Persistent B Cell–Derived MHC Class II Signaling Is Required for the Optimal Maintenance of Tissue-Resident Helper T Cells

Abstract Emerging studies have identified the critical roles of tissue-resident memory CD8+ T (TRM) and B (BRM) cells in the protection against mucosal viral infections, but the underlying mechanisms regulating robust development of TRM and BRM cells remain incompletely understood. We have recently shown that tissue-resident helper CD4+ T (TRH) cells, developed following influenza virus infection, function to sustain the optimal maintenance of TRM and BRM cells at the mucosal surface. In this study, we have explored the cellular and molecular cues modulating lung TRH persistence after influenza infection in C57BL/6 mice. We found that TRH cells were colocalized in tertiary lymphoid structures (TLSs) with local B cells. Abolishing TLSs or the depletion of B cells impaired lung TRH cell numbers. Of note, we found that persistent TCR signaling is needed for the maintenance of TRH cells after the clearance of infectious influenza virus. Furthermore, selective ablation of B cell–derived MHC class II resulted in partial reduction of lung TRH cell number after influenza infection. Our findings suggest that the interaction between lung-resident TRH cells and B cells, along with persistent Ag stimulation, is required to maintain TRH cells after respiratory viral infection.


INTRODUCTION
A fundamental characteristic of the adaptive immune system is its capacity to establish immunological memory following the initial encounter with Ags.Upon secondary infection caused by the same virus or viruses harboring conserved T cell epitopes, memory T cells rapidly activate, undergo secondary effector T cell expansion and differentiation, and expeditiously mediate the clearance of pathogens before they can spread systemically.
Besides the circulating memory T and B cells that patrol the whole body, studies in the past decade have also established the presence of mucosal-residing tissue-resident memory T (T RM ) and B (B RM ) cells, which provide immediate and superior protection against reinfection at the pathogen entry site (1,2).For instance, we and others have previously reported the powerful effects of mucosal protection mediated by T RM and B RM cells against influenza viral infections (38).Notably, recent studies have also indicated that robust induction of mucosal T RM and B RM cells after mucosal vaccination may provide superior protection against SARS-CoV-2 reinfection (912).
Previously, we have found a population of lung PD1 hi FR4 hi tissue-resident helper CD4 1 T (T RH ) cells following influenza virus infection and demonstrated that T RH cells play a vital role in supporting the development and/or maintenance of CD8 1 T RM and B RM cells in the respiratory tract (5).T RH cells coexhibit features of follicular helper T cells and resident memory T cells, and their development is dependent on the transcription factors BCL6 and Bhlhe40 (5).Interestingly, T RH cells are colocalized with B cells in lung tertiary lymphoid structures (TLSs) after influenza infection (5,13).TLSs are ectopic lymphoid organs that develop in nonlymphoid tissues at sites of inflammation (14,15).In the respiratory tract, certain types of TLSs have been reported as inducible bronchus-associated lymphoid tissue (iBALT), which can be detected in the lungs after exposure of pathogens, allergens, and harmful particulates (16,17).
Given the important roles of T RH cells in the development local CD8 and B cell immunity in the respiratory mucosa, it is critical to further dissect the underlying cellular and molecular mechanisms regulating T RH development and/or maintenance.Previously it was shown that B cells and persistent MHC class II (MHCII) signaling are critical for T RH maintenance (13), but the cell types that provide the persistent MHCII signaling to sustain T RH cells are currently unknown.Furthermore, despite their localization in iBALT, the roles of iBALT in maintaining T RH cell development and phenotypes are unknown.In this study, we have gone on to examine the roles of Ag persistence, B cells, and TLSs in regulating the development and maintenance of T RH cells after influenza infection.We found that T RH cells were colocalized mainly in iBALT with B cells, and abolishing TLSs or the depletion of B cells impaired lung T RH cell persistence.Furthermore, we found that persistent TCR signaling was needed for the maintenance of T RH cells, and selective ablation of B cellderived MHCII caused partial reduction of lung T RH levels after influenza infection.Our findings have revealed new insights into the regulation of T RH cells in respiratory traction after viral infection.
Intravascular labeling with anti-CD45 and preparation of lung cell suspension Mice were i.v.injected with 2 mg of anti-CD45 (clone 30-F11; Tonbo Biosciences), which was diluted in 300 ml of sterile PBS 5 min before sacrificing the mice.To prepare single cells from the lung tissue, the lung was cut into small pieces, digested with type 2 collagenase (Worthington Biochemical), and dissociated at 37 C for 30 min using gentleMACS (Miltenyi Biotec).The cells were further processed through a 70-mm cell strainer (Falcon) and washed with plain IMDM (Gibco).After red blood cell lysis, the cells were centrifuged and resuspended in cold FACS buffer (PBS, 2 mM EDTA, 2% FBS, and 0.09% sodium azide) for flow cytometry analysis.Lung circulating immune cells are i.v.Ab 1 , and lung tissue immune cells are defined as i.v.Ab À .

Ab administration in vivo
Influenza-infected WT mice were treated with control IgG or various neutralizing or depleting Abs as described in the corresponding results sections.Lymphotoxin-b receptor Ig fusion (LTbR-Ig; 250 mg) obtained from Dr. Yangxin Fus laboratory (20) was administered for TLS elimination, and 500 mg of CD20 Ab (clone 5D2, Genentech) was administered for B cell depletion by i.p. injection at 14 and 21 days postinfection (dpi).For neutralizing MHCII signaling, mouse MHCII (clone M5/114, Bio X Cell) Ab was injected into WT mice.The first dose was 1 mg at 14 dpi, and the second dose was 0.5 mg at 21 dpi FTY720 (1 mg/kg; Cayman Chemical) administrated via i.p. injection daily from 13 dpi onward to block lymphocyte migration until the mice were sacrificed (5).

Tamoxifen treatment
To induce gene recombination in MHCII fl/fl Ubc CreERT2 mice, tamoxifen (Sigma-Aldrich) was diluted in warm sunflower oil (Sigma-Aldrich) and daily administered (2 mg per mouse) via the i.p. route for 5 consecutive days.

Immunofluorescence
The left lobe of the whole lung was harvested and fixed in 4% paraformaldehyde solution overnight at 4 C.The fixed sample was sequentially incubated in 15 and 30% sucrose solutions in PBS for 12 h each.Subsequently, the sample was embedded with OCT compound (Sakura Finetek) and stored at 80 C. For Ab staining and immunofluorescence imaging, lung sections were blocked with SuperBlock blocking buffer (Thermo Fisher Scientific) for 1 h at room temperature.B220 eFluor 660 (clone 4SM95, Invitrogen), CD4 eFluor 570 (clone RA3-6B2, Invitrogen), and/or GL7 Alexa Fluor 488 (clone GL7, BioLegend) Abs were stained on the lung tissue sections overnight at 4 C.After washing in 0.1% PBST (PBS with Tween 20), the slides were counterstained with DAPI and mounted.Tissue staining was observed, and representative images were captured using a Zeiss LSM 780 confocal system (Carl Zeiss).

B cell Ags
The influenza PR8-hemagglutinin (HA) protein was a gift from M.C.Crank (National Institutes of Health).PR8-nucleoprotein (NP) was purchased from Sino Biological.Purified Ags were biotinylated using an EZ-Link sulfo-NHS-LC biotinylation kit (Thermo Fisher Scientific) with a biotin-to-Ag ratio of 1:1.3 M. To create tetramers, biotinylated Ags were combined with streptavidin-PE (PJ27S; ProZyme) at the predetermined ratio or a 5:1 ratio based on the biotin concentration provided by the manufacturer, as described previously (21).After a 30-min incubation on ice, any unconjugated biotinylated Ag was removed by several rounds of dilution and concentration using a 100-kDa Amicon ultra (MilliporeSigma) or 300-kDa Nanosep centrifugal devices (Pall).The tetramers were stored at 1 mM in 1× Dulbeccos PBS at 4 C before use.

Mixed bone marrow chimera generation
For the generation of mixed bone marrow chimeric mice, CD45.1 recipient mice were lethally irradiated (1100 rad).Mixed donor cells were prepared by mixing 1 × 10 6 bone marrow cells from MHCII fl/fl Ubc CreERT2 or WT mice with 4 × 10 6 bone marrow cells from mMT mice.These donor cells were i.v.injected into the irradiated CD45.1 recipient mice.Experimental chimeric mice were infected with PR8 at 8 wk after reconstitution.

Quantitative RT-PCR
To measure the expression levels of Il21, Bcl6, Cxcr5, and Foxp3, WT or IL-21 VFP reporter mice were infected with influenza PR8 for 28 d.Then, the indicated cells were sorted out from CD45 i.v.

Statistical analysis
Graphs were generated using the GraphPad Prism software.Statistical significance was evaluated by calculating p values using a paired or unpaired Student t test (two-tailed).Differences with p values <0.05 were considered statistically significant.

TLS is required for IL-21 hi CD4 1 T RH cell maintenance
We have previously demonstrated that CD45 i.v.
PD1 hi FR4 hi T RH cells expressed BCL6 and high levels of IL-21 following influenza virus infection (5).Furthermore, BCL6 1 T RH cells have been identified inside TLSs, whereas T-bet 1 T H1 cells were located outside of this structure at 3060 dpi (13).However, whether TLS is required for T RH cell maintenance is not clear.To address the question, we first assessed the characteristics of T RH cells following influenza virus infection.Lung total T RH and non-T RH (CD45 i.v.À CD4 1 CD44 1 PD1 lo FR4 lo ) cells were sorted (Supplement Fig. 1A), and the gene expression levels of Il21, Bcl6, Cxcr5, and Foxp3 were measured by quantitative RT-PCR.In line with previous studies (5, 13), Il21, Bcl6, and Cxcr5 were highly expressed in T RH cells, but these cells also expressed higher levels of Foxp3, compared to nonT RH cells, potentially due to contamination of small percentages of regulatory T cells in this gating strategy (Fig. 1A).
Next, we sorted IL-21 lo , IL-21 int , and IL-21 hi cells (Supplement Fig. 1B) from CD45 i.v.À CD4 1 GITR À CD44 1 lung cells from influenza-infected IL-21-VFP reporter mice (at 28 dpi).We found that IL-21 hi cells exhibited the highest expression levels of Il21, Bcl6, and Cxcr5 but not Foxp3, compared with other cell populations (Fig. 1B), suggesting that T RH cells but not regulatory T cells express IL-21 in the lung after influenza infection.
To assess the colocalization of IL-21 1 T RH and local B cells, we conducted confocal microscope imaging.Our observations revealed that most IL-21 1 CD4 1 T cells were located near B cells within the TLS (Fig. 1C).This led us to investigate the requirement of a TLS for the maintenance of T RH cells after influenza infection.To this end, we eliminated the TLS using LTbR-Ig treatment, which is known to be able to destroy TLSs in various models (2325).We administered LTbR-Ig at 14 and 21 dpi and observed a clear removal of TLSs in the lung at 28 dpi compared to the IgG-treated control group (Fig. 1D).Notably, influenza nucleoprotein epitope (NP 311325 )specific T RH cells were significantly decreased in the LTbR-Igtreated group compared to the control group, while Ag-specific nonT RH cells and spleen follicular helper T (T FH ) cells showed no significant differences between the two groups (Fig. 1EF).In our previous study, we showed that T RH cells supported the formation of germinal center (GC) B cells (GL7 1 CD38 À ) and HA-specific tissue-resident memory B cells (B RM : IgD À IgM À CD38 1 HA 1 ), while aiding the maintenance of CD8 1 T RM cells.Correspondingly, decreased T RH cells resulted in impaired adaptive immunity, rendering the host significantly vulnerable to heterologous influenza reinfection (5).Therefore, we examined tissue B cell persistence and found a significant decrease of lung-resident B (CD45 i.v. À B220 1 ), GC B, and HA 1 B RM cells in lungs from LTbR-Igtreated mice (Supplement Fig. 2).These results suggest that TLS contributes to the maintenance of Ag-specific T RH cells following influenza virus clearance.

Tissue-resident B cells are involved in the maintenance of T RH cells
The diminished T RH cell numbers following TLS ablation led us to examine whether lung B cells are required for the maintenance of T RH cells.To this end, we conducted B cell depletion after influenza infection.To exclude the potential effects of circulating B cells in affecting T RH cells, we treated the mice with anti-CD20 in the presence of daily injection of FTY720, which prevents lymphocyte egress from lymphoid tissues (5), starting from 13 dpi (Fig. 2A).Treatment with anti-CD20 dramatically depleted both lung-resident B and spleen B cells, while not affecting the lung and spleen total CD4 1 T cell population (Fig. 2B).However, B cell depletion in the lung led to a significant decrease in the number of lung NP-specific T RH cells compared to the control group, whereas no significant differences in the lung NP-specific non-T RH and spleen T FH were observed between the IgG and anti-CD20 groups (Fig. 2C).These results indicate that local tissue-resident B cells are involved in the maintenance of Ag-specific T RH cells.
Persistent A stimulation is required to maintain T RH cells in the lung Infectious influenza virus is usually cleared in the respiratory tract by the immune system within 10 dpi (26,27), but influenza Ag, particularly the NP protein, appears to be persistent for a couple of months after infection.Because prior studies have suggested that persistent MHCII signaling may be required for the maintenance of T RH cells (13), we explored whether persistent TCR signaling is required for the generation or maintenance of T RH cells.Reanalysis of our previously published bulk RNA sequence data (Gene Expression Omnibus: https://www.ncbi.nlm.nih.gov/geo/, GSE153226) (5) found that lung T RH cells were enriched with TCR signaling-related genes compared to lung non-T RH or spleen T FH cells (Fig. 3A).To further investigate whether T RH cells continuously receive TCR signaling after viral clearance, we infected Nur77-GFP mice with influenza virus and measured Nur77-GFP expression levels.Nur77 is a downstream signaling molecule activated following TCR stimulation (3), and its expression was widely used as a surrogate of TCR signaling (3,28).Both lung total T RH and NP-specific T RH cells exhibited significantly higher levels of Nur77-GFP expression compared to nonT RH cells (Fig. 3B, 3C).These results support the hypothesis that lung T RH cells exhibit ongoing TCR stimulation.
To explore this hypothesis further, we conducted repeated Ag stimulation test and measured the endogenous NP-specific T RH cells and transferred OTII-specific T RH cells (Supplement Fig. 3).CD45.1 1 OTII cells were transferred into CD45.1 1 /2 1 host mice, and the mice were infected with PR8 virus expressing the chicken OVA 323339 (OTII) peptide (PR8-OTII) (19) 1 d later.OVA or PBS was intranasally injected every 4 d from 14 to 26 dpi (Fig. 3D).The total numbers of lung-resident endogenous NP-specific and transferred OTII-specific CD4 1 T cells have shown no differences between the OVA-or PBStreated group in either the lung or spleen (Fig. 3E).The number of endogenous NP-specific lung T RH cells and spleen T FH cells also showed no differences between the PBS-and OVAtreated groups.Interestingly, OTII-specific lung T RH cells were not detected in the PBS injection group likely due to the lack of Ag persistence, as the OTII peptide was inserted to the less abundant H1 protein locus rather than the NP locus (which is a more abundant protein during infection) (19,29,30).Nevertheless, repeated OVA treatment in the lung fostered T RH development within OTII cells (Fig 3FG ), suggesting that persistent Ag stimulation promotes T RH development after respiratory viral infection.

Persistent local MHCII signaling is required to maintain Ag-specific T RH cells following virus infection
Ag-loaded MHCII is the major signal to stimulate CD4 1 T cells, and the deficiency of MHCII signaling reduced the number of T RH cells in the lung (13).However, whether lungresident MHCII signaling is required for T RH cell maintenance is unclear.Therefore, to examine the roles of local MHCII signaling in sustaining lung Ag-specific T RH cells, we blocked the ImmunoHorizons MHCII signaling at 14 and 21 dpi together with FTY720 treatment (Fig. 4A).The numbers of lung NP-specific T RH cells and HA 1 B RM cells were significantly reduced in the group with MHCII signaling blockade compared to those of the control group (Fig. 4B, 4C).These findings indicate that local MHCII signaling contributes to the formation of lung Ag-specific T RH and B RM cells.

MHCII signaling from B cells is required for the optimal maintenance of Ag-specific T RH cells
To specifically investigate the role of the MHCII signal from B cells in the maintenance of Ag-specific T RH cells, we generated a mixed bone marrow chimera mouse model, in which we can specifically deplete MHCII in B cells in an inducible fashion (Fig. 5A).Lethally irradiated mice were reconstituted with bone marrow cells from mMT mice (lacking B cells [31]) mixed with MHCII fl/fl Ubc CreERT2 (termed the MHCII À/À group) or WT mice bone marrow (WT group) at 4:1 ratio.After 8 wk of reconstitution, the mice were infected with influenza virus.Tamoxifen was administered consecutively for 5 d at 1216 dpi to deplete MHCII in B cells (Fig. 5A).The expression of MHCII was dramatically reduced at lung-resident B, lung GC B, spleen B, and spleen GC B cells in the MHCII À/À group, whereas dendritic cells (DCs) still expressed comparable levels of MHCII between the WT and MHCII À/À groups (Fig. 5B), confirming the successful achievement of B cellspecific MHCII deficiency.In this animal model, we found that lung GC B cells were decreased in the MHCII À/À group compared to the WT group, and lung NP-specific T RH cells were moderately affected after MHCII ablation in B cells (Fig. 5C, 5D).Thus, we

DISCUSSION
T RH cells are essential for local adaptive immunity, especially after respiratory viral infections.They help maintain T RM cells after viral clearance and support local GC B and optimal B RM cells.Consistent with this notion, there is evidence of IL-21 from local CD4 1 T cells influencing the development of T RM cells in the brain (32).Interestingly, we found IL-21 VFP 1 T RH cells interacting with B cells in the TLS at 28 days after influenza infection.The importance of TLS in the respiratory tract, especially the iBALT generated after viral infection, has been emphasized recently (17).We noted a marked reduction in T RH cell numbers when TLS was disrupted, suggesting that T RH cell development and/or maintenance require the support of local environment at the TLS.
Persistent TCR-MHC signaling has been suggested to impact the formation of T RM cells.Blocking MHCI or lacking Nurr77 results in fewer T RM cells after influenza infection (3).Furthermore, continuous TCR signaling aids T RM cell tissue migration while inhibiting their exit to blood (33).Analyzing RNA sequencing data, we observed that T RH cells displayed high levels of TCR signaling molecules such as Nur77, hinting at the necessity of persistent Ag stimulation for T RH cell maintenance.We further delved into the cellular mechanisms that uphold T RH cells within the TLS after viral infections and found that B cell MHCII was required for the optimal maintenance of T RH cells.This is not particularly surprising given that B cell Ag presentation is needed for the generation and maintenance of T FH cells in the secondary lymphoid organ.Notably, B cell MHCII deficiency only partially impaired T RH cell maintenance, while the blockade of MHCII signaling from all cell types greatly abrogated T RH cell preservation (13,34,35).These data suggest that persistent Ag presentation by other cells is likely also involved in the maintenance of T RH cells.Studies have pinpointed CD11c hi DCs in TLS formation and lung epithelial cells in forming T RM cells (16,36).Therefore, it is possible that DCs or the epithelial cellderived Ag/MHCII complex may also be involved in maintaining T RH cells.Such possibilities require future investigations.In conclusion, in this study, we found that sustaining T RH cells after local viral clearance relies on the presence of TLS and continuous Ag stimulation.Given the importance of mucosal immunity in rapid constraining respiratory viral dissemination and the emerging roles of T RH cells in regulating mucosal immune cells, we propose that innovative immunization strategies, offering TLS biogenesis and prolonged local Ag release, may be the key for the success of mucosal vaccines against respiratory viral infections through the bolstering of local T RH and memory B and CD8 1 T cells.

Limitations of the study
Although we used OTII peptide administration to induce persistent TCR signaling in OTII cells after PR8-OTII infection, we have not confirmed whether persistent TCR signaling was indeed induced in OTII cells and, if so, how long the signaling lasted.Further studies using the transfer Nur77-GFP OTII cells to report TCR signaling in vivo after OTII peptide inoculation could address this limitation.Additionally, B cells are the major cell types forming TLSs in the respiratory tract, and B cell depletion with anti-CD20 treatment is expected to impair TLS formation (PMID: 28355561).Thus, it is possible that the diminished T RH cells after anti-CD20 treatment are due to the absence of TLS, rather than the effects of the absence of direct BT cell communication (such as Ag presentation).This possibility necessitates further studies in the future.

DISCLOSURES
The authors have no financial conflicts of interest.

FIGURE 1 . 1 CD44 1
FIGURE 1. Features and localization of IL-21 + T RH cells.(A)IL21, Bcl6, Cxcr5, and Foxp3 were measured by quantitative RT-PCR in the T RH (CD45 i.v.À CD4 1 CD44 1 PD1 hi FR4 hi ) or non-T RH (CD45 i.v.À CD4 1 CD44 1 PD1 lo FR4 lo ) cells sorted from influenza-infected mice (pooling lung cells of 12 mice).(B) Expression of the indicated genes was examined in IL-21 hI , IL-21 int or IL-21 lo cells that were sorted from lung CD45 i.v.À CD4 1 CD44 1 GITR À cells of influenza-infected mice (pooling cells of 10 mice).(C) Lung IL-21 1 CD4 1 T cells were detected with B cells in TLSs from influenza-infected IL-21 VFP mice (green, IL-21 VFP; red, CD4; blue, B220).The representative image is from at least two independent experiments.(D-F) Control IgG or LTbR-Ig was i.p. injected into C57BL/6 WT mice at 14 and 21 dpi, after which the number of lung NP-specific T RH , non-T RH , and spleen T FH cells were observed.The representative image (D) and dot plot (E) from at least two independent experiments (three to four mice per group) are shown.Scale bars, 10 mm (C), 50 mm (D).All experiments were conducted at 28 dpi.Statistical analysis was performed with an unpaired Student t test.**p < 0.01.

FIGURE 2 .
FIGURE 2. Tissue-resident B cells are required for the maintenance of T RH cells.WT mice were infected with influenza.(A) WT mice were treated with anti-CD20 or IgG at 14 and 21 dpi and FTY720 was administered daily from 13 dpi onward.(B) Top, Lung B and CD4 1 T cells.Bottom, Spleen B and CD4 1 T cells.(C) Lung NP-specific T RH , non-T RH , and spleen T FH cells were measured by flow cytometry.The mice were sacrificed at 28 dpi.Pooled results from two independent experiments (each group n 5 3-4) are shown.Statistical analysis was performed with an unpaired Student t test.*p < 0.05, ***p < 0.001.

FIGURE 3 .
FIGURE 3. Persistent Ag stimulation is required for T RH cell maintenance.(A) Enrichment of TCR signaling-related molecule expression between (left) lung T RH versus lung non-T RH cells and (right) lung T RH versus spleen T FH cells.Original bulk RNA sequencing data were extracted from GSE153226.(B and C) Expression of Nur77 was measured by flow cytometry from influenza-infected Nur77 GFP mice at 28 dpi.Pooled results from three independent experiments (each group n 5 2-3) are shown.(D) CD45.1 1 OTII cells were transferred into CD45.1 1 /2 1 host mice.One day later, mice were intranasally infected with recombinant PR8 virus with an OTII epitope (PR8-OTII).Mice were treated with OVA or PBS every 4 d from day 14 to day 26.The mice were then sacrificed at 28 dpi.(E) NP-specific or OTII-specific CD4 1 T cells in the lung (top) or spleen (bottom) were measured.(F and G) NP-specific or OTII-specific cells were analyzed in the lung (top, T RH ) and spleen (bottom, T FH ).Pooled results from two independent experiments (each group n 5 2-4) are shown.Statistical analysis was performed with a paired (C) or unpaired (G) Student t test.*p < 0.05, ***p < 0.001.

FIGURE 4 .
FIGURE 4. Requirement of local MHCII signaling for maintenance of lung Ag-specific T RH cells.WT mice were infected with influenza PR8.(A) WT mice were injected with anti-MHCII or IgG at 14 and 21 dpi together with FTY720 treatment.The mice were sacrificed at 28 dpi.(B and C) NP-specific lung T RH or spleen T FH cells, lung/spleen GC B cells, or HA 1 B RM /memory B (B MEM ) cells were measured by flow cytometry.Pooled results from two independent experiments (each group n 5 2-4) are shown.Statistical analysis was performed with an unpaired Student t test.*p < 0.05, **p < 0.01.