Ajuga bracteosa (AB) has been widely used in folk medicine in Asian countries against gout, hepatitis, pneumonia, rheumatism, and various neuro inflammatory disorders. The aim of this study was to investigate the possible immunoregulatory effects of the ethanolic extract of Ajuga bracteosa (ABEE) on systemic Th1/Th2 immunity in SRBC immunized Balb/C mice. Animals were orally administered with graded doses of ABEE from 6.25 mg/kg to 100 mg/kg. Post sub-cutaneous immunization with SRBCs and circulating antibody titers, DTH responses and splenocyte proliferation was monitored as markers of Th2 and Th1 responses. Cyclophosphamide and levamisole were used as controls. Lymphocyte immunophenotying (CD4/CD8 cell counts) and intracellular Th1/Th2 cytokine concentrations were determined using flow cytometry. Treatment with ABEE demonstrated significant biphasic immunostimulation of effector T-helper immunity. ABEE at 50 mg/kg dose resulted in maximal increase in antibody titers, DTH responses and CD4+/CD8+ T-cell percentages indicating maximal activation and proliferation of T and B lymphocytes at this dose. ABEE, at the same dose, also showed maximal up regulation of LPS and CON A stimulated splenocyte proliferation and also maximal up-regulation of both Th1 (IL-2, IFN-γ) and Th2 (IL-4) cytokines which suggest its mixed Th1/Th2 immunostimulatory activity. Comparatively at higher doses (100 mg/kg), significant down regulation of all these effector T-helper (Th) immune responses was observed. The study therefore suggests mixed biphasic immunostimulatory Th1/Th2 activity of ABEE that could support its immunoadjuvant potential.

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Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop lifethreatening toxicities associated with cytokine release syndrome which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR T cells by humanizing the framework region of single-chain variable fragment (scFv) derived from a murine FMC63 mAb and combining it with CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3z signaling domain (h1CAR19-8BBz). Docking studies followed by molecular dynamics simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared with murine scFv (mCAR19). Ex vivo studies with CAR T cells generated from healthy donors and patients with relapsed/ refractory B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable antitumor activity and proliferation. More importantly, h1CAR19-8BBz T cells produced lower levels of cytokines (IFNg, TNFa) upon antigen encounter and reduced the induction of IL6 cytokine from monocytes than mCAR19-8BBz T cells. There was a comparable proliferation of h1CAR19-8BBz T cells and mCAR19-8BBz T cells upon repeated antigen encounter. Finally, h1CAR19-8BBz T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.

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