DOCK2 Sets the Threshold for Entry into the Virtual Memory CD8 + Compartment by Negatively Regulating Tonic TCR Triggering This information is current as of October 1, 2021. Vinay S. Mahajan, Ezana Demissie, Faisal Alsufyani, Sudha Kumari, Grace J. Yuen, Vinayak Viswanadham, Andrew Huang, Johnson Q. Tran, James J. Moon, Darrell J. Irvine and Shiv Pillai

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 18, 2019, doi:10.4049/jimmunol.1900440 The Journal of Immunology

DOCK2 Sets the Threshold for Entry into the Virtual Memory CD8+ T Cell Compartment by Negatively Regulating Tonic TCR Triggering

Vinay S. Mahajan,*,†,1 Ezana Demissie,*,1 Faisal Alsufyani,*,‡ Sudha Kumari,x Grace J. Yuen,* Vinayak Viswanadham,* Andrew Huang,* Johnson Q. Tran,{ James J. Moon,{ Darrell J. Irvine,*,x and Shiv Pillai*

The control of cytoskeletal dynamics by dedicator of cytokinesis 2 (DOCK2), a hematopoietic cell–specific actin effector , has been implicated in TCR signaling and T cell migration. Biallelic mutations in Dock2 have been identified in patients with a recessive form of combined immunodeficiency with defects in T, B, and NK cell activation. Surprisingly, we show in this study that certain immune functions of CD8+ T cells are enhanced in the absence of DOCK2. Dock2-deficient mice have a pronounced Downloaded from expansion of their memory T cell compartment. Bone marrow chimera and adoptive transfer studies indicate that these memory T cells develop in a cell-intrinsic manner following thymic egress. Transcriptional profiling, TCR repertoire analyses, and cell surface marker expression indicate that Dock2-deficient naive CD8+ T cells directly convert into virtual memory cells without clonal effector T cell expansion. This direct conversion to memory is associated with a selective increase in TCR sensitivity to self-peptide MHC in vivo and an enhanced response to weak agonist peptides ex vivo. In contrast, the response to strong agonist peptides remains unaltered in Dock2-deficient T cells. Collectively, these findings suggest that the regulation of the http://www.jimmunol.org/ actin dynamics by DOCK2 enhances the threshold for entry into the virtual memory compartment by negatively regulating tonic TCR triggering in response to weak agonists. The Journal of Immunology, 2020, 204: 000–000.

e have previously described a loss-of-function Dock2 are polyclonal virtual memory cells and demonstrate that they are allele (Dock2hsd) that had been inadvertently introduced generated by direct conversion of naive T cells into memory as a W into multiple mouse lines (1). Using this knockout allele result of cell-intrinsic hyperresponsiveness of Dock2hsd/hsd T cells of Dock2 extensively in this study, we find that the responsiveness of to weak agonists. Mice with other engineered Dock2 mutations Dock2-deficient CD8+ T cells to weak agonists is unexpectedly also exhibit the same phenotype. These findings suggest that the enhanced. Thus, although dedicator of cytokinesis 2 (DOCK2) absence of DOCK2 lowers the threshold of self-peptide triggering by guest on October 1, 2021 may promote TCR responses to strong agonists, it appears to required to enter the virtual memory T cell compartment. constrain the responsiveness to weak TCR agonists. In vivo, the Aside from homeostatic cytokine signaling and tonic TCR loss of DOCK2 results in an enhanced conversion to virtual memory triggering, very little is known about the regulators of the CD8+ T cells. Virtual memory T cells can provide Ag-independent, T cell virtual memory compartment. Fyn binding protein 1 (FYB1) innate-like bystander protection in the context of intracellular has been proposed to function as a negative regulator of the size of infection against some pathogens, such as Listeria monocytogenes (2), the CD8+ virtual memory compartment by limiting the response and contribute to protective immunity in mice (3, 4). In this pre- to IL-15 (5). This study shows that DOCK2 functions as a novel sent study, we show that the memory phenotype (MP) T cells, negative regulator of the CD8+ virtual memory compartment. previously shown to be expanded in the absence of DOCK2 (1), DOCK2 activates the actin effector Rho GTPase Rac by catalyzing the transition from the inactive GDP-bound state to the active GTP-bound state (6). DOCK2 localizes to the via *Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139; †Brigham and Women’s Hospital, Boston, MA 02115; ‡King Fahad Specialist Hospital, Dammam its DHR1 domain–mediated interactions with PIP3 and polybasic 32253, Saudi Arabia; xThe Koch Institute for Integrative Cancer Research at MIT, amino acid cluster–based interactions with phosphatidic acid and, { Cambridge, MA 02139; and Center for Immunology and Inflammatory Diseases, thus, ensures spatially controlled activation of GTPase Rac at the Massachusetts General Hospital, Boston, MA 02129 1 plasma membrane (6–8). GTP-bound RAC1 subsequently drives V.S.M. and E.D. contributed equally. actin polymerization, enabling cytoskeletal rearrangements required ORCIDs: 0000-0001-7082-2825 (S.K.); 0000-0002-3253-9575 (G.J.Y.); 0000-0003- for (6, 9, 10), T cell interstitial motility (11), 2131-9398 (A.H.); 0000-0002-5263-6321 (J.Q.T.); 0000-0001-8246-931X (J.J.M.). plasmacytoid cytokine secretion (12), and TCR ac- Received for publication April 18, 2019. Accepted for publication October 15, 2019. tivation (13, 14). This study suggests that DOCK2-dependent This work was supported by the National Institutes of Health. remodeling of actin cytoskeletal may limit the responsiveness of Address correspondence and reprint requests to Dr. Shiv Pillai, Ragon Institute of CD8 T cells to weak agonists such as self-peptides, thereby reg- MGH, MIT and Harvard, 400 Technology Square, Cambridge, MA 02139. E-mail address: [email protected] ulating the size of the virtual memory compartment. The online version of this article contains supplemental material. Abbreviations used in this article: DOCK2, dedicator of cytokinesis 2; FYB1, Fyn Materials and Methods binding protein 1; GSEA, gene set enrichment analysis; GSVA, gene set variation Mice analysis; MP, memory phenotype; WT, wild-type. Dock2hsd/hsd mice were purchased from Harlan Laboratories and main- Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 tained as a separate colony in a specific pathogen-free environment in

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900440 2 DOCK2 AND VIRTUAL MEMORY CD8+ T CELLS accordance with institutional guidelines. C57BL/6J, OT-I, and RAG1 contact interface between T cell and dendritic cell, and the F-actin in- knockout mice were purchased from The Jackson Laboratory. Unless tensity was quantified using MetaMorph software. All images presented in otherwise specified, all experiments were conducted using 8–12-wk-old this study are raw images displayed at identical contrast settings. mice. Bone marrow chimeras Results Bone marrow was isolated from the indicated mice. Resuspended cells from Absence of DOCK2 results in the expansion of virtual memory the bone marrow were labeled using biotinylated anti-CD3 Ab and T cells Streptavidin MicroBeads (Miltenyi Biotec). Cells were then resuspended in ∼ + PBS, and 1 3 106 cells were transferred to each mouse. In an earlier study, we identified an 3-fold expansion of MP CD8 T cells in mice carrying a spontaneous loss-of-function mutation Cell transfers in the guanine exchange factor DOCK2 (Dock2hsd/hsd) (1). This For lymphopenia-induced proliferation experiments, mice were irradiated at expansion is not specific to this particular allele of Dock2,asitis a dose of 600 cGy. Six hours later, a total of 1 3 106 CFSE-labeled naive also present in gene-targeted Dock2-deficient mice (6) (Fig. 1A). hsd/hsd T cells from Dock2 and wild-type (WT) mice were coinjected into The increase in the percentage of Dock2hsd/hsd MPcellsisalso irradiated congenic hosts. One week later, transferred cells were recovered and assessed for CFSE dilution and CD44 upregulation. mirrored by a similar increase in the total numbers of these cells For experiments in lymphoreplete mice, a total of 2 3 106 naive T cells (Fig. 1B). Notably, these cells lack surface expression of NK1.1 from Dock2hsd/hsd and WT mice were coinjected into unmanipulated and express both CD8a and CD8b (data not shown). congenic hosts. Three weeks later, transferred cells were assessed for Recent studies have identified cognate Ag-independent memory CD44 and CD122 upregulation. cells that arise in unmanipulated lymphoreplete mice (25–27).

RNA sequencing and TCR repertoire analysis Such virtual memory cells can be distinguished from conventional Downloaded from memory cells by their low expression of CD49d (26). Based RNA from 50,000 cells for each condition was isolated with QIAGEN RNA hsd/hsd isolation kits according to the manufacturer instructions. RNA sequencing on CD49d staining, the majority of expanded MP Dock2 libraries were then prepared using the Smart-seq2 protocol (15). Libraries splenic T cells resemble virtual memory cells (Fig. 1C). The ratio were sequenced on an Illumina NextSeq550.Paired-endreadswere of virtual to true memory is also significantly increased in the aligned to the mm10 reference genome, and expected transcript counts absence of DOCK2 (Fig. 1C). were estimated using the RSEM package. The transcriptomic data are hsd/hsd available at the National Center for Biotechnology Information Gene Dock2 virtual memory cells are functional, and their http://www.jimmunol.org/ Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/query/ presence correlates with protection from intracellular infection acc.cgi?acc=GSE135594). Gene set variation analysis (GSVA) as well as gene set enrichment analysis (GSEA) were used to determine if the naive A key feature of memory cells from Ag-inexperienced mice is the + CD8 T cell gene expression program matched known immunological innate-like propensity for the rapid and cognate Ag-independent gene expression signatures (16, 17, 18). Differentially enriched gene sets production of IFN-g following intracellular bacterial infection in were identified using the linear models for microarray data (Limma) package (19). From each mouse, 50,000 naive or MP CD8 T cells were response to proinflammatory cytokines such as IL-12 and IL-18 or sorted, and the extracted RNA was used to prepare TCR repertoire libraries NKG2D ligands (3, 4, 28, 29). Virtual memory T cells are depen- using a commercially available iRepertoire kit for mouse TCRb and se- dent upon IL-15 for their generation, and also require the continued quenced on an Illumina MiSeq instrument. V, D, and J segment assignment

presence of IL-15 to maintain the levels of effector molecules by guest on October 1, 2021 and clonotype identification were performed using MiXCR (20). Reper- necessary for Ag-independent bystander protection (2). Consistent toire sequencing metrics are included in Supplemental Table III. VDJtools hsd/hsd + was used for postanalysis determination of convergence, diversity, and with these studies, a higher proportion of Dock2 CD8 hierarchical clustering of samples based on TCR Vb usage (21). T cells respond rapidly to in vitro polyclonal stimuli (PMA plus ionomycin or IL-12 plus IL-18) by secreting IFN-g (Fig. 1D, 1E). T cell stimulation As virtual memory cells can robustly traffic to the liver (2), we Ex vivo polyclonal stimulation was performed by incubating T cells in an hypothesized that increased IFN-g production by Dock2-deficient eBioscience Cell Stimulation Cocktail (catalog 00-4975-03) for 4 h. Cy- T cells could be protective against infection with L. monocytogenes, tokines were purchased from PeproTech and used at a concentration of 10 ng/ml for 18 h. For OT-I TCR stimulation, altered peptide ligands were which replicates extensively in the liver. Indeed, expansion of synthesized by AnaSpec and used at the indicated concentrations to virtual memory cells in the absence of DOCK2 correlated with stimulate cells for 4 h. increased resistance to L. monocytogenes infection, as Dock2hsd/hsd Listeria infection mice showed significantly lower bacterial burden 3 d after i.v. in- fection (Fig. 1F). Importantly, there were no differences between 4 Mice were i.v. infected with 2 3 10 CFUs of LM10403S. At the indicated WT and Dock2hsd/hsd liver bacterial CFUs 18 h postinfection, con- times, livers were homogenized with 0.05% Triton X-100 in PBS, followed sistent with published studies showing that the protective effect of by plating of serial dilutions on brain heart infusion agar plates containing + streptomycin. CD8 -derived IFN-g was manifest only 3 d postinfection (3). hsd/hsd Flow cytometry Dock2 MP T cells arise in a hematopoietic-intrinsic manner following thymic egress The following Abs were used for surface staining: CD8 (53-6.7), CD44 (IM7), CD49d (R1-2), CD122 (TM-b1), IFN-g (XMG1.2), CD69 (H1.2F3), CD25, Some studies have identified key roles for radio-resistant stromal Ki67 (16A8), TCR, Vb5 (MR9-4), Va2 (B20.1), CD45.1 (A20), CD45.2 cells in maintaining peripheral T cell homeostasis (30). With this in (104), CD90.1 (OX-7), and CD90.2 (30-H12). Cells were permeabilized mind, we sought to evaluate the contribution of nonhematopoietic for intracellular staining using the Foxp3 permeabilization kit (catalog 00-5523-00; eBioscience). cells in the expansion of virtual memory T cells by transferring bone marrow into irradiated RAG-deficient recipients. We Microscopy and image analysis found that only recipients of Dock2hsd/hsd bone marrow had a OT-I T cells tagged with fluorescently labeled anti-CD8 nanobodies were robust expansion of memory cells, whereas mice that received cocultured for 4 h in the presence of antigenic peptide-loaded syngeneic WT WT bone marrow had a much smaller virtual memory compart- bone marrow–derived dendritic cells generated from B6 bone marrow (22, 23). ment (Fig. 2A). These experiments could not be performed in the The cells were fixed, stained, and imaged using confocal microscopy as previously described (24). Image processing was carried out using ImageJ setting of competitive reconstitution, as Dock2-deficient he- and MetaMorph software. To assess F-actin intensity, the region of interest matopoietic progenitors exhibit a severe defect in bone marrow was defined as corresponding to the zone of actin polymerization along the reconstitution under competition from WT cells because of an The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Dockhsd/hsd mice have an expanded virtual T cell compartment and are resistant to intracellular bacterial infection with L. monocytogenes. (A) The proportion of CD8+ MP cells in the spleens of Dock2hsd/hsd, Dock2Fukui/Fukui, and mice as measured by flow cytometry. (B) Number of naive (CD44loCD1222), MP (CD44hiCD122hi), and total CD8+ T cells in the spleens of WT and Dock2-deficient mice. (C) Percentage of CD8+ virtual memory (VM) (CD44hiCD49dlo) T cells among total CD8+ T cells as well as the ratio of VM to true memory (TM) (CD44hiCD49dhi) CD8+ T cells in the spleens of WT and Dock2-deficient mice. (D and E) Intracellular staining for IFN-g in WT and Dock2-deficient CD8+ T cells stimulated for 4 h with PMA and ionomycin (D) or for 18 h with IL-12 and IL-18 (E). (F) Bacterial burden in the livers of Listeria-infected WT and Dock2-deficient mice at 18 and 72 h postinfection (data from two experiments with groups of three to four mice each). In the above experiments, statistical significance was assessed using unpaired two-tailed Student t test. impaired response to CXCL12 (31). Indeed, mixed bone mar- CD8 memory T cell differentiation were among the top 20 pathways row chimeras, in which WT and Dock2hsd/hsd mice bone marrow that were differentially enriched between the four conditions. were coinjected into the same Rag12/2 recipient, resulted in However, we were surprised to find that the same three gene ∼20:1 hematopoietic reconstitution despite a 1:1 transfer of sets associated with memory CD8+ Tcells,aswellasonegene bone marrow precursors. set associated with day 15 effectors in the LCMV–Armstrong In contrast to innate CD8+ T cells that are generated prior to infection model were also among the top 10 pathways that were thymic egress (32, 33), Dock2hsd/hsd virtual memory cells arise in differentially enriched between WT (C57BL/6J) and Dock2hsd/hsd the periphery, as mature CD8+CD42CD44hi cells are not present naive CD8+ T cells using both GSVA and GSEA (Fig. 3B, 3C). To in the (Fig. 2B). Dock2hsd/hsd naive T cells also show no assess whether a bona fide effector signature was enriched in signs of early effector activation (surface CD69 and CD25 Dock2-deficient naive CD8+ T cells, we performed GSEA using expression) or proliferation (as seen by Ki-67 expression) that custom gene sets that were unique to or shared among the above accompany conventional true memory cell generation following four gene sets (Fig. 3D). We found that only the genes that were thymic egress (34) (Fig. 2C, 2D). common to all four gene sets, or the three memory cells gene + hsd/hsd sets, were enriched in Dock2-deficient naive CD8 T cells. Fur- Dock2 naive T cells directly convert into MP cells thermore, the gene signature that is specific to day 15 effectors To explore the mechanism underlying the enhanced generation of was not enriched in the Dock2-deficient naive CD8 T cells. This Dock2hsd/hsd virtual memory cells, we examined the immu- suggests that CD8+ memory-linked genes are likely to be upreg- nological gene signatures in the whole-transcriptome profiles ulated in the day 15 effector CD8 T cell gene set, perhaps because of Dock2-deficient and WT naive and MP CD8+ T cells using the day 15 effectors in the Kaech and Cui (34) LCMV infec- GSVA (Fig. 3A) (16, 17). Unsurprisingly, genes involved in tion model include memory cell precursors or have initiated the 4 DOCK2 AND VIRTUAL MEMORY CD8+ T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 2. Dockhsd/hsd virtual memory T cells arise in a cell-intrinsic manner following thymic egress. (A) Bone marrow cells from either Dock2hsd/hsd or WT mice were injected into irradiated Rag12/2 mice. The spontaneous generation of memory CD8+ T cells (CD44hi CD122hi) was examined in the recipient mice at 12 wk after cell transfer. (B) CD44 levels on the Thy1.2+ CD8+ CD42 single-positive from WT and Dock2-deficient mice. (C) Levels of activation markers (CD25 and CD69) on splenic CD8+ T cells from WT and Dock2-deficient mice. (D) Percentage of Ki-67+ cells in the CD44lo and CD44hi CD8+ T cell compartments in WT and Dock2-deficient mice. All experiments were performed twice in groups of three to four mice. Statistical significance was assessed using unpaired two-tailed Student t test. by guest on October 1, 2021 upregulation of memory-linked genes. Full details of this analysis that the TCR Vb expression profile of Dock2hsd/hsd virtual memory as well as complete gene lists have been provided in Supplemental cells bore remarkable similarity to naive T cells and is quite Tables I, II. Consistent with the direct conversion of these mutant distinct from WT virtual memory cells (Fig. 3I). naive CD8+ T cells to memory T cells and the bypassing of ef- To determine whether Dock2hsd/hsd naive CD8+ Tcellshave fector T cell clonal expansion, we observed no enrichment of any an increased cell-intrinsic propensity to convert to virtual memory, other gene sets associated with early T cell activation. Genes we investigated how adoptively cotransferred Dock2hsd/hsd and WT associated with activation or exhaustion such as CD137(4-1BB), naive T cells respond to homeostatic signals in vivo. Dock2hsd/hsd PD1, TIM3, and LAG3 were also expressed at extremely low T cells transferred into irradiated lymphopenic mice proliferate at levels in from naive CD8 T cells from WT and Dock2hsd/hsd mice. a strikingly faster rate than cotransferred WT T cells with con- The complete RNA sequencing dataset is available at National comitant CD44 upregulation (Fig. 4A). Importantly, this increased Center for Biotechnology Information Gene Expression Omnibus rate of conversion to memory was also observed when naive (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE135594). Dock2hsd/hsd T cells were transferred into lymphoreplete mice TCR sequencing analysis suggested that the repertoire of the naive without any irradiation (Fig. 4B). CD8+ T cells was comparably diverse in both Dock2-deficient and Surface CD5 levels, a proxy for tonic TCR signaling (35, 36), WT mice (Fig. 3E). However, the Dock2-deficient virtual memory were significantly higher on naive T cells from Dock2hsd/hsd mice CD8+ cells were more diverse than WT virtual memory cells, than WT mice (Fig. 4C). In addition, Dock2hsd/hsd naive T cells suggesting that the conversion to virtual memory cells was a also exhibit lower surface TCR expression (Fig. 4C). These highly polyclonal process in Dock2-deficient mice (Fig. 3E–G). findings suggest that an increase in self-peptide MHC triggering One measure of antigenic selection in a TCR repertoire is con- may be associated with the expansion of MP cells. Interestingly, vergence, the number of unique CDR3 sequences encoding the increased CD5 levels are also seen in Dock2hsd/hsd CD8 single- same amino acid sequence (21). In the context of virtual memory positive thymocytes, suggesting that the Dock2-deficient cells may T cells, convergence can be interpreted as selection by self-antigens receive stronger positively selecting signals. However, this is not in the periphery. Analysis of the Dock2hsd/hsd memory T cell accompanied by dramatic changes in the proportions of thymic repertoire revealed a significantly lower number of unique precursors (Supplemental Fig. 1A). CDR3 nucleotide sequences that encode the same amino acid sequence, compared with WT memory cells (Fig. 3H). This Responsiveness to weak agonists is selectively enhanced in decrease in convergence suggests that the self-antigen affinity Dock2-deficient T cells threshold for entering the virtual memory compartment is lowered As our experiments implicated tonic TCR signaling in driving in the absence of DOCK2, allowing more naive T cells to enter the conversion to virtual memory, we generated Dock2-deficient this compartment. Further supporting this conclusion, we found OT-I transgenic mice (OT-I Dock2hsd/hsd) to interrogate this The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Naive Dock2hsd/hsd T cells exhibit memory-like characteristics. (A) Top 20 differentially enriched immunological gene signatures among naive and MP CD8+ T cells from WT and Dock2hsd/hsd mice. (B) Top 10 differentially enriched immunological gene signatures between WT and Dock2hsd/hsd naive CD8+ T cells. Gene sets linked to memory T cell differentiation are marked with a star in (A) and (B). (A and B) The heatmaps are colored by the GSVA enrichment score. (C) GSEA of genes upregulated in CD8+ naive (CD44loCD1222) Dock2hsd/hsd T cells compared with WT naive CD8+ T cells. Significantly enriched pathways linked to memory T cell differentiation are shown. (D) Overlap among the enriched gene sets linked to memory CD8+ T cell differentiation shown in (A)–(C). Only the starred overlaps are significantly enriched in naive CD8+ (CD44loCD1222) Dock2hsd/hsd T cells compared with WT by GSEA. (E–G) The repertoire diversity of naive and MP CD8+ T cells from Dock2-deficient and WT mice assessed using the Shannon–Wiener diversity index (E) and visualized by scaling the area and color of the clonotypes by their abundance (F) or using a rarefaction plot (G). (H) Repertoire convergence (F) (i.e., number of unique CDR3 nucleotide sequences that encode the same amino acid sequence) in naive (CD44loCD1222) and MP (CD44hiCD122+) T cells from WT and Dock2hsd/hsd mice. Statistical significance was assessed using unpaired two-tailed Student t test. (I)A hierarchically clustered heatmap of the frequency of TCR Vb gene segment usage in naive (CD44loCD1222) and MP (CD44hiCD122+) CD8+ T cells from Dockhsd/hsd and WT mice. 6 DOCK2 AND VIRTUAL MEMORY CD8+ T CELLS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Naive Dock2hsd/hsd T cells undergo spontaneous conversion into virtual memory T cells. (A) Naive T cells from CD45.2+ Thy1.2+ Dock2hsd/hsd and CD45.2+ Thy1.1+ WT mice were cotransferred into irradiated CD45.1+ Thy1.2+ lymphopenic mice and assessed for CFSE dilution and CD44 upregulation after 1 wk. (B) Naive T cells from CD45.2+ Thy1.2+ Dock2hsd/hsd and CD45.2+ Thy1.1+ WT mice were cotransferred into unma- nipulated lymphoreplete CD45.1+ Thy1.2+ mice and assessed for upregulation of memory markers after 3 wk. (C) Surface levels of CD5 and TCR on naive CD8+ T cells from Dock2hsd/hsd and WT mice as assessed by flow cytometry. Statistical significance was assessed using unpaired two-tailed Student t test. phenomenon in the context of a defined TCR repertoire. OT-I SIIQFEKL (Q4), and SIINFEKL (N4) (37). We used CD69 up- Dock2hsd/hsd mice were born at Mendelian ratios and exhibited no regulation (38) and cytoskeletal remodeling (24, 39) to evaluate obvious developmental defects. However, a close examination of TCR-dependent responses. Incubation of splenocytes with strong T cells in the spleens of these mice revealed a striking accumu- and intermediate affinity agonists (N4, T4, and Q4) consistently lation of virtual memory cells, as compared with OT-I mice with activated OT-I T cells as measured by CD69 upregulation, re- normal expression of DOCK2 (Fig. 5A, Supplemental Fig. 1B). gardless of DOCK2 expression (Fig. 5B, 5C). However, weak Thus, the Dock2-dependent expansion of CD8+ T cells does not agonist (G4) stimulation resulted in significantly more CD69+ require a polyclonal repertoire. Dock2-deficient OT-I T cells when compared with cells from We next measured the responses of naive Dock2-deficient OT-I OT-I Dock2+/hsd littermates (Fig. 5C). Dock2-deficient OT-I T cells T cells to altered peptide ligands with various degrees of af- also exhibited greater reactivity to both weak and intermediate finity for the OT-I TCR using the following peptides, listed in affinity agonists when TCR reactivity was measured in terms of increasing order of affinity: SIIGFEKL (G4), SIITFEKL (T4), TCR downregulation (Fig. 5D). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. OT-I Dock2hsd/hsd mice have an expansion of MP T cells and show increased ex vivo responses to TCR stimulation. (A) The proportion of CD8+ CD44hi CD122+ MP cells in the spleens of OT-I TCR-transgenic mice in the WT and Dock2hsd/hsd background. (B–D) T cell activation in response to 4 h of ex vivo stimulation with peptides of various signal TCR affinity. Representative histograms in response to stimulation with 1 mM peptide (B). Percentage of CD69+ cells observed (C) or degree of TCR downregulation (D) in response to a range of peptide concentrations. Statistically significant differences (p , 0.001) assessed using an unpaired t test are marked with asterisks (*). (E and F) Confocal microscopy (original magnification 31000) of F-actin (green) at the immunological synapse in OT-I WT or OT-I Dock2hsd/hsd T cells cocultured with syngeneic WT splenocytes in presence of (C) the high-affinity SIINFEKL (N4) peptide or (D) the low-affinity SIIGFEKL (G4) peptide. OT-I T cells were prestained with a fluorescently labeled anti-CD8 nanobody (red). (G) Quantification of F-actin intensity in OT-I WT and Dock2hsd/hsd CD8+ T cells upon stimulation with high (N4) or low (G4) affinity peptides shown as integrated fluorescence intensity. The Mann–Whitney unpaired t test was used to assess statistical significance (N.S., p . 0.05). The bars charts depict mean 6 SEM.

TCR signaling is associated with actin polymerization and the TCR signaling events (24, 39). The high affinity (N4) peptide enrichment of F-actin at the immunological synapse. Remod- induced comparable levels of polymerized actin at the synapse in eling of F-actin at the immunological synapse is essential for both Dock2-deficient, as well as WT, OT-I T cells (Fig. 5E, 5G). 8 DOCK2 AND VIRTUAL MEMORY CD8+ T CELLS

However, in the Dock2-deficient OT-I T cells, but not WT OT-I present in distinct microclusters in resting T cells, and the disruption T cells, the lowest affinity peptide (G4) induced prominent actin of actin polymerization results in their activation-promoting ag- polymerization (Fig. 5E, 5F). Collectively, these findings suggest gregation (43). DOCK2 is localized to the cell membrane via its that Dock2-deficient OT-I T cells have a significantly reduced interactions with the , PIP3, and phosphatidic acid and threshold of TCR activation in response to low-affinity peptides. promotes RAC1-mediated actin polymerization (6–8). Therefore, we speculate that disruption of cortical actin in Dock2-deficient Discussion mice may contribute to the increased TCR responsiveness to weak We had previously shown that the loss of DOCK2 results in a agonists by promoting the enhanced diffusion of segregated trans- prominent expansion of CD8+ blood MP cells, and had used this membrane such as the TCR and LAT and promote their phenotype to identify a loss-of-function Dock2 genetic variant association with downstream signaling mediators. Further studies (Dock2hsd/hsd) in the commercially available C57BL6/NHsd sub- on the role of Rac, the GTPase activated by DOCK2, in TCR strain of C57BL/6 mice (1). In this study, we show that a similar signaling, and virtual memory differentiation following egress into expansion of CD8+ memory T cells is not specific to this Dock2 the periphery could also be informative as current studies of this mutant allele and is also seen in gene targeted Dock2 knockout GTPase have been, largely focused on thymic development and mice (6). In contrast to conventional Ag-experienced memory homing (44, 45). cells, the majority of Dock2hsd/hsd memory T cells exhibit low In conclusion, we have demonstrated a novel role for DOCK2 in surface expression of CD49d, originate in the periphery, lack restricting the size of the virtual memory T cell compartment most activation makers, and have a highly diverse repertoire without likely by setting the threshold for responses against weak agonists. any dominant clones. It can thus be concluded that the MP cells in DOCK2 deficiency is a rare cause of severe immunodeficiency Dock2hsd/hsd mice represent bona fide virtual memory cells. and early onset infections in humans (46). However, the effect Downloaded from Virtual memory T cell generation and maintenance is dependent of DOCK2 on virtual memory cells in humans is yet to be on TCR triggering by self-peptide MHC and common g-chain determined as the flow cytometric markers for virtual memory cytokine–derived signals. However, there is little known about in humans are not well established (25, 47). Our findings negative regulators of the virtual memory compartment (5). In this suggest that any efforts to dampen immune responses using a study, we have provided evidence that DOCK2 sets a threshold small molecule inhibitor of DOCK2 should be tempered by an for direct entry of naive CD8+ T cells into the virtual memory understanding that this protein has pleiotropic effects on periph- http://www.jimmunol.org/ compartment. We have shown that there is reduced TCR se- eral T cell homeostasis (48). quence convergence and greater naive-like TCR Vb usage in the Dock2hsd/hsd virtual memory TCR repertoire, suggesting that more Disclosures naive TCRs are able to enter this compartment. We found a The authors have no financial conflicts of interest. striking enrichment in gene sets associated with memory differ- hsd/hsd entiation in the genes that are upregulated in Dock2 naive References CD8+ T cells relative to WT naive T cells. Notably, there was no 1. Mahajan, V. S., E. Demissie, H. Mattoo, V. Viswanadham, A. Varki, R. Morris, enrichment of gene sets associated with effector differentiation.

and S. Pillai. 2016. Striking immune phenotypes in gene-targeted mice are by guest on October 1, 2021 We observed an increased propensity of adoptively cotransferred driven by a copy-number variant originating from a commercially available Dock2hsd/hsd naive T cells to convert to an MP in response to C57BL/6 strain. Cell Rep. 15: 1901–1909. 2. White, J. T., E. W. Cross, M. A. Burchill, T. Danhorn, M. D. McCarter, homeostatic signals. We also demonstrated that Dock2 deficiency H. R. Rosen, B. O’Connor, and R. M. Kedl. 2016. Virtual memory T cells de- increases TCR sensitivity to weak agonists, and a higher level of velop and mediate bystander protective immunity in an IL-15-dependent manner. hsd/hsd Nat. Commun. 7: 11291. CD5 observed on Dock2 T cells is consistent with enhanced 3. Berg, R. E., E. Crossley, S. Murray, and J. Forman. 2003. Memory CD8+ T cells responsiveness to self-peptide MHC in vivo. provide innate immune protection against Listeria monocytogenes in the absence A prior study by Sanui et al. (13) showed that, in the absence of cognate antigen. J. Exp. Med. 198: 1583–1593. + 4. Chu, T., A. J. Tyznik, S. Roepke, A. M. Berkley, A. Woodward-Davis, of DOCK2, MHC class II restricted 2B4 TCR-transgenic CD4 L. Pattacini, M. J. Bevan, D. Zehn, and M. Prlic. 2013. Bystander-activated T cells exhibit a reduced response to weak agonist peptides. However, memory CD8 T cells control early pathogen load in an innate-like, NKG2D- the interpretation of this experiment may be complicated by the dependent manner. Cell Rep. 3: 701–708. 5. Fiege, J. K., B. J. Burbach, and Y. Shimizu. 2015. Negative regulation of observation that DOCK2 may negatively influence lymphocyte memory phenotype CD8 T cell conversion by adhesion and degranulation- proliferation independent of AgR signaling (40). Besides DOCK2, promoting adapter protein. J. Immunol. 195: 3119–3128. 6. Fukui, Y., O. Hashimoto, T. Sanui, T. Oono, H. Koga, M. Abe, A. Inayoshi, FYB1 has been shown to negatively regulate the size of the virtual M. Noda, M. Oike, T. Shirai, and T. Sasazuki. 2001. Haematopoietic cell-specific memory compartment (5). However, the relationship between CDM family protein DOCK2 is essential for lymphocyte migration. Nature 412: DOCK2 and FYB1 has not yet been studied, and it is formally 826–831. 7. Nishikimi, A., H. Fukuhara, W. Su, T. Hongu, S. Takasuga, H. Mihara, Q. Cao, possible that DOCK2 and FYB1 function in the same or parallel F. Sanematsu, M. Kanai, H. Hasegawa, et al. 2009. Sequential regulation of pathways. DOCK2 dynamics by two phospholipids during chemotaxis. Science It is currently unclear how the loss of DOCK2 sets the threshold 324: 384–387. 8. Nishikimi, A., M. Kukimoto-Niino, S. Yokoyama, and Y. Fukui. 2013. Immune for weak agonist TCR stimulation. One possibility is that the regulatory functions of DOCK family proteins in health and disease. Exp. Cell decreased interstitial motility (11) observed by Dock22/2 Res. 319: 2343–2349. 9. Terasawa, M., T. Uruno, S. Mori, M. Kukimoto-Niino, A. Nishikimi, T cells results in increased TCR–MHC contact duration. It is F. Sanematsu, Y. Tanaka, S. Yokoyama, and Y. Fukui. 2012. Dimerization of possible that increases in APC residence time results in increased DOCK2 is essential for DOCK2-mediated Rac activation and lymphocyte amounts of TCR triggering and concomitant conversion into memory migration. PLoS One 7: e46277. 10. Gotoh, K., Y. Tanaka, A. Nishikimi, A. Inayoshi, M. Enjoji, R. Takayanagi, cells. Another possibility is that the loss of DOCK2 disrupts the T. Sasazuki, and Y. Fukui. 2008. Differential requirement for DOCK2 in mi- cortical network lowering the threshold for activation by weak gration of plasmacytoid dendritic cells versus myeloid dendritic cells. Blood 111: ligands. Cortical actin forms a dense 100-nm-thick layer lining the 2973–2976. 11. Nombela-Arrieta, C., T. R. Mempel, S. F. Soriano, I. Mazo, M. P. Wymann, plasma membrane and can act as a barrier to TCR signaling by E. Hirsch, C. Martı´nez-A, Y. Fukui, U. H. von Andrian, and J. V. Stein. 2007. A restricting the access of intracellular domains of LAT and CD3 to central role for DOCK2 during interstitial lymphocyte motility and sphingosine- 1-phosphate-mediated egress. J. Exp. Med. 204: 497–510. cytosolic signaling mediators such as PLC-g1 in the absence of 12. Gotoh, K., Y. Tanaka, A. Nishikimi, R. Nakamura, H. Yamada, N. Maeda, CD28-dependent costimulation (41, 42). The TCR and LAT are T. Ishikawa, K. Hoshino, T. Uruno, Q. Cao, et al. 2010. Selective control of type The Journal of Immunology 9

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Supplementary Figure 1 A) Flow cytometric analysis of the CD5 expression levels (left) on thymic T cell subsets and their respective proportions (right) in the thymi of WT and Dock2hsd/hsd mice. B) The proportion of CD8+ memory phenotype cells (MP) in the spleens of OT-I TCR transgenic mice in the Dock2hsd/hsd and WT backgrounds, as well as Dock2hsd/hsd and WT mice lacking the OT-I TCR transgene. The proportion of true memory (TM) and virtual memory (VM) as determined by flow cytometric analysis of CD49d staining is shown on the right.

Supplementary Table 1: Gene set enrichment analysis of MSigDB immunological gene signatures comparing Dock2-deficient and wild-type naïve CD8+ T cells. Significantly enriched gene sets in Dock2-deficient CD8+ T cells (FDR < 0.25) are shown. Memory-linked gene sets are marked with an asterisk.

Supplementary Table 2: Genes shared among or unique to the memory-linked gene immunological gene signatures in Dock2-deficient naïve CD8+ T cells. All possible intersections are shown below. Number Gene set intersection Genes of genes EMP1 KRTCAP2 CHPT1 CD44 ANXA2 ATF6 FASLG FGL2 CRTAM DOCK5 MDFIC CCL5 ANXA1 GZMM DENND5A KLRK1 PHF13 AQP9 CCR2 MCART6 ITGB1 MYO1F NRP1 UNC119B CLDND1 PGLYRP1 GOLDRATH_NAIVE_VS_MEMORY_CD8_TCELL_DN FCGR2B S100A4 CASP1 CCR5 ST3GAL6 CD160 CXCR3 CAPN2 GSE9650_NAIVE_VS_MEMORY_CD8_TCELL_DN 71 LGALS1 CCL4 KLRG1 DAPK2 PRF1 ELL2 IFNG TXNDC5 ERRFI1 KAECH_NAIVE_VS_DAY15_EFF_CD8_TCELL_DN PLSCR1 CASP4 S100A6 EOMES IL18R1 RECK F2R GZMB ACOT7 KAECH_NAIVE_VS_MEMORY_CD8_TCELL_DN GABARAPL2 PTPN13 EEA1 KCNJ8 CCND3 BHLHE40 ID2 SNX10 IL18RAP S100A13 GZMK GLRX AHNAK BCL2A1 ITGAX MAPRE2 S100A10 IFITM10 KLRC1 GOLDRATH_NAIVE_VS_MEMORY_CD8_TCELL_DN KAECH_NAIVE_VS_DAY15_EFF_CD8_TCELL_DN 3 GGH FCGRT STARD10 KAECH_NAIVE_VS_MEMORY_CD8_TCELL_DN XDH IL10RA POLR2L PRR13 FGR SH2D1A RORA RNF19B HLA-A GSE9650_NAIVE_VS_MEMORY_CD8_TCELL_DN ETFB EFHD2 TRAPPC1 GSG2 CTLA4 TXN KLF10 LYPLA2 CTSD KAECH_NAIVE_VS_DAY15_EFF_CD8_TCELL_DN 34 NUCB1 COX17 MAP7D1 MBD2 TSPAN31 CSDA GBP4 SNTB2 S100A11 KAECH_NAIVE_VS_MEMORY_CD8_TCELL_DN HMGB2 TTC7B STMN1 H1F0 MX2 HOPX ITGA4 GOLDRATH_NAIVE_VS_MEMORY_CD8_TCELL_DN PGAM1 GATA3 GSTO1 PRSS12 N4BP1 RPGR VKORC1 HCFC1R1 GSE9650_NAIVE_VS_MEMORY_CD8_TCELL_DN 17 CST7 CTSW TNFRSF1B RNF138 POU6F1 TMEM37 ST3GAL4 ODC1 KAECH_NAIVE_VS_MEMORY_CD8_TCELL_DN PPP3CC GOLDRATH_NAIVE_VS_MEMORY_CD8_TCELL_DN GSE9650_NAIVE_VS_MEMORY_CD8_TCELL_DN 4 NBEAL2 ENPP1 SLC35E4 LPIN1 KAECH_NAIVE_VS_DAY15_EFF_CD8_TCELL_DN KAECH_NAIVE_VS_DAY15_EFF_CD8_TCELL_DN 5 PON1 MTMR7 RACGAP1 RPAP1 TMED10 KAECH_NAIVE_VS_MEMORY_CD8_TCELL_DN GOLDRATH_NAIVE_VS_MEMORY_CD8_TCELL_DN 4 CTNNA1 BAG3 CISH ARL6 KAECH_NAIVE_VS_MEMORY_CD8_TCELL_DN HS1BP3 MOGS PFKP DPM3 JUND RBMX FGF13 SORL1 SHC1 RDBP PCNA GDNF CD7 DPP7 CDC34 ATN1 SEPT1 LRRC8C XRCC5 UNC119 GSE9650_NAIVE_VS_MEMORY_CD8_TCELL_DN CAPNS1 ST8SIA2 FOSB FAM46C PACS1 IGF2R FRMD5 CDK4 FOS 54 KAECH_NAIVE_VS_MEMORY_CD8_TCELL_DN TNFSF10 BCL2 SLCO3A1 ADAM19 CRIP2 TBL2 CYFIP2 YES1 TNFAIP3 LYSMD2 ZMAT3 TOB1 DYM WEE1 CYB5R3 GADD45B UBC FGF19 PIM1 PRDX2 KLF6 SH2D2A KLF4 ITGB7 IQGAP2 GOLDRATH_NAIVE_VS_MEMORY_CD8_TCELL_DN SOCS2 VMP1 GZMA SERPINB9 DSTN LGALS3 BCL2L2 SMYD1 LITAF 11 KAECH_NAIVE_VS_DAY15_EFF_CD8_TCELL_DN PRDM1 MYADM GSE9650_NAIVE_VS_MEMORY_CD8_TCELL_DN 1 PTTG1 KAECH_NAIVE_VS_DAY15_EFF_CD8_TCELL_DN GOLDRATH_NAIVE_VS_MEMORY_CD8_TCELL_DN 2 AIM1 GPC1 GSE9650_NAIVE_VS_MEMORY_CD8_TCELL_DN IL7R RAD51D CECR5 SH3YL1 SF3A1 CD97 BCHE TOM1 ITGB2 KAECH_NAIVE_VS_MEMORY_CD8_TCELL_DN* 12 KIAA0101 SYT9 OPRK1

ACTB CARHSP1 MED12L IL13RA2 GALNT3 ENTPD1 CHL1 MTM1 C7orf73 SLA ODZ3 CD4 LXN NR4A2 ARF6 SSPN GJA1 CR1L INSM1 LAPTM5 C16orf61 CD47 DNMT1 RAB33B RALY SORBS1 GPM6B NT5E CCNB2 FAM89B DNAJC1 ALG2 FANCM ZNF821 RPA2 LPIN2 TERF1 KAECH_NAIVE_VS_DAY15_EFF_CD8_TCELL_DN* 71 MAN2A1 IDE RRBP1 SRGN CASP7 ALCAM C17orf79 UBE2K SEC61G C9orf16 HK2 ALAD HIST1H1C ADORA2A EMP3 ZNF398 ANXA4 GMFG H2AFX CYP3A43 PRDX1 LRP10 PRDX4 C10orf58 KCTD9 LAMC1 KLRD1 ITGAL GSTM3 RAB5C RHOQ PERP RSU1 CASP3

CHCHD7 EI24 GPHN KIAA1737 TK1 RASA4 TRAF3IP2 IER3 MAP3K8 ABHD5 MS4A1 GOLM1 GK DENND4C GCAT SEMA4F PTPN22 CYB5R4 ACY1 FHIT TMEM55A ART3 CAPG CD22 ANKH POLR1B SEMA4A SAMHD1 EYA4 DBNDD2 PLAT SSX2IP PLP2 STX7 IRF8 OSTF1 EVI2A TMEM159 LPGAT1 CYFIP1 DNAJC5 TMEM141 RAB3D IL15 ITM2C GOLDRATH_NAIVE_VS_MEMORY_CD8_TCELL_DN* 88 PLCD1 MAPK12 MGST3 KCTD12 TSPAN4 SERPINB6 MYL1 IL15RA GCLM XIST KIAA1274 PLEKHB2 HSD17B11 LIMD1 SOS2 PLEKHA5 CPNE3 ECI1 PLBD1 IL10RB CCDC130 MED10 CYBB AIF1 TKTL1 TRAF1 PRKCA MCOLN2 ANTXR2 TUSC2 RNASE4 PBX3 KLHL7 GOLIM4 ASAH1 PQLC3 ABCB1 NOTCH4 SOAT2 CYP4V2 NCKAP1 SKAP2 HIP1R TNNI1 PAM FAM207A MNS1 TULP4 KITLG RPS6KA4 PHYH FRAT1 GSE9650_NAIVE_VS_MEMORY_CD8_TCELL_DN* 17 C9orf3 EPN1 MID2 AARS GNPTG CNR1 NFKBIB ZFP36L2 * genes unique to the particular gene set

Supplementary Table 3: TCR repertoire sequencing metrics

Cells (CD8 Total Successfully Successfully Total reads Final CDR3 Replicate Genotype naïve vs sequencing aligned aligned, used in CDR3 clonotype memory) reads reads percent clonotypes count 1 WT CD44lo 3439 3153 91.68% 3068 2109 2 WT CD44lo 3638 3009 82.71% 2913 2344 3 WT CD44lo 2881 2693 93.47% 2618 1900 4 WT CD44lo 2745 2590 94.35% 2507 1662 1 Harlan CD44lo 3851 3761 97.66% 3630 2698 2 Harlan CD44lo 4646 4486 96.56% 4345 3111 3 Harlan CD44lo 2565 2503 97.58% 2411 1853 1 WT CD44hi 17152 16576 96.64% 16438 2729 2 WT CD44hi 19629 17809 90.73% 17660 3238 3 WT CD44hi 13017 12765 98.06% 12613 2510 4 WT CD44hi 18465 17983 97.39% 17839 2609 1 Harlan CD44hi 5964 5322 89.24% 5209 2758 2 Harlan CD44hi 7673 6848 89.25% 6706 2966 3 Harlan CD44hi 3470 3211 92.54% 3170 1525