP/MMF prophylaxis duration provides more effective protection against severe GVHD, and is associated with more favorable outcome in patients receiving NM fludarabine/TBI conditioning than in patients receiving fludarabine/TBI conditioning with CSP and MMF without MTX.

CYP1A1 polymorphisms and risk of lung cancer in non-smoking Chinese women: influence of environmental tobacco smoke exposure and GSTM1/T1 genetic variation.

OBJECTIVE: We examined whether polymorphisms of CYP1A1, which plays a role in the metabolic activation of polycyclic aromatic hydrocarbons (PAHs), confer an increased risk of lung cancer in lifetime non-smoking Chinese women. METHODS: A total of 126 incident lung cancer cases, of which 87.7 were pathologically confirmed, and 162 age-matched hospital controls were included. CYP1A1 MspI and Ile(462)Val polymorphisms were genotyped and tested for association with this disease. RESULTS: An elevated risk of lung cancer was observed among individuals with the MspI CC (OR=1.7, 95 CI=0.9-3.3) and Ile(462)Val ValVal genotypes (OR=2.8, 95 CI=1.1-7.6). After stratifying by environmental tobacco smoke (ETS) exposure, the risk of lung cancer associated with both polymorphisms was higher among individuals with lower exposure to ETS, compared with those who reported at least weekly exposure. Individuals with the MspI CC genotype showed a two-fold higher risk of lung cancer if they were also null for either GSTM1 or T1 (OR=2.3, 95CI=1.0-5.0 and OR=2.7, 95 CI=1.1-6.9, respectively, compared to other genotype combinations combined). CONCLUSIONS: CYP1A1 is a susceptibility gene for lung cancer among non-smoking Asian women and this association can be influenced by ETS exposure and genetic variation at GST genes.