Detecting Tumor Antigen-Specific T Cells via Interaction-Dependent Fucosyl-Biotinylation

Abstract

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach, intratumoral TSA-reactive CD4⁺, CD8⁺ T cells, and TSA-suppressive CD4⁺ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype, TSA-reactive CD8⁺ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle, FucoID should have the potential of accelerating the pace of personalized cancer treatment.

Keywords: TCR sequencing; antigen-specific T cell; bystander T cell; cancer; cell-cell interaction; glycosylation; immunotherapy.

Figure 1.. Illustration of probing cell-cell interactions via interaction dependent fucosyl-biotinylation (FucoID).

(A) Schematic representation of FucoID for the labeling of cell-cell interactions. Conjugation of H. pylori α(1,3)fucosyltransferase (FT) onto the bait cell surface was achieved via the FT-mediated chemoenzymatic glycan labeling using GDP-Fuc-FT as a self-catalyst. (B) Synthesis of GDP-Fuc-FT. (C) Experimental design and representative fluorescence microscopy images of the interaction dependent labeling mediated by FT-functionalized CHO cells (CHO-FT). CHO-FT cells stained with CFSE were mixed (cell ratio 1:5) with unfunctionalized CHO cells, followed by the addition of GDP-Fuc-Biotin (50 μM). Cells were stained with DAPI and streptavidin-APC for fluorescent microscopy imaging. Scale bar: 10 μm.

Figure 2.. Demonstration of the specificity of FucoID in probing DC-T cell interactions.

(A) Workflow for analyzing the FucoID enabled labeling of naïve OT-I CD8⁺ T cells using LCMV GP_33–41 and OVA_257–264 primed iDCs. (B) Flow cytometric analysis showing antigen-specific fucosyl-biotinylation of CD8⁺ T cells under iDC:T ratio of 1:1. (C) Flow cytometric analysis of antigen-specific fucosyl-biotinylation in the mixture of OT-I and P14 CD8⁺ T cells when incubated with iDC-FT primed with LCMV GP_33–41 or OVA_257–264, n=3.

Figure 3.. Probing antigen-specific DC-T cell interactions via FucoID in splenocytes.

(A) Workflow for analyzing the interactions of antigen-primed iDCs with naïve OT-I T cells in OT-I splenocytes. (B) Flow cytometric analysis of antigen-specific fucosyl-biotinylation of CD8⁺ T cells in OT-I splenocytes by iDC-FT loaded with OVA_257–264 at the iDC:T cell ratio = 1:1. (C) Flow cytometric analysis of OT-I T cells selective labeling in C57BL/6J splenocytes (doped with OT-I splenocytes, OT-I T cells at a final ratio of 2% in total cells) by iDC-FT. (D) Flow cytometric analysis of OT-I CD8⁺ T cells Fuc-biotinylation by iDC-FT loaded with altered peptide ligands (APLs) derived from the original OT-I peptide SIINFEKL N4 (OVA_257–264). (E) Flow cytometric analysis of CD4⁺ T cells specific fucosyl-biotinylation by iDC-FT primed with OVA_323–339 in OT-II splenocytes (iDC:T ratio = 1:1), n=3.

Figure 4.. Identification and characterization of endogenous TSA-reactive CD8⁺ T cells from a B16-OVA melanoma model.

(A) Schematic illustration of the detection and enrichment of TSA-reactive TILs via FucoID. (B) Workflow of using FucoID to identify TSA-reactive and bystander CD8⁺ TILs from a B16-OVA tumor. (C) Representative flow cytometric analysis of TSA-dependent fucosyl-biotinylation of CD8⁺ TILs in B16-OVA tumors, n=3. (D) Experimental scheme for validating the isolated PD-1⁺Bio⁺ TILs as bona fide TSA-reactive T cells. Three isolated TIL subsets (CD45.1^+/+) were transferred to normal C57BL/6 mice (CD45.2), immunized with LM-OVA on the next day. Blood and splenocytes were analyzed after another 7 and 37 days, respectively. The isolated splenic CD8⁺ T cells were re-transferred to C57BL/6 mice (CD45.2), immunized with LM-OVA on the next day, and blood was analyzed after another 7 days. Representative flow cytometry plots are from three biological replicates. See also Figure S4B and C.

Figure 5.. Identification and characterization of TSA-reactive and bystander CD8⁺ T cells in distinct murine tumor models.

(A) Workflow for the detection of TSA-reactive CD8⁺ T cells from syngeneic murine tumor models via FucoID. (B) Representative flow cytometric analysis of tumor antigen dependent fucosyl-biotinylation of CD8⁺ TILs in B16 melanoma, E0771 TNBC and MC38 colon cancer models, n=3. (C) IFN-γ ELISpot showing distinct reactivity of expanded PD-1⁻, PD-1⁺Bio⁻ and PD-1⁺Bio⁺ CD8⁺ TILs upon tumor antigen re-stimulation, n=3; (D) Comparisons of the expanded total PD-1⁺ CD8⁺ TILs and PD-1⁺Bio⁺ CD8⁺ TILs for inducing specific lysis of the relevant cancer cells; n=3. (E) In vivo antitumor immunity evaluation of PD-1⁺Bio⁺ and PD-1⁺ CD8⁺ TILs in a murine B16 metastasis tumor model. C57BL/6J mice were intravenously injected with 0.5×10⁶ B16-luc cells. TIL transfer was performed as described in Method on day 3 of tumor inoculation. On day 8 of TIL transfer, luciferin was administrated, and light emission was recorded. Representative bioluminescence images are shown; HBSS: 7 mice; total PD-1⁺: 8 mice; PD-1⁺Bio⁺: 6 mice. (F) In vivo antitumor immunity evaluation of PD-1⁺Bio⁺ and PD-1⁺ CD8⁺ TILs in a murine MC38 s.c. tumor model. MC38 cells were s.c. injected into the right flanks of male C57BL/6J mice (0.5×10⁶ cells per mice). Mice were irradiated (5 Gy) on day 2 of tumor inoculation. Then TIL transfer was performed on day 3 of tumor inoculation. Tumor volumes were measured every 2 days. HBSS: 7 mice; anti-PD-1: 7 mice; total PD-1⁺: 7 mice; PD-1⁺Bio⁺: 8 mice.

Figure 6.. Gene-expression and functional marker characterizations of CD8⁺ TIL subsets isolated via FucoID from MC38 tumors.

(A) PCA of the transcriptome of PD-1⁺Bio⁺ (red), PD-1⁺Bio⁻ (blue) and PD-1⁻ (green) CD8⁺ TILs isolated from murine MC38 tumors. Dots represent samples of the three different populations (grouped by colors) from a total of three biological replicates (grouped by shapes). PCA 1 and 2 represents the largest source of variation. (B) Volcano plot of up- (red) and down-(blue) regulated genes between PD-1⁺Bio⁺ and PD-1⁺Bio⁻ TILs. Significance was determined as Benjamini–Hochberg FDR (p.adjust) < 0.05 and |log₂(fold change)| ≥ 0.6. (C) Biological processes (GO terms) enriched in the up– and down–regulated genes identified in Figure 6B. (D) Gene set enrichment analysis (GSEA) of activation/dysfunction CD8 gene module (Singer et al., 2016) in the transcriptome of PD-1⁺Bio⁺ vs. that of PD-1⁺Bio⁻ CD8⁺ TILs. See also Figure S5 and Table S1. (E) GSEA of up- and down-regulated tumor specific CD8 gene signature (Schietinger et al., 2016) in the transcriptome of PD-1⁺Bio⁺ vs. that of PD-1⁺Bio⁻ CD8⁺ TILs. See also Figure S5 and Table S1. (F) The comparison of representative gene expression of PD-1⁺Bio⁺, PD-1⁺Bio⁻ and PD-1⁻ CD8⁺ TILs. n=3. (G) The expression of PD-1, CD137, TIM-3, CD39 and CD103 in PD-1⁺Bio⁺, PD-1⁺Bio⁻ and PD-1⁻ CD8⁺ TILs from murine MC38 tumor according to flow cytometric analysis. Data obtained from at least two independent replicates.

Figure 7.. TSA-suppressive and -reactive CD4⁺ TILs play opposite roles in regulating the antitumor immunity of TSA-reactive CD8⁺ TILs in a Pan02 pancreatic cancer model.

(A) and (B) Flow cytometric analysis of tumor antigen-specific fucosyl-biotinylation of CD8⁺ TILs (A) and CD4⁺ TILs (B) in the Pan02 model. (C) Flow cytometric analysis of functional markers on Bio⁻ CD4⁺ TILs and Bio⁺ CD4⁺ TILs in Pan02 tumors. (D) IFN-γ ELISpot analysis of tumor-suppressive and -reactive functions of the isolated TIL subsets. Five subsets of TILs (Bio⁺ CD8⁺, Bio⁻ CD8⁺, CD25⁻Bio⁺ CD4⁺, CD25⁺Bio⁺ CD4⁺ and Bio⁻ CD4⁺) isolated from Pan02 tumor digests were re-stimulated by DCs primed with tumor lysates. Re-stimulations were conducted using the individual TIL subsets and a combination of two subsets as specified, n=3.