Expression PatternRare, Antibody-Dependent SLAM-Associated Protein Detailed Phenotypic and Functional Characterization of a

Lee Yap, Asma Mustafa, Sabah Kadri, Wei Huang and Xiaotian T. ZhengAaruni Khanolkar, Jeffrey D. Wilks, Guorong Liu, Edward A. Caparelli, Michelle De Moura, Kai

http://www.immunohorizons.org/content/4/4/153https://doi.org/10.4049/immunohorizons.1900060doi:

2020, 4 (4) 153-164ImmunoHorizons 

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Detailed Phenotypic and Functional Characterization of a Rare,Antibody-Dependent SLAM-Associated ProteinExpression Pattern

Aaruni Khanolkar,*,† Jeffrey D. Wilks,* Guorong Liu,* Edward A. Caparelli,* Michelle De Moura,* Kai Lee Yap,*,† Asma Mustafa,*Sabah Kadri,*,† Wei Huang,* and Xiaotian T. Zheng*,†

*Department of Pathology, Ann and Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL 60611; and †Department of Pathology, Feinberg

School of Medicine, Northwestern University, Chicago, IL 60611

ABSTRACT

SLAM-associated protein (SAP) is an adaptor molecule that facilitates critical effector functions in immune cells, and its deficiency

causes X-linked lymphoproliferative disease type 1 in which effector responses directed against EBV are severely compromised. The

primary objective of this study was to phenotypically and functionally characterize a rare, CD8 T cell–restricted bimodal SAP

expression pattern observed in healthy, human donors with the widely used 1C9-SAP mAb clone. We initially observed this pattern

during the clinical validation of our flow cytometry–based assay to diagnose X-linked lymphoproliferative disease type 1 in our

laboratory. For this validation study, we used multiparameter flow cytometry to identify cytosolic SAP expression in lymphocyte

subsets, and CD8 T cells from the donors displaying the rare SAP expression pattern mentioned above were separately further

evaluated by intracellular cytokine and CD107a staining to examine polyfunctionality following PMA/ionomycin and HLA class I

allele–restricted EBV peptide epitope–induced T cell activation. Our data revealed that SAP 1C9-hi CD8 T cells clearly displayed

higher polyfunctional responses versus SAP 1C9-lo CD8 T cells following PMA/ionomycin stimulation. Furthermore, polyfunctional

EBV-specific CD8 T cell responses segregated with the SAP 1C9-hi CD8 T cells and not the SAP 1C9-lo CD8 T cells. Additionally, and

rather intriguingly, short- and long-term T cell stimulation selectively diminished the signal for the 1C9-hi subset. Overall, our data

suggest that although rare, this unique SAP expression pattern merits further evaluation as it has the potential to provide some insight

into fundamental processes as they might relate to host–pathogen dynamics. ImmunoHorizons, 2020, 4: 153–164.

INTRODUCTION

SAP is an adaptor molecule that plays a vital role in ensuring theoptimal function of T cells and NK cells, and loss of functionmutations in the gene that encodes this protein cause X-linkedlymphoproliferative disease type 1 (XLPD-Type 1), a very rare(affecting one in one million newborn males) and life-threateningimmune disorder in which three fourths of the affected patients

fail to progress beyond the first decade of life without a bonemarrow transplant (1–5). Morbidity in XLPD-Type 1 is oftenassociated with primary EBV infection, a g-herpesvirus thatinfects over 90% of the population worldwide and is effectivelycontrolled by cytotoxic CD8 T cells in immunocompetentindividuals (3, 6). Given the role SAP plays in orchestratingT cell and NK cell responses, XLPD-Type 1 patients display arange of clinical phenotypes indicative of widespread immune

Received for publication August 5, 2019. Accepted for publication March 23, 2020.

Address correspondence and reprint requests to: Dr. Aaruni Khanolkar, Department of Pathology, Ann and Robert H. Lurie Children’s Hospital of Chicago, 225 EastChicago Avenue, Box 82, Chicago, IL 60611. E-mail address: akhanolkar@luriechildrens.org

ORCIDs: 0000-0003-3923-8510 (M.D.M.); 0000-0003-3926-7645 (A.M.); 0000-0003-0365-6550 (S.K.).

This work was supported by philanthropic funds provided by the Jeffrey Modell Foundation to the Ann and Robert H. Lurie Children’s Hospital of Chicago.

Abbreviations used in this article: MFI, median fluorescence intensity; SLE, systemic lupus erythematosus; VCA, viral capsid Ag; XLPD-Type 1, X-linkedlymphoproliferative disease type 1.

The online version of this article contains supplemental material.

This article is distributed under the terms of the CC BY 4.0 Unported license.

Copyright © 2020 The Authors

https://doi.org/10.4049/immunohorizons.1900060 153

RESEARCH ARTICLE

Clinical and Translational Immunology

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dysregulation, including fulminant inflammation, irregularities inthe Ig repertoire, andBcell–associatedmalignancies (6).X-linkedrestriction of the gene encoding for SAP (SH2D1A) andlyonization allows the lymphocyte pool in XLPD-Type 1 femalecarriers to randomly segregate along the lines of SAP sufficiency(SAP+) and deficiency (SAP2), which has enabled the directexamination of themechanismunderpinning the cytotoxicT cellresponse directed at EBV-infected cells (7). Interestingly, studiesperformed in systemic lupus erythematosus (SLE) patients havealso noted a significant downregulation of SAP expression incirculating CD4 T cells (but not CD8 T cells) compared withhealthy control samples, and this downregulation reduced IL-2production by the CD4 T cells (8). Additionally, related to theability of SAP in regulating lymphocyte responses, investigatorshave also examinedhow the expression of SAP itself ismodulatedin response to lymphocyte activation, and this remains an area ofcontinued investigation in the field (6, 9).

We are a Clinical Laboratory Improvement Amendments–certified and College of American Pathologists–accredited di-agnostic immunology laboratory, and XLPD-Type 1 is one ofseveral rare primary immunodeficiency disorders for which weoffer a rapid flow cytometry–based, clinically validated diagnostictest. As part of this validation process, we screened 54 healthydonors to determine the normal range of SAP expression indefined lymphocyte subsets using thewidely reported 1C9-SAPAbclone (5, 7, 10–16). During this effort, we encountered two healthydonors (one adultmale andone adult female, both over 30 y of age,and the femaledonorhas threehealthymale and femaleoffspring),in whom, specifically, the CD8 T cells consistently displayed aunique bimodal pattern of SAP expression observedwith the 1C9-SAP Ab clone. We completed our validation in June of 2014, andsince then, we have observed this identical CD8 T cell–restrictedbimodal SAP expression pattern in only one additional patient, a15-y-oldmalewith a confirmedhypomorphicmutation in the geneencoding theX-linked inhibitorof apoptosis protein (whichcausesX-linked lymphoproliferative disease type 2) and a positive historyof EBV infection early in childhood (unfortunately, we were notable to consent this individual for this study). Interestingly, thissame 1C9 Ab clone–associated CD8 T cell–restricted bimodalexpressionpatternwas also observed in three subjects evaluated ina recent study examining humoral and cellular immune responsesin XLPD-Type 1 patients displaying spontaneous somatic re-version of their SAP mutations, which can occur in the face ofselective pressure mediated by EBV exposure (14, 16). And thisbimodal pattern is clearly distinct fromwhat is observed inXLPD-Type 1 female carriers that harbor SAP+ and SAP2 lymphocytesubsets (7). This bimodality observed with 1C9-SAP Ab cloneaffordedus theopportunity to propose and examine thehypothesisthat 1C9-hi CD8 T cells would demonstrate superior polyfunc-tional responses. We tested our hypothesis by directly compar-ing the ability of in vitro–stimulated 1C9-hi and 1C9-lo CD8T cells to display polyfunctional responses as defined by theirability to coproduce effector cytokines, such as IFN-g, TNF-a,and IL-2 (17–19). Published reports have demonstrated thatSAP-deficient CD8 T cells exhibit defective lytic activity against

EBV-infected targets; hence, we also measured surface mobili-zation of CD107a (lysosomal-associated membrane protein-1), amarker of degranulation potential, following in vitro stimula-tion of both 1C9-hi and 1C9-lo CD8 T cell subsets (6, 19). Ourinvestigations revealed that 1C9-hi CD8 T cells displayedunequivocally superior polyfunctional responses and degranu-lation potential in response to mitogenic stimulation, andpolyfunctional EBV-specific CD8 T cells resided exclusivelywithin this subset. And intriguingly, T cell activation induced aselective diminution of the signal for the 1C9-hi CD8 T cellsubset, whereas that of the 1C9-lo subset remained essentiallyunchanged. As mentioned previously, the concept of T cellstimulation–induced modulation of SAP expression currentlyremains fluid, and our findings add an additional layer ofcomplexity to this issue (6, 9).

MATERIALS AND METHODS

All clinical laboratory tests described in this report, includingimmunological, EBV, and CMV serology and HLA analyses, havebeen clinically validated in-house and are routinely ordered byclinicians within our health system to evaluate both in-patients aswell as out-patients. This studywas carried out in accordancewiththe recommendationsof theHumanResearchProtectionProgramGuidelines of our Institutional Review Board. This protocol wasapproved by our Institutional Review Board (no. 2015-328; StudyTitle: To compare the functionalfitness of SAP-hi andSAP-loCD8T cells), and all subjects gave written informed consent inaccordance with the Declaration of Helsinki.

Flow cytometry assaysMajority of the Abs were purchased from BD Biosciences (SanJose, CA), BioLegend (San Diego, CA), and Thermo FisherScientific (San Diego, CA). Abs purchased from other vendorsare listedalongside their specificdescriptionsbelow.Sampleswereacquired on FACSCanto II instruments (Becton Dickinson,Franklin Lakes, NJ), and data were analyzed using FlowJosoftware (Version $ 10.1) (FlowJo, BD Biosciences, Ashland,OR). Phosflow analysis was performed using Cytobank software(Cytobank, Mountain View, CA) (20). Conventional hierarchicalgating approaches were used to analyze lymphocytes and theirsubsets. Briefly, lymphocytes were gated based on light scattercharacteristics followed by gating on CD3+ and CD32 populationsas appropriate. CD4 and CD8 T cells were identified within theCD3+ population, whereas the CD19+ B cells and CD56+ NK cellswere detected within the CD32 population. All additional subsetswere similarly delineated based on surface or cytosolic markerexpression within the indicated parent population.

Detection of SAP by flow cytometryThis was performed as per published guidelines (10). Briefly,aliquots of whole blood samples collected in sodium EDTA–treated blood collection tubes were analyzed within 24 h of blooddraw.Theywerefirst treatedwithHumanTruStainFcX(BioLegend),

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fixed, permeabilized, and then incubated with either the isotypecontrol Ab (mouse IgG2a, k; cloneMOPC-173) (BD Biosciences)or purified mouse anti-human SAP mAb (clone 1C9) (Abnova,Taipei, Taiwan) (5, 7, 10–16). Both the isotype control Ab and 1C9clone were used at the recommended dosage (1 mg/test). Thesampleswere thenwashed and incubatedwithFITC-conjugatedrat anti-mouse IgG2a, k mAb (clone R19-15) (BD Pharmingen,San Jose, CA). In some experiments, SAP expression was alsoassessed using the PE-conjugated rat anti-human SAP Ab (cloneXLP-1D12) (1 and 0.5 mg/test) (Thermo Fisher Scientific) (8). Inthe XLP-1D12 Ab clone experiments, the background signal wasdetermined by staining the samples with rat IgG2a, k-PE (1 and0.5 mg/test) (clone eBR2a) (Thermo Fisher Scientific). Surfacestaining was performed with Abs directed against human CD3,CD8, CD19, and CD56markers (BDBiosciences). Banked PBMCsobtained froma genotypically confirmedXLPD-Type 1 patient usedin our clinical assay validation studies were kindly provided byDr. A. Klion at the National Institutes of Health (Bethesda, MD).

Staining for intracellular cytokines and CD107a by flowcytometry using whole blood samplesThis was done as per published guidelines (21). Briefly, wholeblood sample aliquots were treated with medium alone orstimulated with PMA (10 ng/ml) and ionomycin (1 mg/ml) orthe EBV peptide epitope (HLA-B08–restricted BZLF-1 epitope[190–197]: RAKFKQLL) depicted in Fig. 5 (10 mg/ml finalconcentration; Bio-Synthesis, Lewisville, TX) for 5 h at 37°C inthe presence of brefeldin A (GolgiPlug) (BD Biosciences) (ormonensin [GolgiStop] [BDBiosciences] for CD107a assays). ForCD107a detection, the relevant isotype control Ab (mouse IgG1,k-PE; clone340; BD Biosciences) or the CD107a-PE Ab (cloneH4A3; BD Biosciences) was added at the initiation of stimulation(19). Staining for SAP was performed prior to the addition of Absdirected at surface Ags and intracellular cytokines.

Phosflow analyses for evaluating ZAP70 phosphorylationThis was performed as previously described (22). Briefly, wholebloodsamplealiquotswere incubatedwitheithermouseanti-humanCD3 mAb (clone UCHT1) (BioLegend) or mouse IgG1, k (cloneMOPC-21;BioLegend) for 30minon ice,washed, and then treatedwith goat anti-mouse IgG (clone Poly4053; BioLegend) on ice foran additional 30min before transferring the tubes to a 37°Cwaterbath for 5 min. The cells were then fixed, permeabilized, andstained per manufacturer’s instructions with an Ab targetingthe phosphoepitope ofZAP70conjugated toAPC (clone n3kobu5;Thermo Fisher Scientific) and with a noncompeting Ab clonetargeting CD3 (clone SK7) as well as with Abs directed againstCD8 and CD45RA.

EBV and CMV serologyIgG and IgM responses to EBV andCMVwere evaluatedwith ourclinically validated assays that use CAPTIA enzyme immunoassaykits manufactured by Trinity Biotech (Jamestown, NY). The EBVkits use 96-well plates coated with the purified viral capsid Ag(VCA), whereas the CMV kits detect responses to the human

AD169CMV strain propagated usingMRC5 cells. The assayswererun on the fully automated, open DS2 platform (Dynex Technol-ogies, Chantilly, VA).

EBV load quantitationEBV DNA was quantified in whole blood specimens using alaboratory-developed, clinically validated real-time PCR assay inour clinical microbiology laboratory. DNA extraction was per-formedusing theEZ1platformbyQiagen (Germantown,MA).Thequantitative amplification was performed with analyte-specificreagents from Roche (Indianapolis, IN) on the company’s Light-Cycler instruments.

Sequencing and bioinformatic analyses of the SH2D1A geneDNA was extracted using Puregene, fragmented to 100–300 bpusing focused ultrasonicator, followed by adapter ligation,amplification, and cleanup using the KAPA HyperPrep Kit. Thelibrary was quantified and sequenced using NextSeq 500/550High-Output Kit v2.5 (150Cycles) on IlluminaNextSeq 500, andraw data were converted to fastq files used bcl2fastq v2.20. Thedata were then processed using an in-house bioinformaticspipeline. In short, the adapters were trimmed using fastp (23)v0.20.0, and alignment to the hg38 human reference genomewas performed using Burrows-Wheeler Aligner MEM (24)v0.7.12-r1039 with options -a and -M. The output sam files weresorted and converted to indexed bam files using picard v2.18.27(http://broadinstitute.github.io/picard/). Indel realignmentwasthen performed using Assembly-Based Realignment v2.22 (25)followed by picard to sort, index, and remove PCR duplicates.The pipeline uses three different variant callers: haplotype callergatk (Ref. R. Poplin, V. Ruano-Rubio, M.A. DePristo, T.J. Fennell,M.O. Carneiro, G.A. Van der Auwera, D.E. Kling, L.D. Gauthier,A. Levy-Moonshine,D.Roazen, K. Shakir, J. Thibault, S. Chandran,C. Whelan, M. Lek, S. Gabriel, M.J. Daly, B. Neale, D.G.MacArthur, andE. Banks,manuscript posted onbioRxiv) v4.1.4.1,VarScan (26) v2.4.3 along with samtools v1.9 mpileup (24) (-Q 13,-q 1, and -B -l piped into varscan mpileup2cns), and last, strelka2(27) v2.9.10. The output of the three variant callerswere combinedusing a custom in-house R script (R version 3.5.2 [2018-12-20])with vcfR package (v1.8.0). The final consensus vcf file wasfurther filtered based on the maximum allele frequency of

TABLE I. Reference ranges for SAP expression established in ourlaboratory

Lymphocyte Subsets Normal Cutoff Valuesa (% SAP+)

CD3+CD8+ T cells .62CD3+CD82 T cells .64CD32CD19+ (B cells) 0–9CD32CD56+ (NK cells) .55

aBackground subtracted values derived from the assessment of 54 healthycontrol donors. Background level of staining was determined by using anisotype control mAb. Assay precision statistics (summary data for intra- andinterassay precision): CD8 T cells (mean coefficient of variation: 2.21%[coefficient of variation range: 0.52–4.33%]); CD4 T cells (mean coefficient ofvariation: 1.99% [coefficient of variation range: 0.65–3.68%]); and CD56+ NKcells (mean coefficient of variation: 3.02% [coefficient of variation range:1.22–5.55%]).

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the three variant callers with 10% allele frequency cutoff. Variantannotation was carried out using Alamut Batch Standalone v1.11(InteractiveBiosoftware, Rouen, France) for all transcripts. Usinga custom R script, annotations from multiple transcripts wereranked and collapsed. We focused on variants in the SH2D1Agene, and all data for this geneweremanually confirmedusing theIntegrative Genomics Browser (Fig. 6) (28).

RESULTS

Clone 1C9–associated CD8 T cell–restricted bimodal patternof SAP expressionFifty four healthy control donor peripheral blood samples wereanalyzed tooutline thenormal reference ranges for SAPexpression

in CD4 and CD8 T cells, B cells, and NK cells (Table I). Histogramoverlay plots depict SAP expression in the indicated cellularsubsets for a representative healthy control donor as well as inbanked PBMCs derived from a genotypically confirmed (de-ceased) case of XLPD-Type 1 (we were unable to visualizeclearly detectable B cells and NK cells in this patient sample)(Fig. 1A). Two of the healthy control donors (labeled as donor 1,an adult male, and donor 2, an adult female) displayed a bimodalSAP expression pattern that was consistently observed using themouse-derived 1C9-SAP Ab clone, and it was restricted to theirCD8T cell subset (both donors have been tested at leastfive timeseach over the past 5 y to ascertain this rare phenotype) (Fig. 1B).Given that this 1C9 Ab clone–associated bimodal pattern was notdetected in our XLPD-Type 1 patient sample or the circulat-ing B cells of all 54 healthy control samples (circulating B cells

FIGURE 1. Analysis of SAP expression in defined lymphocyte subsets.

(A) SAP expression in lymphocyte subsets in a representative healthy control sample and a genotypically confirmed XLPD-Type 1 patient. The

numbers in the plots indicate the background-subtracted frequency (%) of the lymphocyte subset expressing SAP. (B) Comparative assessment of

SAP expression in lymphocyte subsets of donors 1 and 2 using the 1C9 and XLP-1D12 SAP Ab clones. The data shown for the 1C9 Ab clone is

representative of at least five independent experiments performed separately for both donors 1 and 2 to confirm the CD8 T cell–restricted bimodal

expression pattern.

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naturally lack SAPexpression), aswell as theCD4Tcells andNKcells of donors 1 and 2, we believe that this CD8T cell–restrictedbimodal pattern does not represent nonspecific staining with1C9Ab clone. An additional argument against nonspecific stainingis the fact that isotype control Ab staining in all cell subsetsevaluated did not display this bimodal pattern. Furthermore, thisclone 1C9–associated CD8 T cell–restricted bimodal pattern ofSAP expression (hereafter referred to as 1C9-hi and 1C9-lo) isclearly distinct from the phenotype observed in XLPD-Type 1female carriers (SAP+ and SAP2) in which the SAP2 populationoverlaps with the isotype control Ab signal (7). Moreover, asmentioned in the introduction, this identical 1C9 Ab clone–associated CD8 T cell–restricted bimodal pattern of SAPexpression is also portrayed, but was not formally evaluated, ina recent publication examining immune responses in threeXLPD-Type 1 patients displaying spontaneous reversion of their

SAPmutations (16).Wealso examinedSAPexpression indonors 1and 2 with the rat-derived XLP-1D12 SAP Ab clone, which unlikethe 1C9 Ab clone, consistently displayed the conventional uni-modal patternof SAPexpression (Fig. 1B).Onepotential explanationfor the 1C9-associated bimodal pattern versus the conventionalunimodal pattern observedwithXLP-1D12 clone in donors 1 and2 could be the difference in the epitopes targeted by these twomAbs. Based on the technical data available for both SAP Abclones, they were generated by immunizing animals (mice forthe 1C9 clone and rats for the XLP-1D12 clone)with the full-length(128 aa) recombinant SAP protein. Both clones can recognizedenatured as well as native protein; however, the exact epitopeinformation for both clones is currently lacking. Interestingly,we also observed some B cell staining with the XLP-1D12 clonewhen we used both the 1C9 clone and the XLP-1D12 clone at1mg/test (Fig. 1B). Themanufacturer-recommended dosage for

FIGURE 2. Surface marker characteristics and signaling properties of 1C9-hi and -lo CD8 T cell subsets and their phenotypic equivalents.

(A) 1C9-hi and -lo CD8 T cell subsets were evaluated for the surface expression of CD45RA and CD45RO. The numbers in the upper-right section of

each plot depict the percentage of the cells expressing the marker indicated along the x-axis for the histogram on the right-hand side of the plot,

and the numbers in the upper-left section of each plot depict the percentage of the cells expressing the marker indicated along the x-axis for the

histogram on the left-hand side of the plot. (B) The surface expression of CD27 and CD62L was also examined for the indicated CD8 T cell subsets

to determine phenotypic similarities between the 1C9-lo and CD45RA+ CD8 T cells as well as between the 1C9-hi and CD45RO+ CD8 T cells. The

numeric values in each quadrant depict the frequency of the gated cells occupying the quadrant. (C) Assessment of CD69 expression on the surface

of 1C9-hi and -lo CD8 T cells was performed at baseline (unstimulated) and following PMA/ionomycin treatment. (D) Phosphorylation of ZAP70, a

proximal signaling node in the TCR signaling axis, was surveyed to ascertain the signaling potential of CD45RA+ (the phenotypic equivalent of a vast

majority of the 1C9-lo) and CD45RA2 (the phenotypic equivalent of a vast majority of the 1C9-hi) CD8 T cells. The numbers in each plot indicate the fold

increase in the MFI of pZAP70 over basal levels (mouse IgG1, k treatment), which is normalized to one. The colors of individual histograms also depict the

change in MFI based on the colorimetric scale placed below the plots. Data depicted are representative of at least two independent experiments.

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the XLP-1D12 clone is 0.5 mg/test; hence, we also tested theXLP-1D12 clone using the 0.5 mg/test dosage (SupplementalFig. 1A). A similar level of B cell staining was evident with bothdoses of the XLP-1D12 clone. Additionally, we tested bankedPBMC from a genotypically confirmed case of XLPD-Type 1with both clones in the same experiment (Supplemental Fig.1B). As previously shown in Fig. 1A, no positive signal for SAPexpressionwas detected in gatedCD8+ andCD82T cell subsetsusing the 1C9-SAP Ab clone. However, some nonspecific stainingwas observed for both the CD8+ and CD82 T cells with the XLP-1D12 clone. Collectively, these data indicate that the XLP-1D12clone may not be suitable for testing clinical samples, at leastin our hands, and that the 1C9-SAP Ab clone–associated CD8T cell–restricted bimodality is not an experimental artifact andmight potentially reveal biologically relevant information.

1C9-hi CD8 T cells display a predominantlymemory phenotypeAnalysesperformed inSLEpatients andcontrols havedemonstratedthat SAP expression levels are higher in effector/memory CD4T cells compared with naive CD4 T cells, and a separate studydemonstrated that memory T cells harbor a greater proportionof SAP+ T cells compared with naive T cells (8, 29). Additionally,an examination of cases of spontaneous somatic reversion of theSAP mutations in XLPD-Type 1 patients has shown that thisreversion is restricted to the memory CD8 T cell and CD4 T cellsubsets (14, 16). Consistent with these previous reports, weobserved that memory (CD45RO+) CD8 T cells from donors 3and 4 that display the conventional unimodal pattern of SAPexpression with the 1C9-SAP Ab clone also expressed higherlevels of SAP in terms of median fluorescence intensity (MFI)

(2.5–4 times greater than the [CD45RO2] CD8 T cells) (SupplementalFig. 2) (29). This trendwas repeatedwhenwe gated on their IFN-g–producing CD8 T cells as the IFN-g+CD45RO+CD8 T cellsdisplayed a higherMFI for SAP comparedwith IFN-g+CD45RO2

CD8 T cells (Supplemental Fig. 2).In light of this information, we were interested in determin-

ing if the 1C9-hi and 1C9-lo subsets would reveal phenotypicproperties that further differentiated these two subsets. Indeed,our initial analyses indicated that themajority of the 1C9-hi subsetsegregatedwith theCD45RO+ (prototypicalmemory) CD8T cells,whereas themajority of the 1C9-lo subset resembled theCD45RA+

CD8 T cell subset (Fig. 2A). Additional characterization based onthe coexpression of CD62L andCD27 exposed further evidence ofphenotypic symmetry between the 1C9-hi and CD45RO+ CD8 Tcells, and 1C9-lo and CD45RA+ CD8 T cells (we also attempted toexamine CCR7 coexpression; however, the fixation and per-meabilization steps required for detecting SAP expressioninterferedwith this specific signal) (Fig. 2B) (30). Interestingly,we also observed that the 1C9-hi subset in both donorsdisplayed higher basal CD69 expression, thereby also suggest-ing a preactivated phenotype (Fig. 2C). Studies have alsoindicated that reduced SAP expression in the CD4 T cells ofSLE patients and the absence of this protein in XLPD-Type 1patients is accompanied by exaggerated early signaling eventsfollowingTCRengagement (8, 31, 32). Therefore,we also assessedthe phosphorylation status of ZAP70 following anti-CD3 stimu-lation by Phosflow to determine if the 1C9-hi and -lo subsetswould differ in their TCR-associated proximal signaling proper-ties (33).However, the harsh cellularfixation and permeabilizationsteps that are required for Phosflow analyses interfered with ourability to simultaneously examine intracellular SAP expression

FIGURE 3. T cell activation selectively affects the detection of the 1C9-hi CD8 T cell subset.

Cytosolic SAP expression was assessed in CD8 T cells with and without PHA (A) and PMA/ionomycin (B) stimulation [numeric values in the plots in

(B) represent the percentages of 1C9-hi and 1C9-lo subsets]. (C) Background-subtracted frequencies (% values) and MFIs (in parentheses) of SAP+

CD8 T cells6 PMA/ionomycin stimulation were also measured after staining the cells with XLP-1D12 SAP Ab clone. Data depicted are representative

of at least two independent experiments.

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in tandem. But because we had previously determined that themajority of the 1C9-hi CD8 T cell subset segregated with theCD45RO+ CD8 T cells and that the majority of the 1C9-lo CD8Tcells resembledCD45RA+CD8Tcells,weexploited thispropertyin our Phosflow analyses to circumvent our inability to simulta-neously costain for SAP (Fig. 2D). We did not observe any majordifference in the pZAP70 signal between the CD45RA+ andCD45RA2 CD8 T cell subsets (Fig. 2D). These findings areconsistent with what has been reported for naive and memoryCD8 T cells in preclinical models andmight also reflect the factthat donors 1 and 2 are healthy subjects, unlike patient samplesevaluated in the studies referenced above (34, 35).

T cell activation selectively affects the detection of the1C9-hi CD8 T cell subsetExisting data suggest that T cell activation affects SAP expression,and thedirection inwhichSAPexpression ismodulated appears to

depend on the T cell differentiation status and how the T cellsare stimulated (6, 9, 36). SAP is downregulated inmurineT cellsfollowing activation, and this was also observed in one studywith human CD8 T cells, whereas another study demonstratedthat SAP expression is upregulated in in vitro–activated T cells(6, 9, 36). Our efforts demonstrated that PHA stimulation for72 h induced a marked diminution of the 1C9-hi subset of CD8T cells, whereas the signal for the 1C9-lo subset of CD8 T cellsremained essentially unaffected (Fig. 3A). The degree of dim-inution was also directly related to the input dose of theactivation stimulus as it was greater with the higher dose ofPHA used in the assay (Fig. 3A). Short-term stimulation (4–5 h)with PMA and ionomycin also demonstrated a similar trend(Fig. 3B). However, we did not observe a similar degree ofdecline in the proportion of SAP-expressing CD8 T cellsstained with the XLP-1D12 Ab clone, and in fact, based onMFI, the level of SAP expression was slightly enhanced after

FIGURE 4. 1C9-hi CD8 T cells display enhanced polyfunctional responses.

(A) Overlays of dot plots depicting cytokine coproduction and degranulation by 1C9-hi and 1C9-lo CD8 T cells with and without PMA/ionomycin

stimulation. The numerical values listed in the plots depict the background-subtracted frequencies of the gated CD8 T cells lying within the specific

quadrants. Green population: unstimulated and stained with mAbs to IFN-g, TNF-a, IL-2, and CD107a; red population (in the cytokine dot plots):

PMA/ionomycin stimulated and stained with isotype control Abs for IFN-g, TNF-a, and IL-2; red population (in the CD107a contour plots): PMA/

ionomycin stimulated and stained with an isotype control Ab for CD107a and an mAb against IFN-g; and blue population: PMA/ionomycin

stimulated and stained with mAbs to IFN-g, TNF-a, IL-2, and CD107a. (B) Comparison of PMA/ionomycin stimulation-induced IFN-g production

between 1C9-hi and -lo CD8 T cells and their subsets based on surface expression of CD45RA+ and/or CD45RO+. The numbers listed in this

histogram overlay plot represent the background-subtracted MFIs of IFN-g production by the indicated CD8 T cell subsets and the background-

subtracted frequencies (%) of the subsets producing IFN-g following PMA/ionomycin stimulation. Data depicted are representative of at least two

independent experiments.

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stimulation (27% increase for donor 1 and 15% increase fordonor 2) (Fig. 3C).

1C9-hi CD8 T cells display superiorpolyfunctional responsesOne of the key functional attributes of CD8 T cells is robustcytokine production and degranulation following recognition oftheir cognate Ag (37–40). Polyfunctionality is described as the

ability of T cells to coproduce multiple effector cytokines, such asIFN-g, TNF-a, and IL-2, and display robust cytotoxic activityfollowing stimulation (17–19, 41). Impaired polyfunctionality is ahallmark of T cell dysregulation observed in the setting of poorlycontrolled viral infections such as those associated with HIV andhepatitis C virus, inwhich a stepwise sequential loss of IL-2, TNF-a, and finally IFN-g production identifies T cells that eventuallyget physically deleted (41–44). Furthermore, there is publishedevidence demonstrating the diminution of IFN-g production bySAP-deficient EBV-specific CD8 T cells as well as impaired IL-2production by CD4 T cells in SLE patients that also displaydecreased SAP expression, suggesting an intimate link betweenSAP expression andT cell effector function (2, 8). Considering thisinformation, we predicted that 1C9-hi CD8 T cells would displayaugmented cytokine production and higher degranulation poten-tial. Indeed, when we assessed polyfunctionality for both donorsby intracellular cytokine secretion assays, we detected markedlyhigher frequencies of cells coexpressing IFN-g, TNF-a, IL-2, andCD107a following PMA/ionomycin treatment in the 1C9-hi subsetof CD8 T cells (Fig. 4A). Specifically, the frequency of CD8 T cellscoproducing IFN-g and TNF-a were at least 3-fold higher in the1C9-hi subset, and similarly, the frequency of CD8 T cellscoproducing IFN-g and IL-2 were at least 2.7-fold higher in the1C9-hi subset versus the 1C9-lo subset. The 1C9-hiCD8Tcells alsoexhibited better surface mobilization of CD107a, a marker ofdegranulation. For both donors, the proportion of CD8 T cellscoexpressing IFN-g and CD107awas clearly higher ($2.8-fold) inthe 1C9-hi subset.These results areconsistentwith thephenotypiccharacteristics, given that the majority of 1C9-hi CD8 T cellsdisplayed a prototypical memory (CD45RO+) phenotype, andsimilarly, amajority of 1C9-loCD8T cells expressedCD45RA (Fig.2A).Thedata alsodemonstrated that althoughonlya small fractionof the 1C9-hi CD8 T cells expressed CD45RA, 61% of this fractionof cells expressed IFN-g upon stimulation. In contrast, themajority of the 1C9-lo CD8 T cells expressed CD45RA, but only15% of this fraction elaborated IFN-g following stimulation (Fig.4B). A likely explanation for this difference is that roughly 15% ofthe 1C9-hiCD8Tcells that coexpressCD45RA lack the expressionof both CD27 and CD62L, indicating the presence of CD45RA+

T effector memory cells in addition to conventional naive T cellswithin this fraction (data not shown) (30). Additionally, following

FIGURE 5. Polyfunctional responses elicited from 1C9-hi and 1C9-lo

CD8 T cells following the treatment of whole blood samples with and

without the HLA class I allele–restricted EBV peptide epitope.

The upper-right quadrant in each plot depicts the CD8 T cells co-

producing the listed molecules. The numeric values listed in the plots

depict the background-subtracted frequencies of the gated CD8 T cells

lying within the specific quadrants. Red population: unstimulated and

stained with mAbs to IFN-g, TNF-a, IL-2, and CD107a; green population

(in the cytokine dot plots): EBV (HLA-B08–restricted BZLF-1 epitope

[190–197]: RAKFKQLL) treated and stained with isotype control Abs for

IFN-g, TNF-a, and IL-2; green population (in the CD107a contour

plots): EBV (HLA-B08–restricted BZLF-1 epitope [190–197]: RAKFKQLL)

treated and stained with an isotype control Ab for CD107a and an mAb

against IFN-g; and black population: EBV (HLA-B08–restricted BZLF-1

epitope [190–197]: RAKFKQLL) treated and stained with mAbs to IFN-g,

TNF-a, IL-2, and CD107a. Data depicted are representative of at least

two independent experiments.

TABLE II. Viral serology and molecular analyses for Donors 1 and 2

Donor 1 Donor 2 Normal Range

EBV-VCA IgM (serum) 0.03 0.24 #0.90a (negative); 0.91–1.09a (equivocal);

$1.10a (positive)EBV-VCA IgG (serum) 6.93 3.95 #0.90a (negative); 0.91–1.09a (equivocal);

$1.10a (positive)EBV DNA (measured in

whole blood)

Below the limit of detection Below the limit of detection Limit of detection is $2000 copies/ml

CMV-IgM (serum) 0.34 0.04 #0.90a (negative); 0.91–1.09a (equivocal);

$1.10a (positive)CMV-IgG (serum) 0.28 5.20 #0.90a (negative); 0.91–1.09a (equivocal);

$1.10a (positive)aArbitrary units.

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stimulation,1C9-hiCD8Tcells thatwereCD45RO+ (andCD45RA2)displayed frequencies and expressed IFN-g levels (in terms ofMFI) that were higher than those observed for the 1C9-lo CD8T cells that coexpressed CD45RO (Fig. 4B). We also examinedepitope-specific CD8 T cell responses for donor 1 who had beenpreviously HLA typed and was strongly EBV seropositive (Fig. 5,Table II). CD8 T cells from donor 1 were stimulated with anHLA-B08–restricted EBV-lytic cycle protein (BZLF-1 [190–197]:RAKFKQLL)peptideepitopeand then tested fordenovocytokineproduction and degranulation by intracellular cytokine secre-tion assays (Fig. 5) (45, 46). Notably, the epitope-specific poly-functional CD8 T cell responses were detected almost exclusivelywithin the 1C9-hi CD8 T cell subset (Fig. 5).

In an attempt to try to understand the evolution of this CD8T cell–restricted bimodal phenotype, we evaluated donors 1 and 2for evidence of active EBV infection (Table II). Both donorsdisplayed strong IgG responses to the EBV VCA but were IgMnegative, and the EBVDNA load was below the limit of detection.Serological assessment for another common herpesvirus, CMV,similarly did not indicate active infection (Table II). Sequenceanalysis of the SH2D1A gene in donors 1 and 2 also did not revealany specific polymorphisms, including synonymous single nucle-otide variants, that might segregate with this bimodal phenotype(Fig. 6).

We also investigated whether the level of SAP expressionmight inform the degree to which CD8 T cells display poly-functionality by comparing coexpression of IFN-g, TNF-a,

and IL-2 between donors 1 and 2 (SAP MFI . 400) anddonors 5 and 6 (SAP MFI, 400; both donors 5 and 6 displaythe conventional unimodal SAP expression inCD8T cells).We didobserve that the proportions of CD8 T cells expressing IFN-g +TNF-a and TNF-a + IL-2 were higher in donors 1 and 2 (Fig. 7A).Both of these donors also harbored greater proportions ofmemoryCD8 T cells, specifically effector memory T cells, which could alsohave skewed the polyfunctionality data (Fig. 7B).

Cumulatively, our findings suggest that this unique bimodalityin SAP expression defined by the 1C9-SAP Ab clone observed inthese two donors highlights critical functional attributes of theCD8 T cells.

DISCUSSION

In this study, we describe the functional attributes of CD8 T cellsfrom two healthy adult donors that display a unique bimodalpattern of SAP expression that is restricted to their CD8 T cellsstained with the 1C9-SAP Ab clone. This pattern is clearly distinctfrom that observed in XLPD-Type 1 female carriers (7), andoverall, our data reveal that 1C9-hi CD8 T cells display superiorpolyfunctional ability and degranulation potential; intriguingly,they become difficult to track following prolonged exposure toactivation stimuli.

There exists a plethora of published evidence linking thequality of the T cell response to how successfully the host exerts

FIGURE 6. Sequence analysis of the SH2D1A gene.

The bam file image displays the sequence of exon 2 of the SH2D1A gene as shown for the XLPD-Type 1 patient as well as donors 1, 2, and 3 (donor 3

displays the conventional unimodal pattern of SAP expression in the CD8 T cells, whereas donors 1 and 2 display the unique bimodal pattern). Exon

2 harbors the stop mutation detected in the XLPD-Type 1 patient. The sequences for exons 1, 3, and 4 are not depicted in this image, and they do

not harbor any specific polymorphisms, including synonymous single nucleotide variants (sSNV).

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optimal viral control in the setting of viral infections (17, 18, 37–39,41–44). Specifically, the long-term preservation of polyfunctionalT cell responses correlates directly with the host’s ability toeffectively induce and maintain viral suppression (17, 18, 37–39,41–44). Studies in XLPD-Type 1 patients have demonstrated adiminution in the ability of EBV-specific cytotoxic T cells toproduce IFN-g, a key effector cytokine, and these cells displayimpaired lytic activity against EBV-infected target cells (2, 6).Furthermore, spontaneous somatic reversion of SAP deficiency inXLPD-Type 1 patients has been described to selectively localize inthe effector memory CD8 T cells that exert optimal virologicalcontrol following EBV exposure (14). Additionally, reduced SAPexpression in CD4 T cells of SLE patients also directly compro-mises IL-2 production by these cells (8). Collectively, these datasuggest that SAP is involved in regulating critical T cell responses.These observations coupled with the unique bimodal 1C9-SAPAb clone-associated pattern restricted to the CD8 T cells indonors 1 and 2 presented us with an opportunity to directlyexamine the link between this phenotype and the functionalfitness of the CD8 T cells. Our data demonstrate that 1C9-hi CD8T cells clearly outperformed their 1C9-lo counterparts based onkey quality indicators that defineT cell polyfunctionality, and thisfunctional validation further challenges the notion that thisbimodal pattern represents nonspecific staining with the 1C9-Abclone. Specifically, the proportion of cells capable of coexpressingthe effector cytokines IFN-g, TNF-a, and IL-2 as well as thecoexpression of IFN-g and CD107a, a marker of degranulation,were unequivocally higher in the 1C9-hi CD8 T cell subset.And this functional advantage inherent to the 1C9-hi subsetwas observed following the treatment of T cells with bothmitogenic stimuli (PMA/ionomycin) as well as an MHC classI–restricted peptide epitope derived from EBV.

Another notable observation of our study was the activation-induced selective diminution of the 1C9-hi CD8 T cell subsetsignal. Previous studies have yielded divergent results on the issueof how SAP levels are modulated following T cell activation. Onestudy revealed that cord blood T cells activated in vitro with PHAor anti-CD3 and anti-CD28 mAbs for 4 d upregulated SAPexpression (9). A subsequent study detailed the opposite findingusing normal donor-derived untransformed CD8 T cell linesmaintained by serial stimulations using allo-PBMC and the EBV+

cell line JY (6). In this latter study, anti-CD3 mAb treatmentreducedSAPexpressionwhen thecellswere evaluated24and72hlater. These data suggest that the mode of T cell activation as wellas the subset distribution of the T cells within the stimulatedpopulation can variably modulate SAP expression. Anotherpotential issue that could account for the variability in theseresults could be the reagents used to target the SAP protein. Thefirst study used the rat anti-humanSAPAb cloneKST-3 (this clonehasnotbeencommercialized) toexamineSAPexpression,whereasthe second study used an anti-human SAP rabbit polyclonal Ab.We used the widely reported mouse anti-human SAP Ab clone1C9 for our validation study as well as the rat anti-human SAPAb clone XLP-1D12 for this specific study, and the CD8T cell–restricted bimodality was observed exclusively with the

FIGURE 7. Comparing CD8 T cell polyfunctionality and subsets

between high and low SAP expressors.

(A) CD8 T cell–associated coexpression of IFN-g, TNF-a, and IL-2 was

compared between donors 1 and 2 (SAP MFI. 400) and donors 5 and 6

(SAP MFI , 400) following PMA/ionomycin stimulation. The numeric

values listed depict the frequencies of CD8 T cells coproducing the

indicated cytokines. Green population: unstimulated and stained with

mAbs to IFN-g, TNF-a, and IL-2; red population: PMA/ionomycin

stimulated and stained with isotype control Abs for IFN-g, TNF-a, and

IL-2; and blue population: PMA/ionomycin stimulated and stained with

mAbs to IFN-g, TNF-a, and IL-2. (B) The relative distribution of CD8

T cell subsets was compared between high and low SAP expressors.

The bar graphs depict the ratios of the indicated CD8 T cell subsets for

donors 1 and 2 (SAP MFI . 400) and donors 5 and 6 (SAP MFI , 400).

Naive: CD45RA+CD45RO2CD62L+CCR7+; effector memory (Tem):

CD45RA2CD45RO+CD62L2CCR72; central memory (Tcm): CD45RA2

CD45RO+CD62L+CCR7+; and effector memory CD45RA+ (TemRA):

CD45RA+CD45RO2CD62L2CCR72. The median values of the fre-

quencies of these CD8 T cell subsets used to calculate these ratios

were determined from multiple independent measurements for each

donor performed between 2014–2020 (43 for donor 1, 38 for donor 2,

24 for donor 5, and 4 for donor 6).

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1C9 clone in donors 1, 2, and a patient with genotypicallyconfirmedhypomorphicX-linked inhibitor of apoptosismutation(who could not be consented for this study) (5, 7, 8, 10–16). T cellactivation induced a selective loss of the 1C9-hi CD8 T cell subsetsignal, whereas there was no change in the signal for the 1C9-loCD8Tcells; in contrast, stainingwith theXLP-1D12 SAPAb clonedemonstrated that the level of SAP expression was slightlyincreased on the cells that stained positively for SAP.Consideredtogether, these observations might indicate activation-inducedconformational changes in the SAP protein that could affectepitope access by the 1C9 and XLP-1D12 mAbs. Moreover, giventhat the 1C9-hi subset of CD8 T cells in our study displayed apredominantly memory phenotype, higher CD69 expression,and greater polyfunctional responses, hence resembling a cellularpopulation that ispracticallypoised for a rapidresponse, it is likelythat if indeed conformational changes are being induced byactivation, the detection of this subset might be more affected.And unfortunately, this confounding issue also prevented us fromevaluatinganyvariations in theproliferationpotentials of the 1C9-hi and 1C9-lo CD8 T cell subsets.

Considering all of the information described in this report enbloc, we speculate that the infection histories, perhaps related toviral pathogens, and associated CD8 T cell subset distributionsspecific to donors 1 and 2 (who are both healthy) could havecontributed to and promoted the emergence of the CD8 T cell–restricted bimodal 1C9-hi and -lo SAP expression pattern. Wefurther speculate that the infection histories of the three out of thefour SAP-revertant XLPD-Type 1 patients described in a recentindependent studymight have similarly influenced the emergenceof this identical 1C9 Ab clone–associated CD8 T cell–restrictedbimodal SAP expression pattern (16).

Our future effortswill be aimed at trying to identify and recruitadditional subjects that display this rare phenotype and flow sortthe 1C9-hi and 1C9-lo CD8 T cell populations in these subjectsto enable downstream molecular characterization of SH2D1Aexpression at a single cell level within these subsets. We believethis approach coupledwith the functional datawehave outlined inthis report might potentially enhance our understanding of therole SAP plays in the setting of host–pathogen interactions.

DISCLOSURES

The authors have no financial conflicts of interest.

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