Biology subjects

Raikar, S. S.

Publications and source records attributed to Raikar, S. S..

3 recordsLinked to original sources

bioRxiv2026

Serum free expansion and transduction of human Vγ9δ2 T cells for adoptive immunotherapy

Background and AimGamma delta T cells are multivalent immune cells with innate and adaptive features that sense a broad spectrum of tumor-associated stress patterns to lyse them without prior potentiation. They are therefore a good option for developing an off-the-shelf therapeutic immune cell product with antitumor functions. Therefore, we aimed to develop an optimized cGMP compatible protocol for expanding V gamma 9-V delta 2 T cells in a serum-free media for adoptive immunotherapy applications. Methods and ResultsPeripheral blood mononuclear cells (PBMC) from healthy donors were activated with Zoledronic Acid (ZOL) and IL-2 for 14 days. Cell proliferation, fold expansion, and phenotype were monitored. Among the regular donors, the baseline levels (day 0) of CD3+Vgamma9+delta2+ T cells were (05.10+/-0.74%). There was a robust expansion of V gamma 9-V delta 2 T cells in the serum-free media (110+/-29.89-fold). During processing, an abrupt reduction of {beta} T cells was observed as early as day +07. After two weeks, 87.82+/-5.11% (n=8) of T cells were CD3+Vgamma9+ in Optimizer with 97.15+/-0.7% of the CD3+Vgamma9+ T cells positive for delta2. The CD3+Vgamma9+ NKG2D+ increased during expansion, reaching 93.76+/-1.55% expression on day +14. V gamma 9-V delta 2 T cells expanded in the serum-free (Optimizer) media had relatively reduced variance and reduced over all yield and fold expansion but comparable percentage of Vgamma9+ and {beta} TCR+ T cells on day 14. A flow cytometry-based tumor-toxicity assay gauged the antitumor functions against K562 cell lines. Pretreatment of K562 cells with ZOL differentially enhanced (2.48+/-0.76 fold) the cytotoxic capacity of V gamma 9-V delta 2 T cells in a donor-dependent manner. The conditions for lentiviral transduction and transgene expressions of V gamma 9-V delta 2 T cells were improvised as gauged by GFP expression driven by CMV promoter (39.09+/- 8.94%) without compromising the viability. ConclusionWe have optimized a cGMP compatible protocol for expansion of human V gamma 9-V delta 2 T cells in a serum-free media with high purity and viability in as early as 07 days. Pretreatment of target tumor cells with ZOL enhanced the cytotoxicity, revealing the impact of V gamma 9-V delta 2 T cell intrinsic factors in tumor lysis. This protocol, may be scaled up for clinical translation for adoptive immunotherapy.

bioengineering↗

bioRxiv2025

Chimeric Antigen Receptors Transmit Piconewton Forces that are Coupled with T Cell Function

Chimeric antigen receptor (CAR) T cells are engineered to display a receptor that binds antigens expressed on the surface of cancerous cells, which leads to cancer cytotoxicity. Recently, the T cell field has come to recognize the role of small, piconewton level forces in establishing specificity and cytotoxicity in naive T cells with the {beta} TCR, raising the possibility that these forces could be present in CAR T cells. Using DNA-based tension probes, we reveal 8-19 pN mechanical forces with [~]1 sec timescales transmitted by CAR T cells to their target antigens. CAR-antigen force magnitude is independent of CAR expression level and shows heterogeneity across different T cell donors, suggesting utility as a biomarker of T cell fitness. Using an established exhaustion model, we show strong correlation between CAR exhaustion, cytotoxic capacity, and CAR-antigen force, suggesting that CAR mechanics provide a biomarker of CAR potency complementary to functional assays. Pharmacological inhibition studies demonstrate that CAR forces are driven by actin, Zap70 and Src family proximal kinases. Titration of dasatinib, a clinically used tyrosine kinase inhibitor also dampens both CAR-antigen tension and CAR function in a dose-dependent manner. Structural engineering of the CAR confirms that force levels are modulated by the scFv receptor and co-stimulatory domains, but force transmission requires CD3{zeta} ITAMs. Taken together, this work shows that CAR T cells transmit pN force to their cognate antigens which holds potential significance in the design and screening of CAR therapeutic candidates and for predicting treatment outcomes in a personalized fashion.

biophysics↗

bioRxiv2022

Single Cell RNA Sequencing Driven Characterization of Pediatric Mixed Phenotype Acute Leukemia

BackgroundMixed phenotype acute leukemia (MPAL) is a rare subgroup of leukemia characterized by blast cells that display both myeloid and lymphoid lineage features, making this cancer difficult to diagnose and treat. A deeper characterization of MPAL at the molecular level is essential to better understand similarities/differences to the more common and better-studied leukemias, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on MPAL bone marrow (BM) samples in an attempt to develop a more granular map of the MPAL microenvironment landscape. MethodsWe analyzed [~]16,000 cells from five pediatric MPAL BM samples collected at diagnosis to generate a single-cell transcriptomic landscape of B/Myeloid (B/My) and T/Myeloid (T/My) MPAL blasts and associated microenvironment cells. Cell clusters were identified using principal component analysis and uniform manifold approximation and projection (UMAP). Unsupervised analysis was performed to determine the overall relationship among B/My MPAL, T/My MPAL, and other acute leukemias - B-ALL, T-ALL, and AML. Supervised differentially expressed gene (DEG) analysis was performed to identify B/My and T/My MPAL blast-specific signatures. MPAL sample transcriptome profiles were compared with normal BM stem and immune cells to identify MPAL-specific dysregulation. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. Comparative analysis was performed on diagnostic samples based on their future minimal residual disease (MRD) and relapse status. ResultsB/My MPAL and T/My MPAL blasts displayed distinct subtype-specific blast signatures. UMAP analysis revealed that B/My MPAL samples had greater overlap with B-ALL samples, while T/My MPAL samples clustered separately from other acute leukemia subtypes. Genes overexpressed in both MPAL subtypes blasts compared to other leukemias and healthy controls included PLIN2, CD81, and UBE2S. B/My MPAL blast-specific genes included IRS2, SMIM3, and HBEGF, whereas T/My MPAL blast-overexpressed genes included IER5, BOD1L1, and HPGD. Sirtuin signaling, p38 MPAK signaling, and PI3K signaling pathways were upregulated in B/My MPAL blasts while oxidative phosphorylation and Rho family GTPases signaling pathways were upregulated in T/My MPAL blasts. Transcriptomic, pathways, and cell communication level differences were observed in the MPAL samples based on future MRD and clinical outcome status. ConclusionsWe have for the first time described the single-cell landscape of pediatric MPAL and demonstrate that B/My and T/My MPAL have unique scRNAseq profiles distinct from each other as well as from ALL and AML.

genomics↗