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B-acute lymphoblastic leukemia (B-ALL) is one of the most common pediatric cancers, wherein regulatory T cells (Treg) and exhausted CD8+ T cells may be important in its development and maintenance. In this bioinformatics study, we evaluated the expression of 20 Treg/CD8 exhaustion markers and their possible roles in patients with B-ALL. The mRNA expression values of peripheral blood mononuclear cell samples from 25 patients with B-ALL and 93 healthy subjects (HSs) were downloaded from publicly available datasets. Treg/CD8 exhaustion marker expression was normalized with that of the T cell signature and correlated with the expression of Ki-67, regulatory transcription factors (FoxP3, Helios), cytokines (IL-10, TGF-β), CD8+ markers (CD8α chain, CD8β chain), and CD8+ activation markers (Granzyme B, Granulysin). The mean expression level of 19 Treg/CD8 exhaustion markers was higher in the patients than in the HSs. In patients, the expression of five markers (CD39, CTLA-4, TNFR2, TIGIT, and TIM-3) correlated positively with Ki-67, FoxP3, and IL-10 expression. Moreover, the expression of some of them correlated positively with Helios or TGF-β. Our results suggested that Treg/CD8+ T cells expressing CD39, CTLA-4, TNFR2, TIGIT, and TIM-3 favor B-ALL progression, and targeted immunotherapy against these markers could be a promising approach for treating B-ALL. [ABSTRACT FROM AUTHOR]