YY1 Upregulates Checkpoint Receptors and Downregulates Type I Cytokines in Exhausted, Chronically Stimulated Human T Cells

Abstract

T cells infiltrate affected organs in chronic infections and malignancy, but they may fail to eradicate virus-infected cells or tumor because of exhaustion. This report describes a Yin Yang-1 (YY1)-centered mechanism for diverse components that have been correlated with exhaustion. Utilizing an in vitro reconstruction of chronic T cell activation, YY1 is shown to positively regulate the checkpoint receptors PD1, Lag3, and Tim3 and to negatively regulate the type I cytokines interleukin-2 (IL-2) (in collaboration with Ezh2 histone methyltransferase) and interferon gamma (IFN-?). Other tests suggest that IL-2 failure drives a large component of cytotoxic functional decline rather than solely checkpoint receptor-ligand interactions that have been the focus of current anti-exhaustion therapies. Clinical evaluations confirm elevated YY1 and Ezh2 in melanoma tumor-infiltrating lymphocytes and in PD1+ T cells in patients with HIV. Exhaustion is revealed to be an active process as the culmination of repetitive two-signal stimulation in a feedback loop via CD3/CD28?p38MAPK/JNK?YY1? exhaustion.

Keywords: Cancer Systems Biology; Immune Response; Immune System Disorder.

Graphical abstract

Figure 1

Persistent T Cell Activation In Vitro Induces IL-2 Shutdown and Checkpoint Receptor Elevation (A) Schematic depicting repeat stimulation model. T cells were stimulated with anti-CD3/CD28 beads for 2 days; cells were counted at the end of 2 days, beads removed, and cells placed in new medium with fresh beads for the next stimulation. Control cells were cultured identically without beads. (B) ELISA shows high secretion and then decline of IL-2 production after repeated stimulations. (C) Flow cytometry of re-stimulated CD4 T cells analyzed at day 8 shows increasing expression of exhaustion markers, PD1, Lag3, and Tim3. CD8 T cells exhibited same pattern (data not shown). Similar results were obtained when the cells were analyzed right after first, second, third, and fourth stimulations. (D) qRT-PCR and (E) IL-2 promoter luciferase assay in CD4 T cells shows fold change of IL-2 mRNA and promoter activity after re-stimulations. Four replicates performed per assay; data from one of three representative experiments. *p < 0.05.

Figure 2

Persistent T Cell Activation Upregulates YY1 and Ezh2 to Epigenetically Silence IL-2 (A) Western blots show increasing expression of YY1 and Ezh2 after re-stimulation of CD4 T cells, analyzed on day 8. Similar results obtained with CD8 T cells (not shown). β-Actin is the loading control. (B) More YY1 and Ezh2 from persistent activation by comparing 1–4 stimulations with results assessed on day 8 days as in (A). (C) Immunofluorescence (IF) performed on four times stimulated exhausted T cells and unstimulated PBMCs analyzed on day 8 showing YY1 and Ezh2 expression. Right panels show magnified single cell single positive for CD3 or double positive for CD3/YY1 and CD3/Ezh2. In unstimulated PBMCs, Ezh2 is seen to be expressed in non–T cells that correspond to the B cells that are present and known to express Ezh2 (van Galen et al., 2004). (D) Western blot shows decreased levels of H3K27me3 with Ezh2 inhibitor. T cells were treated continuously with Ezh2 inhibitor or mock reagent before third stimulation and analyzed after four stimulations. (E) qPCR for IL-2 promoter shows high H3K27me3 methylation with recurrent stimulation that is decreased with Ezh2 inhibitor or YY1 shRNA. Chromatin immunoprecipitation (ChIP) with anti-H3K27me3 antibody or IgG in twice-stimulated CD4 T cells, transduced before third stimulation with plasmid for control-shRNA (lane 2) or YY1 shRNA (lane 3), or maintained in the presence of Ezh2 inhibitor (lane 4). (F) ELISA shows sustained IL-2 production from CD4 T cells stimulated repeatedly in vitro without or with Ezh2 inhibitor. (G) ELISA assay shows recovered IL-2 production from exhausted T cells with YY1 shRNA. CD4 T cells stimulated twice produced high IL-2 but afterward these cells were exhausted. Twice-stimulated exhausted cells were transduced with plasmids for YY1-specific shRNA and control shRNA or no plasmid just before the third or fourth stimulations. Western blot shows effective YY1 knockdown by shRNA, analyzed 2 days after shRNA before the fourth stimulation. (H) Co-immunoprecipitation (coIP) confirms YY1 interaction with Ezh2 in repeatedly stimulated T cells. Immunoprecipitation (IP) was with Ezh2 antibody, and the blot was probed with YY1 (upper panel) or Ezh2 antibody (bottom panel). No Ezh2 or complex was recovered by Ezh2 IP from unstimulated T cells. (I) Model showing YY1 acting in association with Ezh2 to repress IL-2 transcription. Graphs in (E)–(G) are from a minimum of three replicates; assays representative of at least three experiments. *p < 0.05.

Figure 3

YY1 Upregulates Checkpoint Receptors in Exhaustion (A and B) Electrophoretic mobility shift assay (EMSA) shows YY1 binding to PD1 and Lag3 promoters. Recombinant YY1 protein was incubated with PD1 and Lag3 probes containing intact (lane 2) or mutated YY1-binding sites (lane 3). Epstein-Barr nuclear antigen (EBNA) extract was the control non-specific extract (lane 4). (C and D) Luciferase reporter assay shows increased activity with PD1 and Lag3 reporter plasmids in re-stimulated CD8 T cells with intact YY1-binding sites and activity loss with YY1-mutated sites. Three replicates per assay; *p < 0.05. (E) Flow cytometry shows suppression of increase of PD1, Lag3, and Tim3 in CD8 T cells expressing lentiviral YY1-shRNA. Samples analyzed after four stimulations. (F) YY1 knockdown achieved with YY1 shRNA compared with control after four stimulations.

Figure 4

cJun/ATF2 Stimulates YY1 Transcription (A) YY1 promoter sequence shows cAMP response element (CRE)-binding site for cJun/ATF2 at −136 relative to transcription start site (TSS). (B) Western blot (upper) and qRT-PCR (lower) show increase of YY1 protein and mRNA in CD8 T cells with re-stimulation. Three replicates per assay. (C) Luciferase reporter gene assay of YY1 promoter shows increased activity with re-stimulation (blue bars) and activity loss with mutation of CRE site (red bars). (D) Western blot shows increased phospho-cJun and phospho-ATF2 in CD4 T cells after re-stimulation. Total cJun and ATF2 levels unchanged. (E) Native gel analysis shows increased phospho-cJun dimer formation with restimulation and loss of monomer detected by specific antibodies. (F) Electrophoretic mobility shift assay (EMSA) confirms binding of phospho-cJun and phospho-ATF2 to site on YY1 promoter. YY1 probe was mixed with nuclear extract from four times stimulated CD8 T cells and incubated with antibody to phospho-cJun or phospho-ATF2 before gel loading. Bottom panel: western blot developed after species-specific secondary antibody to detect primary antibodies bound to the probe.

Figure 5

p38MAPK/JNK Signaling Pathway Regulates YY1 Expression (A) Model depicting p38MAPK/JNK signaling to cJun/ATF2 to regulate YY1 in T cells exposed to antigen-rich environment. T cell activation initiates phosphorylation of tyrosine residues within the ITAMs of the TCR/CD3 complex and CD28 co-stimulatory molecules by Src family tyrosine-protein kinases, Lck and Fyn (Arbour et al., 2002), to serve as docking site for ZAP70. Phospho-ZAP70^{(Tyr319/Tyr352)} amplifies the downstream signaling cascade. CD28 co-stimulation complements TCR stimulation by activating and integrating serine-threonine kinase AKT with MEKK kinase (Kane et al., 2001) to phosphorylate MKK3/6 and MKK4/7. Phoshpho-MKK3^(Ser189)/MKK6^(Ser207) and phospho-MKK4^{(Ser257/Thr261)} activate p38 MAPK and the cJun-N-terminal kinase (JNK) pathways, respectively (Kang et al., 2006, Raingeaud et al., 1995). Phospho-p38 MAPK^{(Thr180/Tyr182)} in turn activates ATF2 and phospho-JNK2 ^{(Thr183/Tyr185)} activates the cJun transcription factor (Freshney et al., 1994, Ichijo, 1999). Phospho-cJun and phospho-ATF2 form heterodimers that bind to the YY1 promoter to initiate transcription. (B) Western blots show increased phosphorylation of kinases in p38MAPK/JNK pathway with T cell re-stimulation. (C) YY1 is suppressed by inhibitors to p38 and JNK by reporter assay (upper) and by western blot (lower). Three replicates per assay; representative of three experiments. *p < 0.05.

Figure 6

Functional Assessment of T Cell Exhaustion (A) Design of CAR-T cells. An anti-CEA sFv-antibody-binding domain is fused with CD3 zeta (signal 1) or CD3 zeta plus CD28 (signal 1 + 2) signaling chains in first- and second-generation CAR-T cells. (B and C) Increased expression of checkpoint receptors with restimulation only on second-generation CAR-T cells. CAR-T cells were created with approximately 50% modification and then co-cultured three times for two days with MIPCEA or MIP101 cell lines and analyzed by fluorescence-activated cell sorting (FACS) for PD1 and Lag3. (All remaining assays are with second-generation CAR-T cells). (D) IL-2 ELISA shows high initial IL-2 and then decline in MIPCEA/CAR-T cell co-cultures. Antigen-negative MIP101 cells do not stimulate IL-2 production. (E) Functional exhaustion shown by live images of MIPCEA target cells before and after co-culture with CAR-T cells. T cells from first co-culture were recovered and re-exposed to second and third batches of target tumor cells. High clearing of plates in first co-cultures indicates high initial killing that shows less clearing (less killing) in subsequent co-cultures. (F) IL-2 ELISA as in (D) except co-culture performed in the presence of Ezh2 inhibitor, now showing sustained IL-2 for MIPCEA + CAR-T. (G) Reversal of functional exhaustion shown by live images of MIPCEA and control MIP101 target cells after co-culture with CAR-T cells in the presence of Ezh2 inhibitor without or with anti-IL-2 antibody that blocks exhaustion reversal. (H) Cell counts obtained from experiment in (E) and (G). Three replicates per assay; representative of three experiments.

Figure 7

New Exhaustion Markers Confirmed in Melanoma TILs (A and B) Human melanoma sections underwent immunofluorescence (IF) staining, showing most infiltrating T cells are PD1+ (CD3 and PD1 double staining) and activated phospho-cJun^{(ser 63/73)} positive (CD3/phospho-cJun^{(ser 63/73)} double staining). The tumor cells themselves are positive for phospho-cJun. Normal skin T cells were negative for PD1 and phospho-cJun (arrows point to CD3 cells negative for PD1 in A and CD3 cells negative for phospho-cJun in B). Magnified panels shown for double staining. (C and D) IHC showing expression of YY1 and Ezh2 in TILs, confirmed on double staining (CD3 is brown, YY1 and Ezh2 are red; merged IHC dark red for double positive). Normal skin T cells were negative for YY1 and Ezh2, bottom panels (arrows). Scale bar represents 20 μm. These stainings were performed five times with a minimum of two melanoma and two skin samples in each test with concordant results.

Figure 8

New Exhaustion Markers Confirmed in HIV T Cells (A and B) CD4 T cells from (A) control subjects and (B) patients with HIV were stained and analyzed by flow cytometry for PD1 and YY1 or PD1 and Ezh2 as in Transparent Methods. (C) Summary of MFI values for YY1 and Ezh2 in PD1- and PD1+ CD4 T cells. MFI for YY1 and Ezh2 is higher in PD1+ than PD1- cells at p<0.02 (paired two-tailed t-test).