This study presents an optimised cultured ELISpot protocol for detecting central memory T-cell interferon gamma (IFNγ) responses against SARS-CoV-2 peptides following an initial priming with either peptides, or whole spike protein.
Key variations optimised include the culture length, timing of exogenous survival signals (IL-2), and endpoint analysis modality and cell density to enhance assay sensitivity without compromising specificity for central memory T-cell IFNγ recall responses to cognate antigen.
We noted a culture duration of 10 days, combined with a delayed IL-2 administration on day 5 to enhance assay sensitivity while maintaining response specificity towards cognate antigen when compared with shorter culture periods or earlier exogenous survival signal provision. With regards to lower-frequency T-cell interactions, as we observed with our donor SARS-CoV-2 epitope responses, our findings suggest Fluorospot to be preferable to the chromogenic ELISpot modality, and an immediate cell washing after culture collection to better facilitate cognate antigen responses. Fluorospot enabled a higher cell density while minimising the generation of visual artefacts, meanwhile immediate cell washing was critical for improving endpoint assay sensitivity. CCR7+ cell depletion was used to demonstrate our optimised protocol to selectively demonstrate central memory T-cell responses. Lastly, we provide evidence for the capacity of our assay to delineate individual responding peptides following peptide pool priming, and to explore cross-reactivity between viral variant peptides.
This work advances the methodology for investigating T-cell immunity, particularly in the context of SARS-CoV-2, and emphasises the balance between enhancing specific cognate central memory responses while limiting non-specific activation.
Copyright © 2025 Jerome, Wilson, Fialho, Goodchild, Prakash, McLeod, Richmond, Apostolopoulos, Flanagan and Plebanski.

Atopic dermatitis (AD) is a chronic and inflammatory disease. According to a recent study, administration of canine MSCs is a potential therapy for immunological diseases. However, most related studies involve short-term experiments and acute atopic dermatitis animal models. Thus, studies of repeated subcutaneous injection of canine MSCs for ameliorating long-term inflammatory skin disorders have not yet been established. In this study, we evaluated the effects of long-term canine amniotic mesenchymal stem cells (cAM-MSCs) and calcineurin inhibitors (CNIs) treatments in mouse AD model for up to 8 weeks and compared the differences in therapeutic effect through canine peripheral blood mononuclear cells (PBMCs). Using a mouse model, we validated the therapeutic impact of cAM-MSCs in comparison to pimecrolimus (Pime), the most widely used CNIs, as a therapy for canine AD. Based on our results, we verified that the cAM-MSC treatment group exhibited substantially lower scores for tissue pathologic alterations, inflammatory cytokines, and dermatologic symptoms than the PBS control group. Importantly, compared with Pime, cAM-MSCs were more effective at preventing wound dysfunction and regulating mast cell activity. Additionally, we confirmed that immune modulation proteins (TGF-β1, IDO1, and COX-2) were increased in the cAM-MSCs treatment group. Furthermore, we examined the immunoregulatory effect of cAM-MSCs through the proliferation of T lymphocytes from activated canine PBMCs. As a result, cAM-MSCs suppressed the proliferative capacity of effector T cells from canine PBMCs more effectively than Pime. In conclusion, this study suggested that the cAM-MSCS could be an effective canine treatment for long-term canine AD through regeneration and immunomodulation.
© 2025. The Author(s).

In this study, we used single-cell sequencing, which can comprehensively detect the type and number of transcripts per cell, to efficiently stimulate peripheral blood mononuclear cells of type 1 diabetic patients with overlapping peptides of GAD, IA-2, and insulin antigens, and performed gene expression analysis by single-cell variable-diversity-joining sequencing and T-cell receptor repertoire analysis. Twenty male patients with type 1 diabetes mellitus participating in the KAMOGAWA-DM cohort were included. Four of them were randomly selected for BD Rhapsody system after reacting peripheral blood mononuclear cells with overlapping peptides of GAD, IA-2, and insulin antigen. Peripheral blood mononuclear cells were clustered into CD8+ T cells, CD4+ T cells, granulocytes, natural killer cells, dendritic cells, monocytes, and B cells based on Seurat analysis. In the insulin group, gene expression of inflammatory cytokines was elevated in cytotoxic CD8+ T cells and Th1 and Th17 cells, and gene expression related to exhaustion was elevated in regulatory T cells. In T cell receptors of various T cells, the T cell receptor β chain was monoclonally increased in the TRBV28/TRBJ2-7 pairs. This study provides insights into the pathogenesis of type 1 diabetes and provides potential targets for the treatment of type 1 diabetes.
Copyright © 2025 JCBN.

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver.
This study aimed to assess serum human telomerase enzyme (hTERT) levels and their relation to the progression of liver disease. Also, it aimed to assess the effect of hepatitis C virus (HCV) core protein on memory T-cells in HCV patients with or without HCC and the correlation between memory cell phenotype and the progression of the disease in the same patients.
HTERT level in serum was assessed through relative quantitative RT-PCR. Flow cytometric analysis was used to assess T-cell responsiveness (as IFN- γ secretion) before and after stimulation with HCV core protein and the memory CD8+ cell phenotype using several differentiation markers.
HTERT was found to be increased in a stepwise manner upon comparing its level in controls, chronic hepatitis patients, cirrhotic patients, and HCC patients. T-cells showed a similar manner of stepwise decrease in response (decreased IFN- γ secretion) in HCC patients compared to HCV patients without HCC and controls. Also, late differentiated memory cells (CD8+, CD27-, CD28-, CD45RA+, and CCR7-) were depleted in HCC patients compared to HCV patients without HCC.
These results suggest a negative correlation between hTERT and IFN- γ secretion by T-cells in HCV patients and that this relationship, along with the depletion of late differentiated memory cells, could help the progression of liver disease to HCC.
© 2023 Indian National Association for Study of the Liver. Published by Elsevier B.V. All rights reserved.

Mesenchymal stem cells (MSCs) are a promising tool for treating immune disorders. However, the effect and mechanism of canine MSCs compared with other commercialized biologics for treating immune disorders have not been well studied. In this study, we investigated the characteristics and immunomodulatory effects of canine amnion membrane (cAM)-MSCs. We examined T lymphocytes from activated canine peripheral blood mononuclear cell (PBMC) proliferation. As a result, we confirmed that cAM-MSCs suppressed the proliferation capacity of T cells and cytotoxic activity. Moreover, we confirmed the therapeutic effect and mechanism of cAM-MSCs compared with oclacitinib, the most commonly used Janus kinase (JAK) inhibitor, as a treatment for canine atopic dermatitis (AD) using a mouse AD model. As a result, we confirmed that scores of dermatologic signs, tissue pathologic changes and inflammatory cytokines were significantly reduced by cAM-MSC treatment. In particular, cAM-MSCs were more effective than oclacitinib in the recovery of wound dysfunction and regulation of mast cell activity. Interestingly, subcutaneous injection of cAM-MSCs induced weight recovery, but oral administration of oclacitinib induced weight loss as a side effect. In addition, it was confirmed that the secretion of TGF-β1 and IDO by cAM-MSCs is directly involved in improving atopic dermatitis. In conclusion, this study suggests that cAM-MSCs can be developed as a safe canine treatment for atopic dermatitis without side effects through effective regeneration and immunomodulation.