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Immunotherapy, including adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) predominantly targets improvement in MHC Class I-mediated anti-tumor immune responses. The primary objective of this study is to better understand the role of CD4+ TIL in ACT as a complementary avenue for therapeutic efficacy. Briefly, CD4+ TIL were isolated by negative selection from metastatic melanoma patients who received TIL therapy at Moffitt Cancer Center on IRB approved protocols. Individual T cell clones were tracked by TCRbeta sequencing to quantify clonal persistence in patients. CD4+ TIL clones were found to be decidedly persistent in a candidate patient who achieved a complete response (CR) after infusion of 88% CD4+ T cells. CD4+ TIL from additional patients were stimulated with anti-CD3/CD28 in vitro and those who were clinical responders demonstrated a pleiotropic cytokine profile marked by an elevated ratio of Th1:Th2 cytokines (p=0.07, n=13). When cultured with APCs loaded with autologous tumor (AT), CD4+ TIL produced high levels of IFN-gamma in an MHC Class II-dependent manner. Induction of MHC Class II on melanoma cell lines and AT determined that CD4+ TIL secreted IFNg and TNFa directly in response to AT. ACT of tumor-reactive CD4+ TIL in immunodeficient (NSG) mice provided significant control of AT growth when compared to non-reactive CD4+ TIL. Preliminary data in syngeneic mouse models also suggests that antigen-specific CD4+ T cells aid in initial tumor rejection, memory formation and epitope spreading, resulting in an overall increased therapeutic efficacy during ACT. This data supports the conclusion that CD4+ TIL are tumor-reactive and instrumental to an effective anti-tumor immune response in cancer patients.

Immunomodulatory drugs, such as lenalidomide, pomalidomide, and CC122 are glutarimide derivatives that alter T cell effector functions, reverse tolerance, and increase cytokine production. We hypothesized that cereblon (CRBN), the molecular target of these drugs, is a negative regulator of T cell function. Therefore, germline Crbn knockout mice were used to investigate its role in T cell regulation. Similar to human T cells treated with immunomodulatory drugs, stimulated Crbn−/− T cells exhibit increased proliferation and cytokine production and maintain this phenotype in the absence of CD28 co-stimulation. Using Gene Set Enrichment Analysis (GSEA) to identify transcriptional drivers of differentially expressed genes, we found that Myc and its family member Max may drive the observed activated phenotype in Crbn−/− T cell. Crbn−/− T cells have Myc-related phenotypic changes, such as increased cell size, CD98 expression, glucose uptake, glutamine and arginine uptake, increased intracellular polyamine biosynthesis, and oxygen consumption. Similar increases in Myc-related processes were observed in activated human T cells treated with immunomodulatory drugs. While c-myc mRNA is similar, Crbn−/− T cells and drug-treated human T cells have prolonged c-Myc protein expression. CRBN, a substrate receptor for the DDB1/Cul4A/Rbx1 E3 ubiquitin ligase complex, has only one known endogenous substrate which is glutamine synthetase (GS). GS interacts with CRBN through acetylated lysine residues yielding a protein that is succeptable to polyubiquitinlyation. Collectively, this suggests that CRBN tunes c-Myc-regulated pathways in activated T cells and that this process is blocked by immunomodulatory drugs.

Druggable targets that reinvigorate T cells may improve the efficacy of immunotherapy in melanoma or other cancers. T cells exist in a hostile tumor microenvironment associated with hypoxia, high lactic acid, and poor nutrient availability which limits their anti-tumor activity. We found that the genetic deficiency of Crbn, a novel E3 ubiquitin ligase substrate receptor, improves effector T cell function. Growth of subcutaneous B16 tumors was slower in Crbn−/− mice and the adoptive transfer of Crbn−/− T cells into mice with established B16 tumors lead to a significant growth reduction. While similar total tumor infiltrating lymphocytes (TIL) numbers and normal splenic populations were present, a significant increase in the proportions of CD44+ and PD1+ CD8+ TIL but not KLRG1+CD8+ TIL were present in Crbn−/− mice. These results suggest that CRBN represses antigen-dependent anti-tumor T cell immunity and represents a novel regulator of T cells in the tumor microenvironment. In melanoma, hypoxia is a mode of T cell suppression. Activated Crbn−/− T cells demonstrated higher extracellular acidification rate, oxygen consumption and glucose uptake compared to Crbn+/+ T cells. Under hypoxic conditions (2% O2), effector cell cytotoxicity is reduced at high densities of B16 melanoma compared to normal atmospheric oxygen (~21% O2) and proliferation is suppressed due to reduction of OxPhos. Using anti-CD3ɛ/anti-CD28 stimulation in hypoxic conditions, Crbn−/− T cells retained their proliferative capacity compared to Crbn+/+ T cells indicating that deletion of Crbn restores metabolic activity of T cells in a hostile tumor microenvironment and may serve as a novel drug target in immunotherapy.

Protein degraders have been developed that can aide in understanding gene function. These chemicals contain complementary bifunctional components that 1) interact with specific proteins, and 2) engage an E3 ligase which leads to polyubiquitination and intracellular degradation by the proteasome. Thalidomide, infamously known for its teratogenic effects, is central to this new technology since it binds cereblon (CRBN) which promiscuously recruits the DDB1/Cul4A/Rbx1 complex to trigger target destruction. Thalidomide, and especially its immunomodulatory derivatives lenalidomide and pomalidomide, induce NK and T-cell activation by degrading IKZF1 which is a transcription factor that suppresses IL-2. Currently, it is debated whether mouse cereblon has conserved function due to a single non-conserved amino acid in mouse CRBN, Ile390 (equivalent to Val388 in human CRBN). First, our work confirms that lenalidomide induces IL-2 and the ubiquitin-dependent degradation of IKZF1 only in human and not mouse T-cells treated with lenalidomide. To further test for species-related effects, a series of theoretical and physical binding assays using mutant CRBN proteins show that the binding of IMiD compounds is not impaired by amino acid differences in mouse CRBN. Using dBET1, a novel thalidomide-JQ1 protein degrader, we show for the first time that mouse CRBN can trigger CRBN-dependent degradation of BRD4 and confirm that the CRBN/DDB1/Cul4A/Rbx1 complex is functional in mouse T-cells. Collectively, our results suggest that IKZF1 has divergent regulation in mouse cells rather than the IMiD complex and provide information relevant to the development of chemical conjugates that induce targeted intracellular protein degradation.

Thalidomide, and its immunomodulatory drug analogs (IMiD) including lenalidomide and pomalidomide, activate T-cells and reverse T-cell anergy in cancer patients. Previously we have shown that cereblon, the molecular target of IMiD compounds, is a negative regulator of T-cell metabolism and signaling following suboptimal antigen stimulation. To explore this mechanistically, differentially-regulated genes were subjected to gene set enrichment analysis (GSEA) for transcriptional regulators and global metabolomic profiling was conducted by mass spectrometry in activated Crbn+/+ versus Crbn−/− T-cells. With results confirmed by 1H-NMR spectra analysis, we show that Crbn deficiency leads to an increase in intermediates of the “Arginine/Proline” pathway and enhanced polyamine (putrescine, spermidine, and spermine) biosynthesis. Wang et al showed previously that c-MYC controls polyamine synthesis downstream of mTORC1 during T-cell activation. In activated Crbn−/− CD8+ T-cells, the kinetics and magnitude of c-Myc mRNA were similar to Crbn+/+ T-cells. However, c-MYC is largely regulated through post-transcriptional mechanisms including ubiquitin proteasome-mediated degradation and regulation of c-Myc mRNA translation. Interestingly, polyamines directly bind U- and AU-rich elements (AREs) in the 3′-untranslated region (3′-UTR) of c-Myc mRNA and spermidine is the substrate for a unique post-translational modification required for the efficient translation of c-MYC. Western blot analysis revealed more rapid and sustained expression of c-MYC in activated Crbn−/− T-cells compared to Crbn+/+ T-cells suggesting that CRBN is a regulator of the MYC-polyamine circuit controlling T-cell metabolic fate.