Functional And Mechanistic Studies Of Lenalidomide-enhanced Antitumor Activity Of T Cells

T cells recognize tumor antigens and eliminate tumor cells,thus heralded as a major target by immunotherapies.Sufficient T cell activation involves signals delivered by T cell receptor（TCR）recognizing peptide-MHC（p MHC）complex and a series of co-stimulatory signals.The co-stimulatory receptor CD28 interacts with its B7 ligands to activate downstream co-stimulation signals,promoting T cell activation and effector function,which is required for effective T cell antitumor immune response.However,CD28-deficient T cells,particularly cytotoxic CD8⁺T cells,accumulate with age and within some solid tumors.Loss of CD28 compromises T cell immune response and limits the clinical benefits of PD-1/PD-L1 immune checkpoint blockade.Immunomodulatory drugs（IMi Ds）,including lenalidomide and its derivatives,have been reported to co-stimulate T cells in the absence of CD28 and B7 interaction,but whether lenalidomide can co-stimulate CD28^-T cells to reinstate T cell immune response and PD-1 blockade efficacy has not been established.In order to investigate the therapeutic significance of lenalidomide-induced co-stimulation in solid tumors,we generated a Crbn^I391V knockin mouse model to recapitulate in mice a co-stimulatory role of lenalidomide in primary human T lymphocytes.Our study reveals that lenalidomide inhibits syngeneic tumor growth by engaging CRBN to co-stimulate CD28^-T cells and reinstates the antitumor activity of CD28^-CD8⁺T cells after PD-1 blockade treatment.Furthermore,lenalidomide co-stimulates CD28^-CD8⁺T cells isolated from colorectal cancer（CRC）patient samples and enhances their response to anti-PD-1 therapy.Mechanistically,it is reported that lenalidomide hijacks CRL4^CRBN ubiquitin ligase to degrade IKZF1 and IKZF3 in T cells,leading to de-repressed expression of interleukin 2（IL-2）.Our study elucidates that lenalidomide also up-regulates some Notch-activated genes in CD8⁺T cells.Blocking IL-2 and Notch signaling simultaneously counteracts lenalidomide-induced CD8+T cell cytotoxicity and tumor growth inhibition by PD-1blockade.These data suggest that PD-1 immunotherapy might benefit from a lenalidomide combination when treating solid tumors infiltrated with abundant CD28^-CD8⁺T cells.We continued to explore the therapeutic implication of the bypassed requirement of CD28 by lenalidomide.We performed in vivo CRISPR library screen in MC38-OVA mouse colon cancer model to systematically identify gene mutations that can sensitize cancer cells to lenalidomide.Our results reveal that genetic mutations in such genes as Ptpn2,Jak1 and Eef2 sensitize cancer cells to lenalidomide-activated host immunity.These gene mutations are potential biomarker for lenalidomide treatment in colon cancer.Moreover,inhibition of these targets might benefit lenalidomide treatment in colon cancer.Finally,we synthesized more IMi D compounds and,by screening the CD8⁺T cells from Cd28^-/-Crbn^I391V mice,found two novel compounds that efficiently co-stimulate T cells.These compounds will be tested as adjuvant drugs in cancer immunotherapy.Highlights:1.Our study indicates that lenalidomide can bypass CD28 requirement to promote CD8⁺T cell activation and cytotoxicity both in vitro and in vivo.2.Our study indicates that lenalidomide can reinstate PD-1 blockade efficacy compromised by loss of CD28.3.Our study reveals that lenalidomide co-stimulates CD28^-CD8⁺T cell in part by increasing the expression of some Notch-activated genes.4.Our study indicates that loss of Ptpn2,Jak1 or Eef2 sensitizes mouse colon cancer cells to lenalidomide treatment.