Aiolos restricts the generation of antigen-inexperienced, virtual memory CD8^+ T cells in mice

Domain: biology
Published: 2025-12-14
Editorial outcome: major_revision

Abstract
CD8+ virtual memory T (T ) cells rapidly respond to infection via antigen-independent bystander effector functions. While it is recognized that T cells arise independently of foreign antigen encounter, the mechanisms governing their development are not fully understood. Here, we identify the Ikaros transcription factor Aiolos as a negative regulator of T cell programming. We observe enhanced frequencies and numbers of T in the spleen, liver, and blood of unchallenged Aiolos-deficient (Ikzf3-/-) mice and in the lungs 1-day post-infection with influenza A virus (IAV). Furthermore, Ikzf3-/- T cells produce elevated IFN-γ and granzyme B in response to cytokine stimulation. Importantly, Aiolos-deficient mice control IAV more rapidly and exhibit reduced morbidity, indicating enhanced T cell functionality. Mechanistically, Aiolos represses the expression of the transcription factor Eomes and the IL-15R subunit CD122, known positive regulators of T gene program. Collectively, these findings establish Aiolos as a molecular repressor of T programming and responses.

Introduction
Host infection results in the generation of diverse, antigen-specific CD8+ memory T cell populations that persist long-term to safeguard against re-infection1–4. These include central memory T (T ), effector memory T (T ), and tissue-resident memory T (T ) cell subsets, among others, which are distinguished by their functional properties, including the ability to recirculate or reside in peripheral tissues1,5,6. In contrast to these antigen-specific populations, a more recently described subset, termed virtual memory T (T ) cells, arises independently of foreign antigen encounter7–9. As T and T cells both express L-selectin (CD62L) and the activation marker CD44, T were previously categorized as a part of the T repertoire. However, recent findings have established that low surface expression of the integrin receptor CD49d, an indicator of foreign antigen-encounter, distinguishes T (CD62L+CD44+CD49dLo) from these other T cell populations10,11. As T cell generation is not dependent upon foreign antigen exposure, they are found in significant numbers in uninfected and unchallenged hosts8. During infection, T cells rapidly infiltrate affected sites and provide bystander protection independent of TCR engagement7,10,12,13. As such, these cells comprise a critical component of the first line of defense against various pathogenic infections14–16.

T effector responses are driven by signals from cytokines such as IL-12, IL-18, and IL-15 and include the production of additional inflammatory cytokines (e.g., IFN-γ) and NKG2D-mediated bystander cytotoxicity7,10,13. T cells can also differentiate into antigen-specific effector and memory T cell subsets, as their polyclonal repertoire allows for clonal expansion7,11,14,17. In addition to infection, T have also been shown to infiltrate tumors and target cancer cells independent of MHC-I18,19. Despite having a high avidity for self-antigen, which could be a concern for autoimmunity, these cells demonstrate high self-tolerance and promote homeostasis20,21. Together, their rapid, antigen-independent responses and ability to differentiate in an antigen-specific manner make T cells a uniquely versatile T cell population and notable therapeutic target for the treatment of a range of diseases.

T development and function are dependent upon signals from the cytokine IL-15, as IL-15-deficient mice (IL-15-/-) lack T cells7,12. The response to IL-15 in T cells is mediated by signals received through the shared receptor subunit CD122 (IL-15Rβ/IL-2Rβ)7. Expression of CD122 is positively regulated by the T-box transcription factor Eomesodermin (Eomes), which is highly expressed in T cells and is essential for their differentiation and survival7,19. While Eomes promotes CD122 expression, IL-15 signaling in turn induces Eomes expression in CD8+ T cells12,22. However, the identity of the upstream regulators of this critical regulatory loop, as well as the comprehensive mechanisms underlying T effector functions, remain unknown.

Here, we identify Aiolos as a repressor of T cell differentiation and function. We find that Aiolos-deficient (Ikzf3-/-) mice have significantly elevated frequencies and numbers of T cells both in secondary lymphoid organs at steady-state, and in the lungs 24h following IAV infection. We further find that Aiolos-deficient T have elevated expression of Eomes and enhanced effector functions in response to cytokine stimulation. Importantly, this results in enhanced viral control and reduced morbidity of IAV-infected Aiolos-deficient mice relative to wild-type (WT). Collectively, these findings highlight Aiolos as a negative regulator of T cell formation and functional responses, suggesting that Aiolos could have therapeutic potential in enhancing antiviral immunity.

Results
Aiolos suppresses Eomes and CD122 expression in CD8+ T cells
Eomes is a key regulator of CD8+ T cell differentiation and effector programs and is essential for T formation23–25. We previously established that loss of Aiolos in effector CD4+ T cells during IAV infection (A/PR/8/34 (H1N1), termed “PR8”) results in augmented expression of the transcription factor Eomes and induction of a cytotoxic gene program26. As such, we sought to determine whether the relationship between Aiolos and Eomes may be a conserved feature in CD8+ T cells and whether this may impact the generation of T .

Initially, we examined Eomes protein expression in CD8+ T cells isolated from the spleens of unchallenged, specific-pathogen free (SPF) WT and Aiolos-deficient mice. Aiolos-deficient mice had elevated frequencies and numbers of Eomes+ T cells, and an overall increase in the abundance of Eomes protein expression compared to WT controls (Fig. 1a–c). Further analysis of CD8+ T cells revealed increased percentages of Eomes+ naive (CD62L+CD44-) and activated (CD44+) T cells in Aiolos-deficient mice compared to WT (Supplementary Fig. 1a–d). Aiolos deficiency similarly impacted the expression of the IL-15R subunit CD122, an Eomes target gene encoded by Il2rb (Fig. 1d, e)25. To further define the role of Aiolos in regulating Eomes and CD122 expression, we overexpressed Aiolos in Ikzf3-/- CD8+ T cells using retroviral transduction (Supplementary Fig. 2a, b). Overexpression of Aiolos resulted in a significant decrease in the expression of both Eomes and CD122 (Supplementary Fig. 2c, d). Collectively, these in vivo and in vitro data demonstrate that Aiolos represses Eomes and CD122 expression in primary CD8+ T cells.

To determine the mechanism by which Aiolos suppresses Eomes and CD122, we analyzed publicly available Aiolos ChIP-Seq data from murine thymocytes [GSM510606527]. Analysis of Aiolos enrichment across the Eomes and Il2rb genes using Integrative Genomics Viewer (IGV) revealed peaks proximal to the Eomes and Il2rb promoter regions, suggesting that Aiolos may directly repress their expression (Fig. 1f, Supplementary Fig. 2e). To assess potential impacts of Aiolos, we generated plasmids to overexpress Aiolos or an Aiolos DNA-binding mutant (AiolosDBM), along with gene reporter constructs containing the identified Eomes or Il2rb regulatory regions. Aiolos overexpression resulted in suppression of both Eomes and Il2rb gene-reporter activity (Fig. 1g, Supplementary Fig. 2f). However, the repressive effect was lost with the AiolosDBM mutant, indicating that direct DNA binding is necessary for the repressive effect of Aiolos (Fig. 1g, Supplementary Fig. 2f). A known driver of Eomes expression in CD8+ T cells is the transcription factor STAT528. Interestingly, sites of STAT5b enrichment [GSM788751229] at the Eomes promoter region overlapped with the Aiolos enrichment sites (Fig. 1f). This is in line with previously published studies indicating that IkZF and STAT factor DNA-binding motifs overlap26,30,31. To determine whether Aiolos may repress Eomes expression by interfering with STAT5 mediated induction of Eomes, we overexpressed a constitutively active form of STAT5b (STAT5bCA) along with Aiolos in EL4 T cells. As expected, STAT5b overexpression alone significantly enhanced Eomes promoter activity. However, co-expression with Aiolos abrogated the STAT5-induced response (Fig. 1h). Together, these findings support Aiolos as a transcriptional repressor of Eomes in CD8+ T cells through direct interaction or via competition with STAT5 for promoter occupancy. Further, our data also support Aiolos-dependent suppression of CD122 in these cells, which could result in their decreased responsiveness to IL-15. Given the interplay between IL-15 signaling and Eomes, these findings are suggestive of a mechanism whereby Aiolos represses the IL-15/STAT5/Eomes regulatory axis and potentially T differentiation.

Aiolos expression inversely correlates with that of Eomes and CD122 in T cells
We next assessed the expression of Aiolos, Eomes, and CD122 in splenic CD8+ T naive, effector (T , CD62L-CD44+), and T cells. To distinguish between circulating (CD45.2(i.v.Pos)) and non-circulating resident cells, we retro-orbitally injected mice with fluorophore-labeled anti-CD45.2 antibody prior to euthanasia (Supplementary Fig. 3a). Analysis of the resident (CD45.2(i.v.Neg)) CD8+ T cell populations revealed increased expression of Eomes and CD122 in T cells relative to T cells, while Aiolos expression exhibited the opposite trend (Supplementary Fig. 3b, c). This inverse correlation between Aiolos expression and that of both Eomes and CD122 supports a role for Aiolos in negatively regulating T formation.

Aiolos deficiency results in an expanded T population
We next sought to determine the impact of Aiolos deficiency on T cell generation. We began by assessing T cell populations in the spleens of unchallenged, SPF WT, and Aiolos-deficient mice7,9. We observed no differences in CD3+ T cells, a slight reduction in CD4+ and CD8+ T cells, and a slight increase in double negative (DN) T cells in the spleen of Aiolos-deficient mice compared to WT, consistent with findings in human patients and mouse models harboring Aiolos variants and genetic mutations (Supplementary Fig. 4a, b)27,32–37. Further characterization of both CD4 and CD8 T cells revealed decreased naive cells with an increased effector population in the absence of Aiolos (Supplementary Fig. 4c, d), again in line with observations in human patients with Aiolos variants27,32–37. However, in the CD8+ T cell population, we observed a difference in the CD62L+CD44+ subset in which there was a substantial increase in Aiolos-deficient mice (Supplementary Fig. 4c, d). As it has been reported that T cells make up the majority of CD62L+CD44+ population in the uninfected host, we next used CD49d expression to distinguish T from T cells10,14,38. Consistent with elevated Eomes and CD122 expression (Fig. 1a–e), we observed a significant increase in both the frequency and numbers of T cells in the spleens of Aiolos-deficient mice compared to WT (Fig. 2a, b). We next examined T populations in the blood, liver, and mediastinal lymph nodes and found similar increases in both frequency and numbers of T in Aiolos-deficient mice compared to WT (Fig. 2c, d). In contrast to the aforementioned tissues, few T were found in the lungs of either WT or Aiolos-deficient mice (Fig. 2d). We also noted that Aiolos-deficient T from these tissues exhibited elevated expression of both Eomes and CD122 relative to WT (Fig. 2e, f, Supplementary Fig. 5a, b).

T cells form in the periphery; however, we considered the possibility that Aiolos may impact their cellular precursors in the thymus7,8,12,38. As such, we next examined thymic CD8+ T cells in WT and Aiolos-deficient mice. Consistent with published results, we observed no difference in the frequency of CD8+ T cells between WT and Aiolos-deficient samples (Supplementary Fig. 5c)36. Notably, frequencies of CD8+ Eomes+ and CD8+ CD122+ were relatively low in the thymus of both WT and Aiolos-deficient mice. There was no difference in the frequency and number of CD8+ Eomes+ T cells, and only a slight increase in frequency, but not numbers, of CD8+ CD122+ cells in the absence of Aiolos (Supplementary Fig. 5d, e). Taken together, these findings support a role for Aiolos in repressing T cell programming, at least in part, through modulation of Eomes and IL-15 responsiveness in the periphery.

Aiolos deficiency promotes T gene programming
To assess potential broader impacts of Aiolos on T programming, we performed RNA-sequencing analysis of T cells isolated from the spleens of unchallenged WT and Aiolos-deficient mice, in comparison to WT naive CD8+ T cells as a control. Principal Component Analysis (PCA) of transcripts for T cells and WT naive CD8+ T cells revealed distinct clusters between the naive and T samples, as has been previously reported7. Importantly, PCA analysis of the T transcripts revealed that Aiolos-deficient T samples formed a cluster distinct from WT T (Supplementary Fig. 6a). Hierarchical clustering of the top 200 differentially expressed genes (DEGs) identified unique gene expression patterns between WT and Aiolos-deficient T cells (Supplementary Fig. 6b). In Aiolos-deficient T cells, genes involved in T transcriptional regulation (Eomes, Tbx21) were upregulated along with cytokine receptors known to promote T homeostasis and responsiveness to IL-15 (Il15ra, Il2rb) (Fig. 3a, b)7,12. In addition, genes involved in T cell migration into infected tissues (Ccr2, Cxcr3, S1pr5) were upregulated in the absence of Aiolos, whereas genes encoding residency markers (Itgae, Cd69) were downregulated in Aiolos-deficient T samples (Fig. 3a, b)7,14. We subsequently validated the protein expression for T-bet (Tbx21) and the chemokine receptor CXCR3 (Cxcr3) in naive CD8+ T and T cells14,25. As expected, we observed increased expression of these factors in T cells compared to the naive counterparts. Importantly, their expression was significantly higher in Aiolos-deficient T cells when compared to WT T , corroborating our findings from the RNA-seq analysis (Fig. 3c, d).

In addition to increased tissue infiltration, CCR2-expressing T cells are reported to exhibit enhanced effector responses compared to the CCR2-deficient T cells14. Indeed, analysis of the transcript data revealed that Aiolos-deficient T exhibited increased expression of natural killer cell-associated (Klrk1, Klrc1, Klra4, and Klra8), cytolytic (Gzma, Gzmb, Prf1, Fasl), and cytokine (Ifng) genes (Fig. 3a, b)9,39,40. Further, GSEA analysis of gene ontology and immunologic signature subsets suggested that genes involved in IL-15 signaling, innate immune responses, and effector and memory responses were upregulated in Aiolos-deficient T cells (Fig. 3e, f, Supplementary Fig. 6c–e). Collectively, these data suggest that T may have enhanced effector responses in the absence of Aiolos.

Aiolos alters chromatin accessibility at regulatory regions for T genes
To determine whether Aiolos impacts the T gene program through alterations in chromatin accessibility, we next performed Assay for Transposase-Accessible Chromatin (ATAC)-seq analysis on T cells isolated from the spleens of WT and Aiolos-deficient mice. Similar to RNA-Seq data, PCA analysis revealed distinct genotype clusters with broad changes in the chromatin accessibility between WT and Aiolos-deficient T samples, with the majority of gene regulatory regions displaying increased accessibility (Supplementary Fig. 7a, b). Consistent with the RNA-seq data, there was increased accessibility at the regulatory regions of key T transcription factors (Eomes, Tbx21), cytokine receptors (Il2rb, Il12rb2, Il18r1), chemokine receptors (Ccr2, Ccr5, Cxcr3), and effector molecules (Ifng, Gzmb, Prf1, Klrk1) in the absence of Aiolos (Fig. 4a–f, Supplementary Fig. 7c–f). In contrast, chromatin accessibility was reduced at the regulatory regions of other genes involved in cytokine responses (Ifngr1, Ifngr2, Tgfbr2), tissue-residency (Itgae, Cd69), and migration (Ccr9, Cxcr5) (Fig. 4a, b, Supplementary Fig. 7g, h). These findings suggest that Aiolos governs T gene programming directly and/or indirectly through alterations in chromatin accessibility at key regulatory and effector genes.

Aiolos deficiency enhances the functional capacity of T cells
One of the functional characteristics of T cells is to provide rapid bystander protection through the production of molecules like IFN-γ, granzyme B, and NKG2D in response to cytokine stimulation (IL-12, IL-18, and IL-15)7,10. We hypothesized that the absence of Aiolos may augment responsiveness to these cytokines via alterations in cytokine receptor subunit expression. Indeed, genes encoding IL-15R subunits (Il15ra, Il2rb), IL-12R subunits (Il12rb1, Il12rb2), and IL-18R subunits (Il18r1 and Il18rap) were upregulated in T cells in the absence of Aiolos, suggesting that Aiolos deficiency may selectively augment T responsiveness to these cytokines (Figs. 3b and 5a). GSEA analysis of the immunologic signatures also revealed that genes related to IL-15/STAT5 and IL-12/STAT4 signaling were upregulated in Aiolos-deficient T cells (Fig. 3e, Supplementary Fig. 6e). To determine whether increased expression of cytokine receptor subunits resulted in a functional difference in cytokine signaling, we assessed the tyrosine phosphorylation status of STAT5 (pY-STAT5) and STAT4 (pY-STAT4) in T cells exposed to a cytokine cocktail (IL-12, IL-18, and IL-15C) ex vivo (Fig. 5b). Indeed, we observed increases in both pY-STAT5 and pY-STAT4 in the Aiolos-deficient T cells compared to WT, suggestive of an enhanced cytokine responsiveness in the absence of Aiolos (Fig. 5c, d, Supplementary Fig. 8a, b). To assess whether increased cytokine signaling had a functional consequence, we examined production of IFN-γ and GzmB and found that Aiolos-deficient T cells produced significantly elevated levels of IFN-γ and GzmB compared to WT (Fig. 5e, f, Supplementary Fig. 8c, d). We similarly examined the expression of NKG2D (encoded by Klrk1), which allows for the innate-like bystander cytotoxic functions of T cells38,41. Again, we observed increased frequency and expression of NKG2D in Aiolos-deficient T cells compared to WT in response to cytokine treatment (Fig. 5g, Supplementary Fig. 8e). We next performed a cytotoxicity assay and found increased killing of target YAC-1 cells by Aiolos-deficient T cells in the presence of stimulating cytokines compared to WT (Fig. 5h, Supplementary Fig. 8f)7,11,42. Notably, blocking NKG2D resulted in a significant reduction in target cell killing, specifically by Aiolos-deficient cells (Fig. 5h). Collectively, these data demonstrate that T have enhanced effector responses in the absence of Aiolos.

Aiolos deficiency results in increased control of IAV infection
To determine the impact of Aiolos on T responses in vivo, we next infected WT and Ikzf3-/- mice with IAV (Supplementary Fig. 9a). Through the course of infection, animal body weights were measured daily as an assessment of infection progression and disease severity. Consistent with past observations, Aiolos-deficient mice exhibited significantly less weight loss compared to WT mice, with differences observed as early as 5 d.p.i. (Supplementary Fig. 9b)26. To determine whether the reduced weight loss was an outcome of reduced viral burden in the absence of Aiolos, we assessed viral load in the lung homogenates at 3 d.p.i. Indeed, both viral RNA and infectious virus titers were significantly reduced in Aiolos-deficient mice relative to WT, suggesting a level of increased antiviral response in the absence of Aiolos (Supplementary Fig. 9c). As Aiolos expression is largely restricted to cells of the lymphoid lineage, these early differences suggest an impact of Aiolos on an early-responding lymphocyte subset, perhaps T 43.

Aiolos deficiency enhances the response of CD8+ T cells to IAV infection
Qian and colleagues demonstrated that T cells can be found in IAV-infected lungs as early as 1-day post-infection (1 d.p.i.)14. To determine whether the absence of Aiolos would augment this response, we infected WT and Aiolos-deficient mice with IAV and assessed T populations at 1 d.p.i. Similar to our observations in uninfected mice, the frequency and number of T cells were increased in the spleen of Aiolos-deficient mice at 1 d.p.i. compared to WT (Fig. 6a, b). Strikingly, there was also a substantial increase in the number of resident CD8+ T cells in the lungs of Aiolos-deficient mice compared to WT (Fig. 6c–e). Consistent with our previous findings, Aiolos-deficient T cells from infected spleens and lungs exhibited elevated expression of both Eomes and CD122 at 1 d.p.i. compared to WT (Supplementary Fig. 10a, b).

Comparative analysis of T cells in the spleens and lungs of WT and Ikzf3-/- mice revealed an average 2-fold increase in the number of T cells of Aiolos-deficient mice compared to WT. However, there was an approximate 12-fold average increase in the number of T cells in the lungs of these mice compared to WT, suggesting baseline differences in T numbers in Aiolos-deficient animals is not sufficient to account for this increase in the lungs (Fig. 6f). To determine whether the increased lung T population had trafficked from outside the lungs or rather had expanded from a lung-resident population, we treated WT and Ikzf3-/- mice with Fingolimod (FTY720) or saline control via the intraperitoneal route daily starting 1 day prior to IAV infection (Fig. 6g). FTY720 suppresses lymphocyte migration and has been widely used to study CD8+ T cell egress to infected tissues44,45. Following FTY720 administration, we infected mice with IAV and examined the presence of resident CD8+ T cells in the lungs at 3 d.p.i. As before, infected animals treated with saline control displayed a significant increase in resident CD8+ T cells in the lungs of Aiolos-deficient mice relative to WT. In contrast, FTY720-treated animals had diminished resident CD8+ T cell populations in the lungs of both WT and Aiolos-deficient mice, indicating that T cells migrated to the lungs in response to infection and that this migration was enhanced in the absence of Aiolos (Fig. 6h).

Early lung-infiltrating Aiolos-deficient T correlate with enhanced control of IAV
To determine whether Aiolos-deficient T harbored an enhanced capacity to combat infection, we initially infected WT and Ikzf3-/- mice with IAV, followed by FTY720 administration from 1 to 5 d.p.i to limit our analyses to the impact of early-responding T (Fig. 7a). Consistent with our previous findings (Supplementary Fig. 9b), Aiolos-deficient animals lost significantly less weight than their WT counterparts, with differences observed as early as 3 d.p.i (Fig. 7b). In fact, Aiolos-deficient mice lost very little weight throughout the 6-day time course. Analysis of the lungs at 3 and 6 d.p.i revealed increased resident CD8+ T cells in the lungs of Aiolos-deficient mice compared to WT, and an assessment of viral RNA at day 3 revealed significantly reduced levels in the lungs of FTY720-treated Aiolos-deficient mice compared to WT (Fig. 7c, d). Notably, there was no difference in viral RNA levels between saline control and FTY720-treated mice at day 3, despite reduced numbers of resident CD8+ T cells in FTY720-treated mice compared to controls (Supplementary Fig. 11a, b). By 6 d.p.i., viral RNA levels were comparable between WT and Aiolos-deficient mice (Fig. 7d). However, there was no difference between the viral RNA present in Aiolos-deficient mice at 3 and 6 d.p.i., again suggesting more rapid control of the virus in these animals relative to WT (Fig. 7d).

To further assess the enhanced protective capacity of Aiolos-deficient T cells, we adoptively transferred WT or Ikzf3-/- CD8+ T cells into recipient Cd8-/- mice and infected recipients with IAV 1-day post-transfer (Fig. 7e). We then measured viral RNA levels in the lungs of the recipient mice at 3 d.p.i. Consistent with our prior findings, we observed reduced viral RNA in the lungs of mice that received Aiolos-deficient T compared to mice that received WT T cells (Fig. 7f). Together, these findings demonstrate that Aiolos-deficient T have an enhanced capacity to control IAV infection relative to their WT counterparts.

Methods
Mouse strains and cell lines
Wild-type CD45.2 C57BL/6J (JAX stock #000664) mice were originally obtained from the Jackson Laboratory. Aiolos-deficient mice were originally obtained from Riken BRC (stock # RBRC05582) and backcrossed onto the CD45.2 C57BL/6J background for more than 10 generations to obtain CD45.2 Aiolos-deficient (Ikzf3-/-) mice. Cd8-/- mice (JAX stock #002665) were generously provided by Dr. Ginny Bumgardner and Dr. Hazem Ghoneim at The Ohio State University. All mice were housed in a specific-pathogen free (SPF) facility. Mice were housed in 12/12 dark/light cycle, at an ambient temperature of 68–79F with 29–33% humidity. Age- and sex-matched mice were used for both in vivo and in vitro studies to avoid any unintentional bias. Littermates were used wherever possible for experiments. The experimental and control animals were housed separately for the duration of the study. WT and Ikzf3-/- mice used in the study had similar starting weights for infection experiments. All studies performed utilizing mice were done with the approval of the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University in Columbus, OH. The EL4 murine T cell lymphoma line (TIB-39) for transfection studies and YAC-1 cells (TIB-160) for cytotoxicity were acquired from the American Type Culture Collection (ATCC) and maintained in complete RPMI (RPMI media [catalog # 61870-036, Thermo Fisher Scientific] containing 10% FBS [catalog # 26140-079, Life Technologies] and 1% penicillin/streptomycin [catalog # 15140-122, Life Technologies]). For transduction studies, the Platinum-E (Plat-E) Retroviral Packaging cell Line (RV-101, Cell Biolabs, Inc.) was maintained in complete DMEM (DMEM [catalog # 11995-065, Thermo Fisher Scientific], 10% FBS, and 1% penicillin/streptomycin). MDCK (Madin–Darby canine kidney cells) cell line (ATCC, NBL-2) for viral titer determination was maintained in complete RPMI (RPMI, 10mM HEPES [catalog # 15630080, Thermo Fisher Scientific], 1X L-glutamine [catalog # 25030081, Thermo Fisher Scientific], 1X sodium pyruvate [catalog # 11360070, Thermo Fisher Scientific], 1X MEM NEAA [catalog # 11140050, Thermo Fisher Scientific], 10% FBS and 1% penicillin/streptomycin).

CD8+ T cell isolation and culture
For overexpression studies, naive CD8+ T cells were isolated from the spleens and lymph nodes of 5–8-week-old Ikzf3-/- mice using the BioLegend MojoSort naive CD8+ T cell isolation kit (catalog # 480044) according to the manufacturer’s recommendations. Naive CD8+ T cells (6×105) in complete IMDM (IMDM [catalog # 12440061, Life Technologies] with 10% FBS, 1% penicillin/streptomycin, and 0.05% (50μM) 2-mercaptoethanol [catalog # M3148, Sigma-Aldrich]) were plated in wells coated with anti-CD3 (5μg/mL, clone 145-2C11, catalog # BDB553057, BD Biosciences) and anti-CD28 (10μg/mL, clone 37.51, catalog # BDB553294, BD Biosciences) in the presence of IL-4-neutralizing antibody (5μg/mL, clone 11B11, catalog # 504122, BioLegend) and IL-2-neutralizing antibody (5μg/mL, JES6-1A12, catalog # 503706, BioLegend). IL-15 complex (IL-15C) was added to the samples at a concentration of 100ng/mL of IL-15. The IL-15C was generated as previously described58. Briefly, soluble recombinant IL-15 (catalog # 447-ML-010, R&D) and recombinant mouse IL-15Rα Fc chimera (catalog # 551-MR-100, R&D) were mixed at a 1:2 ratio and incubated at 37°C for 45 min immediately prior to use. Cells were cultured in a 37°C incubator for the indicated times before downstream analyses.

Primary T cell transduction
Aiolos overexpression experiments in primary CD8+ T cells were performed as previously described26. Briefly, pMIG-Aiolos was generated by cloning the Aiolos (Ikzf3) coding sequence into the pMSCV-IRES–GFP II (pMIG II, catalog # 52107, Addgene) vector backbone. The cloning of Aiolos was confirmed by Sanger sequencing, and overexpression was validated via both qRT-PCR and immunoblot using anti-V5 antibody for Aiolos (clone SV5-Pk1; 1:20,000; catalog # R960-25, Thermo Fisher Scientific). Plat-E cells were used to package pMIG-Aiolos virus per the manufacturer’s instructions. Viral supernatants were collected 48h post-transfection and snap frozen. Viral supernatants were thawed immediately before use and added to murine CD8+ T cells, as detailed above, in the presence of polybrene (8μg/mL, catalog # 107689, Sigma Aldrich). Transduction was performed at 24 and 48h after plating CD8+ T cells via spinfection for 2h at 800xg at room temperature. At 48h, cells were treated with anti-IL-2 and IL-15C and harvested 24h after the second round of transduction for analysis.

Assessment of cytokine response and cytotoxic capacity
For functional assessment of T cells, CD8+ T cells were isolated from the spleens of 5–8-week-old WT and Ikzf3-/- mice. Cells were purified using BioLegend MojoSort CD8+ T cell isolation kit (catalog # 480035) according to the manufacturer’s recommendations. Cells were then treated with rmIL-12 (10ng/mL, catalog # 419-ML-010, R&D), rmIL-18 (10ng/mL, catalog # 9139-IL-010, R&D), and IL-15C (100ng/mL) for 30min to determine phosphorylation of STAT5 and STAT4 or for 16h to determine the production of IFN-γ and granzyme B, along with NKG2D expression. To preserve the phosphorylation of STAT proteins, cells were immediately fixed with 4% formaldehyde (37% formaldehyde, catalog # BP531, Fisher Scientific) post-stimulation and harvested for further analysis. For IFN-γ and granzyme B, cells were treated with protein transport inhibitors (PTI, catalog # 00-4980-93, Invitrogen) 4h prior to harvest for downstream analysis.

To determine T cytotoxic potential, cells were incubated with the cytokine mix as above and were co-cultured with YAC-1 cells (50,000) labeled with carboxyfluorescein succinimidyl ester (CFSE, 1μM; catalog # C34554, Thermo Fisher) at an effector-to-target (E:T) ratio of 10:1 for 6h at 37°C. Cells were then stained with the ghost viability dye (Red 780; 1:750; catalog # 13-0865-T100, Tonbo Biosciences) to determine % of dead target YAC-1 cells. The ratio of target cells killed/T was calculated as the number of dead cells in cytokine treated samples—untreated samples (background)/number of T cells. For NKG2D blocking, cells were treated with anti-NKG2D antibody (10μg/mL, clone: HMG2D; catalog # BE0111, BioXCell) or isotype control (catalog # BE0091, BioXCell) for 30min prior to YAC-1 cells addition.

Promoter-reporter assay
To generate the Eomes (pGL3-Eomes) and Il2rb (pGL3-Il2rb) promoter-reporter constructs, the regulatory region of the Eomes gene (~2kb upstream of the transcriptional start site [TSS], Primers: Forward: 5′–GATCGGTACCGGCTTCGCTGCATCAAAAC–3′, Reverse: 5′–GATCGAGCTCGCTTTAGCGAATCGCAGACG –3′), and Il2rb gene (~805bp proximal to the TSS, Primers: Forward: 5′–GATCGGTACCGGTGATGGGAAAAGAAGGGGCC –3′, Reverse: 5′–GATCACGCGTCACTCAGATCAACCACCACCTG–3′) were respectively cloned into the Promega pGL3-Promoter vector (catalog # PAE1761, Promega). Expression vectors for Aiolos, Aiolos DNA-binding mutant (AiolosDBM), and a constitutively active form of STAT5b (STAT5bCA) were generated as previously described26,59. Briefly, the coding sequence of Aiolos was cloned into the pcDNA3.1/V5-His-TOPO vector (catalog # K4800, Life Technologies). The coding sequence was confirmed by Sanger sequencing with T7 and BGH primers and then transferred into the pEF1/V5-His vector (catalog # V920-20, Life Technologies). A similar technique was utilized for the generation of AiolosDBM and STAT5bCA in conjunction with the Agilent QuikChange Site-Directed Mutagenesis kit (catalog # 200519). For AiolosDBM, point mutations were introduced into the first and second N-terminal zinc finger domains. For STAT5bCA, point mutations were introduced into STAT5b (H298R and S715F), resulting in constitutive phosphorylation of the Y699 residue. Using the Lonza 4D nucleofection system (program CM-120, buffer SF), EL4 T cells were nucleofected with the indicated vectors (V5-tagged Aiolos constructs, STAT5bCA, or empty vector control), along with pGL3-Eomes or pGL3-Il2rb and a SV40-Renilla vector as a control for transfection efficiency. Following 22–24h of recovery, samples were harvested, and luciferase expression was analyzed using the Dual-Luciferase Reporter Assay System (catalog # E1910, Promega) according to the manufacturer’s instructions. Overexpression of the vectors was confirmed by Immunoblot analysis using an antibody against the V5 epitope tag (clone SV5-Pk1; 1:20,000; catalog # R960-25, Thermo Fisher Scientific) for Aiolos vectors and anti-STAT5b antibody (clone G-2; 1:10000; catalog # sc-1656, Santa Cruz Biotechnology) for STAT5bCA.

RNA-Seq analysis
CD8+ T cells were isolated from the spleens of WT and Ikzf3-/- mice as described above. Cells were then stained for CD44 (V450; 1:300; clone IM7; catalog # 560452, BD Biosciences), CD62L (APCe780; 1:300; clone MEL-14; catalog # 47-0621-82, Thermo Fisher), CD49d (AF488; 1:100; clone R1-2; catalog # 103611, BioLegend) and viability dye (V510; 1:400; catalog # 13-0870-T100, Tonbo Biosciences) to identify T populations, and were sorted using the Sony MA900 cell sorter for RNA extraction. For naive RNA samples, CD8+ T cells from the spleens of WT mice were isolated using the BioLegend MojoSort naive CD8+ T cell isolation kit as mentioned above. RNA was isolated from the sorted WT and Aiolos-deficient T cells, as well as WT naive CD8+ T cell, s using the Machery-Nagel Nucleospin RNA kit (catalog # 740955.5) as per the manufacturer’s instructions. Purified RNA samples were submitted to Azenta Life Sciences for polyA selection, library preparation, and Illumina 2×150bp sequencing.

Data were pre-processed with Trimmomatic (0.36) and aligned with STAR (2.5.2b) to GRCm38. Transcript counts were generated with Subread (1.5.2) and quantified with DESeq2 (v1.44.0) for analysis. Before analysis, expression data were filtered to exclude non-protein coding transcripts and lowly expressed genes (less than 100 counts). Genes with a DESeq2 adjusted p<0.05 were considered differentially expressed. Principal Component Analysis (PCA) plots were generated with DESeq2 and exported to GraphPad Prism for visualization. Morpheus software (https://software.broadinstitute.org/morpheus) was used for heatmap generation and clustering by Euclidean distance using normalized log2 counts from DESeq2 analysis. Volcano plot was generated using −log10 (adjusted p value) and log2 fold change values from DESeq2 analysis in the VolcaNoseR software (https://huygens.science.uva.nl/VolcaNoseR/)60. For gene set enrichment analysis (GSEA), differentially expressed genes were pre-ranked by multiplying the sign of the fold-change by −log10 (adjusted p value) and analyzed using the Broad Institute GSEA software (v 4.3.3) for comparison against “gene ontology,” and “immunologic signature” gene sets.

ATAC-seq analysis
WT and Ikzf3-/- splenic T cells were isolated as described above. ATAC-seq analysis was performed as previously described61. Briefly, 5×104 cells at >90% viability were processed using the Illumina-compatible Tn5 Transposase (catalog # C01070012-30), followed by a cleanup using the Qiagen MinElute PCR Purification Kit (catalog # 28004) prior to amplification with NEBNext® High-Fidelity 2X PCR Master Mix (catalog # M0541S) and Illumina primers, according to the manufacturer’s instructions. Purified DNA were submitted to CD Genomics for sequencing by Illumina Novaseq X. Resultant sequences were trimmed and aligned to mm10 using Bowtie 2 (v2.5.1), then processed with SAMtools (v1.21) and converted to bigwigs with deepTools (v3.5.6). Peaks were overlapped with ENCODE regulatory elements, and counts at peaks were determined using deepTools. Regions with adjusted P values <0.05 were considered statistically significant. PCA, significant peaks, and data for heatmaps were generated with DESeq2. PCA values were exported to prism for visualization. Heatmap generation and hierarchical clustering of the regions with significant differential accessibility was performed using the Morpheus software (https://software.broadinstitute.org/morpheus). Volcano plot was generated using log10 (adjusted p value) and log2 fold change values in the VolcaNoseR software (https://huygens.science.uva.nl/VolcaNoseR/)60. CPM-normalized tracks were visualized using Integrative Genomics Viewer (IGV, v2.18.2).

Influenza A virus infection and tissue preparation
IAV strain A/PR/8/34 (H1N1, termed “PR8”) was propagated in 10-day-old specific pathogen free embryonated chicken eggs (Charles River Laboratories) and titered on MDCK cells (BEI Resources, NIAID, NIH: Kidney [Canine], NR-2628). WT and Aiolos-deficient mice between 8–12 weeks of age were infected intranasally with 30 or 100 plaque-forming units (PFU) of IAV. For the adoptive transfer studies, WT and Ikzf3-/- T cells were sorted as described above and retro-orbitally transferred into recipient Cd8-/- mice. The recipient mice were then infected via the intranasal route with 100 PFU of IAV. At designated times after infection, spleen and lung tissues were harvested following euthanasia with CO2 inhalation as previously described26. To distinguish circulating from resident cells, mice were retro-orbitally injected with fluorophore-labeled anti-CD45.2 antibody (PE-anti CD45.2, 2μg, clone # 104, catalog # 109808, BioLegend) 3min prior to euthanasia14.

For spleen, mediastinal lymph nodes, and thymus, single-cell suspensions were generated in tissue processing media (IMDM+4% FBS) by passing the tissue through a 100µm nylon mesh strainer followed by erythrocyte lysis in 0.84% NH4Cl. For lungs, the whole tissue was incubated in HBSS (catalog # 14175103, Gibco) with 1.3mM EDTA. Lung as well as liver tissues were dissociated in Collagenase IV (catalog # LS004189, Worthington Biochemical) supplemented media (RPMI+4% FBS) using a gentleMACS Dissociator (Miltenyi Biotech) according to the manufacturer’s instructions. Samples were then filtered through a 40μm nylon mesh strainer and layered with a Percoll (catalog # 17-0891-01, Sigma) density gradient to isolate lymphocytes. Cells were centrifuged at 500×g for 20min with brakes off to allow for separation of mononuclear cells. Cells were then collected, and erythrocyte lysis was performed as previously described. Blood was collected into tubes containing 4% sodium citrate solution, and Ficoll (catalog # 17144003, Cytiva) was added for density separation. Cells were centrifuged as above with brakes off to allow mononuclear cell separation. Cells were resuspended in FACS buffer (PBS [catalog # 14190-136, Thermo Fisher Scientific] + 4% FBS) prior to staining for flow cytometry.

FTY720 administration
Mice were injected with saline control or Fingolimod (FTY720, catalog # S5002, Selleckchem) at 1mg/kg in saline daily via the intraperitoneal route at the indicated time points. Tissues were then collected post-euthanasia at the indicated times for processing and analysis.

Flow cytometry
For flow cytometry analysis, cells were first incubated with anti-mouse CD16/32 Fc block (clone 93; catalog # 101320; BioLegend) for 5min at 4°C. Cells were then incubated for 30min at 4°C for extracellular staining with following antibodies: anti-CD8α (BV570, 1:300; clone 53-6.7; catalog # 100740, BioLegend), anti-CD3 (BV605; 1:100; clone 17A2; catalog # 100237, BioLegend), anti-CD44 (BV785; 1:300; clone IM7; catalog # 103059, BioLegend), anti-CD62L (BV650; 1:300; clone MEL-14; catalog # 104453, BioLegend), anti-CD49d (BUV395; 1:100; clone R1-2; catalog # 740219, BD Biosciences), anti-CD122 (FITC/APC; 1:00; clone 5H4; catalog # 554452, BD Biosciences/105912, BioLegend), anti-Cxcr3 (BV750; 1:100; clone CXCR3-173; catalog # 747298, BD Biosciences), anti-NKG2D (PE-Dazzle 594; 1:100; clone CX5; catalog # 130214, BioLegend) and Ghost viability dye (Red 780; 1:750; catalog # 13-0865-T100, Tonbo Biosciences). Cells were then washed twice with FACS buffer prior to intracellular staining. For intracellular staining, cells were fixed and permeabilized using the eBioscience Foxp3 transcription factor staining kit (catalog # 00-5523-00, Thermo Fisher Scientific) for 30min or overnight at 4°C. After fixation, samples were stained with the following antibodies in 1X permeabilization buffer (catalog # 00-5523-00, Thermo Fisher Scientific) for 30min at room temperature: anti-Aiolos (PE/AF647; 1:100; clone S48-791; catalog # 564874/565265, BD Biosciences), anti-Eomes (PE-Cy7; 1:100; clone DAN11MAG; catalog # 25-4875-82, Thermo Fisher Scientific), anti-T-bet (PerCP-Cy5.5; 1:100; clone 4B10; catalog # 644806, BioLegend) anti-IFN-γ (APC; 1:500; clone XMG1.2; catalog # 505810, BioLegend) and anti-granzyme B (PE; 1:250; clone GB11; catalog # GRB04, Thermo Fisher Scientific). Cells were then washed with 1X permeabilization buffer and FACS buffer before resuspending in FACS buffer for analysis. To determine the phosphorylation levels of STAT proteins, cells were immediately fixed with 4% formaldehyde post-stimulation to preserve the phosphorylation of STAT proteins. Cells were then harvested and washed with 1X PBS prior to incubation with anti-CD49d (AF488) and anti-CD44 (V450) antibodies for 30min at 4°C. Following the extracellular staining, cells were permeabilized with cold methanol overnight at −20°C. These cells were then incubated with antibodies against pY-STAT5 (Y694, APC; 1:12.5; clone: SRBCZX; catalog # 17901042, Fisher Scientific), pY-STAT4 (PY933, PerCP-Cy5.5; 1:12.5; clone: 38/p-Stat4; catalog # 561217, BD Biosciences), and CD8α (PE, 1:300; clone 53-6.7; catalog # 100708, BioLegend), for 30min before resuspending in FACS buffer for analysis. Samples were run on a BD FACSCanto II or BD FACS Symphony and analyzed using FlowJo software (version 10.8.1).

Virus titers and viral RNA determination
Virus titers and virus RNA were determined as previously described62–64. Briefly, mice were euthanized at designated time points post-infection, and lungs were harvested in 1mL PBS+0.5% BSA in Precellys tubes (catalog # 50-154-2932, Bertin Corp) for homogenization using the Precellys homogenizer. The lung supernatants were collected, snap frozen, and stored at −80°C prior to use. For the determination of virus titers, MDCK cells were plated at a density of 7×105 cells/well in a 6-well plate. Virus supernatants were 10-fold serially diluted and added to MDCK cells in the presence of infection media (1X PBS, 35% BSA [catalog # ICN810061, Fisher Scientific], 1X Ca/Mg). Following 1h incubation at 37°C the the viral supernatant was removed, and cells were washed with 1X PBS. An overlay (2% Oxoid agar [catalog # LP0028, Thermo Fisher Scientific], 2X MEM [catalog # 12-684F, Lonza], TPCK Trypsin [catalog # 20233, Thermo Fisher Scientific], DEAE-Dextran [catalog # MP 021951331, Thermo Fisher Scientific], 5% sodium bicarbonate, and H2O) was added to the wells. After 48h of incubation, the overlay was removed, and cells were fixed with 3% paraformaldehyde (PFA). Cells were blocked with 5% non-fat milk in 1X PBS and incubated with chicken anti-PR8 antibody (catalog # NR-3098, BEI Resources) and rabbit anti-chicken peroxidase (catalog # 303-035-003, Jackson ImmunoResearch). The number of plaques formed were quantified following the addition of TrueBlue Peroxidase (catalog # KPL5510-0030, Thermo Fisher Scientific).

For viral RNA determination, lungs were harvested in 1mL Trizol (catalog # 15596026, Thermo Fisher Scientific) and processed as above. 1-bromo-3-chloropropane (catalog # B62404, Sigma Aldrich) was added to the clarified virus supernatant. The aqueous layer was collected post-centrifugation, ethanol was added, and the mixture was transferred onto a spin column from the Machery-Nagel Nucleospin RNA kit. RNA was extracted according to the manufacturer’s instructions. cDNA was prepared, and qRT-PCR analysis was then performed to determine virus RNA levels.

RNA isolation and Quantitative Real-Time PCR
RNA was isolated as described above utilizing the Machery-Nagel Nucleospin RNA kit per the manufacturer’s guidelines. cDNA was then generated using the Superscript IV First Strand Synthesis System (catalog # 18091050, Thermo Fisher Scientific). SYBR Select Mastermix for CFX (catalog # 4472942, ThermoFisher) was used for qRT-PCR reactions with 10ng cDNA per reaction on the CFX Connect (Bio Rad). The following primers were used: (IAV NP primers- Forward: 5′ –GATTGGTGGAATTGGACGAT–3′, Reverse: 5′ –AGAGCACCATTCTCTCTATT – 3′, Rps18 primers- Forward: 5′ –GGAGAACTCACGGAGGATGAG–3′, Reverse: 5′ –CGCAGCTTGTTGTCTAGACCG-3′). Outputs were normalized to Rps18 and are presented as relative to their respective control, as indicated.

Immunoblot analysis
For immunoblot analysis, cells were harvested, lysed in 1X SDS loading dye, (50mM Tris [pH 6.8], 100mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), and boiled for 15min. Lysates were separated via SDS-PAGE on 10% Bis-Tris Bolt gels (catalog # NW00102BOX, Thermo Fisher Scientific) and then transferred onto a 0.45µm nitrocellulose membrane (catalog # 10600002, Cytiva). Membranes were blocked with 2% nonfat dry milk in 1X TBST (10mM Tris [pH 8.0], 150mM NaCl, 0.05% Tween-20). Membranes were then incubated with the following antibodies to detect proteins: α-V5 (1:20,000; clone SV5-Pk1, catalog # R960-25, Thermo Fisher Scientific), α-STAT5b antibody (1:10,000; clone G-2, catalog # sc-1656, Santa Cruz Biotechnology), goat α-mouse HRP (1:10,000; catalog # 115-035-174, Jackson Immunoresearch), α-β-actin-HRP (1:15,000; clone: 2D1D10, catalog # A00730, GenScript). Clarity Max Western ECL substrate (catalog # 1705062, BioRad) was used for the visualization of protein bands.

Statistics and reproducibility
All statistical analyses were performed using the GraphPad Prism software (version 10.3.1). Unpaired Student’s t-test was performed for single comparisons. For multiple comparisons, one-way or two-way ANOVA with Tukey’s multiple comparisons test were performed, as appropriate. Error bars indicate the standard error of the mean. P values ≤0.05 were considered statistically significant. DESeq2 was used for RNA- and ATAC-seq analysis.

Software summary
Data were collected utilizing the following open-source or commercially available software programs: BD FACSDiva (version 8.0.2), Sony MA900 cell sorter software (version 3.1.2), BioRad Image Lab (version 6.0.1, build 34), BioRad CFX Manager (version 3.1). Analyses and/or manuscript preparation were conducted using Microsoft Office Suite (including Microsoft Word, Microsoft Excel, and Microsoft PowerPoint), BD FlowJo (version 10.8.1), Integrative Genomics Viewer (version 2.18.2), Bowtie 2 (version 2.5.1), SAMtools (version 1.21), deepTools (version 3.5.6), Morpheus (Broad Institute), Gene Set Enrichment Analysis (GSEA, version 4.3.3; Broad Institute) and VolcaNoseR (https://huygens.science.uva.nl/VolcaNoseR/)60. All statistical analyses were performed using the GraphPad Prism software (version 10.3.1). Data preparation for this manuscript did not require the use of custom code or software. Schematics in the manuscript were created in Microsoft PowerPoint.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study by by Pokhrei S and the colleagues demonstrates the crucial role of the transcription factor Ikzf3 in the\ndevelopment and function of virtual memory T (Tvm) cells. Given that the mechanisms underlying Tvm cell development and\nfunctional acquisition remain largely unexplored, this research contributes significantly to advancing our understanding of\nTvm biology. The overall findings appear to be well-supported by the presented data. However, the proposed mechanism\nseems to apply not only to Tvm cells but also to total CD8⁺ T cells as a common regulatory pathway. Therefore, further\nstudies are needed to elucidate more Tvm-specific regulatory mechanisms. Additionally, a more in-depth analysis is\nrequired to clarify how the increased number and enhanced function of Tvm cells in Ikzf3⁻/⁻ mice lead to improved outcomes\nin PR8 infection. Moreover, further immunological characterization is essential to support these claims.\nSpecific Comments:\n1. In Figure 2, the authors analyze only splenic CD8⁺ Tvm cells. It is necessary to examine whether Tvm cell numbers and\nthe expression of Eomes and CD122 also increase in Tvm cells in other organs such as lymph nodes, liver, and lungs in\nIkzf3-deficient mice. Additionally, analyzing peripheral Tvm cells in the blood would provide valuable insights. Furthermore,\nin Figure 2c and 2d, a comparison with true memory CD8⁺ T cells induced by vaccination or infection would strengthen the\nfindings. In Figure 2c, do Tvm cells from Ikzf3⁻/⁻ and WT mice exhibit differences in the expression of genes associated with\ntissue residency?\n2. Figures 4 and 5 can be merged for better clarity. Additionally, an in vitro killing assay should be included in Figure 5 to\ndemonstrate whether the increased granzyme B levels in Ikzf3⁻/⁻ Tvm cells actually enhance target cell killing. Furthermore,\nit is particularly important to distinguish whether Tvm cells from Ikzf3⁻/⁻ mice exhibit increased TCR-dependent cytotoxicity\nor NKG2D-dependent innate-like cytotoxicity.\n3. In Figures 4 and 5, the authors could analyze responses to IL-12/IL-18 and IL-15 stimulation separately. Additionally, the\nsample size appears to be small throughout the study. Increasing the number of samples is necessary to achieve statistical\nsignificance in non-parametric tests.\n4. In Figure 6, the authors analyze only the 1 dpi time point. A longitudinal analysis from 1 dpi to 8 dpi is necessary to\ncapture the dynamics of viral clearance and immune response.\n5. In Figure 7, was there any difference in survival rate between WT and Ikzf3⁻/⁻ mice?\n6. In Figure 3i, showing the lung viral titer is essential (as done in Figure 3c). More importantly, the authors present viral titers\nonly at 3 dpi (Figures 3c, 3d, 3e, and 3i). Including viral titer measurements at 5 dpi and 7 dpi would further substantiate the\nrole of Tvm cells in viral clearance.\n7. The authors attribute the lower viral titers and reduced weight loss in Ikzf3⁻/⁻ mice following PR8 infection to enhanced\nTvm function. However, additional experiments are required to determine whether this enhanced antiviral activity is due to\nTCR cross-reactivity (TCR-dependent mechanism) or innate-like cytotoxicity (TCR-independent mechanism).\n8. In Figure 7d, the viral titer data should be presented on a log scale for better visualization and comparison.", "reviewer_2": "The manuscript Pokhrel et al examines the role of Aiolos in restricting Tvm CD8 T cells. The study is rigorous, methodology\nsound and the paper well written and straight forward. Overall, the results and interpretations are compelling.\nMinor concerns and comments.\n1) The influenza infection model in mice is weight dependent. In fact mice weighing more the 4 grams in difference often\nhave a major difference in susceptibility and outcomes to the infection. It is not clear in this study if the WT and Ikzf3-/- mice\nhave similar starting weights. For instance, does the KO weigh more or less than the WT which would make it less or more\nsusceptible to the influenza infections. Many KOs do have different unexpected weight changes relative to their WT\ncounterparts. Therefore, it would be helpful for the authors to add the starting weight range (in grams) for the WT and KO\nmice to the methods or comment that no differences in starting weights were observed.\n2) It would be helpful to the analysis and interpretation to report what the level of Tvms are in the lungs in the steady-state\nbefore the influenza virus challenge. The numbers and fractions in the spleen are reported but I missed if the KO had higher\nlevels at baseline in the lungs vs the WT in the steady-state. The FTY720 + infection data suggest that they may be minimal,\nbut it is unclear what the levels would have been before the treatment, especially if the Tvm are cycling transitly through the\nlungs at a higher number in the steady state within the KO.\n3) The KO has reduced viral titers in the lungs after influenza virus challenge. In the murine influenza model while CD8 T\ncells control virus infection they are also linked to immunopathology. Given comments in the discussion related to targeting\nAiolos to modulate disease outcome, it would helpful to discuss if the authors know or think increased Tvm #s early after\ninfection also result in 1) increased immunopathology and/or 2) alter the establishment of Trms in the lungs and nasal\npassages which are critical for protection against subsequent heterologous influenza virus exposures.\n4) The authors should clarify the statement on lines 316-318 related to the lung Tvm not being related to the spleen given\nthat the FTY720 experiments reduced the lung Tvm #s and the spleen could be a source for those lung Tvms.\n5) It is unclear if the authors think the reduction in viral titers in the KO relate to Tvm killing of virally infected cells and/or due\nto IFNg limiting viral replication.\n6) In Supplementary Figure 3, the distribution of Eomes expression in the Teff appears bimodal with many of them\nexpressing Eomes at a similar level as the Tvm. Do these low Eomes Teff cells have lower Aiolos and higher CD122 than\nthe Eomes high Teff. If not, how does that alter the interpretation.", "reviewer_3": "CD8 Virtual Memory T cells are memory-like CD8 T cells that are rapidly recruited to site of infections where they provide\nantigen-independent bystander effector function such as production of inflammatory cytokines and cytotoxic factors in\nresponse to IL12 and IL18.\nCD8 TVM cells develop without encountering foreign antigens and the primary pathway for their differentiation is thought to\ninvolve the activation of naïve CD8 T cells by cytokines such as IL-4, IL-15, and IL-7, independent of TCR signaling. It is also\nsuggested that a subset of CD8 T cells receiving relatively strong self-peptide-MHC signals during positive selection in the\nthymus acquire TVM-like properties. However, this pathway is not considered the predominant route for TVM cell\ndevelopment as most TVMs are produced in the periphery.\nIn this manuscript the authors address the role of the Ikaros family member Aiolos (Ikzf3) in the generation and function of\nCD8+ TVM cells. They show that the number of CD8 CD62+CD44+CD49dlo TVM cells in the spleen is significantly\nincreased compared to WT mice in uninfected Aiolos mutant mice. Using a combination of molecular, biochemical, and\nfunctional analyses they establish that Aiolos deficient TVM cells exhibit enhanced functionality. They show that Eomes a\nkey regulator of TVM function is increased in expression as are many of its downstream targets such as CD122 that\nmediates IL15 signaling. Given these well documented findings it is proposed that AIOLOS modulates TVM function by\nrepressing the Eomes and STAT5 transcriptional networks, a very good hypothesis. However, these exciting findings need to be\nfurther substantiated with studies on the precise role of Aiolos in CD8 TVM development and function.\nThere are several points to address:\na) Eomes expression is already increased in Ikzf3⁻/⁻ naïve CD8 T cells (Supplementary Figure 1a) indicating that intrinsic\ndifferences exist at this stage and raising the possibility of changes in other cell types that may influence TVM differentiation\nas well as potential CD8 T cell developmental effects. Is this difference already seen in thymic CD8 single-positive (CD8-\nSP) cells? As reported in the study by Wang et al, Immunity 1998, in addition to B cell proliferative responses, T cell\nproliferative responses are also elevated in Aiolos KO compared to WT. A comprehensive analysis of splenic lymphoid\npopulations including naïve and effector CD8 and CD4 T cells in these mice should be shown.\nb) The difference in Luc reporter activity (assay for suppression) between Aiolos plus and minus conditions performed in the\nEL4 cell line although statistically significant is minimal to be considered biologically relevant (Fig.1g and h). Given the key\nrole of the Ikaros family members in regulating chromatin configuration that may affect the output of transcription networks a\nmore relevant study would address chromatin accessibility (ATAC) or changes in transcriptionally permissive histone\nmodifications. For example, ChIP-seq analysis for H3K27Ac, H3K4me3, Aiolos, and STAT5b in naïve and CD8 T VM cells\nfrom WT and Ikzf3⁻/⁻ mice can be attempted. If the number of TVM cells is limiting, ChIP-seq analysis using cultured (anti-IL-\n4/anti-IL-2/IL-15C-stimulated) TVM cells as shown in Figure 5 should be performed."}, "author_response": "Authors’ Summary: We thank the Reviewers for their efforts in reviewing our manuscript and\ntheir overall enthusiasm and positive comments. We now present a revised manuscript in which\nwe have made a sincere effort to incorporate the recommendations and address the thoughtful\ncritiques provided by the Reviewers. Specifically, we would like to highlight the addition of the\nfollowing pieces of impactful data.\n1. In our initial manuscript, we found elevated frequencies and numbers of T in the spleens of\nVM\nunchallenged Aiolos-deficient animals relative to their WT counterparts (Figures 2a,b). We\nhave extended those findings and now present data showing that increased T can also be\nVM\nfound in the mediastinal lymph nodes, blood, and liver of Aiolos-deficient mice (Figures 2c,d).\nFurthermore, we do not observe significant differences in CD8+Eomes+ T cells in the thymus\n(Supplementary Figures 5d), suggesting that the emergence of the Aiolos-deficient T\nVM\noccurs in the periphery.\n2. We present new Assay for Transposase Accessible Chromatin (ATAC)-seq data\ndemonstrating that Aiolos-deficient T have enhanced accessibility at the regulatory regions\nVM\nof key genes associated with T programming and effector functions (Figure 4 and\nVM\nSupplementary Figure 7). Notably, enhanced accessibility is observed at the promoters of\nEomes and Il2rb which correspond to gene reporter data demonstrating Aiolos-dependent\nrepression of these regions (Figures 4a-d, 1g,h, and Supplementary Figure 2f). We also\nobserve increased accessibility at genes regulating key T effector functions including\nVM\nIl12rb2, Ccr2, Cxcr3, Klrk1, Ifng, Gzmb, Tbx21 and Prf1 (Figures 4a-b,e-f and\nSupplementary Figures 7c-f).\n3. We present new data demonstrating that Aiolos-deficient T have superior cytotoxic killing\nVM\ncapacity and that this is at least partially dependent on elevated NKG2D expression (Figures\n5g,h and Supplementary Figures 8e,f).\n4. We present new data demonstrating a role for Aiolos-deficient T in providing increased\nVM\ncontrol of Influenza A Virus (IAV) infection as assessed by weight loss and decreased IAV\nnucleoprotein RNA (Figures 7a-d).\nCollectively, these new findings along with our prior data support our primary conclusion that\nAiolos restrains the generation and function of foreign antigen-inexperienced, virtual memory CD8\nT cells. Further details of our revisions are presented in the point-by-point response below.\nReferee #1 (Remarks to the Author):\nThis study by by Pokhrei S and the colleagues demonstrates the crucial role of the transcription\nfactor Ikzf3 in the development and function of virtual memory T (Tvm) cells. Given that the\nmechanisms underlying Tvm cell development and functional acquisition remain largely\nunexplored, this research contributes significantly to advancing our understanding of Tvm biology.\nThe overall findings appear to be well-supported by the presented data. However, the proposed\nmechanism seems to apply not only to Tvm cells but also to total CD8⁺ T cells as a common\nregulatory pathway. Therefore, further studies are needed to elucidate more Tvm-specific\nregulatory mechanisms. Additionally, a more in-depth analysis is required to clarify how the\nincreased number and enhanced function of Tvm cells in Ikzf3⁻/⁻ mice lead to improved outcomes\nin PR8 infection. Moreover, further immunological characterization is essential to support these\nclaims.\nSpecific Comments:\n1. In Figure 2, the authors analyze only splenic CD8⁺ Tvm cells. It is necessary to examine\nwhether Tvm cell numbers and the expression of Eomes and CD122 also increase in Tvm cells\nin other organs such as lymph nodes, liver, and lungs in Ikzf3-deficient mice. Additionally,\n1\nanalyzing peripheral Tvm cells in the blood would provide valuable insights. Furthermore, in\nFigure 2c and 2d, a comparison with true memory CD8⁺ T cells induced by vaccination or infection\nwould strengthen the findings. In Figure 2c, do Tvm cells from Ikzf3⁻/⁻ and WT mice exhibit\ndifferences in the expression of genes associated with tissue residency?\nIn our initial manuscript, we found elevated frequencies and numbers of T in the spleens of\nVM\nunchallenged Aiolos-deficient animals relative to their WT counterparts (Figures 2a,b). We have\nextended those findings and now present data showing that increased T can also be found in\nVM\nthe mediastinal lymph nodes (mLN), blood, and liver of Aiolos-deficient mice (Figures 2c,d). We\nalso find that these T have increased expression of Eomes and CD122 consistent with our\nVM\nobservations of splenic T from Aiolos-deficient mice (Supplementary Figures 5a,b). With\nVM\nregards to the lungs, we observe very few T in the lungs of either unchallenged WT or Aiolos-\nVM\ndeficient mice. However, this number increases significantly in the lungs of Aiolos-deficient\nanimals within 24 hrs post-IAV infection (Figures 2d and 6c-f).\nWe appreciate the Referees question pertaining to antigen-specific memory cell populations and\nresidency programming. Indeed, we are pursuing this line of inquiry in a separate study. It is\ninteresting to note that genes encoding features of tissue-residency (e.g. Ccr9, Cd69, Itgae) are\ndownregulated in the absence of Aiolos (Figures 3a,b). However, we would respectfully submit\nthat a thorough interrogation of pathways and functional programs related to antigen-experienced\nmemory T cell subsets is outside the scope of the current study.\n2. Figures 4 and 5 can be merged for better clarity. Additionally, an in vitro killing assay should be\nincluded in Figure 5 to demonstrate whether the increased granzyme B levels in Ikzf3⁻/⁻ Tvm cells\nactually enhance target cell killing. Furthermore, it is particularly important to distinguish whether\nTvm cells from Ikzf3⁻/⁻ mice exhibit increased TCR-dependent cytotoxicity or NKG2D-dependent\ninnate-like cytotoxicity.\nWe thank the Referee for the suggestion and have now combined figures 4 and 5 (now Figure\n5). As requested, we have now performed an in vitro killing assay and find that Aiolos-deficient\nT have superior cytotoxic killing capacity compared to WT T and that this is at least partially\nVM VM\ndependent on elevated NKG2D expression in the absence of Aiolos (Figures 5g,h and\nSupplementary Figures 8e,f).\n3. In Figures 4 and 5, the authors could analyze responses to IL-12/IL-18 and IL-15 stimulation\nseparately. Additionally, the sample size appears to be small throughout the study. Increasing the\nnumber of samples is necessary to achieve statistical significance in non-parametric tests.\nWe agree with the Referee and have expanded sample size throughout the study. We also\nappreciate the suggestion to examine each cytokine independently. However, in the spirit of\nlimiting animal numbers whenever possible, we have opted to use combined cytokine treatment\nand to interrogate the activation of individual pathways (pY-STAT4, pY-STAT5) and the combined\nimpact of their activation in Aiolos-deficient and WT T cells (e.g. IFN-g, GzmB, NKG2D). We\nVM\nhope the Referee agrees that our collective data convincingly demonstrate that Aiolos-deficient\nT have enhanced sensitivity to signals from IL-12 and IL-15 and this results in increased\nVM\npropagation of their effector functions relative to WT.\n4. In Figure 6, the authors analyze only the 1 dpi time point. A longitudinal analysis from 1 dpi to\n8 dpi is necessary to capture the dynamics of viral clearance and immune response.\nWe appreciate the suggest from the Referee and agree that such an analysis will provide more\ninsight into the dynamics of the T response. In order to study the early T response, we infected\nVM VM\nWT and Aiolos-deficient mice with IAV and then administered FTY720 at 1-day post-infection and\nthen everyday afterwards for 5 days (Figure 7a). We then assessed weight loss starting at the\nday of infection to day 6, and the CD8 T cell response and viral RNA at both days 3 and 6 post-\n2\ninfection. Strikingly, Aiolos-deficient mice were largely protected as they lost very little weight\ncompared to their WT counterparts (Figure 7b). Consistent with this exciting finding, Aiolos-\ndeficient animals had both increased frequencies and numbers of CD8 T cells in their lungs and\nreduced viral burden (Figures 7c,d).\n5. In Figure 7, was there any difference in survival rate between WT and Ikzf3⁻/⁻ mice?\nMice were infected with a sublethal dose of IAV (PR8), and we did not detect any difference in\nsurvival between WT and Aiolos-deficient mice during any of our infection studies.\n6. In Figure 3i, showing the lung viral titer is essential (as done in Figure 3c). More importantly,\nthe authors present viral titers only at 3 dpi (Figures 3c, 3d, 3e, and 3i). Including viral titer\nmeasurements at 5 dpi and 7 dpi would further substantiate the role of Tvm cells in viral clearance.\nWe agree with the Referee that viral titer is an important component when assessing the impact\nof the immune response on infection and have provided both titer and viral RNA data that agree\nwith each other (Supplementary Figure 9c). However, studying viral titer, viral RNA, and the\nCD8 T response for an increasing number of timepoints and adoptive transfer approaches\nVM\nultimately leads to the use of an extensive number of animals. In an effort to provide increased\ninsight into the functional importance and dynamics of the CD8 T cell response without\ndramatically increasing mouse numbers, we have now performed additional experiments as\noutlined above (Referee 1, point 4) to assess the immune response and impact on infection at\nmultiple timepoints. We hope the Referee agrees that our findings support our conclusion that\nAiolos-deficiency results in 1) an enhanced population of T in multiple tissues prior to infection\nVM\nand in the lungs post-infection (Figures 2 and 6), 2) increased effector responses (Figure 5), and\n3) diminished disease severity as assessed by weight loss and reduced viral RNA (Figure 7).\n7. The authors attribute the lower viral titers and reduced weight loss in Ikzf3⁻/⁻ mice following\nPR8 infection to enhanced Tvm function. However, additional experiments are required to\ndetermine whether this enhanced antiviral activity is due to TCR cross-reactivity (TCR-dependent\nmechanism) or innate-like cytotoxicity (TCR-independent mechanism).\nAs discussed in Point 2, we have performed in vitro killing assays and find that Aiolos-deficient\nT have superior cytotoxic killing capacity compared to WT T (Figure 5h and Supplementary\nVM VM\nFigure 8f). Importantly, the observed increase in cytotoxicity is reduced upon blocking NKG2D\nactivation suggesting that innate-like pathways are at least partially responsible (Figure 5g,h).\nFurthermore, our data convincingly demonstrate that Aiolos-deficient T harbor heightened\nVM\nsensitivity to cytokine signals (in the absence of TCR) which result in the elevated production of\nIFN-g and GzmB (Figures 5a,c-f). We believe these collective data strongly indicate that Aiolos-\ndeficient T possess innate-like cytotoxic features characteristic of T populations.\nVM VM\n8. In Figure 7d, the viral titer data should be presented on a log scale for better visualization and\ncomparison.\nWe agree with the Referee and have made this alteration.\nReferee #2 (Remarks to the Author):\nThe manuscript Pokhrel et al examines the role of Aiolos in restricting Tvm CD8 T cells. The study\nis rigorous, methodology sound and the paper well written and straight forward. Overall, the\nresults and interpretations are compelling.\nMinor concerns and comments.\n1) The influenza infection model in mice is weight dependent. In fact mice weighing more the 4\ngrams in difference often have a major difference in susceptibility and outcomes to the infection.\nIt is not clear in this study if the WT and Ikzf3-/- mice have similar starting weights. For instance,\ndoes the KO weigh more or less than the WT which would make it less or more susceptible to the\n3\ninfluenza infections. Many KOs do have different unexpected weight changes relative to their WT\ncounterparts. Therefore, it would be helpful for the authors to add the starting weight range (in\ngrams) for the WT and KO mice to the methods or comment that no differences in starting weights\nwere observed.\nWe agree and thank the Referee for mentioning this important point. No significant differences in\nstarting weights were observed and we have now added this information to the methods.\n2) It would be helpful to the analysis and interpretation to report what the level of Tvms are in the\nlungs in the steady-state before the influenza virus challenge. The numbers and fractions in the\nspleen are reported but I missed if the KO had higher levels at baseline in the lungs vs the WT in\nthe steady-state. The FTY720 + infection data suggest that they may be minimal, but it is unclear\nwhat the levels would have been before the treatment, especially if the Tvm are cycling transitly\nthrough the lungs at a higher number in the steady state within the KO.\nWe agree with the Referee and are pleased to provide this data (Figures 2c,d). As recommended\nby Referee 1, we have included an enhanced analyses of T in tissues throughout WT and\nVM\nAiolos-deficient mice. This included a comparison of Spleen, mLN, Liver, Blood, and Lungs\n(Figures 2a-d). Importantly, there are very few T in the lungs of either WT or Aiolos-deficient\nVM\nmice prior to infection with IAV (Figure 2d). The Referee is correct in that our FTY720 data prior\nto infection also indicate that T are infiltrating the lung post-infection rather than the expansion\nVM\nof a small lung-resident population (Figures 6g,h).\n3) The KO has reduced viral titers in the lungs after influenza virus challenge. In the murine\ninfluenza model while CD8 T cells control virus infection they are also linked to immunopathology.\nGiven comments in the discussion related to targeting Aiolos to modulate disease outcome, it\nwould helpful to discuss if the authors know or think increased Tvm #s early after infection also\nresult in 1) increased immunopathology and/or 2) alter the establishment of Trms in the lungs and\nnasal passages which are critical for protection against subsequent heterologous influenza virus\nexposures.\nThe Referee raises an interesting set of possibilities. We haven’t assessed potential\nimmunopathology, but we suspect that Aiolos-deficient animals are reasonably healthy given our\nobservation that they lose significantly less weight relative to WT controls, notably also when\nlymphocyte migration to the lung is limited to the first 24hrs post-infection upon FTY720 treatment\n(Figures 7a-d). With regards to tissue-residency features of T and bona fide T , our collective\nVM RM\ndata suggest that Aiolos-deficient T may have a compromised ability to establish long-term\nVM\nresidency. Our data demonstrate that Aiolos-deficient T have an enhanced capacity to migrate\nVM\nand infiltrate the lungs shortly after infection (Figures 6c-f). However, our RNA- and ATAC-seq\ndata also reveal that many genes associated with tissue residency programming (e.g. Cd69,\nItgae, Tgfrb2, Ccr9, Cxcr6) have reduced expression and/or chromatin accessibility in the\nabsence of Aiolos (Figures 3 and 4). We are pursuing this line of inquiry in a separate manuscript\nand look forward to sharing our findings in the future.\n4) The authors should clarify the statement on lines 316-318 related to the lung Tvm not being\nrelated to the spleen given that the FTY720 experiments reduced the lung Tvm #s and the spleen\ncould be a source for those lung Tvms.\nWe thank the Referee for pointing this out and have added the requested clarifying language.\n5) It is unclear if the authors think the reduction in viral titers in the KO relate to Tvm killing of\nvirally infected cells and/or due to IFNg limiting viral replication.\nWe thank the Referee for this question and agree that it is possible that both enhanced production\nof IFN-g and increased cytotoxic capacities of Aiolos-deficient T plays a key role in limiting viral\nVM\n4\nreplication and disease severity. We now discuss this as well as other possibilities in the\ndiscussion section of the manuscript.\n6) In Supplementary Figure 3, the distribution of Eomes expression in the Teff appears bimodal\nwith many of them expressing Eomes at a similar level as the Tvm. Do these low Eomes Teff cells\nhave lower Aiolos and higher CD122 than the Eomes high Teff. If not, how does that alter the\ninterpretation.\nWe thank the Referee for pointing out this interesting observation. Upon analyses, there are not\nclear trends in Eomes and Aiolos expression in the effector population. Our interpretation is that\nthis may be consistent with a heterogenous effector population that is representative of and\ncapable of giving rise to many different cell types (e.g. SLECs, MPECs, T , T , long-lived\nCM RM\neffector cells, and others). Regardless, we are confident that these data demonstrate that T\nVM\nhave elevated expression of Eomes and relatively lower levels of Aiolos expression\n(Supplementary Figures 3b,c). Ongoing studies in the lab are aimed at identifying additional\nroles for Aiolos in regulating the differentiation decisions of antigen-specific effector and memory\ncell populations and we look forward to presenting these findings in subsequent manuscripts.\nReferee #3 (Remarks to the Author):\nCD8 Virtual Memory T cells are memory-like CD8 T cells that are rapidly recruited to site of\ninfections where they provide antigen-independent bystander effector function such as production\nof inflammatory cytokines and cytotoxic factors in response to IL12 and IL18.\nCD8 TVM cells develop without encountering foreign antigens and the primary pathway for their\ndifferentiation is thought to involve the activation of naïve CD8 T cells by cytokines such as IL-4,\nIL-15, and IL-7, independent of TCR signaling. It is also suggested that a subset of CD8 T cells\nreceiving relatively strong self-peptide-MHC signals during positive selection in the thymus\nacquire TVM-like properties. However, this pathway is not considered the predominant route for\nTVM cell development as most TVMs are produced in the periphery.\nIn this manuscript the authors address the role of the Ikaros family member Aiolos (Ikzf3) in the\ngeneration and function of CD8+ TVM cells. They show that the number of CD8\nCD62+CD44+CD49dlo TVM cells in the spleen is significantly increased compared to WT mice\nin uninfected Aiolos mutant mice. Using a combination of molecular, biochemical, and functional\nanalyses they establish that Aiolos deficient TVM cells exhibit enhanced functionality. They show\nthat Eomes a key regulator of TVM function is increased in expression as are many of its\ndownstream targets such as CD122 that mediates IL15 signaling. Given these well documented\nfindings it is proposed that AIOLOS modulates TVM function by repressing the Eomes and STAT5\ntranscriptional networks, a very good hypothesis. However, these exciting findings need to be\nfurther substantiated with studies on the precise role of Aiolos in CD8 TVM development and\nfunction.\nThere are several points to address:\na) Eomes expression is already increased in Ikzf3⁻/⁻ naïve CD8 T cells (Supplementary Figure\n1a) indicating that intrinsic differences exist at this stage and raising the possibility of changes in\nother cell types that may influence TVM differentiation as well as potential CD8 T cell\ndevelopmental effects. Is this difference already seen in thymic CD8 single-positive (CD8-SP)\ncells? As reported in the study by Wang et al, Immunity 1998, in addition to B cell proliferative\nresponses, T cell proliferative responses are also elevated in Aiolos KO compared to WT. A\ncomprehensive analysis of splenic lymphoid populations including naïve and effector CD8 and\nCD4 T cells in these mice should be shown.\nWe thank the Referee for raising this important point. We have now assessed thymic CD8 T cells\nand find no significant differences in CD8 single-positive T cells or in CD8+Eomes+ T cells\n5\n(Supplementary Figures 5c,d). There is a slight increase in the frequency of CD8+CD122+ T\ncells, but this does not translate into a corresponding significant increase in numbers\n(Supplementary Figure 5e). Collectively, these findings suggest that the emergence of the\nAiolos-deficient T population occurs in the periphery. We have also assessed splenic CD8 and\nVM\nCD4 T cells in these animals. While there is little difference between bulk CD8 and CD4 T cell\npopulations, we do observe reduced frequencies in naïve CD8 and CD4 T cells (Supplementary\nFigures 4b-d). Correspondingly, there is an overall increase in activated CD8+CD44+ and\nCD4+CD44+ T cells (Supplementary Figures 4c,d) which is consistent with the published\nliterature examining Aiolos deficient mice and human patients with Aiolos variants1, 2, 3, 4, 5, 6.\nImportantly, however, we only find an expansion of the CD44+CD62L+ population in Aiolos-\ndeficient CD8 T cells, consistent with the observed increase in the T subset (Supplementary\nVM\nFigures 4c,d).\nb) The difference in Luc reporter activity (assay for suppression) between Aiolos plus and minus\nconditions performed in the EL4 cell line although statistically significant is minimal to be\nconsidered biologically relevant (Fig.1g and h). Given the key role of the Ikaros family members\nin regulating chromatin configuration that may affect the output of transcription networks a more\nrelevant study would address chromatin accessibility (ATAC) or changes in transcriptionally\npermissive histone modifications. For example, ChIP-seq analysis for H3K27Ac, H3K4me3,\nAiolos, and STAT5b in naïve and CD8 TVM cells from WT and Ikzf3⁻/⁻ mice can be attempted. If\nthe number of TVM cells is limiting, ChIP-seq analysis using cultured (anti-IL-4/anti-IL-2/IL-15C-\nstimulated) TVM cells as shown in Figure 5 should be performed.\nWe appreciate and agree with the Referee’s comments. We are pleased to provide new ATAC-\nseq data from in vivo-generated WT and Aiolos-deficient T demonstrating that the latter have\nVM\nenhanced accessibility at the regulatory regions of key genes associated with T programming\nVM\nand effector functions (Figure 4 and Supplementary Figure 7). Notably, enhanced accessibility\nis observed at the promoters of Eomes and Il2rb which correspond to gene reporter data\ndemonstrating Aiolos-dependent repression of these regions (Figures 4a-d, 1g,h, and\nSupplementary Figure 2f). We also observe increased accessibility at genes regulating key T\nVM\neffector functions including Il12rb2, Ccr2, Cxcr3, Klrk1, Ifng, Gzmb, Tbx21 and Prf1 (Figures 4a-\nb,e-f and Supplementary Figures 7c-f).\nEditor\nWe consider it particularly important that you better characterize the mechanisms of Ikzf3⁻/⁻ Tvm\nmediated cytotoxicity (Ref#1 and #2), improve the analysis of the effects of AIOLOS depletion on\nthe transcriptional network by ATAC or CHIP-seq experiments (Ref#3), and investigate the\npotential effects on other cell types (Ref#3). Furthermore, please expand the analysis of Tvm cells\nin the lungs and other organs, as requested by Ref#1 and 2 and consider the influence of other\nconfounding factors such as changes in weight, as indicated by Ref#2. In parallel, to ensure the\nvalidity and reproducibility of the results and conclusions, and in observation of the complex and\nvariable nature of most immunological studies, we ask that all experiments should be performed\nin at least two to three independent batches, or have at a minimum 5-10 independent biological\nreplicates, barring clear exceptions such as case reports or non-human primate studies. Please\nnote that technical replicates (e.g. triplicate wells in qPCR or tissue culture, or duplicate western\nblots run with the same lysates) count only as one data point. Lastly, the number of data points\nand independent experiments should be stated clearly in all figure legends.\nOur revised manuscript includes a stronger characterization of the cytotoxic capabilities of Aiolos-\ndeficient T as well as the underlying effector mechanisms. We have also provided ATAC-seq\nVM\ndata demonstrating the impact of Aiolos deficiency on the chromatin accessibility of regulatory\nregions governing the differentiation and functional programs of T . We have provided an\nVM\n6\nenhanced assessment of the impact of Aiolos-deficiency on T cells in many tissues throughout\nVM\nthe body, including the spleen, mLN, liver, blood, lungs and thymus. Finally, we have added\nexperimental replicates and other requested analyses that have enhanced the rigor of our work\nand strengthened our overall conclusions.\nWe thank the Referees and Editors for their time and careful attention to our manuscript. We feel\nstrongly that the work detailed above has strengthened our manuscript and hope that it will now\nbe found acceptable for publication in Nature Communications.\nReferences\n1. Chang, J., Yamashita, M., Padhi, A.K., Zhang, K.Y.J. & Taniuchi, I. Impaired tissue homing\nby the Ikzf3(N159S) variant is mediated by interfering with Ikaros function. Front Immunol\n14, 1239779 (2023).\n2. Kuehn, H.S. et al. T and B cell abnormalities, pneumocystis pneumonia, and chronic\nlymphocytic leukemia associated with an AIOLOS defect in patients. J Exp Med 218\n(2021).\n3. Quintana, F.J. et al. Aiolos promotes TH17 differentiation by directly silencing Il2\nexpression. Nat Immunol 13, 770-777 (2012).\n4. Wang, J.H. et al. Aiolos regulates B cell activation and maturation to effector state.\nImmunity 9, 543-553 (1998).\n5. Yamashita, M. & Morio, T. AIOLOS Variants Causing Immunodeficiency in Human and\nMice. Front Immunol 13, 866582 (2022).\n6. Yamashita, M. & Morio, T. AIOLOS-Associated Inborn Errors of Immunity. J Clin Immunol\n44, 128 (2024).\n7"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made a sincere effort to address the concerns raised in the initial review, and the revised manuscript is\nsignificantly improved with the addition of new data and analyses. The inclusion of extended tissue analyses, functional\nassays, and chromatin accessibility profiling has strengthened the study and enhanced the overall impact.\nNevertheless, certain aspects remain insufficiently addressed. In particular, regarding Specific Comment #1, the authors\nindicated that a comparison with antigen-specific memory CD8⁺ T cells will be pursued in a separate study. While this future\ndirection is appreciated, I believe that such a comparison is still essential for establishing the robustness of the conclusions\nin the current manuscript.\nIn addition, regarding Specific Comment #2, while the newly presented data in Figure 5h demonstrate that the cytotoxic\ncapacity of Aiolos-deficient TVM cells is partially reduced upon NKG2D blockade, the extent of this reduction appears to be\nrather minor. This suggests that additional mechanisms beyond NKG2D-mediated pathways likely contribute to the\nenhanced cytotoxicity observed. The authors should therefore provide further clarification regarding alternative mechanisms\nthat might account for this effect.", "reviewer_4": "The authors have sufficiently addressed concerns raised.", "reviewer_5": "I have carefully reviewed the response to reviewers and believe the authors have done an excellent job addressing these\nconcerns and present a strong well written manuscript. I found the results very intriguing and I believe they will be of broad\ninterest to the field."}, "author_response": "AUTHORS’ SUMMARY: We very much appreciate the thoughtful feedback provided by the\nReviewers and were pleased to note that they felt we addressed the majority of their concerns.\nWe thank Reviewer 1 for providing a few additional suggestions designed to improve the clarity\nof the data presented and conclusions drawn. We have now addressed the remaining concerns\nas outlined in the following point-by-point response:\nReviewer #1 (Remarks to the Author):\nThe authors have made a sincere effort to address the concerns raised in the initial review, and\nthe revised manuscript is significantly improved with the addition of new data and analyses. The\ninclusion of extended tissue analyses, functional assays, and chromatin accessibility profiling has\nstrengthened the study and enhanced the overall impact.\nNevertheless, certain aspects remain insufficiently addressed. In particular, regarding Specific\nComment #1, the authors indicated that a comparison with antigen-specific memory CD8⁺ T cells\nwill be pursued in a separate study. While this future direction is appreciated, I believe that such\na comparison is still essential for establishing the robustness of the conclusions in the current\nmanuscript.\nIn addition, regarding Specific Comment #2, while the newly presented data in Figure 5h\ndemonstrate that the cytotoxic capacity of Aiolos-deficient TVM cells is partially reduced upon\nNKG2D blockade, the extent of this reduction appears to be rather minor. This suggests that\nadditional mechanisms beyond NKG2D-mediated pathways likely contribute to the enhanced\ncytotoxicity observed. The authors should therefore provide further clarification regarding\nalternative mechanisms that might account for this effect.\nWe thank the Reviewer for their complementary and thoughtful feedback. We understand the\nReviewer’s interest in antigen-specific memory subsets (specific comment #1) and have provided\ndata reflecting the suggested comparison for Eomes expression. As expected, we observe an\noverall increase in Eomes expression in T cells compared to other antigen-specific T cell\nVM\nsubsets. Further, Aiolos-deficient T have the highest Eomes expression compared to central\nVM\nmemory T (T ) and tissue-\nCM\nresident memory T (T )\nRM\ncell subsets. While these\nfindings further bolster our\nidentification of an\nAiolos/Eomes regulatory\naxis operative in T cells, we\nrequest this comparison be\nexcluded from the current\nmanuscript as we are\ncurrently pursuing the role\nof Aiolos on antigen-\nspecific memory subsets in\na separate study.\nWith regards to specific comment\n#2, we have included discussion\nof additional mechanisms beyond\nelevated NKG2D/IFN-g/GzmB/\nPerforin expression that may\nfurther contribute to the enhanced\ncytotoxic features observed in\nAiolos deficient T cells\nVM\n(Discussion, lines 316-324).\nNotably, RNA-seq analyses of WT\nand Aiolos-deficient T revealed\nVM\nthat Fas ligand (Fasl) expression\nis similarly enhanced in the\nabsence of Aiolos (Fig. 3b).\nReviewer #4 (Remarks to the Author):\nThe authors have sufficiently addressed concerns raised.\nWe sincerely thank the Reviewer for their time and effort in review of our manuscript.\nReviewer #5 (Remarks to the Author):\nI have carefully reviewed the response to reviewers and believe the authors have done an\nexcellent job addressing these concerns and present a strong well written manuscript. I found the\nresults very intriguing and I believe they will be of broad interest to the field.\nWe thank the Reviewer for their complementary words and their efforts in the review of our\nmanuscript."}]

Decision letter