MHC class I antigen processing distinguishes endogenous antigens based on their translation from cellular vs. viral mRNA Brian P. Dolan, Aditi A. Sharma, James S. Gibbs, Tshaka J. Cunningham, Jack R. Bennink, and Jonathan W. Yewdell1 Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Edited by Thierry Boon, Ludwig Institute for Cancer Research, Brussels, Belgium, and approved March 23, 2012 (received for review July 29, 2011) To better understand the generation of MHC class I-associated presented at high (15–20%) efficiency in a macrophage-like cell peptides, we used a model antigenic protein whose proteasome- line whether synthesized by VV or intracellular Listeria (8), showing mediated degradation is rapidly and reversibly controlled by Shield- that two very different systems of antigen introduction into the 1, a cell-permeant drug. When expressed from a stably transfected cytosol need not differ widely in processing efficiency. gene, the efficiency of antigen presentation is ∼2%, that is, one cell- Taken together, these findings demonstrate system-dependent- surface MHC class I–peptide complex is generated for every 50 folded wide variation in the efficiencies of generating complexes from source proteins degraded upon Shield-1 withdrawal. By contrast, full-length proteins and oligopeptides. To better understand these when the same protein is expressed by vaccinia virus, its antigen differences, here we measure the context dependence of antigen- presentation efficiency is reduced ∼10-fold to values similar to those processing efficiency using a number of antigens, cells, and ex- reported for other vaccinia virus-encoded model antigens. Virus in- pression vectors. fection per se does not modify the efficiency of antigen processing. Rather, the efficiency difference between cellular and virus-encoded Results antigens is based on whether the antigen is synthesized from trans- Kb–SIINFEKL Is Generated from SCRAP at Remarkably High Efficiency gene- vs. virus-encoded mRNA. Thus, class I antigen-processing ma- in EL4 Cells. Building on the work of the Wandless laboratory (9), chinery can distinguish folded proteins based on the precise details of we recently described SCRAP, a chimeric antigen whose stability IMMUNOLOGY their synthesis to modulate antigen presentation efficiency. is precisely controlled by the cell-permeant drug Shield-1 (10). SCRAP consists of a Shield-1 interaction domain NH2-terminally D8+ T lymphocytes play a key role in immunosurveillance by fused to SIINFEKL and GFP (10) (Fig. 1A). In the absence of Ceradicating tumor cells and cells harboring viruses and other Shield-1, nascent SCRAP is degraded with a t1/2 of 16 min. In the intracellular pathogens. Recognition is based on the interaction presence of Shield-1, a stable pool of SCRAP accumulates that is of the clonally restricted T-cell receptor with MHC class I mol- degraded with a t1/2 of 30 min upon removal of Shield-1 (10). ecules bearing oligopeptides. Peptides are predominantly gener- Fig. 1 B and C illustrate the behavior of SCRAP when expressed b ated from defective ribosomal products (DRiPs), nascent as a permanently transfected gene in EL4 cells, an H-2 T-cell translation products that are degraded rapidly either by design lymphoma. Via flow cytometry, properly folded fluorescent b (i.e., dedicated for antigen presentation) or necessity (defective SCRAP and K –SIINFEKL are measured simultaneously in live forms of proteins that can interfere with cell function) (1). The cells based on, respectively, GFP and directly conjugated 25-D1.16 use of DRiPs greatly speeds recognition of virus-infected cells, mAb (11). Because GFP fluorescence requires proper folding and because many peptides derive from otherwise highly stable pro- not all SCRAP is properly folded, only measuring fluorescent teins. For viral infections, the efficiency of recognition afforded SCRAP will underestimate the amount of SCRAP present in cells. by DRiPs is of the essence, because viruses can replicate within Via immunoblotting, however, we can measure all SCRAP forms in hours, and killing must be fast to be effective. Time is not limiting total cell extracts that interact with a mAb specific for GFP (Fig. for tumor cells but efficiency is critical, because T cells often 1D). Using purified GFP of known concentration to generate recognize antigens derived from gene products expressed at ex- a standard curve, we could calculate the average number of im- tremely low levels. Indeed, T cells may be the only means of munoreactive SCRAP molecules expressed per cell (Fig. 1D). We detecting expression of the source antigen (2). simultaneously determined absolute numbers of directly conju- Only a handful of studies have addressed the efficiency of pep- gated 25-D1.16 bound to cells using flow cytometry standardization tide generation, a critical issue with broad implications for the ef- beads as described in Materials and Methods. At the start of the ficiency of protein synthesis and cytosolic compartmentalization of efficiency measurement period, we treated cells with mild acid (pH antigen processing (3). Pamer and colleagues seminally reported 3) to destroy preexisting Kb–SIINFEKL complexes, thereby im- that class I–peptide complexes are generated with an efficiency of proving the signal-to-noise ratio. 3–25% (i.e., 3–25 complexes generated per 100 proteins degraded) The SCRAP system enabled us to determine the efficiency of from Listeria monocytogenes proteins secreted into the cytosol of peptide generation from nascent proteins vs. retirees (12). mouse macrophage-like cells (4, 5). Using recombinant vaccinia Retirees are folded, functional proteins that are selected for viruses (rVVs) to express a rapidly degraded full-length chimeric degradation either stochastically according to the classical view protein, we reported that the efficiency of complex (Kb–SIIN- (13) or, in this case, due to unfolding based on Shield-1 with- FEKL) generation was much lower in a variety of mouse fibroblast drawal. For nascent proteins, we determined the amount of and macrophage/dendritic cell (DC)-like cell lines, 0.25%–0.05% (1/400–1/2,000), with an efficiency of ∼2% for SIINFEKL synthe- sized as the MSIINFEKL minigene product (6). Fruci et al. devised Author contributions: B.P.D., J.R.B., and J.W.Y. designed research; B.P.D. and A.A.S. per- a clever method for quantitating minimal peptides released into the formed research; J.S.G. and T.J.C. contributed new reagents/analytic tools; B.P.D., A.A.S., cytosol based on liberation from chimeric GFP-ubiquitin fusion J.R.B., and J.W.Y. analyzed data; and B.P.D., A.A.S., J.R.B., and J.W.Y. wrote the paper. proteins by ubiquitin hydrolases, reporting an efficiency of 0.2% (1/ The authors declare no conflict of interest. 500) for EBV-transformed human B cells expressing permanently This article is a PNAS Direct Submission. transfected genes (7). Extending this approach, Wolf and Princiotta Freely available online through the PNAS open access option. demonstrated that ubiquitin hydrolase-liberated SIINFEKL is 1To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1112387109 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. Model antigens and a schematic of SCRAP presentation and quantification. (A) The various constructs used in this study are depicted. The red “S” box denotes the SIINFEKL peptide, influenza A virus (IAV) nucleoprotein is in orange, and the green “Ub” box represents ubiquitin. (B) EL4/SCRAP cells were washed in a mild citric acid buffer (pH 3.0) to remove existing Kb–SIINFEKL complexes and cultured in the presence or absence of 5 μM Shield-1. At the indicated times, Kb–SIINFEKL complexes and GFP levels were determined by flow cytometry. (C) Same as in A, except Shield-1 was removed following an initial 3.5-h incubation and, following a second acid wash, cells were cultured in the presence of cycloheximide (CHX). (D) An example of a Western blot for GFP using recombinant GFP standards and lysates from EL4/SCRAP cells treated with or without Shield-1 for 3 h. SCRAP rescued over a 4-h period by Shield-1 and the concomi- cytometry as cells infected with rVV expressing NP-S-GFP, and tant decrease in Kb–SIINFEKL expression (Fig. 1B). For retirees, generated essentially identical amounts of Kb–SIINFEKL. In the we accumulated a pool of SCRAP by treating for 4 h with Shield- absence of Shield-1, nearly identical amounts of fluorescent GFP 1, and then induced degradation by removing Shield-1 while were detected compared with Ub-R-S-GFP (6), and peptides measuring the increase in Kb–SIINFEKL expression concomitant were generated at half the rate from SCRAP vs. rapidly degraded with SCRAP degradation (Fig. 1C) in the presence of cyclohexi- NP. Therefore, an intrinsic difference between SCRAP and NP-S- mide to prevent newly synthesized and rapidly degraded SCRAP GFP (or Ova, also reported in the original study) does not account from entering the antigen presentation pathway. for the high efficiency of peptide generation from SCRAP in The efficiency of generating Kb–SIINFEKL complexes was EL4 transfectants. statistically indistinguishable between Shield-1–sensitive nascent and retiree pools (Tables 1 and 2), and was calculated to be ∼2%. Remarkably, this is 40-fold more efficient than generation Table 1. Efficient antigen presentation of peptides derived of Kb–SIINFEKL complexes from VV-expressed rapidly de- from rapidly degraded self-proteins graded nucleoprotein (NP)- SIINFEKL (S)-GFP [ubiquitin Kb–SIINFEKL GFP Calculated fi (Ub)-R-S-GFP in Fig. 1A], and 10-fold more ef cient than Nascent Experiment complexes molecules efficiency (%) misfolded NP-S-GFP due to the insertion of a KEKE sequence in the NP domain (6, 14). 1 5,445 2.54 × 105 2.1 These large differences could be related to differences in the cell 2 2,958 5.62 × 105 0.5 type studied (EL4- vs.