Introduction
One of the major challenges in cancer immunotherapy remains the persistence of high affinity T cells specifically targeting tumor-associated antigens within an immunosuppressive tumor microenvironment. A good candidate target for immunotherapy should confer a high tumor selectivity with minimal adverse events. In addition, the target should preferably play a pivotal role in promoting tumor development and progression and/or impairing anti-tumor immunity, hence increasing the success rate of the therapeutic intervention to eradicate the tumor. Based on these criteria, the cancer testis antigen (CTA) lactate dehydrogenase C (LDHC) could be considered a novel promising immunotherapeutic target.
LDHC belongs to the lactate dehydrogenase family that catalyzes the interconversion of pyruvate and
l-lactate and plays important roles in aerobic glycolysis [
1]. Lactate dehydrogenase isozymes exist as homo- or hetero-tetramers composed of two major subunits, LDH-M and LDH-H that are encoded by
LDHA and
LDHB, respectively. Different combinations of these subunits assemble into 5 distinct isozymes with different tissue specificity; LDH1/LDHB (4H), LDH2 (3H1M), LDH3 (2H2M), LDH4 (1H3M) and LDH5/LDHA (4M). While LDHA is predominantly expressed in skeletal muscle and preferentially converts pyruvate to lactate, LDHB is mainly expressed in the heart and brain where it catalyzes the interconversion of lactate to pyruvate. LDHC, encoded by the
LDHC gene, assembles into a homotetramer of LDHC subunits, also known as the LDHC or LDHX isoform [
2]. Gene evolution models indicate that LDHC arose from gene duplication of the
LDHA gene in mammals with 75% sequence homology with LDHA and 70% with LDHB [
2]. LDHC expression is restricted to mature testis and spermatozoa, with low expression in oocytes and early embryos [
3]. LDHC deficiency has been linked to male infertility, partly caused by diminished spermatozoa motility, whereas female
null mice are fertile [
4,
5]. Hence, the role of LDHC in spermatogenesis, oogenesis, fertility and early development remains unclear.
Although LDHC expression is tightly controlled and suppressed in normal somatic tissues, it is re-expressed in various malignant tissues, making its expression highly tumor specific [
6]. Furthermore, increased LDHC expression has been associated with poor prognosis in renal cell carcinoma [
7]. Very little data are available on the role of LDHC in cancer. Based on the observations of LDHA- and LDHB-mediated cancer progression, we can speculate that LDHC could be involved in metabolic reprograming of cancer cells. It is well established that growing tumors can bypass oxidative phosphorylation in favor of aerobic glycolysis to support their increasing metabolic need, which involves metabolic enzymes such as lactate dehydrogenases [
8]. Indeed, dysregulation of LDHA and LDHB expression has been observed in tumors with increased glycolysis [
9]. Hence, altered expression of LDHC could be involved in maintaining an alternative energy source by contributing to the metabolic switch in cancer cells. In addition, increased LDHA and decreased LDHB expressions facilitate tumor formation and progression through remodeling of the tumor microenvironment, increasing proliferation, and inducing epithelial-to-mesenchymal transition, cell migration and invasion, and angiogenesis [
10‐
20]. In line with this, two studies to date demonstrate that enhanced expression of LDHC induces epithelial-to-mesenchymal transition, matrix metalloproteinase-9 (MMP9) expression and promotes cancer cell migration and invasion [
7,
21].
Targeting LDHC could be a promising novel approach for cancer immunotherapy. First, given its restricted expression profile, it is likely that LDHC-specific immune-based interventions will result in the generation of LDHC-specific T cells with high affinity and low off-target effects. Moreover, targeting LDHC would not only inhibit LDHC-mediated cancer progression and specifically eradicate LDHC positive tumor cells, but could also induce reversal of the acidic tumor microenvironment, thereby releasing anti-tumor immunity. It is important to note that lactate and the concomitant tumor acidity negatively influence the anti-tumor immune response by skewing the immune cell compartment towards an immunosuppressive environment [
22‐
24]. More specifically, LDHA has been found to promote upregulation of PD-L1 on tumor cells, impeding effector T cell activity [
25]. Furthermore, elevated serum LDHA levels are associated with tumor burden as well as poor clinical outcome to PD-1 and CTLA-4 immune checkpoint blockade therapy [
26]. Therefore, targeting key players of lactate metabolism including LDHC could aid to re-establish anti-tumor immunity and is a largely unexplored area of research.
In the present study, we generated several T cell responses against LDHC using either in vitro stimulation of T cells with synthetic peptides or priming of T cells with autologous peptide-pulsed dendritic cells. Using both approaches, we found that several peptide pools and individual peptides could elicit a cellular immune response, as determined by IFN-γ secretion. More in-depth analysis of the responses in HLA-A*0201 healthy donors enabled us to identify two HLA-A*0201-restricted immunogenic epitopes, LDHC41−55(LKDLADELALVDVAL) and LDHC288−303 (LSIPCVLGRNGVSDV), that could possibly be targeted by adoptive T cell therapy. Using different breast cancer cell lines, we demonstrated that LDHC41−55and LDHC288−303 specific T cells were capable of recognizing and eradicating HLA-A*0201 positive/LDHC positive tumor cells, while no specific cytolytic activity was detected against HLA-A*0201 negative/LDHC positive tumor cells. Interestingly, we found that reduction of LDHC expression in the HLA-A*0201 positive cancer cells attenuated the T cell responses against LDHC, suggesting a plausible threshold of LDHC expression to elicit immune reactivity. To conclude, we demonstrate for the first time that LDHC exhibits immunogenicity and our findings warrant further study into the potential of LDHC as a novel therapeutic target for cancer immunotherapy.
Materials and methods
Cell culture
MDA-MB-453, MDA-MB-468, and MDA-MB-231 breast cancer cells were maintained in DMEM (Gibco-BRL, Waltham, MA, USA) supplemented with 10% (v/v) FBS (Hyclone US origin, GE Healthcare Lifesciences, Pittsburg, PA, USA), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco-BRL, Waltham, MA, USA). MDA-MB-436 breast cancer cells were maintained in DMEM (Gibco-BRL, Waltham, MA, USA) supplemented with 10% (v/v) FBS (Hyclone US origin, GE Healthcare Lifesciences, Pittsburg, PA, USA), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco-BRL, Waltham, MA, USA), 10 mg/ml insulin and 16 mg/ml glutathione (Sigma-Aldrich, St. Louis, MO, USA). BT549 breast cancer cells were maintained in American Tissue Culture Collection (ATCC)-formulated RPMI-1640 medium (Gibco-BRL, Waltham, MA, USA) supplemented with 10% (v/v) FBS (Hyclone US origin, GE Healthcare Lifesciences, Pittsburg, PA, USA), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco-BRL, Waltham, MA, USA), and 0.023 IU/ml insulin (Sigma-Aldrich, St. Louis, MO, USA). HCC1500 breast cancer cells and T2 cells were maintained in ATCC-formulated RPMI-1640 medium (Gibco-BRL, Waltham, MA, USA) supplemented with 10% (v/v) FBS (Hyclone US origin, GE Healthcare Lifesciences, Pittsburg, PA, USA), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco-BRL, Waltham, MA, USA). All cell lines were maintained at 37 °C, 5% CO2 and regular mycoplasma testing was performed using a PCR-based assay of culture supernatants (Forward primer 5′-gggagcaaacaggattagataccct-3′ and reverse primer 5′-tgcaccatctgtcactctgttaacctc-3′).
LDHC breast cancer cell lines
Adherent HCC1500 and BT549 cells were transduced at 80% confluency with purified GFP-positive shLDHC lentiviral particles (SMARTvector Lentiviral Human LDHC hCMV-TurboGFP shRNA, #V3SH11240-229943916, Dharmacon, Lafayette, CO, USA) or purified GFP-positive negative control shCTR lentiviral particles (SMARTvector non-targeting hCMV-TurboGFP control particles, #S-005000-01, Dharmacon, Lafayette, CO, USA). After 6 days, transduced HCC1500 and BT549 cells were maintained under 0.5 ug/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA) selection. In addition, the MDA-MB-468 cell line was transduced with the GFP positive shCTR lentiviral particles.
LDHC peptides
A synthetic peptide library consisting of 81 individual 15-mer peptides with a 11-residue overlap was purchased as a custom-made service (JPT Peptide Technologies, Berlin, Germany). Individual peptides were reconstituted at 2 ug/ul in DMSO. Peptide pools of 10 (peptide pools 1–7) or 11 peptides (peptide pool 8) were generated with a final concentration of 200 ug/ml of each individual peptide or 2 mg/ml total peptide content (Table
1). Finally, peptides were used at 2 ug/ml to stimulate dendritic cells or T cells.
Table 1LDHC synthetic peptide library
PP1 | P1 | H-MSTVKEQLIEKLIED-OH | PP5 | P41 | H-SGCNLDSARFRYLIG-OH |
| P2 | H-KEQLIEKLIEDDENS-OH | | P42 | H-LDSARFRYLIGEKLG-OH |
| P3 | H-IEKLIEDDENSQCKI-OH | | P43 | H-RFRYLIGEKLGVHPT-OH |
| P4 | H-IEDDENSQCKITIVG-OH | | P44 | H-LIGEKLGVHPTSCHG-OH |
| P5 | H-ENSQCKITIVGTGAV-OH | | P45 | H-KLGVHPTSCHWIIG-OH |
| P6 | H-CKITIVGTGAVGMAC-OH | | P46 | H-HPTSCHGWIIGEHGD-OH |
| P7 | H-IVGTGAVGMACAISI-OH | | P47 | H-CHGWIIGEHGDSSVP-OH |
| P8 | H-GAVGMACAISILLKD-OH | | P48 | H-IIGEHGDSSVPLWSG-OH |
| P9 | H-MACAISILLKDLADE-OH | | P49 | H-HGDSSVPLWSGVNVA-OH |
| P10 | H-ISILLKDLADELALV-OH | | P50 | H-SVPLWSGVNVAGVAL-OH |
PP2 | P11 | H-LKDLADELALVDVAL-OH | PP6 | P51 | H-WSGVNVAGVALKTLD-OH |
| P12 | H-ADELALVDVALDKLK-OH | | P52 | H-NVAGVALKTLDPKLG-OH |
| P13 | H-ALVDVALDKLKGEMM-OH | | P53 | H-VALKTLDPKLGTDSD-OH |
| P14 | H-VALDKLKGEMMDLQH-OH | | P54 | H-TLDPKLGTDSDKEHW-OH |
| P15 | H-KLKGEMMDLQHGSLF-OH | | P55 | H-KLGTDSDKEHWKNIH-OH |
| P16 | H-EMMDLQHGSLFFSTS-OH | | P56 | H-DSDKEHWKNIHKQVI-OH |
| P17 | H-LQHGSLFFSTSKITS-OH | | P57 | H-EHWKNIHKQVIQSAY-OH |
| P18 | H-SLFFSTSKITSGKDY-OH | | P58 | H-NIHKQVIQSAYEIIK-OH |
| P19 | H-STSKITSGKDYSVSA-OH | | P59 | H-QVIQSAYEIIKLKGY-OH |
| P20 | H-ITSGKDYSVSANSRI-OH | | P60 | H-SAYEIIKLKGYTSWA-OH |
PP3 | P21 | H-KDYSVSANSRIVIVT-OH | PP7 | P61 | H-IIKLKGYTSWAIGLS-OH |
| P22 | H-VSANSRIVIVTAGAR-OH | | P62 | H-KGYTSWAIGLSVMDL-OH |
| P23 | H-SRIVIVTAGARQQEG-OH | | P63 | H-SWAIGLSVMDLVGSI-OH |
| P24 | H-IVTAGARQQEGETRL-OH | | P64 | H-GLSVMDLVGSILKNL-OH |
| P25 | H-GARQQEGETRLALVQ-OH | | P65 | H-MDLVGSILKNLRRVH-OH |
| P26 | H-QEGETRLALVQRNVA-OH | | P66 | H-GSILKNLRRVHPVST-OH |
| P27 | H-TRLALVQRNVAIMKS-OH | | P67 | H-KNLRRVHPVSTMVKG-OH |
| P28 | H-LVQRNVAIMKSIIPA-OH | | P68 | H-RVHPVSTMVKGLYGI-OH |
| P29 | H-NVAIMKSIIPAIVHY-OH | | P69 | H-VSTMVKGLYGIKEEL-OH |
| P30 | H-MKSIIPAIVHYSPDC-OH | | P70 | H-VKGLYGIKEELFLSI-OH |
PP4 | P31 | H-IPAIVHYSPDCKILV-OH | PP8 | P71 | H-YGIKEELFLSIPCVL-OH |
| P32 | H-VHYSPDCKILVVSNP-OH | | P72 | H-EELFLSIPCVLGRNG-OH |
| P33 | H-PDCKILVVSNPVDIL-OH | | P73 | H-LSIPCVLGRNGVSDV-OH |
| P34 | H-ILVVSNPVDILTYIV-OH | | P74 | H-CVLGRNGVSDVVKIN-OH |
| P35 | H-SNPVDILTYIVWKIS-OH | | P75 | H-RNGVSDVVKINLNSE-OH |
| P36 | H-DILTYIVWLISGLPV-OH | | P76 | H-SDVVKINLNSEEEAL-OH |
| P37 | H-YIVWKISGLPVTRVI-OH | | P77 | H-KINLNSEEEALFKKS-OH |
| P38 | H-KISGLPVTRVIGSGC-OH | | P78 | H-NSEEEALFKKSAETL-OH |
| P39 | H-LPTVRVIGSGCNLDS-OH | | P79 | H-EALFKKSAETLWNIQ-OH |
| P40 | H-RVIGSGCNLDSARFR-OH | | P80 | H-KKSAETLWNIQKDLI-OH |
| | | | P81 | H-KSAETLWNIQKDLIF-OH |
Blood samples and peripheral blood mononuclear cell isolation
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat samples from 14 healthy individuals visiting the blood donation unit at Hamad Medical Corporation. Buffy coat samples were diluted five times with Dulbecco’s Phosphate-Buffered Saline (Gibco-BRL, Waltham, MA, USA), after which 10 ml was layered on top of 10 ml Lymphoprep™ (Stem Cell Technologies, Vancouver, Canada) followed by separation into layers by density gradient centrifugation. The interphase containing the PBMCs was carefully collected and transferred to new tubes and washed twice with serum free RPMI-1640 media (Gibco-BRL, Waltham, MA, USA). The cells were counted and 10 × 10
6 PBMCs were frozen per vial. On average, 500 × 10
6 PBMCs were isolated with > 90% cell viability after cryopreservation. HLA typing of PBMCs from all 14 healthy individuals was performed at the Department of Laboratory Medicine & Pathology, Hamad Medical Corporation. HLA typing was obtained for nine different HLA loci (A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1) and the information on class I alleles is summarized in Table
2.
Table 2Demographics of study cohort
D02 | M | Arab | A*33, B*14, C*08 | NA | 21/18 | NA | 11/16 |
D03 | F | Arab | A*01, B*35, C*04 | 16/14 | 14/13 | 10/8 | 11/10 |
D04 | F | Arab | A*03, B*41, C*03 | 18/22 | 24/15 | 22/23 | 4/2 |
D05 | F | Filipina | A*11, B*07, C*01 | 11/15 | 15/14 | 25/16 | 12/18 |
D06 | F | Asian | A*24, B*18, C*07 | 13/12 | 18/16 | 04/03 | 2/5 |
D07 | M | Asian | A*01, B*35, C*04 | 16/14 | 14/13 | 10/8 | 11/10 |
D08 | F | Asian | A*24, B*15, C*03 | 13/12 | 17/13 | 04/03 | 18/12 |
D09 | M | Arab | A*02, B*18, C*07 | 28/24 | 18/16 | 18/25 | 2/5 |
D10 | M | Arab | A*23, B*27, C*02 | NA | 16/12 | NA | 15/6 |
D11 | M | Asian | A*02, B*35, C*03 | 28/24 | 14/13 | 18/25 | 11/10 |
D12 | F | Arab | A*03, B*39, C*12 | 18/22 | 15/21 | 22/23 | 8/7 |
D13 | M | Arab | A*30, B*15, C*17 | NA | 17/13 | NA | 18/12 |
D14 | F | Asian | A*02, B*15, C*04 | 28/24 | 17/13 | 18/25 | 18/12 |
D15 | F | Arab | A*02, B*50, C*06 | 28/24 | 20/10 | 18/25 | 1/1 |
In vitro stimulation (IVS) of T lymphocytes
PBMCs were seeded at 2 × 105 cells/well in 96-well U bottom plates in complete RPMI-1640 medium (Gibco-BRL, Waltham, MA, USA), containing either 20 ug/ml individual peptide, 20ug/ml peptide pool or no peptides (control). Every 2 days, half of the medium was replenished with complete RPMI-1640 medium (Gibco-BRL, Waltham, MA, USA), supplemented with 250 IU/ml of IL-2 (rhIL-2, #202-IL-050/CF, RnD systems, Minneapolis, MN, USA), and 50 ng/ml of IL-15 (rhIL15, #247-ILB-025/CF, RnD systems, Minneapolis, MN, USA). After 18 days, LDHC-specific T cell responses were determined by IFN-γ ELISpot assay.
Differentiation and maturation of autologous dendritic cells (DCs)
PBMCs from HLA-A*0201 positive healthy individuals were used for the differentiation and maturation of dendritic cells. PBMCs were seeded at a density of 5 × 106/well in a 24-well plate and after 2 h the non-adherent fraction (peripheral blood lymphocytes) was removed and cryopreserved for future incubation with mature autologous DCs. Differentiation of the adherent cells into dendritic cells was induced by GM-CSF (1000U/ml, #300-03, PeproTech, Rocky Hill, NJ, USA) and IL-4 (1000U/ ml; #200-04, PeproTech, Rocky Hill, NJ, USA) with replenishment every 2 days. On day 5, maturation of 0.5 × 106 DCs was induced using 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) and maturation was checked on day 8 by flow cytometry (BD LSRFortessa X-20; Software Diva) using markers for CD83 (Anti-Human CD83 APC; #551073, BD Biosciences, Franklin Lakes, NJ, USA) & CD86 (Anti-Human CD86 FITC; #555,657, BD Biosciences, Franklin Lakes, NJ, USA).
Generation of LDHC-specific T cells by dendritic cell stimulation
Mature dendritic cells (1 × 105) were pulsed with either 20 ug/ml individual peptide, 20 ug/ml peptide pool or no peptides (control) for 2 h at 37 °C. Next, peptide-pulsed DCs were used to prime the previously cryopreserved autologous peripheral blood lymphocytes (PBL) in a 96-well U bottom plate at a DC:PBL ratio of 1:20 (25,000 DCs:500,000 PBLs) using complete RPMI-1640 media (Gibco-BRL, Waltham, MA, USA), supplemented with 250 IU/ml rhIL-2 (rhIL-2, #202-IL-050/CF, RnD systems, Minneapolis, MN, USA) and 50 ng/ml rhIL-15 (rhIL15, #247-ILB-025/CF, RnD systems, Minneapolis, MN, USA). Half of the feeding medium was replenished every 2 days. After 7 days, PBLs were re-stimulated with freshly pulsed autologous DCs for another 7 days. After the second cycle of priming, T cells were collected for functional analyses (IFN-γ ELISpot or co-culture with breast cancer cells).
T2 cell loading assay
HLA-A*02 specificity of peptides P11 and P73 was determined using loaded T2 cells as antigen presenting cells in co-culture with primed T cells. T2 cells were loaded with 20 ug/ml of P11, P73, the non-reactive peptide P78 peptide or no peptide (control) for 2 h. Peptide loaded-T2 cells were incubated overnight with their respective DC pulsed-T cells at an E:T ratio of 50:1, followed by measurement of the number of IFN-γ spot forming units (SFU) by ELISpot.
Co-culture of expanded T cells with breast cancer cells
The cytotoxic ability of LDHC-specific T cells (HLA-A*0201 positive) was determined by co-culture with several breast cancer cell lines in comparison to control-T cells (no peptides). For this purpose, we used several breast cancer cell lines:MDA-MB-468 (HLA-A*0201 negative) cells with endogenous LDHC expression (A2−/high) and two LDHC loss-of-function HLA-A*0201 cell line models (HCC1500 and BT549) with endogenous LDHC expression and transduced with shCTR (A2 + /high) or transduced with shLDHC (A2 + /low). Co-cultures were maintained for 4 h at 37 °C in 96 well U-bottom plates at an E:T ratio of 50:1. Production of IFN-γ was determined by ELISpot and cytolytic activity was assessed by viability assay as described below.
CD4 + T cell depletion
After co-culture of control- (no peptides), P11- and P73-specific T cells with A2 + /high HCC1500 cells, cells were subjected to CD4 + T cell depletion using human CD4 microbeads (#130–045-101, Miltenyi Biotec, Bergisch Gladbach, Germany) and the autoMACS Pro Separator (Miltenyi Biotec, Bergisch Gladbach, Germany), as per the manufacturer’s instructions. T cell reactivity of the CD4 + depleted and CD4 + fractions were determined by IFN-γ ELISpot.
IFN-γ ELISpot
IFN-γ release was determined using the Human IFN-γ ELISpotPLUS HRP assay (#3420-4HST-10, Mabtech, Nacka Strand, Sweden) following the manufacturer’s guidelines. Wells were washed four times with PBS (Gibco-BRL, Waltham, MA, USA) and pre-conditioned with complete RPMI-1640 media (Gibco-BRL, Waltham, MA, USA) for 30 min. Expanded T cells were seeded in the wells at 5 × 104 with either 20 ug/ml individual peptide, 20 ug/ml peptide pool, no peptides (control) or anti-human anti-CD3 antibody as a positive control (mAb CD3-2, #3420-4HST-10, Mabtech, Nacka Strand, Sweden) and left to incubate overnight at 37 °C. The number of IFN-γ SFUs was determined using the AID iSpot ELISpot reader (Autoimmun Diagnostika GmbH, Strasburg, Germany).
Viability assay
Following 4 h of co-culture, all cells were collected, washed with PBS, and stained with 7-Aminoactinomycin D (7-AAD) (#00–6993-50, eBiocience, San Diego, CA, USA) at 4 °C for 20 min in the dark. Next, cells were washed with PBS and resuspended in staining buffer (#554656, BD Biosciences, Franklin Lakes, NJ, USA). Within the GFP positive cancer cells, the percentage of 7-AAD positive cells was determined using the BD LSRFortessa X-20 instrument and FlowJo (BD Biosciences, Franklin Lakes, NJ, USA).
RNA isolation and cDNA synthesis
Total RNA was isolated from the breast cancer cell lines using the PureLink RNA Mini kit (#12183018A, Ambion, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. The RNA quantity and purity was assessed by A260/A230 and A260/A280 absorbance measurement (Nanodrop Technologies, Wilmington, DE, USA). Reverse transcription of 1 µg RNA was performed using Moloney Murine Leukemia Virus (MMLV)-Superscript (#28025013, Thermo Fisher Scientific, Waltham, MA, USA) and random hexamers (#SO142, Thermo Fisher Scientific, Waltham, MA, USA) resulting in a final concentration of 50 ng/µl cDNA.
Quantitative real-time RT-PCR
Real time qRT-PCR was conducted using 50 ng cDNA and specific 5′FAM-3′MGB Taqman gene expression primer/probe sets to determine the mRNA expression of LDHC (Hs01022301_m1, Applied Biosystems, Foster City, CA, USA) and the housekeeping gene 60S acidic ribosomal protein P0 (RPLPO, #4333761F, Applied Biosystems, Foster City, CA, USA).
LDHC western blot
Breast cancer cells were harvested at 80% confluency and lysed on ice using Radioimmunoprecipitation assay (RIPA) lysis buffer (#89900, Thermo Fisher Scientific, Waltham, MA, USA) containing Halt™ EDTA-free protease inhibitor cocktail mix (#78425, Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates were centrifuged at 12,000 rpm for 25 min, supernatants were collected and total protein content was determined using the Bicinchoninic acid assay (BCA) protein assay (#23225, Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were denatured in 4 × Laemmli sample buffer (#1610747, Bio-Rad Laboratories, Hercules, CA, USA) at 60 °C for 10 min and equal amounts of protein (80ug) were loaded onto a 4–15% Tris-Glycine eXtended (TGX) protein gel (#4561084, Bio-Rad Laboratories, Hercules, CA, USA). Proteins were transferred onto 0.2 µm polyvinylidinedifluoride membranes (#1704156, Bio-Rad Laboratories, Hercules, CA, USA) followed by blocking in 5% non-fat dried milk/Tris-buffered saline and 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were incubated overnight at 4 °C with the following primary antibodies diluted in 5% non-fat dried milk/TBST: rabbit anti-LDHC (1:500, #ab52747, Abcam, Cambridge, UK) and rabbit anti-β-actin (clone 13E5, #4970, 1:1000, Cell Signaling technologies, Danvers, MA, USA). Membranes were washed three times each with TBST and Tris-buffered saline (TBS) for 5 min each and probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5000, # 111-035-003, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature followed by washes as before. Bound antibodies were detected using Enhanced ChemiLuminescence (ECL) Plus (#32132, Thermo Fisher Scientific, Waltham, MA, USA) or ECL Supersignal-West Femto (#34095, Thermo Fisher Scientific, Waltham, MA, USA) on the Chemidoc XRS + Imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Images were acquired and processed with the Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA).
LDHC expression by flow cytometry
Flow cytometry was used to determine the protein expression of LDHC across breast cancer cell lines. Approximately 1 × 106 cells were fixed and permeabilised (#554714, BD Biosciences, Franklin Lakes, NJ, USA), and resuspended in 100 µL of staining buffer containing 2.5 µg of Human BD Fc Block™ (#130-059-901, Miltenyi Biotec, Bergisch Gladbach, Germany). After 10 min at room temperature, the cells were incubated with anti-human LDHC antibody (#ab52747, Abcam, Cambridge, UK) at a concentration of 1:50 for one hour followed by two washes with PBS. Next, anti-rabbit secondary antibody (Alexa-Flour 647, #A31573, Thermo Fisher Scientific, Waltham, MA, USA) was added at a concentration of 1:1000 for 1 h followed by two washes with PBS. LDHC expression was analyzed using the BD LSRFortessa X-20 instrument and FlowJo (BD Biosciences, Franklin Lakes, NJ, USA).
Immunophenotyping of generated T cells
Multiparametric flow cytometry was performed to characterize the generated T cell responses after peptide- or control (no peptides)-stimulation or priming with peptide- or control (no peptide)-pulsed DCs. T cells were washed and resuspended in 100 µL of staining buffer containing 2.5 µg of Human BD Fc Block™ (#130-059-901, Miltenyi Biotec, Bergisch Gladbach, Germany). Cell surface staining of various markers was obtained using the following antibodies: CD3-APC-Cy7 (#560176; clone SK7; BD Biosciences, Franklin Lakes, NJ, USA), CD4-PE-efluor 610 (#61-0049-42; clone RPA-T4; eBioscience, San Diego, CA, USA), CD45RA-APC (#304112; clone HI100; BioLegend, San Diego, CA, USA), CD45RO-BUV395 (#564291, clone UCHL1, BD Biosciences, Franklin Lakes, NJ, USA), CD62L-BV786 (#565312, clone SK11, BD Biosciences, Franklin Lakes, NJ, USA) and CCR7-BV711 (#563712, clone 3D12, BD Biosciences, Franklin Lakes, NJ, USA). Dead cells were gated out using the 7-AAD viability dye (#00-6993-50, eBiocience, San Diego, CA, USA). Data analysis was performed using the BD LSRFortessa X-20 instrument and FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA).
Statistics
Gaussian distribution of data was assessed using the Shapiro–Wilk test. Non-parametric analyses were conducted using Kruskal–Wallis test, while parametric analyses were performed using unpaired, 2-tailed t test or 1-way ANOVA. Data are represented as mean ± SEM unless stated otherwise.
Discussion
Cancer testis antigens are gaining interest as targets for adoptive T cell therapy with numerous preclinical studies and clinical trials focusing on NY-ESO-1, MAGE-A3 and PRAME [
30‐
32]. To date, there are no published data on the immunogenicity and targetability of the cancer testis antigen LDHC for cancer immunotherapy except for a Master’s thesis that is deposited in the public domain [
33]. LDHC expression has been detected in different tumor types at varying degrees with frequencies up to 100% in lung adenocarcinoma, 83% in cervical cancer, 76% in high-grade serous ovarian carcinoma (HGSC), 44% in melanoma, and 35% in breast cancer [
6,
33,
34]. As aforementioned, to the best of our knowledge, the immunogenicity of LDHC has been investigated in one study only that demonstrated the presence of LDHC-peptide reactive T cells in the ascites of three of five patients with high-grade serous ovarian carcinoma [
33]. Following expansion of LDHC-reactive T cells of 2 out of 3 patients; one patient displayed specific T cell responses against one peptide pool. Further analysis revealed that these responses were elicited by the 11-mer YTSWAIGLSVM peptide, corresponding to peptide p62. However, p62-specific T cells were not able to recognize autologous ascites, autologous B cells transfected with LDHC or tumor cell lines with endogenous LDHC. Of note, the 11-mer peptide identified in their study was also included in our peptide pool 7 as P62. In our study, we did not observe any strong T cell responses against P62, as determined by IFN-γ release, using in vitro peptide stimulation of T cells isolated from 14 healthy donors, rather than patients. HLA prediction analysis of the P62 peptide sequence (KGYTSWAIGLSVMDL) did not reveal strong nonamer or decamer binders for the reported HLA haplotype of the HGSC patient (HLA-A*02, HLA-B*08, HLA-B*057, HLA-C*06, and HLA-C*07) which may in part explain the lack of endogenous LDHC-reactive T cell responses in the study. In addition, it is unclear whether the LDHC-derived peptide is processed and presented as a cognate peptide and to what extent LDHC is expressed in the patient’s tumor cells and the tumor cell lines. Furthermore, it is likely that pre-existing high-affinity LDHC-specific T cells are partially exhausted or are only present in the tumor microenvironment and not in the ascites of patients.
In our study, we found a wide range of T cell responses against synthetic 15-mer LDHC-derived peptides using T cells from 14 healthy donors. Further analysis of four HLA-A*02 healthy individuals demonstrated the immunogenic potential of two individual peptides, P11 (LDHC
41−55) and P73 (LDHC
288−303), and the functional activity of the respective-primed T cells against breast cancer cell lines. HLA binding prediction and peptide-T2 cell experiments support the HLA-A*0201 specificity of both peptides. LDHC
41−55 and LDHC
288−303-primed T cells exhibited increased IFN-γ secretion and cytolytic activity against HLA-A*0201 breast cancer cell lines with endogenous LDHC expression. In contrast, no specific T cell responses were observed against a HLA-A*0201 negative breast cancer cell line. Depletion assays demonstrated a predominant CD8 + T cell response against both peptides. Moreover, our results suggest that there is a plausible threshold of LDHC expression to elicit an anti-tumor immunity since we observed attenuated T cell responses against the HLA-A*0201 breast cancer cell line following LDHC silencing. More specifically, we observed reduced IFN-γ secretion and a complete lack of cytolytic activity of T cells in co-culture with low LDHC expressing cancer cells, which was confirmed, in a second breast cancer cell line model. Furthermore, LDHA expression is not altered in LDHC high versus LDHC low expressing cancer cells (unpublished data), suggesting that the difference in peptide-specific T cell responses in LDHC high versus LDHC low expressing cells might not be affected by cross-reactivity with LDHA cognate peptides. However, future studies are required to study cross-reactivity with LDHA and LDHB in more detail and to ensure LDHC specificity of the peptide-induced cytotoxic T cell responses. Our findings are in line with previous observations of a recognition threshold for expression of the cancer testis antigen PRAME and of different signaling thresholds for CD8
+ T cell IFN-γ secretion and acquisition of cytolytic activity [
35,
36]. The potential existence of an expression threshold for an effective anti-tumor response has important implications for LDHC-specific immunotherapy. In accordance, current efforts are directed towards increasing the expression of CTAs, including NY-ESO-1 and PRAME, through combination treatment of demethylating agents and histone deacetylase inhibitors [
30,
32]. Using this combination treatment, both the intra-tumor heterogeneous expression of CTAs and the expression threshold could be addressed. Interestingly, we found that the majority of P11 (LDHC
41−55) and P73 (LDHC
288−303) specific T cells displayed an effector memory phenotype. This is of importance since an effective long-term anti-tumor response requires multiple T cell subpopulations, including memory and effector cells. We found an increase in the number of CD4
+ and CD8
+T effector memory cells after priming with the immunogenic LDHC-derived peptides P11 (LDHC
41−55) and P73 (LDHC
288−303), and after co-culture with HLA-A*0201/LDHC positive breast cancer cells, suggesting that the CD8
+ T effector memory cell population was responsible for the cancer cell killing in our in vitro model. Likewise, it has been reported that the majority of redirected T cells against NY-ESO-1 is comprised of CD8
+ effector memory cells [
37]. However, it might be beneficial to also expand the smaller population of LDHC-specific T central memory cells to more readily sustain in vitro proliferation and in vivo persistence after antigen re-encounter [
38,
39]. Indeed, secondary activation or re-stimulation of NY-ESO-1-specific T central memory cells in vitro induced differentiation into functional effector T cells which may be able to generate an anti-tumor immune response against minimal residual disease [
37].
To conclude, this is the first study to induce T cell responses against numerous LDHC-derived peptides and identified two HLA-A*0201 restricted LDHC-specific peptides, P11 (LDHC41−55) and P73 (LDHC288−303), with immunogenic potential. Moreover, we were able to demonstrate the functional activity of the peptide-specific CD8 + T cells against breast cancer cell lines with endogenous LDHC expression, albeit with a constraint of an antigen threshold. Given the expression of LDHC in breast tumors, future studies are needed to evaluate the presence of pre-existing LDHC41−55 and LDHC288−303-reactive T cells in the peripheral blood of breast cancer patients.