Optimizing the cryopreservation of murine splenocytes for improved antigen-specific T cell function in ELISPOT

Gad, Ekram; Rastetter, Lauren; Herendeen, Dan; Curtis, Benjamin; Slota, Meredith; Koehniein, Marlese; Disis, Nora

doi:10.1186/2051-1426-1-s1-p211

Cited by 7 publications

(3 citation statements)

References 3 publications

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“…This demonstrates that the capability of BCMA.CAR-T cells to produce cytokines did not decline over time. Some previous studies reported similar results for CD19.CAR-T cells [21,22]. The data also showed a constant trend toward a lower frequency of INFγproducing cells than TNFα-producing cells over the entire time of cryopreservation of up to one year in all CAR-T cells.…”

Section: Discussionsupporting

confidence: 80%

In Vitro Functionality and Endurance of GMP-Compliant Point-of-Care BCMA.CAR-T Cells at Different Timepoints of Cryopreservation

Jiang,

Neuber,

Hückelhoven-Krauss

et al. 2024

IJMS

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The search for target antigens for CAR-T cell therapy against multiple myeloma defined the B-cell maturation antigen (BCMA) as an interesting candidate. Several studies with BCMA-directed CAR-T cell therapy showed promising results. Second-generation point-of-care BCMA.CAR-T cells were manufactured to be of a GMP (good manufacturing practice) standard using the CliniMACS Prodigy® device. Cytokine release in BCMA.CAR-T cells after stimulation with BCMA positive versus negative myeloma cell lines, U266/HL60, was assessed via intracellular staining and flow cytometry. The short-term cytotoxic potency of CAR-T cells was evaluated by chromium-51 release, while the long-term potency used co-culture (3 days/round) at effector/target cell ratios of 1:1 and 1:4. To evaluate the activation and exhaustion of CAR-T cells, exhaustion markers were assessed via flow cytometry. Stability was tested through a comparison of these evaluations at different timepoints: d0 as well as d + 14, d + 90 and d + 365 of cryopreservation. As results, (1) Killing efficiency of U266 cells correlated with the dose of CAR-T cells in a classical 4 h chromium-release assay. There was no significant difference after cryopreservation on different timepoints. (2) In terms of endurance of BCMA.CAR-T cell function, BCMA.CAR-T cells kept their ability to kill all tumor cells over six rounds of co-culture. (3) BCMA.CAR-T cells released high amounts of cytokines upon stimulation with tumor cells. There was no significant difference in cytokine release after cryopreservation. According to the results, BCMA.CAR-T cells manufactured under GMP conditions exerted robust and specific killing of target tumor cells with a high release of cytokines. Even after 1 year of cryopreservation, cytotoxic functions were maintained at the same level. This gives clinicians sufficient time to adjust the timepoint of BCMA.CAR-T cell application to the patient’s course of the underlying disease.

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Section: Discussionsupporting

confidence: 80%

In Vitro Functionality and Endurance of GMP-Compliant Point-of-Care BCMA.CAR-T Cells at Different Timepoints of Cryopreservation

Jiang,

Neuber,

Hückelhoven-Krauss

et al. 2024

IJMS

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“…Thus, cryopreservation of ex vivo ‐expanded T cells at an early stage could constitute a beneficial strategy to store functional T cells for prolonged periods and would allow for their delayed or repeated use during the course of therapy, following lymphodepletion or in the event of relapse or recurrence, without extended in vitro culture. Previous studies have focused on optimising the cryopreservation and recovery of peripheral blood mononuclear cells from human patients 12 , 13 or splenocytes isolated from mice, 14 by challenging them with mitogens that stimulate leukocytes in a nonspecific manner. In a recent inconclusive clinical trial, a cohort infused with freshly isolated T cells had to be interrupted due to severe adverse patient responses, though the study indicated that cryopreservation may attenuate T‐cell function 15 .…”

mentioning

confidence: 99%

Antigen‐specific T cells fully conserve antitumour function following cryopreservation

et al. 2016

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Immunotherapies based on the autologous adoptive transfer of ex vivo-manipulated T cells are rapidly evolving for the treatment of both metastatic and primary malignancies. However, extended ex vivo culturing reduces the functionality of isolated T cells. Cryopreservation of rapidly expanded T cells for subsequent use throughout an immunotherapeutic regimen is a highly desirable recourse, thus far encumbered by a lack of studies investigating its effects on effector T-cell functionality. Here we directly compare murine tumour-reactive CD8+ T cells cryopreserved during ex vivo expansion to freshly isolated populations. We show that cryopreservation fully conserves the differentiation potential of effector T cells, secretion of pro-inflammatory cytokines, cytotoxic function and does not impair the three-dimensional scanning motility of T cells or their capacity to infiltrate and reject tumours.

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“…Cells were then pelleted at 400 x g for 5 min at 4 °C, stained with trypan blue, and enumerated using a Nexcelom Bioscience cellometer. Mouse splenocytes were cryopreserved in 1.0 ml of freezing medium (90% FBS, 10% DMSO) [61].…”

Section: Methodsmentioning

confidence: 99%

VirB10 vaccination for protection against Anaplasma phagocytophilum

et al. 2018

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BackgroundHuman granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the etiologic agent Anaplasma phagocytophilum. HGA was designated a nationally notifiable disease in the United States in 1998. Currently there are no vaccines available against HGA. Conserved membrane proteins that are subdominant in Anaplasma species, such as VirB9 and VirB10, may represent better vaccine targets than the variable immunodominant surface proteins. VirB9 and VirB10 are constituents of the Type 4 secretion system (T4SS) that is conserved amongst many intracellular bacteria and performs essential functions for invasion and survival in host cells.ResultsImmunogenicity and contribution to protection, provided after intramuscular vaccination of plasmid DNA encoding VirB9-1, VirB9-2, and VirB10 followed by inoculation of homologous recombinant proteins, in a prime-boost immunization strategy was evaluated in a murine model of HGA. Recombinant VirB9-1-, VirB9-2-, and VirB10-vaccinated mice developed antibody responses that specifically reacted with A. phagocytophilum organisms. However, only the mice vaccinated with VirB10 developed a significant increase in IFN-γ CD4+ T cells and partial protection against challenge with A. phagocytophilum.ConclusionsThis work provides evidence that A. phagocytophilum T4SS VirB10 is partially protective in a murine model against infection in an IFN-γ-dependent fashion and suggests that this protein may be a potential vaccine candidate against this and possibly other pathogenic bacteria with a T4SS.

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Optimizing the cryopreservation of murine splenocytes for improved antigen-specific T cell function in ELISPOT

Cited by 7 publications

References 3 publications

In Vitro Functionality and Endurance of GMP-Compliant Point-of-Care BCMA.CAR-T Cells at Different Timepoints of Cryopreservation

In Vitro Functionality and Endurance of GMP-Compliant Point-of-Care BCMA.CAR-T Cells at Different Timepoints of Cryopreservation

Antigen‐specific T cells fully conserve antitumour function following cryopreservation

VirB10 vaccination for protection against Anaplasma phagocytophilum

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