Xuefeng Wang ^1,2* Peijun Tang ³ Xinghua Shen ⁴ Jianling Gao ⁵ Jianping Zhang ³ Yanjun Feng ³ Ji Zhang ⁶ Ziyi Huang ⁷

• ¹ Soochow University, Suzhou, China
• ² Independent researcher, Suzhou, China
• ³ Department of Tuberculosis, Fifth People's Hospital of Suzhou, Suzhou, China
• ⁴ Department of Critical Care Medicine, Fifth People's Hospital of Suzhou, Suzhou, China
• ⁵ Department of Emergency and Critical Care Medicine, Suzhou Dushu Lake Hospital, Suzhou, China
• ⁶ Suzhou University, Suzhou, China
• ⁷ Jiangsu Institute of Clinical Immunology, First Affiliated Hospital of Soochow University, Suzhou, China

Introduction: Pulmonary tuberculosis (PTB) remains one of the deadliest infectious diseases. Understanding PTB immunity is of potential value to explore immunotherapy for treating chemotherapy-resistant PTB. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are key players impairing immune responses to Mycobacteria tuberculosis (MTB). Currently, the intrinsic factors governing Treg expansion and influencing Treg immunosuppressive attributes in PTB patients are far from clear. Methods: Here, we employed flow cytometry to determine the frequency of Tregs, the expression of B and T lymphocyte attenuator (BTLA) and its ligand herpesvirus entry mediator (HVEM) on Tregs as well as conventional T cells and the expression of programmed death-ligand 1 (PD-L1) and programmed death-1 (PD-1) on Tregs in patients with active PTB. Then we dissected the characteristics of BTLA/HVEM expression and its correlation with Treg frequency and PD-L1 and PD-1 expression on Tregs in PTB patients. Results: The frequency of Tregs was increased in PTB patients and it had a relevance to PTB progression. Intriguingly, the axis of cosignal molecules, BTLA and HVEM, were both downregulated on Tregs of PTB patients compared with healthy controls (HC), which was the opposite of their upregulation on conventional T cells. Unexpectedly, their expression levels were positively correlated with frequency of Tregs, respectively. These seemly contradictory results may be interpreted as that downregulation of BTLA and HVEM may alleviate BTLA/HVEM cis-interaction-mediated coinhibitory signal pressing on naïve Tregs helping for their activation while BTLA/HVEM axis on effector Tregs induces a costimulatory signal promoting their expansion. Certainly, the mechanism underlying such complex effects remains further exploration. Additionally, PD-L1 and PD-1, being regarded as two of markers characterizing the immunosuppressive attributes and differentiation potential of Tregs, were upregulated on Tregs of PTB patients. Further analysis revealed that the expression levels of BTLA and HVEM were positively correlated with frequency of PD-1+Tregs and PD-L1+Tregs, respectively. Conclusion: Our study illuminated distinct characteristics of BTLA/HVEM axis expression on Tregs and uncovered its impact on expansion and attributes of Tregs in patients with active PTB. Therefore, blockade of BTLA/HVEM axis may be a promising potential pathway to reduce Treg expansion for improvement of anti-MTB immune responses.