Fig 1: Functional ability of candidate aptamers to inhibit cellular transcriptional response to LT⍺(A) Illustration of signaling pathways induced by LT⍺/TNF⍺-TNFR1. Left: Engagement induces TNFR1 trimerization and recruitment of the adaptor protein TRADD, along with TRAF2, RIPK1, and cellular inhibitors of apoptosis (cIAP1/2), forming the membrane-associated signaling complex I. This complex activates TAK1, which in turn phosphorylates the IKK complex (IKK⍺, IKKβ, and IKKƔ/NEMO). Activated IKK phosphorylates IκB⍺, targeting it for degradation and releasing the canonical NF-κB heterodimer RelA (p65)/p50. RelA/p50 translocate to the nucleus to drive expression of acute inflammatory and survival-associated genes, exemplified here by Cxcl1, Ccl2, and Ccl7. Right: Signaling initiated by binding of LTβR by LT⍺1β2 recruits TRAF2, TRAF3, and cIAP1/2, forming a regulatory complex that controls the stability of NF-κB-inducing kinase (NIK). Ligand engagement leads to TRAF3 degradation and consequent NIK accumulation, enabling activation of an IKK⍺ homodimer independent of IKKβ and NEMO. Activated IKK⍺ phosphorylates p100, promoting its proteolytic processing into p52. The resulting RelB/p52 heterodimer then translocates to the nucleus, driving a distinct transcriptional program associated with lymphoid tissue organization and stromal cell activation, including Cxcl13, Ccl19, and Ccl21, and the transcription of genes involved in lymphoid tissue organization, stromal cell differentiation, and chemokine expression. (B–J) L929 murine fibroblasts were stimulated with LT⍺3 (1 ng/ml) alone or after 1 h preincubation with pateclizumab (PzMAb, 1 μg/ml), LT⍺-specific aptamers (LTa1, LTa5, LTa9, LTa12 at 100 nM), or a scrambled aptamer control (AptScram 100 nM). Temporal induction of cytokine and chemokine transcripts were determined to identify peak induction, while bar graphs show data from a single measured time point (indicated as 1 or 24 h) and the impact of specific inhibitors. Both panels display gene expression changes as mean ± SD ΔΔCt normalized to housekeeping controls. (B–D) (left) Time course and (right) 1-h qPCR analysis of (B) Cxcl1, (C) Ccl7, and (D) Ccl2 expression following LT⍺3 stimulation. (E–G) Expression of non-canonical NF-κB-associated lymphoid chemokine transcripts: (E) Cxcl12, (F) Ccl21, and (G) Ccl19 following LT⍺1β2 stimulation. (Left) Temporal expression kinetics and (right) alterations in 24-h peak cytokine transcript expression levels under the indicated inhibitory conditions. (H and I) (left) Time course and (right) 1-h endpoint expression of (H) Cxcl1, (I) Ccl7, and (J) Ccl2 following TNF-⍺ stimulation in the presence of indicated inhibitors. Statistical significance was determined using two-way ANOVA with Tukey’s multiple-comparison post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 2: Dynamic regulation of lymphotoxin in TIL-melanoma cocultures.A, UMAP projection of expanded CD8+ TIL pre- and post-coculture with cancer cells for 24 hours (n = 31,413 cells) with 9 unique populations identified from scRNA-seq analysis. B, Percentage of LTA+CD8+ TIL before and after coculture with control (sgNC) melanoma cells. Individual open circles represent one TIL (n = 4; 2-sided paired t-test). C, Dot plot showing expression of selected genes before and after coculture with control (sgNC) melanoma cells. D, Bar plot examining the log2 fold change (mean CPM) of the LTA/LTB ratio in TIL before and after coculture with sgNC. E, Schematic of experimental design. CD8+ OT-1 T cells were isolated from the spleens of OT-1 transgenic mice, activated by αCD3 and αCD28 antibodies, and expanded in vitro with IL-2 (50 ng/mL) for 48 hours. Activated OT-1 T cells were then co-cultured for 0, 24, and 48 hours with B16-OVA cells pretreated with IFNγ (E:T = 8:1) and named as OT-1_0, OT-1_24, and OT-1_48 after dead cell removal. Next, these OT-1 cells were cocultured with B16-OVA or B16-B2m KO cells pretreated with IFNγ, respectively. F, UMAP embedding of scRNA-seq data for OT-1_0, OT-1_24, and OT-1_48 cells. G, Dot plot showing the expression of selected genes among OT-1_0, OT-1_24, and OT-1_48 cells. H, Bar plot examining the log2 fold change (mean CPM) of Lta/Ltb ratio in OT-1_0, OT-1_24, and OT-1_48 cells. I-J, Cell viability of B16-OVA+IFNγ and B16 B2m KO+IFNγ cocultured with OT-1_0, OT-1_24, and OT-1_48 cells. Mean +/− s.d. (bar) is shown (n = 3; Two-way ANOVA with Tukey correction of multiple comparisons). K-L, Normalized fluorescence intensity of viable melanoma cells (10049, sgNC-GFP) cocultured with autologous TIL at the E:T ratio of (K) 2:1 and (L) 1:1 with or without the indicated neutralizing antibodies (anti-LTα, 10 μg/mL; anti-IFNγ, 10 μg/mL) over indicated time points. Mean +/− s.d. (shaded region) is shown (n = 3, two-way ANOVA with Tukey correction of multiple comparisons; ns, not significant). M, Schematic showing the dynamic change of LTA, LTB, and IFNG expression as well as LTA/LTB ratio upon coculture with cancer cells.
Fig 3: LTB+CD8+ T cells are enriched in TIL products of patients responsive to lifileucel.A, UMAP embedding of scRNA-seq data from CD8+ TIL (n = 116,540 cells) from lifileucel TIL products (n = 34) identifying 15 unique clusters. B, Dot plot showing the representative marker genes that are differentially expressed across different clusters. C, UMAP projection of LTB expression in lifileucel TIL products. D-E, Bar plots showing the percentage of (D) TIL-LTBhi_c0 and (E) LTB+CD8+ TIL of all CD8+ TIL grouped by best overall response (BOR); responder (R), stable disease (SD), or progressive disease (PD). Patient-level pseudobulk profiles were generated from the scRNA-seq data and gene set scoring was performed as described in the methods. Mean +/− s.d. (bars) and individual values (open circles) are shown (Kruskal-Wallis test with Dunn’s multiple comparisons test; ns, not significant). F-H, Bar plots showing (F) stem-like signature enrichment scores, (G) cytolytic activity scores, and (H) terminal differentiation enrichment scores for LTBhi cluster 0 (TIL-LTBhi_c0) compared to all other CD8+ TIL clusters. Mean +/− s.d. (bars) and individual values (open circles) are shown (Kruskal-Wallis test with Dunn’s multiple comparisons test).
Fig 4: LTB+CD8+ TIL are expanded from putative neo-antigen reactive CD8+ T cells.A, UMAP embedding of scRNA-seq data from CD8+ T cells (n = 4,401 cells) in baseline tumor digests (n = 7). B, UMAP projection of LTB expression in baseline tumor digests. C, Selected differentially expressed genes between baseline tumor digests and TIL product as indicated. D, Percentage of LTB+CD8+ cells in TIL products compared to matched baseline tumor digests (n = 7, 2-sided paired t-test). E, Projection of TCRs shared with TIL-LTBhi_c0 (green) compared to TCRs from all other CD8+ TIL clusters (light blue) on the UMAP embedding of CD8+ T cells of the baseline tumor digests. F, NeoTCR8 signature expression projected on the UMAP embedding of CD8+ T cells of the baseline tumor digests. G, Proportions of CD8+ T cell clusters in baseline tumor digests with TCRs shared with TIL-LTBhi_c0 compared to all other CD8+ TIL clusters (ordered by NeoTCR8 signature enrichment). H-I, Projection of TCRs shared with (H) Baseline_LTBhi and (I) Baseline_NeoTCR8hi CD8+ T cells on the UMAP embedding from CD8+ TIL (lifileucel TIL products). J, Bar plot showing the percentage of cells expressing the LTBhi or NeoTCR8hi signature in baseline tumor digests, and the percentage of cells expressing the same TCRs in lifileucel products. Mean +/− s.d. (bars) and individual values (open circles) are shown (n = 7, 2-sided paired t-test, ns, not significant). K, Dot plot showing expression of indicated genes in NeoTCR8hi CD8+ T cells in baseline tumor digests compared to CD8+ TIL in lifileucel with shared TCRs. L, Schematic showing the dynamic change of LTBhi and NeoTCR8hi (also LTBlo) CD8+ T cells during expansion. M, Schematic showing the proposed working model.
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