Healthy cells functionally present TAP-independent SSR1 peptides: implications for selection of clinically relevant antigens

Summary Tumors with an impaired transporter associated with antigen processing (TAP) present several endoplasmic reticulum-derived self-antigens on HLA class I (HLA-I) which are absent on healthy cells. Selection of such TAP-independent antigens for T cell-based immunotherapy should include analysis of their expression on healthy cells to prevent therapy-induced adverse toxicities. However, it is unknown how the absence of clinically relevant antigens on healthy cells needs to be validated. Here, we monitored TAP-independent antigen presentation on various healthy cells after establishing a T cell tool recognizing a TAP-independent signal sequence receptor 1-derived antigen. We found that most but not all healthy cells present this antigen under normal and inflammatory conditions, indicating that TAP-independent antigen presentation is a variable phenomenon. Our data emphasize the necessity of extensive testing of a wide variety of healthy cell types to define clinically relevant TAP-independent antigens that can be safely targeted by immunotherapy.


HIGHLIGHTS
The ER-resident SSR1 holds an antigenic peptide that is processed independently of TAP TAP-independent peptide presentation is functional in healthy cell types TAP-independent SSR1derived antigen presentation varies between healthy cells This exposes safety and efficacy risks of clinical TAP-independent peptide targeting INTRODUCTION HLA class I (HLA-I) (neo)antigen presentation by tumor cells can induce activation of CD8 + cytotoxic T cells (Neefjes et al., 2011). This natural immunological pressure forces various tumors to downmodulate HLA-I antigen presentation through genomic or epigenetic alterations (Campoli and Ferrone, 2008;Khong and Restifo, 2002). In addition, T cell-based immunotherapies increase mutation rates in genes associated with antigen presentation such as B2M, tapasin, TAP1, and TAP2 (Gettinger et al., 2017;Jiang et al., 2010;Restifo et al., 1996;Sade-Feldman et al., 2017).
HLA-I is generated and folded in the ER, assisted by several lectin chaperones and stabilized by the beta-2 microglobulin (B2M) light chain, enabling the loading of small peptides through the collaborative effort of tapasin, calreticulin, and ERp57 (Rock et al., 2016). Conventional peptide generation is mediated by the proteasome in the cytosol. These peptides are transported into the endoplasmic reticulum (ER) by the heterodimeric transporter associated with antigen processing (TAP) complex.
Alterations in the antigen presentation machinery affect the composition of the peptide repertoire presented by HLA-I (van Hall et al., 2006). Perhaps the largest impact on the repertoire is the functional disruption of the TAP transporter (hereafter referred to as TAP-deficient), interrupting the supply of cytosolic peptides. Without TAP, HLA-I presented peptides are either derived from ER-resident proteins or enter the ER through routes other than the canonical TAP pathway (Oliveira and van Hall, 2015). Several of these socalled TAP-independent peptides are not presented on healthy TAP-proficient cells, therefore representing a potential specific immunotherapeutic target on TAP-deficient tumors (van Hall et al., 2006). Since studies targeting TAP-independent peptides in mice show low toxicity, the first proof-of-concept study targeting TAP-independent peptides in patients with non-small cell lung cancer will soon be initiated (Brolsma, 2019;Doorduijn et al., 2018). Potential target antigens for such therapy were recently identified in humans by comparing the HLA-I presented peptidome eluted from TAP-deficient and -proficient cells (Marijt et al., 2018). Because a considerable proportion of TAP-independent peptides can also be presented by at least some TAP-proficient cells (Man et al., 1992;Weinzierl et al., 2008), clinical targeting of TAP-independent antigens potentially involves significant safety and efficacy risks. Although caution is needed in designating peptides as solely being presented on TAP-deficient cells, efforts are largely lacking to validate that antigens are not at all presented by any healthy cell type with native TAP expression.
To define a validation roadmap, it would be instrumental to have a tool-such as a defined T cell clone-to monitor functional TAP-independent peptide presentation both on tumor and healthy cells. However, isolation of T cells targeting TAP-independent antigens on healthy cells may be challenging since they are likely deleted during thymic development (Takaba and Takayanagi, 2017). Therefore, human T cells that recognize a molecularly defined TAP-independent antigen on healthy cells have not been identified to date.
To bypass potential thymic deletion issues, we here investigated TAP-independent antigen recognition on healthy cells using CD8 + T cell clones derived from allogeneic repertoires Van Bergen et al., 2010). We identified that the cognate antigen of one of these clones, derived from the ER-resident protein signal sequence receptor 1 (SSR1), is presented both by TAP knockout (KO) and TAP-proficient tumor cells. This ubiquitously expressed antigen is effectively presented under normal and inflammatory conditions on several but not all healthy primary cells, indicating that TAP-independent antigen presentation is a variable phenomenon. Thus, a broad healthy cell expression analysis of TAP-independent targets improves the safety profile of their immunotherapeutic application.

The protein SSR1 encodes a TAP-independent peptide
To study the effect of the TAP transporter on the functional presentation of antigens, we genetically knocked out TAP1 in human HAP1 cells. After lentiviral introduction of CRISPR/Cas9 machinery targeting the first exon of TAP1, 20% of the transduced cells still displayed surface HLA-I levels comparable to wild type cells (Figures 1A and S1). In order to prevent interference of remaining wild-type cells in T cell coculture assays, we generated five clonal TAP1 KO cell lines which showed decreased HLA-I surface expression ( Figure 1B). In addition, Western blot analysis confirmed a complete lack of TAP1 expression in clone #1 . Sequence analyses showed a lack of at least one amino acid in the first transmembrane domain of TAP1 in each clone (Figures S2A and S2B) (Schrodt et al., 2006).
To analyze functional presentation of antigens in the absence of the TAP transporter, we utilized several available alloreactive CD8 + T cell clones that we previously isolated from patients following allogeneic stem cell transplantation Van Bergen et al., 2010). These T cell clones recognize defined antigens, one of which is derived from the ER-localized protein SSR1 and may therefore be processed independently of TAP-mediated cytosol-to-ER transport (Pfeffer et al., 2017). Two other T cell clones recognize antigens derived from the cytosolic proteins USP11 or VPS13B (Koike and Jahn, 2019; Zhou et al., 2017). Since these antigens are presented by HLA-A2, we first confirmed that this specific allele was affected in the TAP1 KO cells ( Figure 1C). Subsequent coculture of T cells with HAP1 target cells showed that USP11-and VPS13B-specific T cells recognized wild type but not TAP-deficient cells ( Figure 1D, right panels). Conversely, SSR1-specific T cells were activated by the TAP-deficient cells demonstrating that the SSR1 peptide is processed independently of TAP ( Figure 1D, left panel). The amount of HLA-A2 on the different TAP1 KO clones ( Figure 1C) did not correlate with their capacity to stimulate SSR1-specific T cells ( Figure S2C). Strikingly, HAP1 wild-type cells were capable of activating SSR1-specific T cells to a similar level as TAP-deficient cells resulting in T cell-mediated lysis of both cell lines, showing that the SSR1 peptide can be functionally presented at the cell surface even in the presence of the TAP transporter ( Figures 1D and 1E). Thus, the SSR1 peptide represents a model antigen to further evaluate TAP-independent antigen presentation by healthy cells.
The SSR1 signal peptide contains the TAP-independent SSR1 antigen SSR1 is the transmembrane spanning alpha subunit of the TRAP complex that translocates proteins cotranslationally in the ER lumen (Pfeffer et al., 2017). The epitope recognized by the SSR1-specific T cell clone is polymorphic in the population as dictated by a nonsynonymous SNP (rs10004) encoding for either a serine or leucine (SSR1-S of SSR1-L) ( Figure S3) . A 14-mer peptide containing this polymorphism (VLFRGGPRGSLAVA) was previously shown to be presented by HLA-A2 and recognized by SSR1-specific T cells, albeit with low avidity (Bijen et al., 2018). As more specific details on functionally presented SSR1 antigens may aid in defining TAP-independent peptide processing, we next investigated the HLA-I mediated presentation and recognition of additional peptides covering the SNP. iScience Article HLA-A2-binding predictions of SSR1 peptides covering the SNP failed to designate strong binders (Table S1). Since predictions for this SSR1 epitope can be inaccurate (Bijen et al., 2018), we biochemically determined whether SSR1 peptides other than the 14-mer are presented by HLA-A2 positive cells. Using LC-MS/MS we analyzed eluted peptides from HAP1 wild-type cells , and from two HLA-A2 positive primary AML samples that genetically encode the SSR1-S peptide. In first instance, we automatically retrieved only SSR1-L peptides (13-mer and 14-mer) from the AML samples (Table 1). In addition, using the MS2 spectrum of a synthetic SSR1-S 14-mer peptide as reference, we identified this peptide in all three samples (Table 1).
To obtain more data on presented SSR1-derived peptides, we analyzed the peptidome from additional HLA-A2 positive primary tumor samples (n = 9) and cell lines (n = 6). As both SSR1-S and SSR1-L are presented at similar levels by HLA-A2 positive cells (Bijen et al., 2018), we further focused on identifying length variants of the SSR1-L peptide. The analysis yielded four length variants of the same core peptide (VLFRGGPRGLLAVA) covering the polymorphic residue ( Table 2). Coculture of SSR1-specific T cells with S-antigen-negative T2 cells loaded with decreasing amounts of synthetic candidate peptides showed superior responses against the 14-mer peptide ( Figure 2A). These data establish the 14-mer peptide as the cognate epitope of the SSR1-specific T cell clone.
The location of an antigen within a transmembrane protein determines the molecular path of presentation, including the requirement for TAP transportation after proteolytic degradation (Oliveira and van Hall, 2013). SSR1 is a type-I transmembrane protein with an intra-ER domain of 189 amino acids and a cytosolic domain of 58 amino acids (Pfeffer et al., 2017). To gain insight into the presentation route of the SSR1 antigen, we analyzed the subcellular localization of the recognized peptide. The 14-mer peptide is located near the N-terminus of the (D) HLA-A2-restricted SSR1-, USP11-or VPS13B-specific T cells were cocultured with three separate batches of HAP1 wild-type or each of the five clonal TAP1 KO cell lines (B) Van Bergen et al., 2010). Culture supernatant was used for indicated cytokine ELISAs. Each datapoint represents the average of triplicate cultures. Dotted lines represent the lower detection limit of the ELISA. Student's t-test, ***p < 0.001, ****p < 0.0001, ns is not significant. (E) The indicated T cells were cocultured with 51 Cr-loaded wild-type, TAP1 KO #1 and HLA-I KO HAP1 cells  at indicated effector:target cell ratios, after which specific lysis (%) was calculated based on 51 Cr release. Each datapoint represents the mean of triplicate cultures with SD. See also Figures S1 and S2.

OPEN ACCESS
iScience 24, 102051, February 19, 2021 3 iScience Article SSR1 protein, spanning part of the H-and the complete C-region of the signal peptide as predicted by Phobius (Kall et al., 2007) and is likely to be C-terminally liberated in the ER by the signal peptidase complex (Figures 2B and S3) (Voldby Larsen et al., 2006;Weinzierl et al., 2008). In summary, the peptides derived from USP11 and VPS13B require TAP translocation before HLA-I binding, whereas the SSR1 peptide is readily localized in the ER and likely processed by local proteases before being loaded on HLA-I ( Figure 2C).

Multiple SSR1 peptides are presented independently of TAP
To establish whether other HLA-I alleles can also present TAP-independent SSR1 peptides, we analyzed the peptidomes of TAP-deficient HAP1 cells and MCF-7 cells expressing the herpes simplex virus TAP inhibitor infected cell protein 47 (ICP47). These analyses confirmed that the antigen recognized by our SSR1specific T cell clone was presented by TAP-deficient HAP1 cells (Table S2). In addition, we identified two SSR1-derived candidate peptides from TAP-inhibited MCF-7 cells, indicating that other SSR1 antigens may also be presented independently of TAP (Table S2 and Figure S3). Because these candidate peptides were also found on wild-type MCF-7 cells (Table S2), we questioned whether even more ER-derived SSR1 peptides can be presented by TAP-proficient cells. Examination of membrane bound and soluble HLA-I presented peptidomes from cells and sera of glioblastoma patients and non-cancerous individuals revealed multiple SSR1-derived candidate peptides (Table S3) . All candidate peptides were derived from the ER-luminal domain of SSR1 ( Figure S3).
Altogether, the identification of a vast number of ER-derived SSR1 candidate peptides presented by a variety of HLA-I alleles on different cells (Tables 1 and 2, S2, and S3) indicates that multiple TAP-independent peptides derived from a single protein can be presented by TAP-proficient cells.

TAP-independent peptide presentation is functional in healthy donor cells
To investigate the capacity of non-transformed cells to present TAP-independent peptides, we determined the reactivity of the SSR1-specific T cell clone against healthy donor material. Monocytes, T cells and B cells, the main constituents of peripheral blood mononuclear cells (PBMCs), express SSR1 according to publicly available RNA-seq data ( Figure 3A). Therefore, we isolated PBMCs from three HLA-A2 positive healthy donors and genotyped rs10004 to evaluate whether these donors were carrying the antigen recognized by the SSR1-specific T cell clone. Two donors (A and B) were heterozygous for rs10004 and thus positive for the SSR1 epitope, and one donor (C) was homozygous negative ( Figure 3B). Coculture of our T cell clones with fresh PBMCs from the three donors largely failed to elicit a detectable response of SSR1-specific T cells toward antigen positive donors, while the positive control USP11-and VPS13B-specific clones were activated ( Figures 3C and S4A). In contrast, monocytes isolated from the genetically positive PBMCs induced an SSR1-directed T cell response ( Figures 3D and S4B). We then interrogated T cell reactivity against human umbilical cord endothelial cells (HUVECs), as a non-hematopoietic healthy cell type that expresses SSR1 ( Figure 3E and S4C). SSR1-specific T cells recognized and killed selected HLA-A2 positive, SSR1-antigen positive HUVECs, but not HLA-A2 negative control HUVECs ( Figures 3F, 3G, and S4D). Together, these data demonstrate that presentation of the TAP-independent SSR1-antigen is functional in multiple but not all healthy TAP-proficient cells.

TAP-independent peptides are presented under inflammatory conditions
Patients receiving immunotherapy regularly experience severe inflammatory reactions and cytokine storm, which may also be the case after immunotherapeutic targeting of TAP-independent peptides (Gangadhar  Henden and Hill, 2015). Inflammatory cytokines such as IFN-g enhance expression of various components of the HLA-I pathway including the TAP transporter, but their influence on the expression and presentation of the antigens studied here is unknown Ma et al., 1997). Therefore, we first analyzed the change in expression of SSR1, USP11, and VPS13B after 24 hr of IFN-g treatment using microarray datasets from the Interferome database (Rusinova et al., 2012). In comparison to the reference gene PPIA (Riemer et al., 2012), the expression of SSR1, USP11 and VPS13B were unaffected by IFN-g treatment, whereas TAP1 and TAP2 expression were increased (Figure 4A). Additionally, proteome analysis after 48 hr IFN-g stimulation revealed no significant change in SSR1 and USP11 protein levels while TAP1 and TAP2 increased eight-fold ( Figure 4B) (Megger et al., 2017).
To examine the effects of inflammatory conditions on TAP-independent peptide presentation, we stimulated HAP1 wild-type and TAP1 KO cells for 48 hr with IFN-g, which resulted in increased HLA-A2 surface expression on both cell types ( Figure 4C). These stimulated cells and their unstimulated controls were then exposed to the different T cell clones. The IFN-g-induced upregulation of classical antigen presentation on wild type cells was characterized by an increased T cell response against control USP11 and VPS13B peptides ( Figure 4D, right panels). These IFN-g stimulated wild-type cells also presented significant amounts of SSR1 peptide ( Figure 4D, left panel). In addition, the SSR1 peptide presentation in the absence of the TAP transporter was similar after IFNg stimulation ( Figure 4D). These results indicate that this TAP-independent antigen presentation pathway is largely unaffected by an inflammatory IFN-g stimulus on an immortal model cell line.

Activated healthy cells functionally present TAP-independent peptides
To evaluate whether healthy tissues present TAP-independent peptides under inflammatory conditions similar as induced by immunotherapy, we evaluated recognition of the TAP-independent SSR1 antigen on activated healthy cells. Although functional SSR1 antigen presentation by unstimulated PBMCs was negligible ( Figure 3C), we observed a significant SSR1-specific T cell response against IFN-g treated PBMCs from the SSR1 antigen-positive donors A and B but not against the antigen-negative donor C (Figure 5A). In order to test the TAP-independent antigen presentation in a broader inflammatory context, we activated B cells of the same three donors using CpG and a BCR-stimulus. The antigen-positive activated B cells were efficiently recognized and killed by SSR1-specific T cells, further substantiating that an inflammatory environment allows for TAP-independent antigen presentation in healthy cells ( Figures 5B and S4E). Finally, the SSR1-specific T cells were also reactive to non-immune cells (HUVECs) pre-exposed to IFN-g iScience Article ( Figure 5C). Together, our data demonstrate that healthy TAP-proficient cells can efficiently present TAPindependent peptides under steady state and inflammatory conditions.

DISCUSSION
Tumor-specific abrogation of the antigen presentation machinery can provide opportunities for new therapeutic approaches, such as targeting (neo)antigens that are presented only in the case of a defective TAP transporter (Marijt and van Hall, 2020; Oliveira and van Hall, 2013;van Hall et al., 2006). In our current study, we identified that an immunogenic T cell epitope originating from the ER-resident protein SSR1 is processed independently of TAP. Using an SSR1-specific T cell clone, we provide evidence that TAP-independent presentation is variable on various healthy cell types under resting and inflammatory conditions. Thus, to ensure the safety and efficacy of a therapy against TAP-independent antigens, our data imply that it is vital for every individual target to map the degree of presentation on healthy tissue.
Therapeutic targeting of non-mutated TAP-independent antigens has potential for broad clinical application, since these antigens are shared among patients with TAP-deficient tumors (Oliveira et al., 2013;van Hall et al., 2006;Voldby Larsen et al., 2006). Such immunotherapies will utilize either vaccination strategies or adoptive transfer of specific T cells. A strict prerequisite for a vaccination strategy to be effective is the lack of presentation of the targeted antigen(s) on healthy cells because otherwise specific T cells will be functionally impaired or deleted during thymic development (Doorduijn et al., 2016;Takaba and Takayanagi, 2017). Importantly, such immune tolerance inherently provides a safety margin for clinical studies in which presentation of targeted antigens would unexpectedly be not restricted to TAP-deficient cells. To increase the chance of raising an effective immune response against TAP-independent antigens, vaccination with TAP-silenced DCs could be a potential strategy (Marijt et al., 2019). Of note, there are multiple reports that in humans vaccination strategies against antigens presented on healthy tissue can break tolerance and induce severe autoimmunity, so caution is warranted (Ludewig et al., 2000;Sultan et al., 2017). The alternative for immunotherapeutic vaccination is adoptive T cell transfer, which can induce a fast anti-tumor response. A combination of adoptive transfer of T cells specific for a tumor-restricted TAP-independent antigen with repeated long peptide vaccination proved effective against murine TAP-deficient tumors (Doorduijn et al., 2016). However, presentation of a target antigen by only a subset of healthy cells can induce lethal autoimmune toxicity (Cameron et al., 2013;Morgan et al., 2013;Yee et al., 2000). Our study emphasizes that TAP-independent peptides may be presented by a subset of TAP-proficient cells. Thus, before clinical targeting of TAP-independent antigens, it is crucial to apply a thorough selection with the goal to exclude antigens that are presented on healthy cells.  Table 2), were cocultured with SSR1specific T cells after which culture supernatant was used for IFN-g ELISA. Each datapoint represents a single measurement. To optimally select target antigens, mechanistic insights into the intracellular process of peptide selection are required. Current knowledge on how certain TAP-independent peptides fail to be selected for presentation in the presence of TAP is limited. Hypotheses are based on the peptide affinity for HLA-I, its abundance in the ER, and the distance between generation site and peptide-receptive HLA-I molecules    Figure S4). Results from triplicate cultures are shown. Dotted lines represent the detection limits of the ELISA.
(D) As in (C) but using monocytes as target cells (IFN-g data in Figure S4). iScience Article (Durgeau et al., 2011;Oliveira et al., 2011;Oliveira and van Hall, 2013). Our human TAP-independent SSR1 antigen and its cognate T cells represent a unique toolset to further investigate existing and novel hypotheses. In support of the affinity hypothesis, the 14-mer SSR1 peptide, which has an HLA-A2 affinity well below the generally accepted threshold for presentation of 500 nM (78 nM), is selected for presentation in the presence of TAP (Bijen et al., 2018;Paul et al., 2013). However, several TAP-independent peptides that are not presented by a TAP-proficient model cell line have a strong predicted HLA-I binding, suggesting that peptide presentation decisions are not solely dictated by HLA-I affinity (Marijt et al., 2018). The second hypothesis suggests that highly abundant peptides pumped into the ER by TAP may be presented at the expense of less abundant TAP-independent peptides (Oliveira et al., 2011). In line with this hypothesis, boosting TAP-mediated competitor peptide supply into the ER by IFN-g seemed to decrease TAP-independent SSR1 antigen presentation on wild-type cells. In contrast, a reduction in competitor peptide abundance by depleting TAP from wild-type cells failed to enhance SSR1 peptide presentation, suggesting that also other factors play a role. Because the general abundance of TAP-dependent peptides positively correlates with the expression level of TAP (Boegel et al., 2018;Durgeau et al., 2011), the presentation of TAPindependent peptides may be more efficient on cells with low TAP expression, as long as sufficient peptide-receptive HLA-I heavy chains are available. Thus, presentation of a putative TAP-independent target antigen depends at least on the expression of the antigen-containing protein, TAP and the restricted HLA-I allele, on its affinity and on the amount of competitor peptides. Therefore, safe TAP-independent clinical target definition requires assessment of a broad panel of resting and inflamed healthy cells, including cells T A P 1 T A P 2 S S R 1 U S P 1 1 V P S 1 3 B P P I A (D) T cell coculture with unstimulated or IFN-g stimulated wild-type or TAP1 KO clones (same as in Figure 1B). Culture supernatants were analyzed for the presence of cytokines using cytokine ELISA. Dotted lines represent the lower and upper detection limits of the ELISA. Repeated measures one-way ANOVA followed by Dunnett's (A), ordinary one-way ANOVA followed by Dunnett's (B) or by Sidak's (D) multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns is not significant. See also Figure S1.  Figure 5B, and can be considered for such a panel.
In addition, other PBMC-derived cells and HUVECS can be included but also other healthy human cells iScience Article such as but not limited to fibroblasts, keratinocytes, melanocytes, differentiated induced pluripotent stem cells or organoids from different organs.

IFN-γ stimulated PBMCs
Taken together, the TAP-independent, polymorphic SSR1 antigen provides opportunities to improve our understanding of TAP-independent peptide presentation through T cell-and MS-based experiments on healthy and TAP-deficient cells. Our data warrant testing presentation of candidate TAP-independent target antigens on a broad panel of healthy cells before clinical application.

Limitations of the study
Although we provide a direct proof of principle of TAP-independent antigen presentation by TAP-proficient cells, we have only tested a limited number of healthy donor-derived cells. Panels focusing on validating TAP-independent antigens for immunotherapy should rely on a broader panel of cell types derived from other vital tissues and organs. A technical limitation of this study is that none of the obtained TAP1 KO clones had a frameshifting mutation on both chromosomes. Although we showed absence of TAP1 by immunoblot , a decrease in surface HLA-I, and a lack of presentation of cytosolic antigens, we cannot rule out residual TAP function in our model cells. The facts that the peptide is derived from a signal sequence in the ER, and that we and others eluted the SSR1 peptide from TAP-deficient T2 and 721.174 cell lines, respectively (Weinzierl et al., 2008), provide further support of the TAP-independence of the SSR1 antigen.

Resource availability Lead contact
Further requests for resources and materials should be directed to and will be fulfilled by the lead contact, Robbert M. Spaapen (r.spaapen@sanquin.nl).

Materials availability
TAP1 KO HAP1 cells generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability
Data of peptidome analyses generated in this manuscript are available from the lead contact on request.

METHODS
All methods can be found in the accompanying Transparent methods supplemental file.     Figure 1C) with their capacity to activate SSR1specific T cell response ( Figure 1D). The location of the peptide recognized by the SSR1-specific T cell clone is depicted in red with the asterisk indicating the SNP (rs10004). The predicted signal peptide is underlined, the predicted transmembrane domain orange and the cytosolic domain in italics. Peptides eluted from various HLA-I alleles (Table S3) are depicted in green.   Figure 3F) or (E) B cells isolated from three healthy donors (see Figure 3B) activated using CpG and B cell receptor crosslinking antibodies at indicated effector:target cell ratios. Specific lysis (%) was calculated based on the 51 Cr release. Each datapoint represents the mean of triplicate cultures with SD.  antibiotics. The HLA-A*02:01 restricted T cell clones were previously described and recognize a peptide derived from the endogenously expressed human proteins USP11, VPS13B or SSR1 Van Bergen et al., 2010). The clones were expanded using irradiated feeder cells (mixed PBMCs and EBV-LCLs exposed to 30 Gy and 50 Gy, respectively) in IMDM with 5% fetal calf serum and 5% human serum (Sanquin), supplemented with 120 U/mL IL-2 (Miltenyi and Chiron), 0.8 µg/mL PHA (HA16 Remel™, ThermoFisher) and antibiotics as described .

Sequencing
DNA was isolated from full PBMCs and HLA-A2 positively sorted HUVECs using the NucleoSpin Tissue kit (Machery-Nagel) according to manufacturer's protocol. DNA was amplified using Taq DNA-polymerase (VWR) with forward primer TTCTTCGGTGCATTGTAATTG and reverse primer GGGTCAATGAAACCTTTTTCC and subsequently Sanger sequenced using the forward primer or a sequencing primer GGTGAACTGGGAGATTCTGTT (HUVECs) and BigDye v1.1 (Applied Biosystems).

Genetic engineering
Genome-editing of HAP1 cells and genomic verification was performed as previously described for TAP1 . In short, cells were transduced with pL.CRISPR.efs.GFP (Addgene) containing a gRNA targeting TAP1 (ACTGCTACTTCTCGCCGACT). Transduced cells were enriched by FACS sort on GFP positivity and clones were subsequently generated by limiting dilution. The targeted region was sequenced using primer (GCTCCCCATGAGATCAGCTC) after PCR on genomic DNA of single cell-derived clonal cell lines using forward primer (CAGCCTGTTCCTGGGACTTT) and reverse primer (ACTGACAACGAAGGCGGTAG). HLA-I KO HAP1 cells were previously described . The MCF-7 cells were transfected with pcDNA3.1 containing the Herpes Simplex Virus-derived TAP inhibitor ICP47 using PEI reagent (PolyScience, Niles, Illinois) followed by G418 selection. Clones were generated of which two positive clones were selected (C#15, C#32) (Dganit Melamed Kadosh, Alan Frey, Ian Mohr, and Arie Admon, in preparation).
T cell activation assays 50,000 or 40,000 (only for activated B cells) target cells were cocultured with T cells in a 1:1 ratio in IMDM ++ for 18 hours as previously described . IFN-γ and GM-CSF release was measured by standard sandwich ELISA (Sanquin and BioLegend, respectively) according to the manufacturer's protocol. For IFN-γ stimulation, target cells were seeded two (HAP1 cells, HUVECs) or three (PBMCs) days prior to the experiment in a culture flask in IMDM ++ containing 20 U/mL recombinant human IFN-γ (Peprotech).

T cell-mediated killing assays
Cytotoxicity experiments were performed as previously described . In short, target cells were loaded with 100 µCi 51 Cr (HAP1 and HUVEC, Perkin-Elmer) or 200 µCi 51 Cr (B cells) for 90 min at 37°C and washed. T cells were cocultured with 5,000 HAP1 cells, 5,000 HUVECs or 10,000 B cells at indicated effector:target (E:T) ratios for 4-6 h after which 30 µL supernatant was harvested, dried and analyzed using a gamma counter (Wallac). A maximum release control was based on 0.1% triton X-100 treatment. A medium only control was used to detect spontaneous release. Specific lysis (%) was calculated using (experimental cpmspontaneous cpm) / (maximal cpmspontaneous cpm)] × 100%. mode), with recording of the MS2 spectrum in the orbitrap with a collision energy at 32 V. For master scan (MS1), the AGD target was 400,000/maximum fill time of 50 ms at a resolution of 60,000 and scan range 300-1,400. Charge states 1-4 were included and the dynamic exclusion was set after n=1 with a duration of 20 sec. For MS2, precursors were isolated using the quadrupole with an isolation width of 1. The HLA-I peptidome analyses of the HAP1 and MCF7 cells shown in Table S3 were done using exactly the HLA immunoaffinity purification and LC-MS/MS procedures as in Komov et al. 2018(Komov et al., 2018.

Synthetic peptide stimulation
Synthetic peptides were titrated on 20,000 T2 cells prior to incubation with 5,000 T cells for 18 hours before cytokine ELISA similar as previously described (Bijen et al., 2018).

Expression analysis
Data on SSR1, CD3E, CD14 and CD19 RNA expression in blood cells and of SSR1, CD45 and PECAM1 in HUVECs was retrieved from the Human Protein Atlas (https://www.proteinatlas.org)  using the RNA HPA blood cell gene and the RNA HPA cell line gene datasets. Data for B and T cells was pooled from the different naïve and memory subsets. Microarray data of IFN-γ stimulated human cells were selected based on equal in vitro treatment with 200 U/mL IFN-γ for 24 hours containing expression data on SSR1 (downloaded from Interferome.org v2.01, datasets 64, 144, 311). PPIA was selected as house-hold control as it is unaffected by interferon signaling and was present in all datasets (Riemer et al., 2012). VPS13B and USP11 expression was only present in datasets 64 and 144, respectively.

Statistical Analyses
All error bars correspond to the standard deviation of the mean. Statistical evaluations were done by a student's t-test for comparison of two independent groups (T cell assay on HAP1 wild type and TAP1 KO cells), a repeated measures one-way ANOVA followed by Dunnett's