| Objective: The clinical prognosis of relapsed/refractory(R/R)central nervous system lymphoma(CNSL)is extremely poor.Recently,a series of studies have indicated some R/R CNSL patients can benefit from chimeric antigen receptor(CAR)T cell therapy,however,failure and relapse are still inevitable.Therefore,investigating the detail mechanism and further improving the clinical outcomes are the main current challenges.In this study,we investigated the phenotype of microglia in the tumor immune microenvironment(TME)of CNSL as well as its influence on the functional effects of CAR-T cells,and innovatively combined autologous hematopoietic stem cell transplantation(ASCT)with CAR-T cells infusion for the treatment of R/R CNSL,aimed to improve the long-term remission and prognosis.Methods: Part I.1.Clinical CNSL specimens were analyzed for immunocytes infiltration;2.Microglia(HMC3 cells)was treated with the conditioned medium(CM)prepared from the lymphoma cells culture supernatant,and the phenotypes were analyzed by flow cytometry and real time polymerase chain reaction;3.CD19 CAR-T cells were constructed and co-cultured with HMC3 cells in vitro,the activation,exhaustion,apoptosis and tumor killing ability of CAR-T cells were detected by flow cytometry.Part II: The clinical data of R/R CNSL patients underwent ASCT combined with CAR-T cells infusion in the Department of Hematology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology were collected,and comprehensively evaluated the clinical efficacy and safety.Results: 1.22 immune cell subsets were identified in 69 CNSL patients from the GEO database,the proportions of M0,M1 and M2 macrophage/microglia were 12.81%,5.48% and 12.44%,respectively.Immunofluorescence showed the proportion of microglia in the tumor tissue of six CNSL patients was 12.89%,of which M1 type accounted for 0.90% and M2 type accounted for 5.54%.2.After 48 h of treatment with CM,the expression of CD206 but not CD86 molecule of HMC3 cells increased.The median fluorescence intensity(MFI)of PD-L1 and SIRPα elevated,and PD-1 MFI didn’t significantly change,relative m RNA expression of TGF-β,CCL-18,MIP-1β and COX-2 also increased.3.The transfection efficiency of CAR-T cells ranged from 70% to 80%,neither normal nor CM-pretreated HMC3 cells altered the macroscopic migration capacity of CAR-T cells.In addition,normal HMC3 cells activated CAR-T cells accompanied by a slight upregulation of exhausted indicators,but CM-pretreated HMC3 cells leaded to the over-activation of CAR-T cells,along with the significantly increased expression of exhausted molecules including LAG-3 and PD-1,resulted in CAR-T cells apoptosis,which interfered with the tumor killing capacity in vitro.4.A total of 38 R/R CNSL patients received sequential CAR-T cells infusion following ASCT,the common adverse events within 30 days included hematotoxicity(100%),hypocalcemia(97.4%)and hypoalbuminemia(92.1%),the incidence of grades ≥3 cytokine release syndrome(CRS)and immune effector cell-associated neurotoxicity syndrome(ICANS)were 18.4% and 10.5%,respectively.Of the enrolled patients,the overall remission rate(ORR)and complete remission rate(CRR)were 73.7% and 52.6%,respectively,and the cumulative progression incidence after remission was 19.0%.After a median follow-up of 20.7 months,the median overall survival(OS)and progression-free survival(PFS)were not reached,and the 1-year OS and PFS rates were 72.1% and 56.2%,respectively.CD79 b mutation,grades ≥3 CRS,any grades of ICANS and therapy tolerance were risk factors of PFS.5.There were 8 and 23 R/R CNSL patients with and without CD79 b mutation separately treated with ASCT in combination with CAR-T cells infusion,respectively,with ORRs of 62.5% and 82.6%(P = 0.335),CRRs of 25.0% and 60.9%(P = 0.113),duration of response was only 3.8 months in the mutated group,but not reached in the non-mutated cohort,the cumulative progression rates were 60.0% and 5.3%(P = 0.003),1-year OS rates were 75.0% and 81.4%(P = 0.033),and 1-year PFS rates were 18.8% and 73.4%(P = 0.005)in the two groups,respectively,patients with CD79 b mutation significantly accompanied with lower duration of response and PFS.Conclusions: 1.Microglia is an important component of CNSL TME,which polarizes toward M2 type and upregulates the expression of immunosuppressive checkpoints and molecules.2.Microglia of CNSL TME increases the exhaustion and apoptosis of CAR-T cells and affects the tumor killing ability in vitro.3.Sequential CAR-T cell therapy following ASCT has promising long-term clinical efficacy and can be a new salvage option for R/R CNSL.4.CD79 b mutation is an important reason of the unsustainable remission in R/R CNSL patients receiving ASCT combined with CAR-T cell therapy. |
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