IFNs Reset the Differential Capacity of Human Monocyte Subsets to Produce IL-12 in Response to Microbial Stimulation.

Human primary monocytes are composed of a minor, more mature CD16+(CD14low/neg) population and a major CD16neg(CD14+) subset. The specific functions of CD16+ versus CD16neg monocytes in steady state or inflammation remain poorly understood. In previous work, we found that IL-12 is selectively produced by the CD16+ subset in response to the protozoan pathogen, Toxoplasma gondii In this study, we demonstrated that this differential responsiveness correlates with the presence of an IFN-induced transcriptional signature in CD16+ monocytes already at baseline. Consistent with this observation, we found that in vitro IFN-γ priming overcomes the defect in the IL-12 response of the CD16neg subset. In contrast, pretreatment with IFN-γ had only a minor effect on IL-12p40 secretion by the CD16+ population. Moreover, inhibition of the mTOR pathway also selectively increased the IL-12 response in CD16neg but not in CD16+ monocytes. We further demonstrate that in contrast to IFN-γ, IFN-α fails to promote IL-12 production by the CD16neg subset and blocks the effect of IFN-γ priming. Based on these observations, we propose that the acquisition of IL-12 responsiveness by peripheral blood monocyte subsets depends on extrinsic signals experienced during their developmental progression in vivo. This process can be overridden during inflammation by the opposing regulatory effects of type I and II IFN as well as the mTOR inhibition.

Production of a cellular product consisting of monocytes stimulated with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for human use.

BACKGROUND: Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood. METHODS: Counter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction. RESULTS: Both iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. CONCLUSION: Large scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.

The IL-12 Response of Primary Human Dendritic Cells and Monocytes to Toxoplasma gondii Is Stimulated by Phagocytosis of Live Parasites Rather Than Host Cell Invasion.

As a major natural host for Toxoplasma gondii, the mouse is widely used for the study of the immune response to this medically important protozoan parasite. However, murine innate recognition of toxoplasma depends on the interaction of parasite profilin with TLR11 and TLR12, two receptors that are functionally absent in humans. This raises the question of how human cells detect and respond to T. gondii. In this study, we show that primary monocytes and dendritic cells from peripheral blood of healthy donors produce IL-12 and other proinflammatory cytokines when exposed to toxoplasma tachyzoites. Cell fractionation studies determined that IL-12 and TNF-α secretion is limited to CD16(+) monocytes and the CD1c(+) subset of dendritic cells. In direct contrast to their murine counterparts, human myeloid cells fail to respond to soluble tachyzoite extracts and instead require contact with live parasites. Importantly, we found that tachyzoite phagocytosis, but not host cell invasion, is required for cytokine induction. Together these findings identify CD16(+) monocytes and CD1c(+) dendritic cells as the major myeloid subsets in human blood-producing innate cytokines in response to T. gondii and demonstrate an unappreciated requirement for phagocytosis of live parasites in that process. This form of pathogen sensing is distinct from that used by mice, possibly reflecting a direct involvement of rodents and not humans in the parasite life cycle.

Heme Oxygenase-1 Regulation of Matrix Metalloproteinase-1 Expression Underlies Distinct Disease Profiles in Tuberculosis.

Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients.

Characterization of CAR T cell expansion and cytotoxic potential during Ex Vivo manufacturing using image-based cytometry.

Since the FDA approval of two Chimeric Antigen Receptor (CAR) T cell therapies against CD19+ malignancies, there has been significant interest in adapting CAR technology to other diseases. As such, the ability to simultaneously monitor manufacturing criteria and functional characteristics of multiple CAR T cell products by a single instrument would likely accelerate the development of candidate therapies. Here, we demonstrate that image-based cytometry yields high-throughput measurements of CAR T cell proliferation and size, and captures the kinetics of in vitro antigen-specific CAR T cell-mediated killing. The data acquired and analyzed by the image cytometer are congruent with results derived from conventional technologies when tested contemporaneously. Moreover, the use of bright-field and fluorescence microscopy by the image cytometer provides kinetic measurements and rapid data acquisition, which are direct advantages over industry standard instruments. Together, image cytometry enables fast, reproducible measurements of CAR T cell manufacturing criteria and effector function, which can greatly facilitate the evaluation of novel CARs with therapeutic potential.

Inflammatory monocytes expressing tissue factor drive SIV and HIV coagulopathy.

In HIV infection, persistent inflammation despite effective antiretroviral therapy is linked to increased risk of noninfectious chronic complications such as cardiovascular and thromboembolic disease. A better understanding of inflammatory and coagulation pathways in HIV infection is needed to optimize clinical care. Markers of monocyte activation and coagulation independently predict morbidity and mortality associated with non-AIDS events. We identified a specific subset of monocytes that express tissue factor (TF), persist after virological suppression, and trigger the coagulation cascade by activating factor X. This subset of monocytes expressing TF had a distinct gene signature with up-regulated innate immune markers and evidence of robust production of multiple proinflammatory cytokines, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and IL-6, ex vivo and in vitro upon lipopolysaccharide stimulation. We validated our findings in a nonhuman primate model, showing that TF-expressing inflammatory monocytes were associated with simian immunodeficiency virus (SIV)-related coagulopathy in the progressive [pigtail macaques (PTMs)] but not in the nonpathogenic (African green monkeys) SIV infection model. Last, Ixolaris, an anticoagulant that inhibits the TF pathway, was tested and potently blocked functional TF activity in vitro in HIV and SIV infection without affecting monocyte responses to Toll-like receptor stimulation. Strikingly, in vivo treatment of SIV-infected PTMs with Ixolaris was associated with significant decreases in D-dimer and immune activation. These data suggest that TF-expressing monocytes are at the epicenter of inflammation and coagulation in chronic HIV and SIV infection and may represent a potential therapeutic target.

IFNs Reset the Differential Capacity of Human Monocyte Subsets to Produce IL-12 in Response to Microbial Stimulation

Human primary monocytes are composed of a minor, more mature CD16+(CD14low/neg) population and a major CD16neg(CD14+) subset. The specific functions of CD16+ versus CD16neg monocytes in steady state or inflammation remain poorly understood. In previous work, we found that IL-12 is selectively produced by the CD16+ subset in response to the protozoan pathogen, Toxoplasma gondii In this study, we demonstrated that this differential responsiveness correlates with the presence of an IFN-induced transcriptional signature in CD16+ monocytes already at baseline. Consistent with this observation, we found that in vitro IFN-γ priming overcomes the defect in the IL-12 response of the CD16neg subset. In contrast, pretreatment with IFN-γ had only a minor effect on IL-12p40 secretion by the CD16+ population. Moreover, inhibition of the mTOR pathway also selectively increased the IL-12 response in CD16neg but not in CD16+ monocytes. We further demonstrate that in contrast to IFN-γ, IFN-α fails to promote IL-12 production by the CD16neg subset and blocks the effect of IFN-γ priming. Based on these observations, we propose that the acquisition of IL-12 responsiveness by peripheral blood monocyte subsets depends on extrinsic signals experienced during their developmental progression in vivo. This process can be overridden during inflammation by the opposing regulatory effects of type I and II IFN as well as the mTOR inhibition.

TLR9 and TLR7 agonists mediate distinct type I IFN responses in humans and nonhuman primates in vitro and in vivo.

Human I-IFNs include IFN-ß and 13 independently regulated subtypes of IFN-α (I-IFNs). TLR7 and -9 induce I-IFNs, but it is unknown whether their subtype repertoire is similar. This study used new PCR arrays that selectively amplify individual I-IFN subtype genes of human and nonhuman primates to characterize the TLR7- and -9-mediated IFN response in vitro and in vivo. We show that in human PBMCs, TLR7 agonists induce a rapid burst of I-IFN transcripts, consisting primarily of IFN-α1/13, -α2, and -α14. In contrast, TLR9 agonists, regardless of the type used (CpG C-, B-, or D-ODN), prompted slower but sustained expression of IFN-α1/13, -α2, -α7, -α8, -α10, -α14, -α16, and -α21. These qualitative differences were translated downstream as differences in the pattern of IFN-inducible genes. In macaque PBMCs, imiquimod produced a short burst of IFN mRNA, dominated by IFN-α8, whereas C- or D-ODN induced a greater than tenfold increase in transcripts for all I-IFN subtypes by 12 h of culture. Differences were more evident in vivo, where TLR7 and -9 agonists induced significantly different levels of I-IFN transcripts in skin. Although the rates of gene transcription differed significantly for individual TLR9 agonists, their IFN-α subtype signature was almost identical, indicating that the type of receptor dictates the quality of the I-IFN response in vitro and in vivo. These results may underlie the differential therapeutic effects of TLR7 and -9 agonists and should inform future clinical studies.