Infection of mice with influenza virus induces differentiation of virus-specific CD8⁺ T cells into CD8⁺ T_RM cells, which persist in the lung and provide protection against subsequent reinfection (5, 21, 22). To investigate lung CD8⁺ T_RM cells arising after influenza virus infection, mice were infected intranasally with HKx31 influenza A virus and CD8⁺ T cells were isolated and analyzed in the memory phase (day 30+ p.i.). Influenza virus infection gave rise to a substantial CD69⁺ CD8⁺ T_RM cell population in the lung, which partially expressed CD103 (Figure 1A). This population was nearly absent in lungs from naïve mice, indicating that the vast majority of CD8⁺ T_RM cells were a direct result of influenza virus infection. In mice, a core signature of gene-expression has been determined in CD8⁺ T_RM cells (12). Transcriptional profiling of CD69⁺ and CD69⁻ memory CD8⁺ T cells isolated from lungs of HKx31-immune mice (day 30+ p.i.) by RNA sequencing confirmed the resident phenotype of the CD69⁺ CD8⁺ T cell population arising after influenza virus infection (Figure 1B). When compared to the T_RM core signature, the obtained transcriptional profiles of lung CD8⁺ T_RM cells showed a good congruency. Genes associated with tissue-residency, including Cdh1, Itga1, Itgae, Rgs1, and Rgs2, were specifically upregulated in the CD69⁺ population, while genes associated with circulating memory CD8⁺ T cells, including tissue-egress factors (e.g., S1pr1 and S1pr5), were substantially downregulated in these cells (Figure 1B). Overall, out of 35 genes of the T_RM core signature, 20 were significantly up- or downregulated in the CD69⁺ CD8⁺ T cell compartment compared to the CD69⁻ CD8⁺ T cell pool from the lung. Importantly, the transcription factor Hobit (encoded by Zfp683), which we have recently identified as a key regulator of tissue-residency (20), was also significantly upregulated in lung CD8⁺ T_RM cells, as compared to the circulating memory CD8⁺ T cell population in the lung (Figures 1B,C). In contrast, expression levels of the related transcription factor Blimp-1 (encoded by Prdm1) were not significantly different between the two memory subsets (Figure 1C). Members of the common γ-chain cytokines, in particular IL-2, IL-7, and IL-15, play an important role in the maintenance of memory CD8⁺ T cells. In the lung, both circulating and resident memory CD8⁺ T cells expressed the individual components of the IL-7 (Il7r, Il2rg) and IL-15 receptor (l2rb, Il2rg) (Figure 1D). In contrast, the alpha chain of the IL-2 receptor was upregulated in lung CD8⁺ T_RM cells compared to CD69⁻ memory CD8⁺ T cells in the lung. Furthermore, in comparison to their circulating counterparts in the lung, the CD69⁺ lung T_RM cells expressed significantly higher levels of pro-inflammatory cytokines, chemokines and cytotoxic mediators, including colony-stimulating factor-1 (Csf1), lymphotactin (Xcl1) granzyme B (Gzmb) and TNF-related apoptosis-inducing ligand (TRAIL, Tnfsf10), indicative of a poised effector state (Figures 1E–G). Taken together, influenza virus infection induced a distinct population of CD69⁺ CD8⁺ T cells in the lungs, which were identified as bona fide T_RM cells by transcriptional analysis. Importantly, these lung-resident CD8⁺ T cells exhibited elevated transcript levels of effector molecules and were characterized by high expression of the T_RM-associated transcription factor Hobit.

Given its selective expression in lung CD8⁺ T_RM cells, we hypothesized that Hobit may contribute to the development of these cells. In other tissues, including the skin, liver, kidney, and small intestine, Hobit regulates the generation and/or maintenance of CD8⁺ T_RM cells together with its homolog Blimp-1 (20). In order to investigate the role of these two transcription factors in the development of lung CD8⁺ T_RM cells, mixed bone marrow (BM) chimeric mice were generated, containing a WT compartment and a compartment lacking functional Hobit and Blimp-1 (double knock-out, DKO) (Figure 2A). An approach with mixed BM chimeric mice was chosen to minimize indirect effects on CD8 T cell differentiation through differences in viral clearance. Mice were infected intranasally with HKx31 virus, and the virus-specific (D^b NP366⁺) CD8⁺ T cell response was analyzed over time. Previous studies have demonstrated a critical role for Blimp-1 in terminal effector cell (TEC) differentiation (24, 25). In line with these findings, analysis of virus-specific D^b NP366⁺ CD8⁺ T cells in the blood at the peak of the anti-viral effector CD8⁺ T cell response (day 10 p.i.) revealed a substantial decrease in KLRG1⁺ CD127⁻ TECs in the DKO compared to the WT compartment (Figures 2B–D). Concomitantly, D^b NP366⁺ cells deficient for both Hobit and Blimp-1 exhibited a sharp increase in CD127⁺ KLRG1⁻ memory precursor effector cells (MPECs) compared to their WT counterparts (Figures 2C,D). In lung tissue, a distinct CD69⁺ population was already observed at the effector stage, while CD103 expression was minimal (Figure 2F). Both the WT and the DKO compartment gave rise to similar frequencies of CD69⁺ CD103⁻ and CD69⁺ CD103⁺ cells at this stage, suggesting little impact of Hobit and Blimp-1 deficiency on the formation of these cells (Figures 2E–G). In contrast, D^b NP366⁺ DKO cells generated less T_RM cells in the lung at the memory phase than their WT counterparts (Figures 2H,I). This defect was most pronounced for CD69⁺ CD103⁺ cells, which were decreased in both frequencies and absolute numbers in the DKO compartment compared to the WT compartment (Figures 2I,J). Interestingly, DKO cells formed CD69⁺ CD103⁻ T_RM cells at near similar frequencies as WT cells, indicating little effect of combined Hobit and Blimp-1 deficiency on the generation of this population (Figures 2I,K). Apart from CD69 and CD103, CD8⁺ T_RM cells across tissues express additional tissue-residency markers, including the chemokine receptor CXCR6 and the integrin CD49a (26–29). Influenza-virus-specific WT CD8⁺ T cells in the lungs co-expressed CXCR6 and CD49a at similar frequencies as the residency marker CD69, suggesting that both molecules also identify CD8⁺ T_RM cells in this tissue (Figures 2L,M). Interestingly, combined deficiency for Hobit and Blimp-1 impaired the formation of CXCR6⁺ CD49a^high cells, which were decreased in both frequencies and absolute numbers in the DKO compartment compared to the WT compartment (Figures 2L,M). In all, these results show that the combined genetic ablation of Hobit and Blimp-1 results in reduced TEC and enhanced MPEC formation during the effector CD8⁺ T cell response, and impairs the generation of CD103⁺ lung T_RM cells in the memory CD8⁺ T cell response.

Apart from CD8⁺ T_RM cells in the lung, influenza virus infection also induces the formation of circulating effector memory (T_EM) and central memory (T_CM) CD8⁺ T cells (30, 31). To assess the impact of co-deficiency of Hobit and Blimp-1 on these circulating memory subsets, we analyzed virus-specific (D^b NP366⁺) WT and DKO cells isolated from secondary lymphoid organs of mixed BM chimeric mice after HKx31 infection. In both spleen and lung-draining lymph nodes (mediastinal lymph nodes, mLN), virus-specific CD44^high CD62L⁺ CD8⁺ T_CM cells were present at elevated levels in the DKO compartment compared to the WT compartment (Figures 3A,B), both in frequencies and in absolute numbers. In contrast, no effect of Blimp-1 and Hobit deficiency was observed in the CD44^high CD62L⁻ CD8⁺ T_EM subset, as these cells were present in both the WT and the DKO compartment in similar numbers (Figure 3C). Taken together, these data suggest that Hobit and/or Blimp-1 not only drive the formation of CD8⁺ T_RM cells, but also suppress the development of CD8⁺ T_CM cells after influenza virus infection.

We next investigated whether Hobit and Blimp-1 collaborated in the development of lung CD8⁺ T_RM cells, as observed in other tissues (20), or whether either one was the dominant transcription factor driving CD8⁺ T_RM formation in the lungs. To this end, three groups of mixed BM chimeric mice were generated, consisting of one control group, containing two WT compartments (CD45.1 and CD45.2) and two experimental groups, containing a WT (CD45.1) compartment next to either a Hobit KO (CD45.2) compartment or a Blimp-1 KO (CD45.2) compartment. The mixed BM chimeric mice were infected intranasally with HKx31 virus, and the virus-specific (D^b NP366⁺) CD8⁺ T cell response was analyzed over time. As expected, similar to cells lacking both functional Hobit and Blimp-1 (Figures 2C,D), D^b NP366⁺ Blimp-1 KO cells contained elevated frequencies of CD127⁺ KLRG1⁻ MPECs at the peak of the anti-viral CD8⁺ T cell effector response (Figures 4A,B). Moreover, D^b NP366⁺ Blimp-1 KO cells nearly lacked KLRG1⁺ CD127⁻ TECs, which was also observed for cells with combined deficiency for Hobit and Blimp-1 (Figures 2C,D). Neither of these phenotypes was observed for D^b NP366⁺ Hobit-deficient cells, which contained TECs and MPECs at similar frequencies as their WT counterparts (Figures 4A–C). These data indicate a dominant role of Blimp-1 over Hobit in regulating the balance between terminal effector and memory precursor cell differentiation during the effector response. After clearance of the infection, the D^b NP366⁺ Hobit KO compartment exhibited no defects in the formation of CD8⁺ T_RM cells in the lung (Figures 4D,E). In contrast, the Blimp-1 deficient compartment of virus-specific cells was severely impaired in the formation of CD69⁺ CD103⁺ CD8⁺ T_RM cells (Figures 4D,E). As previously observed for cells with combined deficiency for Hobit and Blimp-1, D^b NP366⁺ Blimp-1 KO cells did not exhibit a substantial defect in generating CD69⁺ CD103⁻ cells in the lung (Figure 4F). Thus, Blimp-1, but not Hobit, appears to be essential for the formation of terminal effector cells and CD69⁺ CD103⁺ CD8⁺ T_RM cells in the lung.

We next considered the role of Blimp-1 and its homolog Hobit in the formation of other memory CD8⁺ T cell subsets. Consistent with previous research demonstrating the repressive role of Blimp-1 in the formation of CD8⁺ T_CM cells (24, 25), we observed elevated frequencies of CD44^high CD62L⁺ D^b NP366⁺ cells in the Blimp-1 KO compartment of mixed BM chimeras already during the effector CD8⁺ T cell response (Figures 5A,B). Importantly, Blimp-1 deficiency resulted in a strong increase in the frequency of D^b NP366⁺ CD8⁺ T_CM cells in the circulation, lung and secondary lymphoid organs during the memory phase (Figures 5C–E). This effect was not observed for cells lacking functional Hobit, neither during the effector nor the memory response (Figures 5B,D). Consequently, these data suggest that Blimp-1 suppresses CD8⁺ T_CM development as early as in the effector response, while Hobit does not appear to have an essential role in this process.

The transcription factors Hobit and Blimp-1 regulate the development of CD8⁺ T_RM cells in part by suppressing genes, which are incompatible with the establishment of tissue-residency (20). A direct target of both Hobit and Blimp-1 is T-cell factor 1 (TCF-1). TCF-1 is a nuclear effector of the canonical Wingless/Integration 1 (Wnt) signaling pathway and constitutes an essential transcription factor for the development of CD8⁺ T_CM cells (32, 33). Given the enhanced formation of CD8⁺ T_CM cells upon genetic ablation of Blimp-1, we analyzed TCF-1 expression in different memory CD8⁺ T cells subsets arising after influenza infection. As expected, WT D^b NP366⁺ CD8⁺ T_CM cells exhibited high uniform expression of TCF-1 at the protein level, while virus-specific WT CD8⁺ T_RM cells in the lung showed markedly lower expression of TCF-1 (Figures 6A,B). Hobit deficiency did not significantly impact TCF-1 expression in both CD8⁺ T_CM and T_RM populations. Loss of Blimp-1 had no effect on the high levels of TCF-1 expression in CD8⁺ T_CM cells. However, Blimp-1 deficiency resulted in strongly increased TCF-1 protein expression in the remaining virus-specific CD8⁺ T_RM cells present in the lung (Figures 6A,B). These results indicate that Blimp-1 mediates suppression of TCF-1 expression in lung CD8⁺ T_RM cells, which may contribute to the instruction of CD8⁺ T_RM development in the lungs.

C57BL/6JRj (CD45.2⁺ WT) mice were purchased from Janvier and B6.SJL-Ptprc^a Pepc^b/BoyJ (CD45.1⁺ WT) mice from the Jackson Laboratory. Both lines were crossed to generate B6.SJL-Ptprc^a Pepc^b/BoyJ x C57BL/6JRj mice. Zfp683^−/− (Hobit KO) (61), Prdm1^flox/flox × Lck Cre (Blimp-1 KO) mice (24) and Zfp683^−/− × Prdm1^flox/flox × Lck Cre mice (20) were maintained on a C57BL/6JRj background. For the generation of mixed bone marrow (BM) chimeras, CD45.1 × CD45.2 (B6.SJL-Ptprc^a Pepc^b/BoyJ × C57BL/6JRj) recipient mice were irradiated (2x 5 Gy) and reconstituted with i.v. transfer of 10^*10⁶ BM cells per genotype. Mixed BM chimeras were used 12–16 weeks after reconstitution and chimerism of lymphocytes was confirmed prior to usage. All mice were maintained under SPF conditions in the animal facility of the Netherlands Cancer Institute (NKI). Animal experiments were conducted according to institutional and national guidelines.

Mice were infected intranasally with 100x TCID₅₀ (median tissue culture infectious dose) of HKx31 influenza A virus in a volume of 50 μl after anesthetization by inhalation of isoflurane. HKx31 virus was kindly provided by Dr. Guus Rimmelzwaan (University of Veterinary Medicine Hannover). At the indicated time points after infection, mice were sacrificed, and tissues were harvested for analysis of CD8⁺ T cell responses.

Single cell suspensions from spleen and lymph nodes were prepared by mechanical disruption via passing of the tissues over a 70 μm cell strainer. Lung tissue was cut into pieces of 1 mm² and enzymatically digested for 30 min at 37°C with 750 U ml⁻¹ Collagenase Type I (Worthington) and 0.31 mg ml⁻¹ DNase I (Roche, from bovine pancreas, grade II) in RPMI 1,640 supplemented with 10% (v/v) fetal calf serum (FCS). Single cell suspensions were generated by filtering over a 70 μm cell strainer and the isolated lymphocytes were purified by density centrifugation on a 66/44% Percoll gradient (GE Healthcare). Lung lymphocytes were extracted from the interphase of the Percoll gradient. Contaminating erythrocytes were removed using red blood cell lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA).

Cells were incubated with antibodies and tetramers for 25 min at 4°C and washed with PBS supplemented with 0.5% (v/v) FCS. The following anti-mouse monoclonal antibodies were used: anti-CD3 (17A2), anti-CD4 (RM4-5), anti-CD8a (53–6.7), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD49a (Ha31/8), anti-CD62L (MEL-14), anti-CD69 (H1.2F3), anti-CD103 (M290), anti-CD127 (A7R34), anti-CXCR6 (SA051D1), anti-KLRG1 (2F1), anti-TCF-1 (S33-966), mouse IgG1, κ isotype control (P3.6.2.8.1). Antibodies were purchased from BioLegend, eBiosciences, BD Biosciences, or BD Pharmingen. Influenza-virus-specific CD8⁺ T cells were detected using H-2 D^b ASNENMETM (NP_366−374) tetramers (D^b NP366), which were a kind gift of Dr. Anja ten Brinke (Sanquin Research). Exclusion of dead cells was performed with live/dead fixable near-IR dead cell stain kit (Thermo Fisher Scientific). For staining of intracellular molecules, the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) was used according to the manufacturer's specifications. Samples were acquired on an LSR Fortessa flow cytometer (BD) and data was analyzed using FlowJo V10 (Tree star) software.

A subset of previously published RNA-seq (CEL-seq) data (geo accession number GSE79774) (23) was reanalysed. DMSO-treated CD69⁺ and CD69⁻ memory (CD44^high CD62L^lo) CD8⁺ T cells isolated from murine lungs at day 30+ after HKx31 infection were analyzed to determine the tissue-resident phenotype of these populations. Reads were first aligned to mm10 genome with STAR, followed by read count quantification with featureCounts using Ensembl's v92 annotation with modification of Zfp683. Since CEL-seq was used, only the 3′ end of the genes is sequenced, and inspecting the alignments manually revealed that the reads that mapped to Zfp683 were outside Ensembl's defined region of the gene. We therefore extended the 3′ end of Zfp683 with 1,057 nucleotides to include all reads of this gene. To find differentially expressed (DE) genes, first low expressed genes were removed (retained genes had more than 1 cpm (counts per million) in at least four samples), followed by limma voom and quantile normalization. A linear model was fitted to each gene using a design that also included mouse (paired) and library (batch) effect, and eBayes moderated t-statistics were used to find DE genes between CD69⁺ and CD69⁻ memory CD8⁺ T cells. Benjamini-Hochberg procedure was used for FDR correction, and adjusted P < 0.05 were considered significant.

For pairwise comparisons, a standard two-sided Student's t-test (paired), was applied with GraphPad Prism 6 software. P < 0.05 were considered statistically significant (^*P < 0.05; ^**P < 0.01; ^***P < 0.001). The Morpheus software (https://software.broadinstitute.org/morpheus), developed by the Broad Institute, was used to generate heat maps. Values were converted to heat map colors using the mean and maximum values for each row.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.