HLA ligandomics identifies histone deacetylase 1 as target for ovarian cancer immunotherapy

Figures & data

Table 1. HLA ligandome analysis and immunohistochemistry of OvCa samples.

OvCa ID	HLA type	# technical replicates	# identified peptides	RMLPHAPGV	EYSKQMQRF	YTTDRVMTV	LPHAPGVQM
OvCa15	A*11, A*24, B*07, B*55, C*03, C*07	5	1426	/	Yes	/	Yes
OvCa41	A*02, A*24, B*18, B*51, C*02, C*12	7	1194	Yes	Yes	Yes	/
OvCa45	A*01, A*23, B*08, B*44, C*04, C*07	9	2762	Yes*	/	Yes*	/
OvCa48	A*02, A*25, B*15, B*41, C*03, C*24	6	1948	Yes	/	Yes	/
OvCa53	A*02, A*03, B*27, B*35, C*02, C*04	5	1746	Yes	/	No	/
OvCa60	A*24, A*25, B*13, B*18, C*06, C*12	5	1443	Yes*	/	/	/
OvCa ID	Source	stage	CD3 epithelium TMA	CD3 stroma TMA	CD8^+ epithelium TMA	CD8^+ stroma TMA	HDAC1 TMA IRS
OvCa15	Serous peritoneal carcinoma	3c	6.3 (1–12)	11.7 (2–25)	5.3 (1–7)	7.3 (1–16)	12
OvCa41	Serous ovarian carcinoma	3c	14 (8–20)	21.7 (12–30)	9 (3–13)	10 (6–12)	9
OvCa45	Serous ovarian carcinoma	1c	35 (28–45)	17 (7–32)	10.7 (7–13)	15.7 (6–29)	12
OvCa48	Serous carcinoma of left fallopian tube	3c	5.3 (4–7)	5 (3–7)	2.3 (2–3)	3.3 (2–5)	9
OvCa53	Serous ovarian carcinoma	3b	27 (5–28)	23.7 (3–32)	13.7 (1–15)	19.3 (1–35)	12
OvCa60	Serous ovarian carcinoma	3c	7.7 (1–12)	5.3 (1–9)	4.3 (0–8)	4 (1–7)	9

Six samples of high-grade serous carcinoma (four ovaries, one fallopian tube, one peritoneum) were analyzed using MS/MS analysis. Depending on peptide concentration in the sample, different numbers of technical replicates were performed. RMLPHAPGV, EYSKQMQRF, YTTDRVMTV and LPHAPGVQM are peptides derived from HDAC1 and HDAC2 and have been represented in different combinations on the six analyzed OvCa samples.

Immunohistochemical analysis of CD3⁺ and CD8⁺ TILs in the stromal or epithelial compartment showed variable numbers. Numbers reflect average count and range of lowest and highest count in different HPFs. HDAC1 was overexpressed in all six samples. Numbers refer to the immunoreactive score (IRS; IRS 9–12 = strongly positive).

/Peptide was not expected in particular tumor due to HLA mismatch.

*Peptide was detected despite HLA mismatch.

Figure 1. Immunogenicity analysis of RMLPHAPGV-primed T cells. CD8⁺ T cells from healthy blood donors were primed in vitro using (A) aAPCs or (B+C) natural APCs. Numbers represent percentages of all T cells. (A) Exemplary tetramer staining of one donor primed with RMLPHAPGV-specific aAPCs. Tetramer staining was performed after three stimulations with aAPCs using control tetramer YLLPAIVHI and RMLPHAPGV tetramer. Numbers represent percentage of all T cells. This figure is representative of nine donors tested positive for RMLPHAPGV-specific T cells after in vitro expansion with aAPCs. (B) Exemplary intracellular IFNγ staining of one donor primed with RMLPHAPGV-presenting natural APCs. Staining was performed after five stimulations with natural APCs using control peptide YLLPAIVHI and RMLPHAPGV peptide. (C) T cells, which have been primed in vitro using natural APCs, (from panel B) were stained with irrelevant YLLPAIVHI and RMLPHAPGV tetramer.

Table 2. Immunogenicity of HDAC-derived HLA ligands.

Peptide sequence	Proposed HLA-restriction	Responding donors/tested donors
EYSKQMQRF	HLA-A*24	0/6
LPHAPGVQM	HLA-B*07	5/7
RMLPHAPGV	HLA-A*02	13/16
YTTDRVMTV	HLA-A*02	0/5

Summary of the in vitro primings of CD8⁺ T cells from healthy blood donors performed for all four HDAC1/2-derived peptides. After three stimulation intervals, HLA tetramer staining was performed. Donors were considered positive if at least 1% of all T cells were stained with the specific tetramer.

Figure 2. Multi-functionality and cytotoxicity of in vitro primed T cells reacting to RMLPHAPGV. (A) T cells show significant expression of TNFα, MIP-1β, IL-2 and CD107a after 6 h incubation with RMLPHAPGV. Numbers in the plot reflect percentage of reactive T cells for the respective marker. (B) Reactive T cells show different degrees of functionality. Only T cells expressing at least one activation marker (60.1% of all RMLPHAPGV-specific T cells) are displayed. 39.9% of RMLPHAPGV-tetramer-specific T cells did not react upon peptide stimulation. (C) Different combinations of activation markers are represented within the reactive T-cell population. (D) Percentage of specific lysis by antigen-specific T cells was determined by ⁵¹chromium release assay after 24 h coincubation with different RMLPHAPGV peptide-loaded or peptide-unloaded OvCa cell lines or HLA-negative K562 cells. Experiments were performed in triplicates with a 30:1 E:T ratio.

Figure 3. Immunohistochemistry of high-grade serous OvCa. Immunohistochemical stainings on TMAs with 136 high-grade serous OvCa samples were performed. (A) Expression of HDAC1: Top: Immunohistochemical staining for HDAC1 of two exemplary OvCa samples with high HDAC1 expression (score 12, left) and low HDAC1 expression (score 3, right). Bottom: Impact of HDAC1 on OS of high-grade serous OvCa stage III and IV (Kaplan Meier analysis, low expression: score < 9, n = 94; high expression score ≥ 9, n = 45). (B) Intraepithelial CD3⁺ TILs: Top: Immunohistochemical staining for CD3⁺ TILs of two exemplary OvCa samples with lower infiltration (left) and higher infiltration (right) of TILs (epithelial compartment is highlighted in yellow). Bottom: Impact of epithelial CD3⁺ TILs on OS of high-grade serous OvCa stage II to IV (Kaplan Meier analysis, low: < 7 cells/HPF, n = 108; high: ≥ 7 cells/HPF, n = 28). (C) Intrastromal CD8⁺ TILs: Top: Immunohistochemical staining for CD8⁺ TILs of two exemplary OvCa samples with lower infiltration (left) and higher infiltration (right) of TILs (stromal compartment is highlighted in green). Bottom: Impact of stromal CD8⁺ TILs on OS of high-grade serous OvCa stage II to IV (Kaplan Meier analysis, low: < 1.5 cells/HPF, n = 91; high: ≥ 1.5 cells/HPF, n = 45).

Figure 4. TILs overcome prognostic effect of HDAC1. Kaplan–Meier analysis was performed to investigate the survival effect of HDAC1 in high-grade serous OvCa stage II–IV with. (A) Low and high numbers of intraepithelial CD3⁺ TILs. (B) Low and high numbers of stromal CD8⁺ TILs.