Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique

Garcia, Luz Milbeth Cumba; Kelcher, April M. Huseby; Malo, Courtney S.; Johnson, Aaron J.

doi:10.1016/j.jim.2016.09.002

Cited by 29 publications

(19 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Brains were harvested and manually homogenized using a dounce homogenizer as previously described (12). Brain samples were filtered through a 70 μm filter to achieve a single cell suspension into a 50% percoll solution.…”

Section: Methodsmentioning

confidence: 99%

Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma

et al. 2018

Self Cite

View full text Add to dashboard Cite

Glioblastoma (GBM) is a lethal cancer of the central nervous system with a median survival rate of 15 months with treatment. Thus, there is a critical need to develop novel therapies for GBM. Immunotherapy is emerging as a promising therapeutic strategy. However, current therapies for GBM, in particular anti-angiogenic therapies that block vascular endothelial growth factor (VEGF), may have undefined consequences on the efficacy of immunotherapy. While this treatment is primarily prescribed to reduce tumor vascularization, multiple immune cell types also express VEGF receptors, including the most potent antigen-presenting cell, the dendritic cell (DC). Therefore, we assessed the role of anti-VEGF therapy in modifying DC function. We found that VEGF blockade results in a more mature DC phenotype in the brain, as demonstrated by an increase in the expression of the co-stimulatory molecules B7-1, B7-2, and MHC II. Furthermore, we observed reduced levels of the exhaustion markers PD-1 and Tim-3 on brain-infiltrating CD8 T cells, indicating improved functionality. Thus, anti-angiogenic therapy has the potential to be used in conjunction with and enhance immunotherapy for GBM.

show abstract

Section: Methodsmentioning

confidence: 99%

Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…Isolation of CNS-ILs was performed using previously described methods ( 23 ). Briefly, at designated time points, post-infection mice were euthanized with CO 2 prior to collection of brain and spinal cord into 5 mL of 4°C RPMI.…”

Section: Methodsmentioning

confidence: 99%

B7-H1 Influences the Accumulation of Virus-Specific Tissue Resident Memory T Cells in the Central Nervous System

et al. 2017

View full text Add to dashboard Cite

Therapies that target the PD-1/B7-H1 axis have revolutionized cancer treatment, yet precise knowledge of how this pathway provides benefit continues to evolve. Here, we report a novel role for the immune checkpoint ligand B7-H1 in the accumulation of tissue-resident memory CD8+ T-cells (TRM). After intracranial infection, Theiler’s murine encephalomyelitis virus (TMEV) generates TRM that are maintained in the central nervous system (CNS) tissues of B7-H1WT animals. Although no differences in acute T-cell responses between B7-H1WT and B7-H1KO are observed, at long-term periods post-infection the maintenance of CD8+ TRM is diminished in B7-H1KO animals. This is accompanied by redistribution of the resident CD8+ population from primarily CD103+ TRM to a diminished population of TRM and a preponderance of non-specified PD-1+ CD103− CD8+ T-cells. T-cell transfer studies demonstrate that host B7-H1 is necessary for maintaining TRM and limiting accumulation of PD-1+ CD103− CD8+ T-cells. The lack of host B7-H1 results in compromised control of a heterologous virus re-challenge demonstrating a functional defect in TRM mediated virus control. This study reveals a new role for B7-H1 in TRM and pro-inflammatory PD-1+ CD103− CD8+ T-cell accumulation in the CNS and gives insight for using B7-H1/PD-1 blockade in modulating long-term T-cell protection.

show abstract

“…Flow cytometric measurements were conducted as previously published [49, 64]. Spleens and thymi harvested from euthanized animals were crushed through a 70um filter into 10mL RPMI to create a single cell suspension.…”

Section: Methodsmentioning

confidence: 99%

Conditional silencing of H-2D^bclass I molecule expression on dendritic cells modulates the protective and pathogenic kinetics of virus-antigen specific CD8 T cell responses during Theiler’s Virus infection

Tritz

Orozco

Malo

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Theiler's murine encephalomyelitis virus (TMEV) infection of the central nervous systemis rapidly cleared in C57BL/6 mice by an anti-viral CD8 T cell response restricted by the MHC class I molecule, H-2D b . While the CD8 T cell response against neurotropic viruses is well characterized, the identity and function of the antigen presenting cell(s) involved in this process is(are) less well defined. To address this gap in knowledge, we developed a novel C57BL/6 H-2D b conditional knockout mouse that expresses an H-2D b transgene in which the transmembrane domain locus is flanked by LoxP sites. We crossed these H-2D b LoxP mice with MHC class I-deficient mice expressing Cre-recombinase under either the CD11c or LysM promoter in order to silence H-2D b restricted antigen presentation predominantly in dendritic cells or macrophages, respectively. Upon challenge with intracranial TMEV infection, we observe that CD11c+ APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2 121-130 presented in the context of the H-2D b molecule. This stands in stark contrast to later time points post TMEV infection where CD11c+ APCs appear dispensable for the activation of antigen-specific T cells; the functionality of these late-arising antiviral CD8 T cells is reflected in the restoration of viral control at later time points. These latearising CD8 T cells also retain their capacity to induce blood-brain barrier disruption. In contrast, when H-2D b restricted antigen presentation was selectively silenced in LysM+ APCs there was no overt impact on the priming of D b :VP2 121-130 epitope-specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model system which enables critical dissection of MHC class I restricted antigen presentation to T cells, revealing cell specific and temporal features involved in the generation of antiviral CD8 T cell responses. Employing this novel system, we established CD11c+ cells as a pivotal driver of acute, but not later-arising, antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2 121-130 , with functional implications both for T cell-mediated viral control and immunopathology. Class I conditional knockout mice were developed through construction of an H-2D b transgene, in which the transmembrane (TM) domain is flanked by LoxP sites. This transgene was introduced into C57BL/6 mice deficient in H-2D b and H-2K b , leaving the floxed D b as the onlyMHC class I molecule expressed by nucleated cells (Fig 1A) [33]. Crossing this transgenic mouse line with mice expressing Cre-recombinase under the CD11c, LysM, or CMV promoter, also on total MHC class I knockout backgrounds, allowed for CD11c+ cell specific, LysM+ cell specific, or ubiquitous ablation of MHC-I expression, respectively ( Fig 1B, Fig 1C, Fig 1D).Resultant mice will be referred to as CD11c D b conditional knockout (cKO), LysM D b cKO, CMV D b cKO, or Cre-littermates depending on whether the F1 generation pups inherited the...

show abstract

Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique

Cited by 29 publications

References 31 publications

Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma

Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma

B7-H1 Influences the Accumulation of Virus-Specific Tissue Resident Memory T Cells in the Central Nervous System

Conditional silencing of H-2D^bclass I molecule expression on dendritic cells modulates the protective and pathogenic kinetics of virus-antigen specific CD8 T cell responses during Theiler’s Virus infection

Contact Info

Product

Resources

About

Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique

Cited by 29 publications

References 31 publications

Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma

Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma

B7-H1 Influences the Accumulation of Virus-Specific Tissue Resident Memory T Cells in the Central Nervous System

Conditional silencing of H-2Dbclass I molecule expression on dendritic cells modulates the protective and pathogenic kinetics of virus-antigen specific CD8 T cell responses during Theiler’s Virus infection

Contact Info

Product

Resources

About

Conditional silencing of H-2D^bclass I molecule expression on dendritic cells modulates the protective and pathogenic kinetics of virus-antigen specific CD8 T cell responses during Theiler’s Virus infection