Therapeutic melanoma vaccine with cancer stem cell phenotype represses exhaustion and maintains antigen-specific T cell stemness by up-regulating BCL6

Figures & data

Table 1. Characteristics of the patients enrolled in this study

	Patient	Sex	Age	Stage	Time of vaccine adm. [months]	Tumor-free period [months]	Pan T cell separation [%CD2+ CD3+]	RNA quality [RIN]	Tested on microarray [y/n]
1	AV_1	M	70	IIIc	182	182	86.1	8.7	y
2	AV _2	M	41	IIIb	193	193	88.2	9.2	y
3	AV _3	F	54	IIIc	141	141	90.0	7.7	y
4	AV _4	F	63	IVa	135	135	79.9	8.1	y
5	AV _5	M	50	IVc	178	178	83.5	6.9	n
6	AV _6	F	60	IVa	167	167	80.2	NA	n
7	AV _7	F	57	IIIb	169	169	80.5	NA	n
8	AV _8	F	53	IIIc	143	143	83.7	7.9	n
9	AV _9	M	70	IIIc	147	147	81.9	8.7	y
10	AV _10	F	53	IIIb	141	141	82.4	7.1	n
11	AV _11	F	69	IVc	13	13	84.6	7.6	n
12	AV _12	M	55	IIIa	132	132	85.9	5.8	n
13	AV _13	F	76	IIIc	171	171	90.6	NA	n
14	AV _14	M	68	IIIb	130	130	81.0	6.9	n
15	AV _15	F	71	IIIc	191	191	80.2	7.6	n
16	AV _16	F	65	IIIc	145	145	80.4	8.7	y
17	AV _17	F	59	IIIc	190	190	89.9	8.4	y
18	AV _18	M	68	IIIb	141	141	87.6	8.8	y
19	C_1	M	37	IIIa	NA	0	80.1	8.8	y
20	C_2	M	61	IVc	NA	0	80.8	8.7	y
21	C_3	F	64	IVc	NA	0	83.2	8.6	y
22	C_4	M	60	ND	NA	0	81.6	8.7	y
23	C_5	M	50	IVc	NA	0	84.3	7.2	n
24	C_6	M	64	ND	NA	0	80.9	8.7	n
25	C_7	M	78	IIIc	NA	0	90	8.5	y
26	C_8	M	61	IVc	NA	0	81.8	NA	n
27	C_9	F	72	IVc	NA	0	82.3	8.8	n
28	C_10	F	78	IVc	NA	0	86.1	6.9	n
29	C_11	M	70	ND	NA	0	83.9	8.5	y
30	C_12	F	33	IIIb	NA	0	80.7	8.8	y
31	C_13	F	59	IVc	NA	0	80.2	8.5	y
32	H_1	F	47	NA	NA	NA	80.3	8.8	y
33	H_2	M	60	NA	NA	NA	82.6	9.2	y
34	H_3	F	59	NA	NA	NA	81.9	9.0	y
35	H_4	F	55	NA	NA	NA	80.9	9.3	y
36	H_5	F	57	NA	NA	NA	84.5	9.5	y
37	H_6	M	35	NA	NA	NA	89.4	9.6	y
38	H_7	F	50	NA	NA	NA	88.3	8.9	y
39	H_8	F	36	NA	NA	NA	87.2	9.3	y

ND – no data, NA – not applicable; y- yes; n – no.

Figure 1. Differential gene expression analysis between peripheral T lymphocytes from patients with melanoma (c) and patients with melanoma treated with AGI-101H 6 days after vaccine administration (AV) using HG U219 microarray data

(a) Principal component analysis (PCA) of HG U219 microarray data for 19,285 markers from all biological replicates of AGI-101H-immunized patients 6 days after AGI-101H administration, patients with melanoma not included in the clinical trial, and healthy controls (n = 12). Numbers in parentheses reflect the percentage of total variance explained by the first and second PCs. Prediction ellipses are such that, with a probability 0.95, a new observation from the same group will fall inside the ellipse. Dots represent samples and are colored accordingly: black – healthy, purple – cancer (non-immunized melanoma), orange – AGI-101H-vaccinated melanoma 6 days after immunization. Samples from healthy subjects cluster together and are clearly demarcated from other groups. (b) Scree plot of the percent variability explained by each principal component. The first five principal components explain 69% of the variation. (c) Volcano plot of gene expression changes between untreated patients with melanoma (c) and patients with melanoma treated with the AGI-101H vaccine 6 days after vaccine administration (AV). The number of significantly differentially expressed genes (DEGs) with adj. p < .05 is shown in the volcano plot; orange – up-regulated, green – down-regulated markers. (d) Heatmap of 538 DEGs between peripheral T lymphocytes from patients with melanoma immunized with AGI-101H and non-immunized patients with melanoma (adj. p < .05). DEGs with |log2FC| > 1.0 are denoted and those selected for RT-qPCR validation are marked in red. Technical replicates in the microarray results are denoted with „r”.

Figure 2. Significantly up-regulated markers in the peripheral T cells of AGI-101H-immunized patients are mainly involved in the regulation of gene transcription

(a) The overrepresentation enrichment analysis (ORA) revealed the enrichment of only two Reactome pathways (FDR < 0.05) in the list of 165 up-regulated markers. Both terms are strictly associated with the regulation of gene transcription. (b) The ORA revealed the enrichment of 22 Reactome pathways (FDR < 0.05) in the list of 373 down-regulated markers. Enriched terms are mainly associated with cell metabolism and cell cycle. Each dot presents a Reactome term. The size and the color of the dot depend on the number of overlapping genes between the tested list of genes and the term-associated genes. (c) The protein-protein interaction network formed between significantly up-regulated markers in the peripheral T cells from AGI-101H-immunized patients. The top-ranking seed genes (BCL6, BIRC2, CUL3, H2AX, HIF1A, MAPK8, NMB, PCNA, PPP2R5C, SERINC1) are depicted as large circles and 4 out of 10 seed genes (circled in red) are either transcription factors or DNA-binding/DNA-associated proteins. Blue – relatively low gene expression, the average for each group, orange – relatively high gene expression, the average for each group.

Figure 3. The expression of BCL6 transcriptional repressor is significantly up-regulated in the peripheral T cells of AGI-101 immunized patients with melanoma and negatively correlates with the expression of BCL6 target genes as well as the expression of “exhaustion markers.”

(a) The relative expression of selected markers (PTGER2, BCL6, SKIL, CHKA, ELL2) verified with RT-qPCR analysis using RNA samples previously tested in the microarray. * p < .05, ** p < .01, *** p < .001; n = 8. (b) The relative expression of selected markers was assessed using all RNA samples from magnetically-separated, untouched peripheral T cells from healthy controls, untreated patients with melanoma, and AGI-101H-vaccinated patients 6 days after immunization. * p < .05, ** p < .01, *** p < .001; H = 8 samples, C = 13 samples, AV = 18 samples. (c) The expression of the BCL6 transcriptional repressor negatively correlates with the level of BCL6 target gene (GPR183, CD69, PRDM1) expression in microarray data as determined using the Pearson correlation test. Red line – linear regression. The Pearson correlation coefficient (r) is denoted in each graph. (d) The relative expression of BCL6 target genes was validated with RT-qPCR analysis using RNA samples previously tested in the microarray. * p < .05, *** p < .001, **** p < .0001; n = 8. (e) Heatmap of selected exhaustion markers (CTLA4, KLRG1, PTGER2, IKZF2, TIGIT) in the peripheral T cells of AGI-101H-immunized patients, untreated patients with melanoma, and healthy controls based on microarray data. Technical replicates in the microarray results are denoted with „r”. Blue – the lowest value in each row, orange – the highest value in each row. (f) The relative expression of selected exhaustion markers (CTL$, KLRG1, IKZF2, TIGIT) was validated with RT-qPCR analysis using RNA samples previously tested in the microarray. * p < .05, ** p < .01, *** p < .001, **** p < .0001; n = 8.

Figure 4. Gene set enrichment analysis (GSEA) revealed significant up-regulation of TNF-a (via NFkβ) and down-regulation of IL2-STAT5 signaling in the peripheral T cells from AGI-101H-vaccinated patients with melanoma

(a) All 19,285 genes were sorted based on their log2FC value between AGI-101H-immunized patients and untreated patients (c) resulting in a pre-ranked gene list that was further used in a GSEA analysis with Molecular Signature Database Hallmark Gene Set Collection as the gene set database. Only biological processes with a nominal p-value < 0.05 and FDR < 10% are presented. (b) Activation of TNF-α in the peripheral T lymphocytes of patients with melanoma 6 days following administration of AGI-101H confirms significant activation of the anti-tumor response and of T cell differentiation into functionally distinct lineages. (c) Down-regulation of IL2-STAT5 signaling in the peripheral T lymphocytes from patients with melanoma 6 days following AGI-101H immunization. (d) Heatmap presenting the expression of TNF-α (via NFkB) signaling hallmark genes (top) and TGF-β signaling hallmark genes (bottom) in the peripheral T cells from healthy subjects, untreated patients with melanoma, and AGI-101H-immunized patients (average expression for each group). Blue – the lowest value in each column, orange – the highest value in each column. (e) Heatmap presenting the expression of IL2-STAT5 signaling hallmark genes in the peripheral T cells from healthy subjects, untreated patients with melanoma, and AGI-101H-immunized patients (average expression for each group). Blue – the lowest value in each column, orange – the highest value in each column.