Apparent Lack of BRAFV600E Derived HLA Class I Presented Neoantigens Hampers Neoplastic Cell Targeting by CD8+ T Cells in Langerhans Cell Histiocytosis

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50–60% of LCH-patients, the somatic BRAFV600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAFV600E can be a source of neoantigens capable of eliciting effective antitumor CD8+ T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8+ T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8+ T cell density in n = 101 LCH-lesions, with BRAFV600E mutated lesions displaying significantly lower CD8+ T cell:CD1a+ LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8+ T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAFV600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAFV600E derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A*03:01 and HLA-A*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAFV600E expressing cell lines with various HLA genotypes. While the HLA-A*02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLATEK was not detected in the HLA class I peptidomes of two distinct BRAFV600E transduced cell lines with confirmed expression of HLA-A*03:01 or HLA-A*11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAFV600E protein are not presented by HLA class I molecules. Given that the BRAFV600E mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH.

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic BRAF V600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAF V600E can be a source of neoantigens capable of eliciting effective antitumor CD8 + T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8 + T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8 + T cell density in n = 101 LCH-lesions, with BRAF V600E mutated lesions displaying significantly lower CD8 + T cell:CD1a + LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8 + T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAF V600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAF V600E derived neopeptide (KIGDFGLATEK), which indeed displayed strong to intermediate binding capacity to HLA-A * 03:01 and HLA-A * 11:01 in an in vitro peptide-HLA binding

INTRODUCTION
Langerhans Cell Histiocytosis (LCH) is a rare neoplastic disorder of hematopoietic origin that primarily affects children, but also involves adults (1). Its clinical manifestation varies from a single bone lesion or benign skin rash to a widely disseminated and lifethreatening condition, similar to acute myeloid leukemia (2). The histopathological hallmark of LCH are phenotypically aberrant CD1a + CD207 + histiocytes (LCH-cells), although not all pathological CD1a + histiocytes co-express CD207 (3). Typically, these LCH-cells are accompanied by a diverse inflammatory infiltrate, often including T cells (2). These T cells have been shown to frequently make intimate contact with LCH-cells (4,5). While patients with high CD8 + T cell density in the tumor infiltrate have a more favorable prognosis across many other neoplastic diseases (6), little is still known about the presence and clinical impact of CD8 + T cells in LCH-lesions (7)(8)(9).
In 2010, universal activation of the mitogen-activated protein kinase (MAPK) signaling pathway in LCH-cells was demonstrated (21,22). Since then, recurrent somatic mutations in genes of the MAPK signaling pathway have been identified in ∼85% of LCH-patients (23,24). Oncogenic driver mutations are essential for tumorigenesis and tend to be clonally conserved. This makes neoantigens derived from proteins encoded by oncogenes highly attractive targets for immunotherapy. In addition, the natural T cell pool should contain T cells expressing high affinity T cell receptors for these neoantigens (25), which may exert potent antitumor function (26)(27)(28)(29). This requires, however, neoantigens to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8 + T cells at tumor sites. Over the past years, several HLA class I presented "public" neoantigens resulting from recurrent hotspot mutations in driver oncogenes have been discovered (30)(31)(32)(33)(34)(35)(36)(37)(38). Approximately 50-60% of LCHpatients carry the somatic BRAF V600E driver mutation (1,21). CD8 + T cells specific for BRAF V600E derived neopeptides have already been reported in vitro and in murine models (39)(40)(41)(42). Thus, activation of LCH-lesional BRAF V600E neoantigenspecific CD8 + T cells could hypothetically lead to the eradication of BRAF V600E expressing LCH-cells. Moreover, the concurrent formation of long-lasting bone-marrow homing memory CD8 + T cells could control new outgrowth of residual BRAF V600E mutated histiocyte precursor cells (43). Immunotherapy specifically aimed at enhancing the number and effector function of these BRAF V600E -specific CD8 + T cells could offer great promise in the treatment of highrisk LCH-patients, given that these patients often bear the BRAF V600E mutation and fail first-line chemotherapy (44). Importantly, the BRAF gene is mutated in ∼7% of human cancers, with the BRAF V600E mutation accounting for >90% of all genetic variations (45,46). Hence, the identification of HLA class I presented "public" neoantigens derived from the BRAF V600E protein would offer great therapeutic opportunity for many patients with other BRAF V600E mutated neoplasms as well (47).
The aim of this study was therefore to (i) assess the presence and clinical impact of lesional CD8 + T cells in (HLA and BRAF V600E ) genotyped LCH-patients, and (ii) to investigate whether BRAF V600E derived neopeptides are presented by HLA class I molecules and could be recognized by such CD8 + T cells.

Patients and Samples
Patient accrual started after approval of the study protocol (CCMO NL33428.058.10) by each local Institutional Review Board. Only patients of whom formalin-fixed-paraffin-embedded (FFPE) first disease onset (FDO) LCH tissue biopsies were available were asked to participate in the study. Informed consent was provided by n = 135 patients and/or their parents/legal guardians. LCH diagnosis was confirmed by a combination of clinical findings and the presence of phenotypically aberrant CD1a + histiocytes in the tissue biopsy. The tissue samples were handled according to the code of conduct for proper secondary use of human tissue of the Federation of Dutch Medical Scientific Societies (FEDERA). Clinical information was collected by each participating center separately using a standardized Case Report Form (CRF) and anonymized data were provided to the researchers of the LUMC. Events were defined as LCH disease progression or reactivation. Progression was defined as (i) progression of existing lesions requiring start or intensification of systemic chemotherapy and/or radiotherapy, or (ii) the development of new lesions when Non-Active Disease (NAD) state had not yet been attained. LCH reactivation was defined as the development of new lesions after NAD had been attained for LCH FDO.

Flow Cytometric Analysis of LCH Tissue Biopsies
Fresh LCH tissue was dissociated using a gentle MACS tissue dissociator (Miltenyi Biotec) and single cells were cryopreserved in DMSO and albumin containing Roswell Park Memorial Institute (RPMI) culture medium. Before flow cytometric analysis, cells were thawed in RPMI + 20% fetal calf serum (FCS) + Penicillin-Streptomycin (P/S) containing 1,600 IU/ml DNAase (Sigma-Aldrich). After washing, the cells were stained with a mixture of different antibodies: CD45 (2D1, 1

HLA Genotyping and Analysis
High-resolution HLA genotyping was performed by DKMS Life Sciences Lab on DNA extracted from buccal swabs obtained from n = 104 LCH-patients using an ampliqon sequencingbased approach, as previously described (48,49). For n = 14 additional patients, low-resolution HLA genotype data were acquired using a sequence specific oligoprimer-based approach (50). Hardy-Weinberg Equilibrium testing and HLA association analyses were performed using the HLA genotype data of Dutch LCH-patients. To evaluate statistical significance, twosided Fisher's exact tests were carried out. The p-values were corrected for multiple comparisons conform the Šidák method (51). Odds ratios and corresponding 95% confidence intervals were calculated according to the method of Woolf with the Haldane correction (52,53). Since a large control group could lead to significant differences that are clinically irrelevant, pvalues were standardized to a smaller control sample size following the method of Good (54). The smaller control sample size was obtained using the following calculation: the total number of LCH-patients plus 3 times the number of patients as maximum allowed size for the control group.

Immunohistochemical Staining of LCH Tissue Sections
FFPE tissue sections (4-10 µm) were deposited on Superfrost TM (Thermo Fisher Scientific) glass slides, dried overnight at 37 • C and stored at 4 • C. Prior to immunohistochemical (IHC) staining, selected 4 µm slides were preheated at 66 • C for 1 h and deparaffinized in xylol. For enzymatic CD1a IHC staining, endogenous peroxidase was blocked using Methanol/0.3% H 2 O 2 for 20 min, before slides were rehydrated in ethanol and demineralized in water baths. Antigen retrieval was performed in boiling citrate buffer (pH 6.0) for 10 min and sections were incubated overnight with mouse IgG1-anti-human CD1a antibody (Clone 010, 1:800, DAKO) diluted in phosphate buffered saline (PBS)/0.5% bovine serum albumin (BSA). The next day, Envision+ System-HRP labeled polymer anti-mouse (DAKO) was applied for 30 min and color development was attained using commercial DAB+ (DAKO) for 10 min in the dark. This reaction was stopped using demineralized water and slides were counterstained with Mayer's hematoxylin (Klinipath) for 5 s prior to mounting with Pertex (Leica Microsystems).

BRAF V600E Mutation Analysis
CD1a + enriched tissue parts were marked by a blinded pathologist on enzymatically CD1a stained LCH tissue slides.
Based on these reference slides, CD1a + enriched tissue parts were manually microdissected from multiple consecutively cut 10 µm tissue sections prepared from the remainder of the LCH tissue blocks. Total nucleic acid was automatically isolated from microdissected tissue using the Siemens Tissue Preparation System (Siemens Healthcare) robot (55). Presence of the BRAF V600E mutation was assessed by allele-specific real-time qPCR, as previously described (56). Of the n = 54 BRAF V600E negative samples, absence of the BRAF V600E mutation was confirmed in 46 samples (85%) by next-generation sequencing (n = 39), whole exome sequencing (n = 1) (57) or BRAF V600E droplet digital PCR (n = 6).

Quantification of T Cell Density in LCH-Lesions
For the manual cell counting method, multiple representative images were taken of each tissue slide at 400× magnification using a conventional Leica DM5500 fluorescent microscope equipped with LAS AF software (Leica Microsystems). Images were solely taken of representative areas containing phenotypically aberrant CD1a + LCH-cells. Using Image J software (version 1.47v) with the public Cell Counter plugin, fluorescently stained CD1a + , CD3 + CD8 − and CD3 + CD8 + cells were manually counted in all images by two independent researchers (PGK and ECS) who were unaware of patient identity and outcome data. The cell counts of the individual images were added to form total CD1a + , CD3 + CD8 − and CD3 + CD8 + cell counts. When total cell counts differed more than 10% between the two researchers, a third researcher (AGSH) reviewed the cell counting results and selected the most appropriate scoring (19/101 cases). Total CD3 + cell counts were obtained by adding total CD3 + CD8 − and CD3 + CD8 + cell counts. To adjust for substantial differences in biopsy size between different patients, which may lead to profound disparities in absolute numbers of counted cells, ratios between the final numbers of total CD3 + and CD3 + CD8 + T cells and CD1a + LCH-cells were calculated for each patient.
For the manual semi-quantitative eyeball estimation method, whole slide images were taken of the same immunostained tissue slides at 400× magnification using a Pannoramic 250 Flash II slidescanner (3DHISTECH). These images were scored semiquantitatively for LCH-lesional CD3 + and CD3 + CD8 + T cell density as has been previously described (58,59): 1+, no, or sporadic T cells; 2+, moderate number of T cells; 3+, abundant occurrence of T cells; and 4+, highly abundant occurrence of T cells. Scoring examples are shown in Figure S1. Unfortunately, n = 21/101 (21%) of the tissue slides could not be reanalyzed due to considerable photobleaching of the fluorophores, induced by the earlier collection of high-power images for the manual cell counting analysis. Slides were scored independently by three researchers (PGK, ECS and AGSH). When scorings between two or more researchers differed more than 1 value (15/80 cases), the scoring was reviewed by all three researchers collectively and a consensus score was attained. Otherwise, the average score of the three scorings determined the final result, rounded to the nearest whole value (1-4+).
Whole slide images of sufficient quality (without significant color casts and/or folded tissue parts that are highly autofluorescent and/or out of focus) from n = 48 LCHpatients were analyzed using a quantitative automated digital image analysis method ( Figure S2). First, the LCH-lesion and its directly adjacent T cells were encircled in the whole slide image in CaseViewer software and exported. In this way, cells that clearly did not belong to the microenvironment of the CD1a + LCH-cells were excluded. Using a custom in-house developed macro in ImageJ software, a white balance was then set for each individual exported image by designating background, foreground and autofluorescence. Next, uniform color thresholds for green (CD1a + ), red (CD3 + CD8 − ), and purple (CD3 + CD8 + ) were applied to all images, so that only green, red, and purple areas with color intensities higher than the threshold remained. Since automated quantification of individual cells was not feasible, the cumulative area of the remaining green, red, and purple areas was measured for each image, representing the total quantity of CD1a + , CD3 + CD8 − , and CD3 + CD8 + cells. Purple and Red (CD3 + ) area/Green (CD1a + ) area and Purple (CD3 + CD8 + ) area/Green (CD1a + ) area ratios could then be calculated for each patient. Comparison of the results obtained using our three separate analysis methods showed substantial concordance ( Figure S3), supporting the validity of the findings in this study.

In vitro Peptide-HLA Class I Binding Analysis
Competition-based peptide-HLA class I binding assays were performed as previously described (60). The HLA binding affinities of the target peptides and strong binding reference peptides are expressed as the concentration that inhibits 50% binding of a fluorescently-labeled standard peptide (IC 50 ). The standard peptides were FLPSDCFPSV for HLA-A * 02:01 and KVFPCALINK for HLA-A * 03:01 and HLA-A * 11:01. Notably, the ratio between the IC 50 of a target peptide and the IC 50 of an established strong binding reference peptide (for example 260:250 vs. 100:5) provides superior information on the true HLA class I binding capacity of the target peptide than the absolute IC 50 of the target peptide.

Generation of BRAF V600E Expressing EBV-LCLs
The full length BRAF V600E sequence incorporated in a pBABE-Puro-BRAF-V600E plasmid was re-cloned into a LZRS-ires-Green Fluorescent Protein (GFP) retroviral vector by introducing the SwaI restriction site and a kozak sequence in front of the ATG start codon at the 5 ′ end of the BRAF V600E sequence using Phusio DNA polymerase. In addition, a stop codon and NotI restriction site was introduced at the 3 ′ end of the BRAF V600E sequence. The original pBABE-Puro-BRAF-V600E plasmid was kindly provided by William Hahn (Addgene plasmid #15269; http:// n2t.net/addgene:15269; RRID:Addgene_15269) (61). Ligation of the BRAF PCR product in the LZRS vector digested with SwaI and NotI was performed overnight at 16 • C. Prior to spin inoculation of Phoenix packaging cells, the correct sequence of the re-cloned BRAF V600E gene was confirmed by Sanger sequencing (data not shown). Retrovirus containing supernatant was subsequently used to transduce Epstein-Barr virus-immortalized B cell lines (EBV-LCLs) with either a control empty LZRS vector (mock transduced EBV-LCL) or with the new BRAF V600E containing LZRS vector (BRAF V600E transduced EBV-LCL). Stably transduced GFP high cells were purified using an ARIA3 flow cytometer prior to bulk expansion in RPMI medium containing 10% bovine serum.
For parallel reaction monitoring (PRM) analyses, the samples were lyophilized and resuspended in buffer A. HLA-eluates were injected together with a mix of 40 fmol of each heavy labeled peptide. The Orbitrap Fusion LUMOS mass spectrometer was operated in PRM-mode. Peptides KIGDFGLATE, KIGDFGLATV, KIGDFGLATEK, and KIGDFGLATVK were monitored. Selected peptides, the transitions and collision energies can be found in Table S1. The isolation width of Q1 was 1.2 Da. MS2 resolution was 35,000 at an AGC target value of 1 million at a maximum fill time of 100 ms. The gradient was run from 2 to 36% solvent B (20/80/0.1 water/acetonitrile/formic acid (FA) v/v) in 120 min. The nano-HPLC column was drawn to a tip of ∼5 µm and acted as the electrospray needle of the MS source. PRM data analysis and data integration were performed in Skyline 3.6.0.10493. Peptide abundances were calculated by comparing the peak area of the eluted (light) and the peak area of the spiked-in heavy peptides.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 8.0.1 and IBM SPSS Statistics version 25. Comparisons of (sub)groups were performed with the Mann-Whitney U test for continuous data and the Fisher exact test for categorical data. The Cox proportional hazards model was used for univariate analysis. Notably, log transformation of the widely differing CD8 + T cell:CD1a + LCH-cell ratios was performed to increase the validity of the univariate analysis. Survival curves were estimated with the Kaplan-Meier method and compared with the Log-rank test. A p-value of <0.05 was considered statistically significant.

LCH-Cells Express Normal Levels of HLA Class I and II Molecules at Their Cell Surface
Since loss or downregulation of HLA expression has been shown to be a major tumor escape mechanism from T lymphocytes in a wide variety of cancers (63), we first evaluated by flow cytometric analysis the levels of HLA class I and HLA-DR expression on the surface of CD1a + (LCH-)cells present in n = 6 LCH-biopsies. The gating strategy applied is shown in Figure S4. The mean fluorescent intensity (MFI) of HLA class I and HLA-DR expression by CD1a + (LCH-)cells was comparable to MFI levels of HLA class I and HLA-DR expression by CD1a − CD14 + (monocytic) cells present in the same LCH-biopsies (Figure 1; HLA class I, p = 0.69; HLA-DR, p = 0.94).

The HLA Genotype of LCH-Patients Does Not Differ From Healthy Controls
Besides HLA expression, HLA subtype is a crucial factor influencing whether a (neo)antigen is actually presented at the surface of nucleated cells. Several earlier published studies have suggested associations between particular HLA subtypes and LCH disease (extension) (64)(65)(66)(67). To investigate this, we compared HLA genotype data from n = 94 Dutch LCH-patients to the HLA genotypes of 5,604 healthy Dutch blood donors reflecting the HLA genotype of the Dutch population (50). To maintain sufficient statistical power, HLA genotype was compared at low resolution level. No significant differences between Dutch LCH-patients and the Dutch reference population were observed (Table S2). Thus, our data do not support previous reports describing excess frequency of HLA-Bw61 and HLA-Cw7 (64), HLA-B7 and HLA-DR2 (65), and HLA-DR4 and/or HLA-Cw7 (66) genotypes in LCH-patients (Tables S2, S3). Moreover, our results neither confirm that LCH-patients with unifocal bone disease have significantly more often HLA-DR4 and/or HLA-Cw7 (66) subtypes nor that patients with single-system LCH have an increased prevalence of HLA-DRB1 * 03 (67) when compared to patients with multisystem LCH (Table S4 and Figure S5, respectively).

BRAF V600E Mutation Correlates With Decreased CD8 + T Cell Density in LCH-Lesions
Assured that LCH-cells express HLA class I (and II) molecules and that there is a normal HLA subtype distribution among LCH-patients, we next investigated the presence of CD8 + T cells in LCH-lesions. Various methods for the quantification of cell numbers in (specific areas of) tissue sections exist, including eyeball estimation, manual cell counting and automated digital image analysis. Although automated digital image analysis is increasingly being applied, manual cell counting is still considered the golden standard (68). Accordingly, we first determined the relative number of total CD3 + and CD3 + CD8 +  Figure S4. The box extends from the 25th to 75th percentiles. The line in the middle of the box is plotted at the median. The whiskers go down to the smallest value and up to the largest. Each individual value is plotted as a point superimposed on the graph.
T cells in LCH-lesions using this method. Fluorescently stained CD1a + , CD3 + CD8 − and CD3 + CD8 + cells (Figure 2A) were manually counted in LCH-biopsies from n = 101 patients collected at first disease onset using the public ImageJ Cell Counter plugin. A median of 1,810 cells (range: 188-9,301) were counted in a median of 16 representative images (range: 2-56) taken at 400× magnification of tissue areas containing phenotypically aberrant CD1a + LCH-cells. Large inter-and intrapatient heterogeneity was seen in the relative number of LCH-lesional CD3 + and CD8 + T lymphocytes (Figure S6 and Figure 2B, respectively). Calculated CD8 + T cell:CD1a + LCHcell ratios (CD8 ratios) ranged from 0.00 to 4.96. The median CD8 ratio was 0.06, corresponding to 1 CD8 + T cell per 16 CD1a + LCH-cells. No significant difference in LCH-lesional CD8 ratios was observed between bone and skin biopsies (p = 0.37) nor between patients with single-or multisystem LCH disease (p = 0.55). Yet, BRAF V600E mutated patients displayed significantly lower LCH-lesional CD8 ratios when compared to BRAF wildtype (BRAF WT ) patients (p = 0.01; Figure 2C). BRAF V600E mutated LCH-lesions had a median CD8 ratio of 0.0316, corresponding to 1 CD8 + T cell per 32 CD1a + LCHcells. In contrast, BRAF WT lesions had a median CD8 ratio of 0.0775, corresponding to 1 CD8 + T cell per 13 CD1a + LCHcells. BRAF V600E mutated lesions also had significantly lower total CD3 + T cell:CD1a + LCH-cell ratios than BRAF WT lesions (p = 0.001; Figure S7). As manual selection of representative tissue areas may introduce bias, we also analyzed whole slide images taken from a subset of immunostained tissue sections using a previously described semi-quantitative eyeball estimation method (58, 59) ( Figure S1) and a quantitative automated digital image analysis method ( Figure S2). The correlation between the BRAF V600E mutation and decreased LCH-lesional CD3 + and CD8 + T cell density was confirmed by these two additional analysis methods ( Figure S8).

Lesional CD8 + T Cell Density Does Not Correlate With Event-Free Survival in LCH
We subsequently assessed whether lesional CD8 + T cell density is of prognostic value in LCH. Using univariate cox regression analysis, no significant association was observed between LCHlesional CD8 ratio and event-free survival (p = 0.46; Hazard Ratio= 0.89; 95% Confidence Interval = 0.66-1.21). In addition, no significant difference was present when patients were divided by a median split, grouped in patients with HIGH or LOW CD8 ratios ( Figure 2B and Table 1) and compared with regard to event-free survival (p = 0.96, Figure 2D). Thus, LCH-lesional CD8 + T cell density did not correlate with disease outcome in this retrospective patient cohort.
The BRAF V600E Derived Neopeptide KIGDFGLATEK Binds to HLA-A * 03:01 and HLA-A * 11:01 To investigate the immunogenicity of the BRAF V600E mutation, we used the online NetMHC 4.0 server (69) to explore putative HLA class I binding 8-12 amino acid long (8-12mer) neopeptides derived from the BRAF V600E protein. In addition, NetCHOP 3.1 software (70) was used to predict proteasomal cleavage motifs and thereby identify peptides that are presumably generated by the human proteasome. From all 8-12mer BRAF V600E derived neopeptides that are generated by the human proteasome according to NetCHOP, only a single neopeptide, the 11mer KIGDFGLATEK, is predicted to bind to one or more of the analyzed HLA class I molecules (Table S5). According to NetMHC, KIGDFGLATEK binds weakly to HLA-A * 11:01 and HLA-A * 03:01, expressed by respectively n = 11/104 (11%) and n = 25/104 (24%) LCH-patients from our cohort. The remainder of the 8-12mer BRAF V600E derived neopeptides are all considered not be generated by the human proteasome and/or to be non-binders. Additional in vitro peptide-HLA binding studies however demonstrated that KIGDFGLATEK binds with comparable affinity to HLA-A * 11:01 as the strong binding reference peptide (QVPLRPMTYK) that was used in our competition-based peptide-HLA binding assay (60). In line with the predicted binding affinity, this neopeptide was shown to also bind, albeit less efficiently, to HLA-A * 03:01 ( Table 2), as evidenced by the small difference in nanomolar concentration that inhibited 50% binding (IC 50 ) of the fluorescently-labeled standard peptide (KVFPCALINK) between KIGDFGLATEK and QVPLRPMTYK (672 vs. 297 nM, respectively). Notably, NetMHCstab 1.0 software (71) predicts that the KIGDFGLATEK-HLA-A * 11:01 complex is highly stable (predicted half-life: 8.87 h) and that the KIGDFGLATEK-HLA-A * 03:01 complex is weakly stable (predicted half-life: 3.29 h). We also assessed the in vitro HLA binding affinity of the 11mer KIGDFGLATVK and 10mer KIGDFGLATV BRAF wildtype peptides and of the 10mer KIGDFGLATE neopeptide ( Table 2). In accordance with the predictions made by NetMHC, KIGDFGLATVK was shown to bind with comparable affinity to HLA-A * 11:01 as the strong binding reference peptide QVPLRPMTYK, and to confer weaker binding to HLA-A * 03:01, just like KIGDFGLATEK. Moreover, the 10mer BRAF wildtype peptide KIGDFGLATV was shown to bind with comparable affinity to HLA-A * 02:01 as the strong binding reference peptide  Figure S9).

KIGDFGLATEK Is Not Detected in the HLA Class I Peptidome of BRAF V600E Expressing Cells
To assess whether KIGDFGLATEK is actually presented on the surface of cells that express BRAF V600E and HLA-A * 03:01 and/or HLA-A * 11:01, we performed mass spectrometry-based targeted peptidomics of HLA class I presented peptides isolated from various EBV-LCL transduced with a LZRS-retroviral vector containing full length BRAF V600E protein and reporter Green  (Table 3). Notably, neither the 11mer BRAF wildtype peptide KIGDFGLATVK was detected in HLA class I peptides isolated from the mock or BRAF V600E transduced SB and MLA EBV-LCL. In contrast, the 10mer BRAF wildtype peptide KIGDFGLATV was detected in the HLA class I peptidomes of 3/3 BRAF V600E transduced and 2/2 mock transduced cell lines expressing HLA-A * 02:01 (Table 3).

DISCUSSION
A large number of studies have demonstrated a positive association between overall CD8 + T cell density in the tumor infiltrate and a favorable clinical prognosis in many different types of cancers (6). In this study, we did not observe such an association in a substantial cohort of LCH-patients with well-documented clinical outcome. This dissimilarity between LCH and other neoplastic disorders may be due to their vast differences in mutational load and, correspondingly, the number of T cell activating neoantigens that can arise from this mutational burden. Furthermore, the immune suppressive microenvironment in LCH-lesions (5,14,15,18,(78)(79)(80)(81) may hamper CD8 + T cell infiltration (non-mutated), antigen recognition and cytolytic function .
In line with an earlier undetailed observation (7), the relative number of LCH-lesional CD8 + T cells appears low in this study. Moreover, we demonstrate with three separate analysis methods that BRAF V600E mutated LCH-patients display lower lesional CD3 + and CD8 + T cell densities than BRAF wildtype patients. Although the clinical significance of this latter observation is not immediately apparent, it does point out that the different MAPK pathway mutations expressed by neoplastic LCH-cells seem to have a distinct impact on their immune microenvironment. A number of studies on BRAF V600E positive melanoma have already suggested that the BRAF V600E mutation promotes immune evasion by upregulating the transcription of many immunomodulatory chemokine and cytokine genes as well as the internalization of cell surface HLA class I molecules (82,83). The presence of many of these immunomodulatory chemokines and cytokines in LCH-lesions has been extensively demonstrated (2). Notably, we did however observe normal HLA class I expression by CD1a + (LCH-)cells in two BRAF V600E positive LCH-biopsies analyzed by flow cytometry (Figure 1A), and showed that transduction of EBV-immortalized B cells with a BRAF V600E encoding retroviral vector does not impair HLA class I expression. Zeng and colleagues recently described that BRAF V600E mutated LCH-patients have significantly higher numbers of lesional Foxp3 + regulatory T cells and increased PD-L1 expression by LCH-cells when compared to BRAF WT patients (80). In accordance with this study, a preliminary report by Chakraborty and others also describes that BRAF V600E expressing LCH-cells display higher expression levels of ligands for inhibitory receptors, including PD-L1/L2 and Galectin-9, when compared to BRAF WT patients (19). Notably, the presence of PD-1 expressing T cells in LCH-lesions has been reported as well (18,19), and was confirmed in (BRAF V600E positive) patients from our cohort ( Figure S11). PD-L1 blockade has been shown to induce expansion of tumor-infiltrating CD8 + T cells (84). Thus, the reported increased PD-L1 expression by BRAF V600E positive LCH-cells (19,80) could explain the decreased LCH-lesional CD8 + T cell density in BRAF V600E mutated patients from our study. In addition, the immune suppressive microenvironment in LCH-lesions (5,14,15,18,(78)(79)(80)(81) may clarify why the rare CD8 + T cells that did make it into these lesions had no significant clinical impact. This is supported by our own observation of low numbers of HLA-DR pos LCH-lesional CD8 + T cells (Figure 1), low numbers of "licensed-to-kill" CD8 + T cells co-expressing the cytolytic enzymes Perforin and Granzyme B (85) (Figure S12), and rare presence of Caspase 3 expressing LCH-cells (data not shown). HLA-DR is widely recognized as a marker of T cell activation (86), and Caspase 3 is the hallmark marker of efficient target cell apoptosis induced by activated CD8 + T cells (87). In line with the recently reported defective response of LCH-lesion infiltrating T cells to allogeneic stimulation (19), these observations collectively suggest that CD8 + T cells in LCH-lesions are often dysfunctional. Future studies using (imaging) mass cytometry, which allows the simultaneous detection of a multitude of cellular markers (with spatial context), are needed to study the phenotypic characteristics of LCH-lesional (CD8 + ) T cells in more detail. Moreover, the alleged distinct impact of the different MAPK pathway mutations on the immune microenvironment of neoplastic LCH-cells should ideally be investigated in a LCH mouse model.
Encouraged by published results suggesting that BRAF V600E protein-derived neopeptides can trigger antitumor immunity (41,82), we used the most recent version of publicly accessible NetMHC software to explore putatively HLA class I binding neoantigens derived from the BRAF V600E protein. Surprisingly, from all 8-12mer BRAF V600E derived neopeptides that are predicted to be generated by the human proteasome by NetCHOP 3.1 software, only a single neopeptide (KIGDFGLATEK) was predicted to bind to one or more of the analyzed HLA class I molecules. In vitro peptide-HLA binding assays confirmed the predicted binding capacity of KIGDFGLATEK to HLA-A * 03:01 and HLA-11 * 01. In contrast to the results generated with an earlier version of Syphpeiti software (41), the NetMHC 4.0 server did not qualify the two (putatively HLA-A * 02:01 binding) neopeptides LATEKSRWSG and LATEKSRWS to be HLA-binders. Using PRM-based targeted peptidomics, KIGDFGLATEK was not detected in the HLA class I peptidomes of 2/2 BRAF V600E expressing EBV-LCL (MLA BRAF V600E and SB BRAF V600E ) that expressed normal levels of HLA-A * 03:01 or HLA-A * 11:01. In contrast, the HLA-A * 02:01 binding BRAF wildtype peptide KIGDFGLATV was traceable in HLA class I peptides isolated from 5/5 cell lines expressing this HLA subtype, verifying normal antigen processing in these cells and adequate sensitivity of our peptidomics approach. Since the 11mer BRAF wildtype peptide KIGDFGLATVK was not detected in mock (empty-GFP) nor BRAF V600E transduced EBV-LCL as well, the apparent lack of KIGDFGLATEK presentation at the cell surface seems not due to a competitive HLA binding disadvantage relative to its wildtype counterpart (88). Instead, both KIGDFGLATEK and KIGDFGLATVK peptides may not be generated by the human proteasome. This could be explained by the fact that both HLA-A * 03:01 and HLA-A * 11:01 molecules exclusively bind peptides with lysine as the C-terminal anchor residue (89). NetCHOP software only produces neural network predictions for proteosomal cleavage. Protein cleavage yielding C-terminal lysine residues is, however, not readily accomplished by the human proteasomes alone. Instead, this process requires the cytosolic endopeptidases nardilysin and thimet oligopeptidase as well (89,90). Another possibility is that the 11mer KIGDFGLATVK and KIGDFGLATEK peptides are expressed at the cell surface, but that they are underrepresented among the large pool of naturally presented ligands eluted from peptide-HLA class I complexes, because of a common peptide length distribution including mostly 9mer peptides and far less 8mer, 10mer, and longer peptides (91). This is also demonstrated by the list of peptides that were detected using data-dependent acquisition-based peptidomics in the HLA class I peptide pools isolated from the mock transduced JY and MLA EBV-LCL ( Table S8). The high sensitivity of targeted peptidomics makes this option however less probable, although it must be noted that lower numbers (28-170 × 10e6) of BRAF V600E transduced SB, MLA, and JY B cells were subjected to analysis as compared to mock transduced B cells (158-1,900 × 10e6). This was because GFP high BRAF V600E transduced cells displayed intrinsically higher apoptosis rates leading to substantially lower yields (data not shown).
In addition to the importance of CD8 + T cells, multiple studies have highlighted the importance of CD4 + T cells in tumor rejection (34,(92)(93)(94)(95)(96). Notably, one study identified BRAF V600Especific CD4 + T cells after repetitive peptide stimulation of peripheral blood mononuclear cells from three melanoma patients whose metastatic tumors harbored the BRAF V600E mutation (40). Moreover, Veatch and colleagues recently identified HLA-DQB1 * 03-restricted BRAF V600E -specific CD4 + T cells in an acral melanoma patient, who nonetheless developed metastases under ipilimumab (anti-CTLA-4) immunotherapy (97). Unfortunately, the precise amino acid sequence of the recognized neoantigen was not reported. Available software tools to predict HLA class II binding peptides are known to be significantly less accurate than available algorithms for predicting HLA class I binding peptides. Moreover, the yield of BRAF V600E transduced B cells expressing HLA-DQB1 * 03:02 (SB EBV-LCL) was far too small to elute sufficient quantities of peptide-HLA class II complexes needed for successful data-dependent acquisition-based peptidomics. We could, therefore, not confirm that this recently identified BRAF V600E protein-derived HLA-DQB1 * 03 binding neopeptide is naturally processed and presented at the cell surface of our BRAF V600E transduced HLA-DQB1 * 03 expressing EBV-LCL. We did however investigate whether BRAF V600E mutated LCH-patients expressing HLA-DQB1 * 03 in general, or HLA-DQB1 * 03:02 and/or HLA-DQB1 * 03:03 in particular [due to their putative strongest binding and/or peptide-HLA complex stability (97)], had increased event-free survival when compared to BRAF V600E mutated patients without these HLA genotypes. Notably, HLA class I subtype has already been demonstrated to influence response to checkpoint blockade immunotherapy in patients with diverse cancers (98). Neither BRAF V600E mutated LCH-patients with HLA-DQB1 * 03 (n = 30, 62.5%) nor with HLA-DQB1 * 03:02 and/or HLA-DQB1 * 03:03 (n = 18, 37.5%) displayed increased event-free survival when compared to patients without these HLA alleles (p = 0.78 and p = 0.57, respectively; data not shown). Thus, although we agree that adoptive cell therapy with T cell receptor-engineered BRAF V600E -specific CD4 + T cells may offer great therapeutic potential, the clinical impact of potentially present BRAF V600E -specific CD4 + T cells in HLA-DQB1 * 03 bearing, BRAF V600E mutated LCH-patients is questionable. Of note, the rare CD4 + BRAF V600E -specific T cells reported in the acral melanoma patient by Veatch et al. were not paralleled by BRAF V600E -specific CD8 + T cells, but by diverse CD8 + T cells reactive to multiple melanoma-associated self-antigens. Whether such non-mutated tumor-associated antigens are also expressed by LCH-cells is of great interest and remains to be determined. This will however be challenging given the (relatively) low numbers of neoplastic LCH-cells that can be obtained for peptidome analysis from fresh or frozen LCH tissue samples, which are in addition very scarce due to the rarity of the disease.
Since the generation of neoantigens is a probabilistic process (47), we can of course not rule out that other somatic mutations in LCH-cells are a source of neoantigens that are naturally processed and presented in (stable) peptide-HLA class I complexes. Based on recent insights, this probability is however very low. With the development of deep-sequencing technologies, comprehensive analyses of neoantigen-specific T cell responses have been carried out for a substantial number of cancer patients since 2013 (25,26,29). The striking conclusion that can now be drawn from these studies is that only a very small fraction of non-synonymous mutations leads to the formation of a neoantigen for which CD4 + or CD8 + T cell reactivity can be detected (25). Most melanomas and a sizable fraction of other high-prevalence cancers in adults have a mutational load above 10 somatic mutations per Mb, corresponding to ∼150 non-synonymous mutations within expressed genes (25,99,100). Even in melanoma patients, neoantigen-specific T cell reactivity is however not always observed (95). Thus, there is a growing awareness that tumor types with a mutational load below 10, and especially below 1 mutation(s) per Mb, are less likely to express neoantigens that can be recognized by autologous T cells (25). Although the total number of LCH samples analyzed by whole-exome sequencing (WES) is still small (101), a remarkably low frequency of somatic mutations in LCHcells was found in the largest WES analysis to date (n = 41), with a median of 1 somatic mutation per patient (0.03 mutations per Mb) (22). Thus, the likelihood of neoantigen formation and concurrent induction of protective neoantigen-specific T cell responses in LCH-patients seems very low (25). Notably, Goyal and others recently demonstrated a low mutational burden in other histiocytic neoplasms as well (102). We therefore question the usefulness of classical immune checkpoint inhibitors for the treatment of relapsed or refractory LCH (or other histiocytic neoplasms), especially given that these LCH-patients often bear the BRAF V600E mutation (44), and that pretherapy intratumoral CD8 + T cell density has been shown to positively correlate with mutational burden, neoantigen load and response to immune checkpoint inhibition in many other neoplastic diseases (103,104).

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Medical Ethical Committee of the LUMC. Written informed consent to participate in this study was provided by the participants or by the participants' legal guardian.

AUTHOR CONTRIBUTIONS
PK, ES, AH, QA, JB, SV, AR, GJ, PV, GH, KF, RE, NS-W, and TW performed experiments and/or analyzed data. DR performed low-resolution HLA genotyping and provided HLA subtypespecific antibodies. LV helped with the automated digital image analysis. RV, CN, AC, and CH provided LCH tissue biopsies and marked CD1a + enriched tissue parts on CD1a stained slides that served as reference slides for the manual microdissection procedure. AB, JL, OA, and CB included patients and supervised the clinical data collection by PK, TZ, VL, and RME. AH, CB, and OA designed the study. PK and AH drafted the manuscript.

FUNDING
This study was financially supported by grants awarded by the Histiocytosis Association of America, Stichting 1000 Kaarsjes voor Juultje, Stichting Kiwanis Run-for-LCH and structural research funding from Histiocytose Nederland (AH). In addition, the peptidomics experiments were financially supported by a research programme Investment Grant NWO Medium with project number 91116004, which is (partly) financed by ZonMw. PK received a Van Walree Travel Grant from the Royal Netherlands Academy of Arts and Sciences.