scholarly journals No cell is an island: circulating T cell:monocyte complexes are markers of immune perturbations

2018 ◽  
Author(s):  
Julie G. Burel ◽  
Mikhail Pomaznoy ◽  
Cecilia S. Lindestam Arlehamn ◽  
Daniela Weiskopf ◽  
Ricardo da Silva Antunes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Immune Responses ◽  
Human Blood ◽  
Significant Proportion ◽  
Complex Frequency ◽  
The Common ◽  
First Time ◽  
High Resolution Microscopy

AbstractOur results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artefacts and thus ignored in data acquisition and analysis, are the result of true biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell:monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be revisited.

scholarly journals Circulating T cell-monocyte complexes are markers of immune perturbations

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Julie G Burel ◽  
Mikhail Pomaznoy ◽  
Cecilia S Lindestam Arlehamn ◽  
Daniela Weiskopf ◽  
Ricardo da Silva Antunes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Immune Responses ◽  
Human Blood ◽  
Significant Proportion ◽  
Complex Frequency ◽  
The Common ◽  
First Time ◽  
High Resolution Microscopy

Our results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artifacts and thus ignored in data acquisition and analysis, are the result of biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell-monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be re-visited.

scholarly journals Neutrophil subtypes shape HIV-specific CD8 T-cell responses after vaccinia virus infection

npj Vaccines ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Mauro Di Pilato ◽  
Miguel Palomino-Segura ◽  
Ernesto Mejías-Pérez ◽  
Carmen E. Gómez ◽  
Andrea Rubio-Ponce ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Vaccinia Virus ◽  
Adaptive Immune System ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Adaptive Immune

AbstractNeutrophils are innate immune cells involved in the elimination of pathogens and can also induce adaptive immune responses. Nα and Nβ neutrophils have been described with distinct in vitro capacity to generate antigen-specific CD8 T-cell responses. However, how these cell types exert their role in vivo and how manipulation of Nβ/Nα ratio influences vaccine-mediated immune responses are not known. In this study, we find that these neutrophil subtypes show distinct migratory and motility patterns and different ability to interact with CD8 T cells in the spleen following vaccinia virus (VACV) infection. Moreover, after analysis of adhesion, inflammatory, and migration markers, we observe that Nβ neutrophils overexpress the α4β1 integrin compared to Nα. Finally, by inhibiting α4β1 integrin, we increase the Nβ/Nα ratio and enhance CD8 T-cell responses to HIV VACV-delivered antigens. These findings provide significant advancements in the comprehension of neutrophil-based control of adaptive immune system and their relevance in vaccine design.

scholarly journals Efficient Induction of Primary and Secondary T Cell-Dependent Immune Responses In Vivo in the Absence of Functional IL-2 and IL-15 Receptors

2003 ◽  
Vol 170 (1) ◽  
pp. 236-242 ◽  
Author(s):  
Aixin Yu ◽  
Jiehao Zhou ◽  
Norman Marten ◽  
Cornelia C. Bergmann ◽  
Michele Mammolenti ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Efficient Induction

scholarly journals Optimized Adenovirus-Antibody Complexes Stimulate Strong Cellular and Humoral Immune Responses against an Encoded Antigen in Naïve Mice and Those with Preexisting Immunity

2011 ◽  
Vol 19 (1) ◽  
pp. 84-95 ◽  
Author(s):  
Jin Huk Choi ◽  
Joe Dekker ◽  
Stephen C. Schafer ◽  
Jobby John ◽  
Craig E. Whitfill ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Immune Responses ◽  
Neutralizing Antibodies ◽  
T Cell Responses ◽  
Transduction Efficiency ◽  
Virus Antibody ◽  
Cell Responses

ABSTRACTThe immune response to recombinant adenoviruses is the most significant impediment to their clinical use for immunization. We test the hypothesis that specific virus-antibody combinations dictate the type of immune response generated against the adenovirus and its transgene cassette under certain physiological conditions while minimizing vector-induced toxicity.In vitroandin vivoassays were used to characterize the transduction efficiency, the T and B cell responses to the encoded transgene, and the toxicity of 1 × 1011adenovirus particles mixed with different concentrations of neutralizing antibodies. Complexes formed at concentrations of 500 to 0.05 times the 50% neutralizing dose (ND50) elicited strong virus- and transgene-specific T cell responses. The 0.05-ND50formulation elicited measurable anti-transgene antibodies that were similar to those of virus alone (P= 0.07). This preparation also elicited very strong transgene-specific memory T cell responses (28.6 ± 5.2% proliferation versus 7.7 ± 1.4% for virus alone). Preexisting immunity significantly reduced all responses elicited by these formulations. Although lower concentrations (0.005 and 0.0005 ND50) of antibody did not improve cellular and humoral responses in naïve animals, they did promote strong cellular (0.005 ND50) and humoral (0.0005 ND50) responses in mice with preexisting immunity. Some virus-antibody complexes may improve the potency of adenovirus-based vaccines in naïve individuals, while others can sway the immune response in those with preexisting immunity. Additional studies with these and other virus-antibody ratios may be useful to predict and model the type of immune responses generated against a transgene in those with different levels of exposure to adenovirus.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

scholarly journals Targeting cryptic epitope with modified antigen coupled to the surface of liposomes induces strong antitumor CD8 T-cell immune responses in vivo

2015 ◽  
Vol 34 (6) ◽  
pp. 2827-2836 ◽  
Author(s):  
YUTAKA HORIUCHI ◽  
AKIRA TAKAGI ◽  
TETSUYA UCHIDA ◽  
TOSHITAKA AKATSUKA
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cell ◽  
Cryptic Epitope ◽  
T Cell Immune Responses

scholarly journals Mapping of the autophagosomal degradome identifies IL-7Rα as key cargo in proliferating CD4+ T-cells

2021 ◽  
Author(s):  
Dingxi Zhou ◽  
Mariana Borsa ◽  
Daniel J. Puleston ◽  
Susanne Zellner ◽  
Jesusa Capera ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Surface Expression ◽  
T Cell Proliferation ◽  
Gamma Chain ◽  
Direct Identification ◽  
The Common ◽  
First Time

CD4+ T cells orchestrate both humoral and cytotoxic immune responses. While it is known that CD4+ T cell proliferation relies on autophagy, direct identification of the autophagosomal cargo involved is still missing. Here, we created a transgenic mouse model, which, for the first time, enables us to directly map the proteinaceous content of autophagosomes in any primary cell by LC3 proximity labelling. IL-7Rα, a cytokine receptor mostly found in naive and memory T cells, was reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy showed increased IL-7Rα surface expression, while no defect in internalisation was observed. Mechanistically, excessive surface IL-7Rα sequestrates the common gamma chain, impairing the IL-2R assembly and downstream signalling crucial for T cell proliferation. This study provides proof-of-principle that key autophagy substrates can be reliably identified with this model to help mechanistically unravel autophagy's contribution to healthy physiology and disease.

scholarly journals 228 Antagonistic pH-selective VISTA antibody SNS-101 potentiates anti-PD-1/PD-L1-induced anti-tumor immunity

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A243-A243
Author(s):  
Thomas Thisted ◽  
Arnab Mukherjee ◽  
Kanam Malhotra ◽  
Zuzana Biesova ◽  
Yuliya Kleschenko ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Activation ◽  
Drug Disposition ◽  
Checkpoint Inhibitors ◽  
Tumor Rejection ◽  
Therapeutic Window ◽  
Drug Candidate

BackgroundImmunotherapies, especially immune checkpoint inhibitors, have become a cornerstone of cancer treatment. Remarkable clinical responses have been observed blocking the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis across a spectrum of indications. However, innate and/or acquired resistance to anti-PD-1 blockade remains a major challenge. V-domain Ig suppressor of T-cell activation (VISTA) is a B7-family member, which promotes T-cell and myeloid quiescence and represents a promising target, particularly in combination with anti-PD-1/PD-L1 treatment. Recently, the interaction of VISTA with its receptor PSGL-1 was demonstrated to be significantly enhanced by the acidic tumor microenvironment (TME). As VISTA is highly expressed on myeloid cells, including those in the blood, antibodies binding VISTA at physiological pH 7.4 could result in rapid elimination from circulation through targeted-mediated drug disposition, making efficacious drug occupancy levels difficult to reach and potentially narrowing the therapeutic window. An antibody engineered to selectively bind and block VISTA at low pH in the TME may therefore be an ideal drug candidate.MethodsIn this study, fully human anti-VISTA antibodies were generated through pH-selective enrichment strategies of a yeast-based display library comprising highly diverse synthetic immune repertoires. The ‘parental’ antibodies have been extensively characterized using in vitro flow-cytometry, surface-plasmon resonance (SPR) and PSGL-1/VISTA inhibition assays in primary human CD4 and CD8 T-cells at pH 6.0 and pH 7.4. Eight parental antibodies were identified and tested for combinatorial efficacy with anti-PD-1 in vivo in human VISTA knock-in mice inoculated with syngeneic MC-38 tumors. These antibodies underwent further optimization for enhanced binding affinity at pH 6.0 and decreased binding at pH 7.4. ‘Progeny’ antibody ranking was based on the same in vitro and in vivo characterization as parental antibodies.ResultsEighty four parental antibodies were initially discovered. Flow-cytometry and SPR analysis revealed candidates displaying pH-dependent binding to endogenously expressed native VISTA on cells, and a PSGL-1/VISTA inhibition assay at pH 6.0 was run to identify and rank potent interface blockers. Eight candidate antibodies were tested in an in vivo intervention study in combination with anti-murine PD-1 demonstrating varied combinatorial efficacy with a subset leading to superior tumor rejection. Characterization of optimized progeny antibodies led to identification of anti-VISTA antibody SNS-101.ConclusionsEnrichment of highly diverse antibody libraries led to the identification of a pH-selective inhibitory anti-VISTA antibody SNS-101, which exerts excellent combinability with anti-PD-1 leading to superior anti-tumor activity in a mouse model.

scholarly journals Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

2020 ◽  
Vol 8 (2) ◽  
pp. e001250
Author(s):  
Benson Chellakkan Selvanesan ◽  
Kiran Meena ◽  
Amanda Beck ◽  
Lydie Meheus ◽  
Olaya Lara ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Immune Cells ◽  
Mouse Tumor ◽  
Epitope Spreading ◽  
Stromal Compartment ◽  
Positive Effects

BackgroundTreatments for pancreatic ductal adenocarcinoma are poorly effective, at least partly due to the tumor’s immune-suppressive stromal compartment. New evidence of positive effects on immune responses in the tumor microenvironment (TME), compelled us to test the combination of gemcitabine (GEM), a standard chemotherapeutic for pancreatic cancer, with nicotinamide (NAM), the amide form of niacin (vitamin B3), in mice with pancreatic cancer.MethodsVarious mouse tumor models of pancreatic cancer, that is, orthotopic Panc-02 and KPC (KrasG12D, p53R172H, Pdx1-Cre) grafts, were treated alternately with NAM and GEM for 2 weeks, and the effects on efficacy, survival, stromal architecture and tumor-infiltrating immune cells was examined by immunohistochemistry (IHC), flow cytometry, Enzyme-linked immunospot (ELISPOT), T cell depletions in vivo, Nanostring analysis and RNAscope.ResultsA significant reduction in tumor weight and number of metastases was found, as well as a significant improved survival of the NAM+GEM group compared with all control groups. IHC and flow cytometry showed a significant decrease in tumor-associated macrophages and myeloid-derived suppressor cells in the tumors of NAM+GEM-treated mice. This correlated with a significant increase in the number of CD4 and CD8 T cells of NAM+GEM-treated tumors, and CD4 and CD8 T cell responses to tumor-associated antigen survivin, most likely through epitope spreading. In vivo depletions of T cells demonstrated the involvement of CD4 T cells in the eradication of the tumor by NAM+GEM treatment. In addition, remodeling of the tumor stroma was observed with decreased collagen I and lower expression of hyaluronic acid binding protein, reorganization of the immune cells into lymph node like structures and CD31 positive vessels. Expression profiling for a panel of immuno-oncology genes revealed significant changes in genes involved in migration and activation of T cells, attraction of dendritic cells and epitope spreading.ConclusionThis study highlights the potential of NAM+GEM as immunotherapy for advanced pancreatic cancer.

scholarly journals Membrane-associated and secreted forms of the Rhesus macaque rhadinovirus-encoded CD200 homologue and cellular CD200 demonstrate differential effects on Rhesus Macaque CD200 Receptor signaling and regulation of myeloid cell activation

2020 ◽  
pp. JVI.01654-20
Author(s):  
Ryan D. Estep ◽  
Aparna N. Govindan ◽  
Kristin Fitzpatrick ◽  
Tiffany C. Blair ◽  
S.A. Rahim Rezaee ◽  
...  
Keyword(s):  
Immune Responses ◽  
Rhesus Macaque ◽  
Cell Activation ◽  
Myeloid Cell ◽  
Functional Protein ◽  
Dna Viruses ◽  
Cd200 Receptor ◽  
First Time

The CD200-CD200R pathway is involved in inhibition of immune responses, and the importance of this pathway to infectious disease is highlighted by the fact that viral CD200 (vCD200) molecules have been found to be encoded by several DNA viruses, including the human gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV), and the closely related rhesus macaque rhadinovirus (RRV). KSHV vCD200 is the most extensively studied vCD200 molecule, however, the only herpesvirus vCD200 molecule to be examined in vivo is that encoded by RRV. Our prior studies have demonstrated that RRV vCD200 is a functional CD200 homologue that is capable of affecting immune responses in vivo, and further, that RRV can express a secreted form of vCD200 (vCD200-Sec) during infection. Despite this information, RRV vCD200 has not been examined specifically for effects on RM CD200R signaling, and the functionality of vCD200-Sec has not been examined in any context. Thus, we developed an in vitro model system in which B cells expressing vCD200 were utilized to assess the effects of this molecule on the regulation of myeloid cells expressing RM CD200R, mimicking interactions that are predicted to occur in vivo. Our findings suggest that RRV vCD200 can bind and induce functional signals through RM CD200R, while vCD200-Sec represents a non-functional protein incapable of affecting CD200R signaling. We also provide the first demonstration of the function of RM CD200, which appears to possess more robust signaling capabilities than RRV vCD200, and also show that KSHV vCD200 does not efficiently induce signaling via RM CD200R.IMPORTANCE Viral CD200 homologues are encoded by KSHV and the closely related RRV. Though RRV vCD200 has been examined, questions still exist in regard to the ability of this molecule to induce signaling via rhesus macaque CD200R, as well as the potential function of a secreted form of vCD200. Further, all previous in vitro studies of RRV vCD200 have utilized an Fc fusion protein to examine functionality, which does not replicate the structural properties of the membrane-associated form of vCD200 that is naturally produced during RRV infection. In this study, we demonstrate for the first time that membrane-expressed RRV vCD200 is capable of inducing signal transduction via RM CD200R, while the secreted form of vCD200 appears to be non-functional. Further, we also demonstrate that RM CD200 induces signaling via RM CD200R, and is more robust than RRV vCD200, while KSHV vCD200 does not appear to induce efficient signaling via RM CD200R.

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