TCR repertoire landscape reveals macrophage-mediated clone deletion in endotoxin tolerance

Endotoxin tolerance (ET) is a protective mechanism in the process of sepsis, septic shock, and their sequelae including uncontrolled inflammation. Accumulating evidence has shown that peripheral T cells contribute to the induction of ET. However, what and how T-cell development contributes to ET inductions remain unclear.

Mice were intraperitoneally injected with LPS at a concentration of 5 mg/kg to establish an LPS tolerance model and were divided into two groups: a group examined 72 h after LPS injection (72-h group) and a group examined 8 days after LPS injection (8-day group). Injection of PBS was used as a control. We performed high-throughput sequencing to analyze the characteristics and changes of CD4⁺SP TCRβ CDR3 repertoires with respect to V direct to J rearrangement during the ET induction. Moreover, the proportion and proliferation, as well as surface molecules such as CD80 and CD86, of F4/80⁺ macrophages were analyzed using FCM. Furthermore, ACT assay was designed and administered by the tail vein into murine LPS-induced mouse model to evaluate the role of F4/80⁺ macrophages on the development of CD4⁺SP thymocytes in ET condition.

We found that the frequency and characteristics of the TCRβ chain CDR3 changed obviously under condition of ET, indicating the occurrence of TCR rearrangement and thymocyte diversification. Moreover, the absolute numbers of F4/80⁺ macrophages, but not other APCs, were increased in thymic medulla at 72-h group, accompanied by the elevated function-related molecules of F4/80⁺ macrophages. Furthermore, adoptively transferred OVA_332-339 peptide-loaded macrophages into Rag-1^-/- mice induced the clone deletion of OVA-specific CD4⁺SP, thereby ameliorating the pathology in lung tissue in LPS challenge.

These data reveal that the frequency and characteristics of the TCRβ chain CDR3 undergo dynamic programming under conditions of LPS tolerance. Furthermore, the peripheral macrophages may be a key factor which carry peripheral antigen to thymic medulla and affect the negative selection of T-cell population, thereby contributing to the formation of ET. These results suggest that the clone selection in thymus in ET may confer protection against microbial sepsis.