Autologous Monocyte-derived Macrophages Research Articles

Abstract PO2-04-08: Translational insights from a phase 1, first-in-human (FIH) clinical trial of the anti-HER2 CAR macrophage CT-0508 in participants with HER2 positive metastatic breast cancer and other HER2 overexpressing solid tumors

Abstract Background: In pre-clinical studies, CAR macrophages (CAR-M) phagocytose tumor cells, activate the tumor microenvironment (TME), recruit T cells, and induce anti-tumor T cell immunity. CT-0508 is a first-in-class CAR-M product comprised of autologous monocyte-derived macrophages expressing an anti-HER2 CAR. In pre-clinical models, anti-HER2 CAR-M was able to control the growth of syngeneic metastatic HER2+ breast cancer. Here we present preliminary clinical results and translational data from Group 1 of the CT-0508 Phase 1 FIH study. Methods: This multi-center, open-label study is evaluating CT-0508’s safety, tolerability, and manufacturing feasibility in 18 participants with advanced solid tumors overexpressing HER2 with progression on prior therapies. Monocytes are isolated from mobilized apheresis products, differentiated into macrophages, and engineered with an anti-HER2 CAR. Group 1 participants (n = 9) receive a fractionated dose on days 1, 3, 5 and Group 2 participants (n = 9) receive the full dose on day 1. CT-0508 is administered without preparative chemotherapy. Serial blood samples and biopsies (baseline and 2 post-treatment) are collected to investigate safety, pharmacokinetics, and mechanism of action. AU565 and 4T1-HER2 cell lines were utilized to model human and murine breast cancer, respectively, in vitro and in vivo. Results: Nine participants (6F/3M) have been treated in Group 1, comprising breast (4), esophageal (2), cholangiocarcinoma, ovarian, and parotid gland cancers, with a median age of 58. Participants had received a median of 3 (range, 2-11) prior lines of therapy; 8 had received prior anti-HER2 therapy. CT-0508 was successfully manufactured and well tolerated with no dose-limiting toxicities. Three related SAEs occurred in 2 participants: grade 1 CRS with hospitalization for monitoring and grade 2 infusion reaction that resolved within 1 hour were reported in one participant. Grade 2 CRS with fever and hypoxia occurred in another participant and resolved within ~ 72 hours. Five additional participants experienced Grade 1-2 CRS and/or infusion reactions with rapid resolution. There were no cases of Grade 3 or 4 CRS. There were no major organ toxicities. Post-infusion cytokines were transiently elevated in most participants enrolled in group 1 and were self-limiting. Four of the 7 participants evaluated had stable disease. CT-0508 was transiently detectable in the blood and was detected in the TME of 8/9 participants. CT-0508 modulated the TME, leading to myeloid cell activation, effector T cell infiltration, activation, and proliferation. TCR sequencing demonstrated newly expanding T cell clones in the blood post-treatment that accumulated within the TME, suggesting expansion of tumor-reactive T cells upon CT-0508 infusion. Data from participants enrolled in Group 1 will be presented. Most of these patients were breast cancer patients with HER2 overexpressing metastatic disease. Conclusions: CT-0508 was feasible to manufacture and had acceptable safety and tolerability. Early correlative data demonstrate trafficking, TME modulation, and induction of anti-tumor T cell immunity in participants with HER2 overexpressing solid tumors including metastatic breast cancer. The study is actively enrolling (NCT04660929). Citation Format: Yara Abdou, Joanne Mortimer, Paula Pohlmann, Melissa Johnson, Richard Maziarz, Jennifer M. Specht, Claire Dees, Naoto Ueno, Yuan Yuan, Mathew Angelos, Saar Gill, Olga Shestova, Jonathon Serody, Saul Priceman, Rehman Qureshi, Poonam Sonawane, Stefano Pierini, Maria Cecilia Oliveira-Nunes, Daniel Cushing, Michael Klichinsky, Thomas Condamine, Ramona Swaby, Kim Reiss. Translational insights from a phase 1, first-in-human (FIH) clinical trial of the anti-HER2 CAR macrophage CT-0508 in participants with HER2 positive metastatic breast cancer and other HER2 overexpressing solid tumors [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-04-08.

Mycobacterium intracellulare induces a Th17 immune response via M1-like macrophage polarization in canine peripheral blood mononuclear cells

Mycobacterium avium-intracellulare complex (MAC) is one of the most prevalent pathogenic nontuberculous mycobacteria that cause chronic pulmonary disease. The prevalence of MAC infection has been rising globally in a wide range of hosts, including companion animals. MAC infection has been reported in dogs; however, little is known about interaction between MAC and dogs, especially in immune response. In this study, we investigated the host immune response driven by M. intracellulare using the co-culture system of canine T helper cells and autologous monocyte-derived macrophages (MDMs). Transcriptomic analysis revealed that canine MDMs differentiated into M1-like macrophages after M. intracellulare infection and the macrophages secreted molecules that induced Th1/Th17 cell polarization. Furthermore, canine lymphocytes co-cultured with M. intracellulare-infected macrophages induced the adaptive Th17 responses after 5 days. Taken together, our results indicate that M. intracellulare elicits a Th17 response through macrophage activation in this system. Those findings might help the understanding of the canine immune response to MAC infection and diminishing the potential zoonotic risk in One Health aspect.

The tetraspanin CD151 is an activation molecule that characterizes M. tuberculosis- specific effector CD4+ T cells

Abstract Tetraspanins are a family of membrane proteins regulating cell morphology, motility, fusion, and signaling. CD151 is expressed on T cells that stabilizes the immune synapse during antigen recognition, engages in integrin signaling, and primes T cell activation. We have recently demonstrated that CD151 expression leads to increased proliferation and activation in human CD4+ T cells. Given its important functional role, we hypothesized that CD151 may be elevated on M. tb-specific effector CD4+ T cells. We assessed CD151 on ex vivo PBMCs from PPD+ donors (Purified Protein Derivative from M. tb) stimulated with M. tb H37Rv whole cell lysate. CD151+ frequencies were elevated on IFN-γ+ (52 ± 6% vs. 17 ± 2%, P<0.001) and TNF-α+ (74 ± 6% vs. 17 ± 2%, P<0.0001) CD4+ T cells compared to overall baseline levels. To assess the functionality of CD151 on M. tb-specific CD4+ T cells, we established an ex vivo culture model with M. tb-infected monocyte-derived macrophages (MDMs) in the presence of autologous PPD+ or PPD− donor CD4+ T cells. Upon day 6 of culture, PPD+ donor cultures, proliferating Ki67+ CD4+T cells were elevated in CD151 and CD25, but diminished in CD57, cell surface expression. Additionally, enhanced frequencies of CD151+ Ki67+CD4+T cells were associated with reduced M. tb growth within autologous MDMs during ex vivo culture. In summary, CD151 is a novel T cell activation marker that is elevated on M. tb- specific CD4+ T cells, and is associated with diminished M. tb growth in culture. CD151 could thus be an interesting candidate to supplement the existing TB-TAM diagnostic (T cell activation marker of tuberculosis) – the first to assess the quality of the CD4+ effector T-cell response to M. tb – and may guide future CD4+ T-cell based vaccine strategies.

Helicobacter pylori Infection Inhibits Phagocyte Clearance of Apoptotic Gastric Epithelial Cells

Increased apoptotic death of gastric epithelial cells is a hallmark of Helicobacter pylori infection, and altered epithelial cell turnover is an important contributor to gastric carcinogenesis. To address the fate of apoptotic gastric epithelial cells and their role in H. pylori mucosal disease, we investigated phagocyte clearance of apoptotic gastric epithelial cells in H. pylori infection. Human gastric mononuclear phagocytes were analyzed for their ability to take up apoptotic epithelial cells (AECs) in vivo using immunofluorescence analysis. We then used primary human gastric epithelial cells induced to undergo apoptosis by exposure to live H. pylori to study apoptotic cell uptake by autologous monocyte-derived macrophages. We show that HLA-DR(+) mononuclear phagocytes in human gastric mucosa contain cytokeratin-positive and TUNEL-positive AEC material, indicating that gastric phagocytes are involved in AEC clearance. We further show that H. pylori both increased apoptosis in primary gastric epithelial cells and decreased phagocytosis of the AECs by autologous monocyte-derived macrophages. Reduced macrophage clearance of apoptotic cells was mediated in part by H. pylori-induced macrophage TNF-α, which was expressed at higher levels in H. pylori-infected, compared with uninfected, gastric mucosa. Importantly, we show that H. pylori-infected gastric mucosa contained significantly higher numbers of AECs and higher levels of nonphagocytosed TUNEL-positive apoptotic material, consistent with a defect in apoptotic cell clearance. Thus, as shown in other autoimmune and chronic inflammatory diseases, insufficient phagocyte clearance may contribute to the chronic and self-perpetuating inflammation in human H. pylori infection.

Human Adipose Tissue Macrophages Display Activation of Cancer-related Pathways

Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.

Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis

The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (MΦ) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDMΦ) as target cells. Various B. abortus antigen preparations were tested including whole γ-irradiated B. abortus bacteria (γBA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDMΦ targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with γBA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4 +, CD8 − cells indicating that the observed cytotoxic responses were most likely due to an inflammatory T h1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle.

Monocyte-derived dendritic cells as a model for the study of HIV-1 infection: productive infection and phenotypic changes during culture in human serum.

Dendritic cells (DC) have been implicated in the initial selection for macrophage-tropic HIV-1 during transmission and in the generation of high-level virus replication during interactions with CD4 T cells. The role of DC as viral reservoirs and the extent of productive infection is unclear, but the ability to generate large numbers of DC from blood monocytes has produced a tractable model for study of DC-HIV-1 interactions. When cultured in granulocyte-macrophage colony stimulating factor and IL-4, sorted CD14+ monocytes rapidly lost phagocytic function for both 93 nm and 977 nm latex particles and developed the surface markers and function of DC. After 7 days, when returned to medium containing human serum without cytokines, some monocyte-derived dendritic cells (MDDC) became adherent, but retained the costimulatory markers CD80 and CD86 and continued to express CD83 and CD40. The MDDC stimulated allogeneic CD4 T cells, did not express new macrophage markers and remained non-phagocytic. With or without TNF-alpha, MDDC generated in cytokines were infected by macrophage and T cell-tropic virus and produced higher reverse transcriptase levels than did the autologous monocyte-derived macrophages (MDM). When added to T cells, the infected MDDC were able to infect T cells with a wider range of viral isolates than were MDM.

The presence and absence of histocompatibility antigens in HIV type 1 produced by autologous blood-derived macrophages and peripheral blood lymphoblasts.

Acquisition of cellular proteins by HIV-1 virions is known to alter the physiology of the virus in vitro. Reported studies of this aspect have been largely limited to transformed T cell lines. In this study, we investigated the incorporation of major histocompatibility antigens (HLAs) on a primary macrophage-tropic isolate, HIV-1ADA, grown from autologous monocyte-derived macrophages (MDMs) and peripheral blood mononuclear cells (PBMCs). A virus precipitation assay (VPA) demonstrated that HIV-1ADA grown from PBMCs incorporated substantial amounts of HLA class I (alpha chain and beta2m) and DR antigens, comparable with a laboratory strain, HIV-1MN, grown from the same host cells. HIV-1ADA, however, grown from MDMs incorporated significantly lower amounts of HLAI and -II antigens despite the fact that the infected MDMs were found to express significant amounts of HLA antigens. The lack of incorporation of these important immunomodulatory cell surface proteins may be yet another unique characteristic of macrophage-tropic isolates and suggests a possible role in their biology and or immunology.

Compensation for decreased expression of B7 molecules on Leishmania infantum-infected canine macrophages results in restoration of parasite-specific T-cell proliferation and gamma interferon production.

Infection of humans and dogs by Leishmania infantum may result in visceral leishmaniasis, which is characterized by impaired T-cell-mediated immune responses to parasite antigens. Dogs are natural hosts of Leishmania parasites and play an important role in the transmission of the parasites to humans. In an effort to characterize the immune response in dogs infected with this intracellular pathogen, we examined how infection with L. infantum affects canine macrophages and the consequences for T-cell activation in vitro. We showed that the proliferation of T-cell lines to cognate antigen decreases to background levels when infected autologous monocyte-derived macrophages are used as antigen-presenting cells (APC). The observed reduction of antigen-specific T-cell proliferation was shown to be dependent on the parasite load and to require cell-to-cell interaction of T cells with the infected APC. In addition, we observed a decreased expression of costimulatory B7 molecules on infected monocyte-derived macrophages. The expression of other surface molecules involved in T-cell activation, such as major histocompatibility complex class I and class II, on these cells did not change upon infection, whereas the expression of intracellular adhesion molecule 1 was marginally increased. Compensation for the decreased expression of B7 molecules by the addition of B7-transfected cells resulted in the restoration of cell proliferation and gamma interferon (IFN-gamma) production by a Leishmania-specific T-cell line. These results showed that for the activation of parasite-specific canine T cells producing IFN-gamma, which are most likely involved in protective immunity, sufficient expression of B7 molecules on infected macrophages is required. Provision of costimulatory molecules may be an approach for immunotherapy of leishmaniaisis as well as for vaccine development.