Identification Of CD4~+T Cell Subpopulations Associated With Obesogenic Memory

Currently,over 1.9 billion people are overweight or obese around the world.There is a substantially risk of developing a series of acute and chronic diseases related with obesity,including Cardiovascular disease（CVD）,hypertension,osteoarthritis,type-2diabetes,late-onset Alzheimer’s disease and cancers¹.Obesity also increases the mortality and morbidity in adults and children.Thereby,obesity causes public concerns regarding the global health,and the global economy².Many researches focused on developing strategies for weight regulation,including bariatric surgery,weight loss medications,exercise,diet restriction and modification.However,these remedies weren’t effective,yet willpower and time-consuming.Even though these methods can achieve short-term weight loss,but they failed to maintain long-term normal body weight.It’s because these patients will suffer a weight regain after or during the weight loss process.Even the patients strictly restricted their diet,most of them still recovered the initial weight or gained more weight in the following2-3 years.The trend of weight regain is defined as“obesogenic memory”.Then patients would use a stricter weight loss strategy to lose weight.This often leads to repeated weight loss and weight regain,which is defined“weight cycling”.Weight cycling is related with increased body fat and increased T cell accumulation in adipose tissue^3,4.Our lab’previous research demonstrated that CD4⁺T cells isolated from spleen contribute to the obesogenic memory.We revealed that C57BL/6J mice with obesity history showed a faster weight gain rate compared with the CTR C57BL/6J mice,while the Rag1^-/-mice with obesity history showed similar body weight gain rates compared with the CTR Rag1^-/-mice.Furthermore,the adoptive transfer experiment showed that CD4⁺T cells isolated from mice with a history of obesity could promote weight gain rate of CTR receptor mice similar to that of the PC mice,and the CD4⁺T cells from CTR group didn’t have the ability.Taken together,our lab’s previous research demonstrated that mice with obesity history still possess obesogenic memory,and the obesogenic memory is contributed by CD4⁺T cells.To investigate the mechanism of obesogenic memory,we conducted the further research:first,we performed the single-cell RNA sequencing analysis.As our previous study showed that CD4⁺T splenocytes stores obesogenic memory and promotes weight regain.So,we isolated CD4⁺T cells from spleen to accomplish the experiments.The obesogenic memory mice model includes three groups:obesogenic memory group（MOH）,control group（CON）and high fat diet group（DIO）.The MOH group was generated through a high fat diet for 8 weeks,then underwent diet restriction for 4weeks to achieve the same body weight as CON group.During the whole period,the DIO group was fed a high fat diet and the CTR group a normal chow diet.After the mouse model was successfully generated,the CD4⁺T cells were isolated from spleen for the single-cell RNA sequencing analysis.The results showed that some subpopulation（cluster 2,3,5,7,10,13）in the CD4⁺CD127^-T cells were enriched in MOH group and DIO group.We further analyzed the enriched genes and specific signature of these clusters.The cluster 10 was double positive for CD44 and CD62L,markers of central memory T cells.And cluster 1,3,13were CD44^highCD62L^low,which were consistent with markers of tissue resident memory T cells(T_{r m}).Secondly,we found that the obesogenic memory is stored in CD4⁺T cells,suggesting there exists a signal molecule to activate CD4⁺T cells.And the activating signals should be increased during obesity and weight regain period.Lipids can store excessive energy,and also participate in mammalian immunity⁵.We performed the serum lipid mass spectrometry experiment to detect lipid changes of MOH group.The experiment results uncovered that the level of serum Phosphatidylethanolamine（PE）was significantly increased.PE is a secondary membrane phospholipid that can possess multiple biological functions,including membrane stabilization,food intake regulation and drug delivery.In addition,it is also the main component of phospholipids in cell membranes.The association of PE and metabolic diseases,including obesity was also highlighted.CD300 family proteins are a receptor family of phosphatidylethanolamine（PE）.Furthermore,we have revealed that the number of CD300 family positive cells was upregulated in cluster 7 and 10 based on the results of single-cell RNA sequencing.And CD300c uniquely up-regulated in cluster 10 is an activating receptor on the cells surface⁶.One of the main ligands for CD300c was PE ^7-9.So,cluster 10 may be the most important cell subgroup of obesogenic memory.We observed that PE can stimulate the number of CD300c positive CD4⁺T cells,consistent with our s c-RNA sequencing results.All the data above suggested that PE can stimulate CD4⁺T cells to generate obesogenic memory.In this research,we further studied function of CD4⁺CD127^-CD300c⁺T cells in obesogenic memory.Meanwhile the level of PE in mouse serum of MOH group was elevated.Our results demonstrated that PE can stimulate CD300c of the primary CD4⁺T cells.Taken together,it can be speculated that CD4⁺CD127^-CD300c⁺T cells were stimulated by PE stores obesogenic memory.