Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8+ T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8+ T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8+ T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8+ T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8+ T cell activation, mediated by the binding of empty HLA-I to CD8.


Introduction
The major histocompatibility complex class I (MHC-I) molecules play a crucial role in adaptive immune responses by presenting antigenic peptides to CD8 + T cells, which enables the immune system to detect transformed or infected cells that display peptides from foreign or mutated self-proteins. Peptides are an integral component of MHC-I molecules. In the MHC-I antigen presentation process, peptides are mainly produced in the cytosol by proteasome and then translocated to the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Peptides are loaded to MHC-I peptide binding groove with the assistance of several ER chaperones, ERp57, calreticulin and tapasin. There are several quality control components to ensure that most cell surface MHC-I molecules are filled with optimal peptide. However, under certain pathophysiological conditions, MHC-I peptide-deficient or open conformers are also detected on the cell surface. Prior evidence suggests that peptide-deficient conformers of MHC-I molecules appear on the cell surface of activated lymphoid cells (Madrigal et al., 1991;Schnabl et al., 1990), TAP-deficient cells (Ljunggren et al., 1990;Ortiz-Navarrete and Hämmerling, 1991) or EBV transformed B cells (Madrigal et al., 1991).
Although the presence of peptide-deficient conformers of MHC-I molecules on the cell surface under certain conditions is established, their functions are poorly understood. In the past few years, peptide-deficient conformers of MHC-I molecules of some allotypes have been shown to be ligands for cell surface receptors such as KIR3DS1 (Burian et al., 2016;Garcia-Beltran et al., 2016), KIR3DL2 (Goodridge et al., 2013), KIR2DS4 (Goodridge et al., 2013) and LILRB2 (Jones et al., 2011). However, most of these studies involved a non-classical HLA-I, HLA-F (Garcia-Beltran et al., 2016), which has a higher propensity to be expressed in a peptide-deficient version compared to classical HLA-I molecules (Goodridge et al., 2010). The paucity of functional studies of peptide-deficient conformers of classic HLA-I could partly be attributed to their general low stability on the cell surface. In a previous study (Rizvi et al., 2014), we tested the refolding efficiencies of several HLA-B allotypes in the absence of peptide and found that peptide-deficient conformers of some allotypes such as B*35:01 are relatively more stable. Higher stability of peptide-deficient B*35:01 is also measurable in this study using a thermal unfolding assay with peptide-deficient HLA-B molecules that were engineered for enhanced stability via leucine zippered sequences ( Figure 1). Peptide-receptive B*35:01 molecules are also detectable on the surface of activated T cells (this study) and TAP-deficient cells (Geng et al, submitted manuscript). Therefore, B*35:01 is a good representative HLA-B to investigate the function of peptide-deficient conformers of HLA-I molecules. In exploring potential binding partners for peptide-deficient conformers of HLA-I molecules, we found that tetramers of peptide-deficient conformers of HLA-B*35:01, in stark contrast to their peptide-filled conformer, stain a majority of blood-derived CD8 + T cells. We hypothesized that the staining is largely CD8mediated and also that peptide-deficient B*35:01 molecules can modulate CD8 + T cell activation. Indeed, we show that CD8 prefers to bind peptide-deficient B*35:01 molecules and that peptidedeficient HLA-B*35:01 molecules on the cell surface enhance cell adhesion to CD8 + T cells. Although they do not directly activate CD8 + T cells, peptide-deficient HLA-B*35:01 molecules on the surface of antigen presenting cells enhance antigen-specific CD8 + T cell responses. Together, these studies indicate key immune regulatory functions for peptide-deficient conformers of HLA-I molecules.
eLife digest The immune system keeps tabs on everything that happens in our body, looking for potential signs of threat. To alert it to any problems, almost every cell produces specific proteins on its surface called human leukocyte antigens class I, or HLA-I for short. These HLA-I molecules are bound to small protein fragments called peptides that have been exported from within the cell and are presented to the cells of the immune system for scanning. When cells are healthy, the peptides all stem from normal proteins. But, if the cell has become infected or cancerous, it contains foreign or abnormal peptides.
Some of the HLA-I molecules, however, are empty. These antigens are unstable, and their role is unclear. Now, Geng et al. investigated this further by studying blood samples from healthy donors. The experiments revealed that empty HLA-I molecules help specialized cells of the immune system, the killer T cells, to bind to the antigens, improving their killing ability.
It is known that these T cells recognize and bind to the antigens through two receptor proteins, one of which is called CD8. It was known that when HLA-I molecules carry a peptide, only a small fraction of T cells with a matching receptor can bind. However, Geng et al. found that when HLA-Is were empty, a much larger proportion of the T cells was able to bind to antigens. This indicates that CD8 'prefers' to attach to empty HLA-Is, maybe because binding sites are more accessible. CD8 also enhances the binding between the T cells and the antigen. Empty HLA-Is did not directly activate the T cells but did enhance their immune response. When both full and empty HLA-I were present, the T cells were even more effective at killing their targets.
Understanding how killer T cells work is essential for the development of immunotherapiestreatments that help to boost the immune system to fight infections and cancer. Increasing the number of empty HLA-I molecules on cancer or infected cells could enhance T cell killing.

Results
Higher relative thermostability of peptide-deficient conformers of some HLA-B allotypes We previously quantified refolding efficiencies of HLA-B heterodimers based on in vitro refolding reactions conducted in the absence of peptides. Significant differences in folding efficiencies were noted (Rizvi et al., 2014). In the present study, we assessed whether peptide-deficient conformers of HLA-B allotypes also differ in their thermal unfolding characteristics ( Figure 1). The NIH tetramer core facility has developed HLA-I molecules with epitope-linked b2m (ELBM), wherein an HLA-I binding peptide is covalently linked to human b2m via a linker peptide that contains a protease cleavage site. HLA-I heavy chain and b2m are further tethered via leucine zippers (LZ) at their C-termini ( Figure 1A). Treatment of the HLA-I molecules with protease is expected to release the linked peptides, which are all C-terminally elongated, and thus sub-optimal for binding. When the cleavage is done in the presence of another HLA-I binding peptide, exchange should occur. Cleavage in the absence of peptide can produce peptide-deficient conformers of HLA-I molecules.
Peptide-deficient conformers of four HLA-B molecules, B*18:01, B*35:01, B*44:02 and B*51:01, were prepared and verified first by SDS-PAGE. The mobility of b2m was increased for all allotypes, consistent with expected reduction in molecular weight after cleavage ( Figure 1B left panels). Formation and peptide loading of peptide-deficient conformers of HLA-B molecules were further validated by native-PAGE ( Figure 1B right panels). In general, the mobilities for the cleaved HLA-B molecules are clearly different from uncleaved proteins. The reduced intensities of complex bands and increased intensities of the free b2m bands, likely because of heterodimer dissociation during electrophoresis, indicate that these heterodimers are less stable than uncleaved proteins. The HLA-B heterodimers were further loaded with allotype-specific peptides. The observed downward mobility shifts of the complex-specific bands for HLA-B*35:01, HLA-B*18:01 and HLA-B*44:02 in the native gels are indicative of peptide binding, and provide further evidence that the cleaved forms of those HLA-B molecules are in fact peptide-deficient. A downshift was not observed of the complex-specific bands for HLA-B*51:01, suggestive of low peptide-loading efficiency, also previously noted (Rizvi et al., 2014).
Thermostabilities of the peptide-deficient HLA-B molecules were assessed by comparing heatinduced unfolding with a thermal shift assay ( Figure 1C). A fluorescent dye (Sypro Orange) was used that displays enhanced binding to proteins following thermal unfolding. Clear cut transitions are observable for peptide-deficient B*18:01 and B*35:01, but not for B*44:02 or B*51:01. These findings indicate important thermostability hierarchies among peptide-deficient conformers of HLA-B; allotypes such as B*35:01 and B*18:01 are more stable in their peptide-deficient conformers compared to allotypes such as B*44:02 and B*51:01, consistent with previously described refolding assay (Rizvi et al., 2014). As a representative peptide-deficient HLA-B with high thermostability, HLA-B*35:01 was used for further functional assessments.
Peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and stain CD8 + T cells in a CD8-dependent manner To explore potential receptors that are responsive to peptide-deficient HLA-B*35:01, tetramers were generated with peptide-deficient HLA-B*35:01 or their peptide-filled versions. Peripheral blood mononuclear cell (PBMC) staining of tetramers of peptide-deficient conformers was compared with the peptide-filled HLA-B*35:01. PBMCs obtained from healthy donors were stained with a panel of lymphocyte markers before tetramer staining. As expected, antigen-specific CD8 + T cell populations were rare or absent in PBMCs from healthy B*35:01 + donors, as assessed by staining with uncleaved B*35:01 (carrying an epitope LPYPQPQPF from Triticum aestivum) tetramers (for example, Figure 2A). In contrast, peptide-deficient HLA-B*35:01 tetramers bound to most (over 70%) of total CD8 + T cells present in the donor ( Figure 2B). These findings suggested that observed tetramer binding was unlikely to be linked to specific TCR. Rather, staining was significantly blocked by anti-CD8 (Clone SK1, BioLegend) ( Figure 2C), suggesting that peptide-deficient B*35:01 tetramers are capable of binding to cell surface CD8 with higher potency compared to the peptide-filled version. In parallel analyses, CD4 + T cells were poorly stained by the peptide-deficient B*35:01 tetramers ( Figure 2D) and staining was not blocked by anti-CD8 ( Figure 2E), consistent with the finding of CD8-dependent binding to CD8 + T cells. Similar results were obtained with cells from a CD8 is also found on the surface of other lymphocytes, such as NK cells, although as a CD8aa homodimeric form instead of CD8ab heterodimeric form (Moebius et al., 1991). We examined the ability of peptide-deficient B*35:01 tetramers to bind to CD8 on the NK cell surface. B*35:01 belongs to Bw6 serotype of HLA-B alleles that lack a binding sequence for engagement of the HLA-B recognizing killer immunoglobulin-like receptor, KIR3DL1 (Gumperz et al., 1997). In fact, we found that peptide-deficient B*35:01 tetramers exclusively bind to NK cells expressing CD8 and further that binding to the cells is fully blocked by anti-CD8 ( Figure 3A). Furthermore, NK cells from different donors have different percentages of CD8 expressing NK cells, and the level of staining with peptide-deficient B*35:01 tetramers is directly proportional to the CD8 expressing fraction of NK cells ( Figure 3B). Further experiments were undertaken to compare the binding of purified FITClabeled CD8aa to peptide-deficient or peptide-filled HLA-B*35:01 conjugated to streptavidin resin ( Figure 3C). Bead-bound CD8 was quantified by fluorimaging analyses of SDS-PAGE-separated samples. Nonlinear curve fitting analyses of the FITC fluorescence signals yielded a K D value of~20 mM for peptide-deficient B*35:01, significantly stronger binding than that for peptide filled B*35:01, for which a K D value could not be accurately estimated ( Figure 3D).
To test cell adhesion mediated by peptide-deficient B*35:01 or its CD8-null version, two CTL lines were used as CD8 expressing cells. Cell conjugation between the SK19 cells and CTLs was investigated by two approaches, confocal microscopy and flow cytometry. In the microscopy assay, SK19 cells were pre-attached to glass-bottomed petri dish. After co-incubation, CTL line A2-AL9 specific for HLA-A*0201 complexed to the HIV-derived AL9 peptide (AIIRILQQL) (Altfeld et al., 2001) showed No direct activation of antigen-specific CD8 + T cell responses by peptide-deficient HLA-B*35:01 Stronger binding of peptide-deficient HLA-B to CD8 (Figure 4) raised the possibility of CD8 + T cell activation regulation by peptide-deficient conformers (Wooldridge et al., 2010). We therefore tested whether tetramers of peptide-deficient conformers directly activated CD8 + T cells. Intracellular staining assays were carried out to examine whether CD8 ligation with peptide-deficient B*35:01 tetramers induces cytokine expression. We found that cross-linking of CD8 with peptide-deficient B*35:01 tetramers at a concentration as high as 40 mg/ml failed to activate primary CD8 + T cells as well as the CTL lines A2-AL9 and B8-RL8, as assessed by the general absence of changes in the expression of cytokine interferon gamma (IFN-g) (Figure 4-figure supplement 1). These findings suggested that the ligation of CD8 by peptide-deficient B*35:01 did not directly induce significant activation signaling.

Peptide-deficient HLA-I is induced on activated CD4 + T cells and enriched in the antigen-dependent immunological synapse
Although ligation of CD8 by peptide-deficient B*35:01 did not induce direct activation of CD8 + T cells, the enhanced cell-adhesion mediated by the interaction (Figure 2-Figure 4) could modulate antigen-specific CD8 + T cell activation. This could not be tested in TAP-deficient SK19 cells, due to very low cell surface expression of both HLA-A*02:01 and HLA-B*08:01, the HLA-I molecules recognized by the two CTL lines used in Figure 4. Lymphocyte activation is previously shown to induce forms of HLA-I molecules that are recognized by HC10 (Matko et al., 1994), the antibody specific for peptide-deficient conformations . Consistent with these prior findings, purified CD4 + T cells from different donors were shown to consistently induce HC10-reactive HLA-I molecules on the cell surface following their activation with PHA ( Figure 5A). As discussed below in Figure 6, the presence of peptide-receptive B*35:01 was directly measurable on activated CD4 + T cells from HLA-B*35:01 + donors. Activated CD4 + T cells were thus used as antigen-presenting cells Molecular clustering within the immunological synapse is emerging as key mechanism for the control of T cell activation. Therefore, we first used a cell-cell contact assay to determine whether peptide-deficient conformers are clustered within the immunological synapse induced by recognition of RL8-HLA-B*08:01 by the B8-RL8 CTL line. CD4 + T cells from a donor expressing both HLA-B*08:01 and HLA-B*35:01 were pre-activated to induce peptide-deficient conformers on the cell surface. B8-RL8 CTLs were co-incubated with the activated CD4 + T cells pulsed with the antigenic peptide RL8. RL8 induces stronger clustering of HLA-I peptide-deficient conformers (measured with the peptidedeficient conformer-specific antibody HC10, Figure 5D-E,H) than peptide-filled HLA-I (measured with the peptide-MHC-I complex-specific W6/32 antibody, Figure 5B-C and H) in the interface between antigen presenting cells (APC) and CTLs. On the other hand, in the absence of RL8 peptide, cell conjugates were strongly reduced and little enrichment was observed in the junctions between CTL and APC ( Figure 5F-G,H). We did not observe strong CD8 clustering in the interface, probably due to different kinetics of MHC-I and CD8 clustering which has been reported previously (Purbhoo et al., 2004). Similar findings were obtained when activated PBMC rather than activated CD4 + T cells were used as the antigen-presenting cells ( Figure 5-figure supplement 1).
HLA-B*35:01 peptide-deficient conformers enhance cognate peptideinduced antigen-specific target cell lysis We further tested whether the peptide-deficient HLA-B*35:01-CD8 interaction has a regulatory effect on cognate antigen-induced target cell lysis. The CTL line specific to the HLA-A2-AL9 complex was first chosen as the effector cell. Primary CD4 + T cells expressing both A*02:01 and B*35:01 were used as target cells. After incubation at effector-to-target ratios of 1:1, 5:1 and 20:1, CTLs exhibited a strong increase in the ability to kill target cells pulsed with the cognate peptide AL9 (Figure 6-figure supplement 1, right panels) compared to target cells pulsed with control peptide SLYNTVATL (SL9) (Figure 6-figure supplement 1, left panels). Next, primary CD4 + T cells expressing both A*02:01 and B*35:01 from Donor 24 were activated and cell surface expression of peptidedeficient conformers were detected with peptide-deficient conformer-specific antibody HC10. Peptide-deficient conformers could be partially blocked by B*35:01-specific peptides YPLHEQHGM (YPL), HPNIEEVAL (HPN), HPV and FPTKDVAL (FPT), but not control peptide (TSTLQEQIGW, TW10) ( Figure 6F-I), indicating that a subset of the HLA-B*35:01 molecules are peptide-deficient. We found that the cognate peptide-induced CD4 + T cell lysis can partly be blocked by B*35:01-specific peptides ( Figure 6A), suggesting that HLA-B*35:01 peptide-deficient conformers do enhance cell lysis induced by cognate peptides. The effect on modulating antigen-specific cell lysis is observed with different HLA-B*35:01 peptides and across different donors ( Figure 6B-C). Similar effects were also observed in cell lysis assays with the B8-RL8 CTL line ( Figure 6D-E).

Discussion
The major functions of MHC-I proteins include presenting antigenic peptides to CD8 + T cells and delivering activation or inhibitory signals to NK cells. It was widely known that the interactions between MHC-I molecules with their receptors are both allotype and peptide dependent. Our studies indicate that peptide-deficient MHC-I molecules are also functional in the immune response. A given peptide-HLA-I complex is typically able to engage only a small percentage of blood-derived CD8 + T cells, those that bear an appropriate TCR. In contrast, peptide-deficient conformers of HLA-B*35:01 engaged a majority of CD8 + T cells from multiple donors (Figure 2). While the pMHC-I/CD8 interaction is generally characterized by very low affinities (Wang et al., 2009;Wyer et al., 1999), we find that peptide-free HLA-B*35:01 binds CD8 with significantly higher affinity than their peptide-filled versions (Figure 3). Thus, unlike peptide-occupied HLA-B that engage CD8 + T cells via a Figure 5. Clustering of peptide-deficient HLA-I in cognate peptide-induced immunological synapses. Expression levels of HLA-I on the surface of activated CD4 + T cells isolated from PBMCs of three healthy donors were assessed by flow cytometry after staining with HC10 (A), confirming that activated lymphocytes express peptide-deficient conformers of HLA-I molecules. The mean ± SEM of two replicates for each donor are shown. CTL line B8-RL8 was incubated with activated CD4 + T cells from Donor 25 (carrying B*08:01 and B*35:01) loaded with peptide RL8 (B, C, D and E) or not (F and G). Cells were fixed and stained with anti-CD8 and W6/32 or HC10 before analysis by confocal microscopy. Peptides were used at a concentration of 100 mM. Anti-CD8 staining (B, D and F) were shown in red and W6/32 (C) or HC10 (E and G) in green. Arrowheads indicate peptide-deficient conformers of HLA-I clustering at the interface between CD4 + T cells and CTL line. The intensity of HLA-I staining of the CD4 + T cells at the interface was compared with the membrane at a noncontact area and plotted as the fold increase above background (H). The results with a total of 20-30 conjugates (mean ± SEM) per condition are shown. CD8 clustering was derived from 30 conjugates under conditions shown in D. Statistical analyses were undertaken using a one-way ANOVA analysis with Fisher's LSD test. ***, p<0.001; ****, p<0.0001. DOI: https://doi.org/10.7554/eLife.36341.008 The following figure supplement is available for figure 5:  TCR-dependent binding mode, peptide-deficient conformers of HLA-B*35:01 engage CD8 + T cells via a binding mode that is largely CD8-dependent.
The MHC-I-binding site for CD8 is spatially separated from the peptide-binding domains that are recognized by the TCR, and this spatial segregation allows both TCR and CD8 to bind a single MHC-I molecule simultaneously. In contrast to peptide-loaded MHC-I molecules, peptide-free MHC I molecules are suggested to possess properties similar to molten globules (Bouvier and Wiley, 1998), and show more protein plasticity based on MD simulations (van Hateren et al., 2013). The stronger binding to CD8 of peptide-deficient HLA-B compared to peptide-filled HLA-B (Figure 3) is likely caused by conformational differences between peptide-occupied and peptide-deficient conformers of HLA-B molecules that determine the accessibility or orientation of the CD8 binding site on HLA-I. Peptide-deficient HLA-I molecules are also preferred by ER chaperones tapasin and TAPBPR, which functions to facilitate peptide loading of MHC-I molecules. Crystal structures of tapasin-MHC-I and TAPBPR-MHC-I complexes highlight some common MHC-I binding sites by tapasin/ TAPBPR and CD8 (Blees et al., 2017;Jiang et al., 2017;Thomas and Tampé, 2017). Residues at the C-terminal immunoglobulin-like domain of tapasin are positioned close to the CD8 recognition loop (especially residues 225 and 226) of the a3-domain of the MHC-I heavy chains (Gao et al., 1997;Wang et al., 2009), suggesting that the sites co-evolved (Blees et al., 2017). A b hairpin of TAPBPR at the N-terminal domain, which reaches under the floor of the peptide-binding groove, is important for sensing the conformation changes of the peptide-binding groove (Thomas and Tampé, 2017). CD8 also interacts with MHC-I at a similar region (including residues 115, 122 and 128) (Gao et al., 1997). Peptide loading reduces binding affinity between MHC-I molecules from tapasin and TAPBPR resulting in release of MHC-I molecules from tapasin and TAPBPR (Rizvi and Raghavan, 2006;Wearsch and Cresswell, 2007). CD8 might share a similar mechanism as tapasin and TAPBPR to distinguish MHC-I molecules with different conformations.
CD8 functions as an adhesion molecule and co-receptor to enhance the formation of TCR/pMHC complexes and the activation of CD8 + T cells. Although ligation of CD8 with non-cognate peptide-MHC-I complex was proposed to augment CD8 + T cell activation levels (Anikeeva et al., 2006;Yachi et al., 2005), generally, cognate peptide loading of MHC-I molecules is indispensable for CD8 T cell activation. Although a previous study showed that MHC-I molecules with super-enhanced CD8 binding properties bypass the requirement for cognate TCR recognition and nonspecifically activate CTLs (Wooldridge et al., 2010), we did not see any direct activation of CTL by HLA-B*35:01 peptide-deficient conformers. Nonetheless, our data suggested that preferential engagement of CD8 by peptide-deficient conformers of HLA-B*35:01 enhances cognate peptide-induced cell lysis ( Figure 6). The enhancement of T cell activation appears to be caused by enhanced cell adhesion induced by the peptide-deficient conformer-CD8 interaction, or enhanced signaling induced by peptide-deficient conformer enriched within the immunological synapse (Figure 7). To escape immune surveillance by CD8 + T cells, several pathogens and tumors block HLA-I antigen presentation pathways to prevent antigenic peptide presentation by HLA-I molecules. Interestingly, many pathogen evasion or tumor progression strategies involve the targeting of the TAP transporter, inducing the cell surface expression of partially peptide-deficient HLA-I, as illustrated for HLA-B*35:01 ( Figure 4). Peptidedeficient conformers of HLA-I are expected to enhance CD8 + T cells responses against TAP-independent epitopes, and thus counter the pathogen evasion strategies that target the HLA-I antigen presentation pathway.
Further studies are needed to quantitatively understand the extent of allele-dependent variations in CD8 binding by both peptide-deficient and peptide-filled conformers of HLA-B, as well as the induction of HLA-I peptide-deficient conformers under different physiological and pathological conditions. Peptide-deficient forms of different HLA-B allotypes were shown to have distinct thermostabilities and are therefore expected to be expressed at different levels on the cell surface. HLA alleles are known to differently associate with disease progression outcomes in major infectious diseases such as acquired immune deficiency syndrome (AIDS) (Carrington and Walker, 2012) and with autoimmune diseases such as ankylosing spondylitis (AS) (Brown et al., 2016), but the general underlying mechanisms are incompletely characterized. AS has been linked to the expression of HLA-B*27:05-free heavy chains (Khare et al., 1996), which can readily be detected on the surface of TAP-deficient cells (Allen et al., 1999). It would be of interest to test whether the interactions between peptide-deficient conformers of HLA-B*27:05 molecules and CD8 are involved in the onset and outcome of these diseases.
In conclusion, our findings indicate that, without interaction with TCR, the peptide-deficient conformers of HLA-B*35:01 are able to interact efficiently with CD8. The preferential interaction between HLA-I peptide-deficient conformers and CD8 described here identifies a previously unknown mechanism by which CTL can be regulated. Finally, HLA-B peptide-deficient conformer-CD8 interactions may also have physiological regulatory functions in NK cell biology, which requires further study. Figure 7. Peptide-deficient conformers of MHC-I molecules enhance CTL activation. CTL activation is generally induced by the recognition of a specific peptide-MHC-I complex on the surface of antigen presenting cells (APC) by a T cell receptor (TCR) and co-receptor CD8. In this study, we show that peptide-deficient conformers of MHC-I molecules have increased binding to CD8 compared with peptide-filled conformers and therefore enhance the cell-cell contact between APC and CTL. Peptide-deficient conformers of MHC-I molecules are enriched in the immunological synapse and augment CTL activation. DOI: https://doi.org/10.7554/eLife.36341.012

Study approval
Blood was collected from consented healthy donors for HLA genotyping and functional studies in accordance with a University of Michigan IRB approved protocol (HUM00071750).

Cell lines
Human melanoma cell line SK-mel-19 (SK19) (RRID: CVCL_6025) (Yang et al., 2003) and ecotropic virus packaging cell line BOSC (RRID: CVCL_4401) were grown in DMEM (Life Technologies) supplemented with 10% (v/v) FBS (Life Technologies) and 1 Â Anti/Anti (Life Technologies) (D10). SK19 cells were gifted by Dr. Pan Zheng and verified for the absence of TAP1 expression. BOSC cells were obtained from the lab of Dr. Kathleen Collins. CTL line A2-AL9 was kindly gifted by Dr. Bruce Walker. CTL line B8-RL8 was generated in the lab by sorting after tetramer staining as previously described (Dong et al., 2010).

Isolation of peripheral blood mononuclear cells (PBMC)
Fresh blood was subjected to centrifugation over a Ficoll-Paque Plus (GE Healthcare Life Sciences) density gradient, washed twice in PBS and resuspended in RPMI1640 (Life Technologies) supplemented with 10% (v/v) FBS (Life Technologies) and 1 Â Anti/Anti (Life Technologies) (R10). Assays were performed either on freshly isolated PBMC used within 2 to 4 hr of cell preparation, or on PBMC cryopreserved in Recovery Cell Culture Freezing Medium (Life Technologies).

HLA typing
DNA was extracted from PBMCs using DNeasy Blood and Tissue Kit (Qiagen). The HLA typing was performed by Sirona Genomics (Mountain View, CA), an Immucor Company. The assay, based on a previous publication (Wang et al., 2012) was performed using the MIA FORA NGS HLA typing assay for the class I loci. The full-length amplicons for the class I loci were amplified and pooled. These samples were then fragmented, and tagged with unique index adaptors. The samples were pooled and sequenced on the Illumina MiSeq, and the HLA type was determined using the MIA FORA NGS HLA typing software. The Sirona Genomic HLA typing method has been validated by the Histocompatibility, Immunogenetics and Disease Profiling Laboratory of the Stanford University School of Medicine using 50 reference cell lines.

Thermal shift assay
LZ-ELBM HLA-B molecules were provided by the NIH tetramer core facility. Peptide-deficient conformers of molecules were prepared by incubation with PreScission protease or thrombin overnight at 25˚C. Cleaved fragments were removed by centrifuging the sample in a 0.5 ml Amicon Ultra filter device for 30 min at 13,000 rpm, 4˚C. Native-PAGE and SDS-PAGE gels were both run to verify that the cleavage was efficient and HLA-B molecules became peptide-deficient. Peptide exchanges were performed by incubating HLA-B molecules with high affinity peptides together with PreScission protease or thrombin overnight at 25˚C. Thermal shift assays were undertaken as previously described (Del Cid et al., 2010;Huynh and Partch, 2015). HLA-B molecules (8 mM) were incubated in buffer (PBS, pH7.4) and 1 Â Sypro Orange Stain (Invitrogen) in a total reaction volume of 20 ml. Thermal scans were performed using an ABI PRISM 7900HT Sequence Detection System with temperature increments of 1˚C. Fluorescence emission was measured at ROX channel. Fluorescence was normalized within wells as percent maximum fluorescence and plotted against the sample temperature.

Tetramer staining
Peptide-deficient conformers of HLA-B*35:01 were prepared by treatment of LZ-ELBM HLA-B*35:01 molecules with PreScission protease (GE Healthcare Life Sciences) for 2 hr at room temperature to release the tethered peptide, while peptide exchange was performed by adding HLA-B*35:01 binding epitopes simultaneously with PreScission protease. HLA-B*35:01 molecules were further dialyzed thoroughly with Amicon Ultra Centrifugal Filter Devices (Millipore) with a 10 kDa cutoff to remove unbound peptides. The peptide-deficient and peptide-exchanged monomers were verified by SDS or native-PAGE gels. The peptide epitope HPV was used in this study to reconstitute B*35:01. Tetrameric HLA-I reagents were constructed by the addition of streptavidin conjugated to PE (Prozyme, PJRS25) or APC (Prozyme, PJ27S) at 4:1 molar ratios following the tetramerization protocol from NIH core facility. PBS + 0.5% dialyzed BSA was used as staining buffer. For CD8 blocking, anti-CD8 (clone SK1; BioLegend) was incubated with PBMCs at 10 mg/ml for 15 min at room temperature. After washing once, freshly prepared tetramers were added typically at 20 mg/ml and anti-CD8-AF700 (clone HIT8a; BioLegend), anti-CD3-pacific blue (clone UCHT1; BioLegend) and anti-CD4-APC-Cy7 (clone RPA-T4; BioLegend) were added at concentrations indicated by the manufactures and incubated for another 30 min at room temperature. Cells were washed 3 times and 7AAD was added as a live/dead marker and samples were then analyzed by flow cytometry on a BD FACS-Canto II flow cytometer. The FACS data were analyzed with FlowJo software version 10.0.8 (Tree Star, San Carlos, CA).

In vitro HLA and CD8 binding assay
Soluble human CD8aa (residues 1-120) with his-tag at the N-terminus was expressed in Escherichia coli, refolded, and purified by gel filtration as a~30 kDa homodimer (Gao et al., 1997). The CD8aa concentration was calculated from the extinction coefficient, which was determined by amino acid analysis to be 37150 M À1 cm À1 at 280 nm. CD8aa was labeled with FITC according to manufacturer's protocol (Thermo Scientific, Rockford, IL, USA). Relevant biotinylated HLA-B monomers from the tetramer core were immobilized onto streptavidin-coated agarose resin. FITC-labeled soluble CD8aa was added at different concentrations (2.5, 5, 10 and 20 mM) to immobilized HLA-B in binding buffer (PBS + 0.5% BSA). CD8aa was pulled down after co-incubation with the resin, and the beads were washed with binding buffer. SDS loading buffer was added and samples were denatured by heating for 10 min. Samples were loaded and resolved by SDS-PAGE and visualized by fluorimaging on a Typhoon scanner (at 520 nm). The binding at each concentration was obtained by subtraction of the control response (resin alone) from the B*35:01 response.

Flow cytometric analysis to assess MHC-I cell surface expression
A total of 1 Â 10 5 -1 Â 10 6 cells were washed with FACS buffer (phosphate-buffered saline (PBS), pH 7.4, 1% FBS) and then incubated with W6/32 or HC10 antibodies at 1:250 dilutions for 30-60 min on ice. Following this incubation, the cells were washed three times with FACS buffer and incubated with GaM-PE at 1:250 dilutions for 30-60 min on ice. Following incubations, the cells were washed three times with FACS buffer and analyzed using a BD FACSCanto II cytometer. For peptide occupancy assay, cells were preincubated with peptides for 2 hr at 37˚C before staining.

Cell adhesion assay
SK19 cells expressing HLA-B*35:01, HLA-B*35:01-CD8 null or lacking HLA-B*35:01 (those infected with a control virus lacking HLA-B) were first labeled with CFSE according to manufacturer's protocol. For microscope-based assays, SK19 cells were plated to glass-bottomed petri dish the day before the adhesion assay. After washing the dish with medium, CTLs were added at 1:1 and incubate at 37˚C for 2 hr. The cells were then washed with 1 Â PBS and fixed with 2% PFA. After staining with anti-CD8-APC, imaged using a Leica SP8 confocal microscope. For flow-cytometry-based assays, CFSE-labeled SK19 cells were incubated in suspension with CTLs at 37˚C for 2 hr. The cells were then washed with 1 Â PBS, fixed with 2% PFA and stained with anti-CD8-APC. CFSE and CD8 double positive cells were quantified by flow cytometry as conjugated cells.

HLA-I clustering in the immunological synapse
PBMCs from donor carrying HLA-B*08:01 and B*35:01 were preactivated with PHA to express peptide-deficient conformers of HLA-I. Cognate peptide RL8 (100 uM) or DMSO was loaded at 37˚C for 2 hr. The PBMCs and HLA-B*08:01-RL8 specific CTL line (CTL B8-RL8) were mixed, centrifuged briefly and incubated for 10 min at 37˚C to allow immunological synapse formation. Cells were fixed and stained with anti-CD8-APC and W6/32-FITC or HC10-FITC antibodies. Cells were imaged using a Leica SP8 confocal microscope. FITC and APC emission were collected in different channels. Data were processed using Leica Imaging software and ImageJ software. The intensity of HLA-I molecules at the interface was compared with the membrane at a noncontact area and plotted as the fold increase above background.

CD8 + T cell activation assay
Primary CD8 + T cells were purified from PBMCs by negative selection by magnetic-activated cell sorting (MACS, Miltenyi Biotec), according to the instructions. Tetramers were incubated with primary CD8 + T cells or CTL lines at indicated concentrations in serum free medium for 30 min at room temperature and 5 mg/ml anti-CD28 was then added. FBS was supplemented to a final concentration of 10%. 2 Â 10 5 CD8 + T cells were stimulated at 37˚C for six hours in the presence of brefeldin A (Golgiplug, 1:1000; BD Biosciences). Intracellular staining assays were performed to test cytokine expression. Briefly, cells were washed and fixed using 100 ml of 4% formaldehyde at RT for 10 min. After being washed, cells were incubated with 100 ml of 0.2% saponin and then stained with fluorochrome-conjugated antibodies specific for intracellular markers at RT for 30 min. After a final wash, flow cytometry measurements were acquired on a BD FACSCanto II flow cytometer. Flow cytometry gates to identify positive cytokine signal were based on unstimulated control tubes.

Cell lysis assay
Primary CD4 + T cells were purified from PBMCs by negative selection by magnetic-activated cell sorting (MACS, Miltenyi Biotec), according to the instructions and activated then with PHA. Cells form donors carrying HLA-A*02:01 or HLA-B*08:01 were pulsed for 2 hr with AL9 or RL8 peptides at 100 mM, respectively, together with or without B*35:01 blocking peptides and then incubated with corresponding CTL lines (A2-AL9 or B8-RL8) at indicated ratios for 5 hr at 37˚C. Cells were then stained with anti-CD4, anti-CD8 and live/dead marker 7AAD or Aqua to test the viability of CD4 + T cells by flow cytometry.

Statistical analysis
Statistical analyses (ordinary one-way ANOVA analysis with Fisher's LSD or Dunnett test) were performed using GraphPad Prism version 7.