Stem-like memory-T maintenance and differentiation into tissue-resident T cells sustain chronic graft-versus-host disease in mice

Domain: chemistry
Published: 2026-02-24
Editorial outcome: minor_revision

Abstract
Pathogenic CD4+ memory T cells (Tm) sustain chronic inflammation, but mechanisms remain undefined. Here, we identify four donor-type CD4+ Tm subsets in the target tissues of autoimmune-like chronic graft-versus-host disease in mice: Ly108+CD69− stem-like memory T cells (Tsm), Ly108+CD69+ resident memory progenitor T cells (Trmp), Ly108−CD69+ terminally differentiated tissue-resident T cells (Trm), and Ly108−CD69− intermediate T cells (Tint). Trm are terminally differentiated but not exhausted and show highly biased clonotypes with high proinflammatory cytokine expression. Tsm cells require TCR-MHCII interactions for their maintenance and expansion and show greater capacity than Trmp cells in self-renewal/expansion, generation of Trm, and pathogenicity in adoptive recipients. The transcription factors TCF1/BCL6 and BHLHE40 differentially regulate the stemness and differentiation of Tsm into Trm, respectively, and their selective targeting reduces the number of Trm in tissues and ameliorates inflammation. Thus, our findings indicate that targeting the Tsm subset, involved in the maintenance of the pathogenic Tm pool, offers an attractive approach to treat T cell-mediated chronic inflammation.

Introduction
Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for hematological malignancies and certain non-malignant lymphohematopoietic disorders1–4, but acute graft-versus-host disease (aGVHD) and chronic GVHD (cGVHD) remain the major obstacles4,5. cGVHD is a systemic lupus-like autoimmune syndrome mediated by donor-type CD4+ T cells in the graft or generated de novo in the thymus damaged by aGVHD, as indicated in publications from our group and others during the past two decades6–13. Although murine model of C57BL/6 donor to MHC-mismatched BALB/c recipient had been considered as a model of aGVHD14, we reported that this model could also develop into cGVHD when aGVHD was not lethal9,13. cGVHD in murine models and in many humans is a sequela of aGVHD2,15. During GVHD pathogenesis, naïve donor CD4+ T cells in the graft are activated by recipient antigen-presenting cells (APC) and differentiate into effector helper T cells to mediate tissue damage16–19. Some of the donor-type CD4+ T cells that possess cross-reactive TCRs that recognized both donor- and host-type MHC-peptide complexes become autoreactive memory T cells8 and long-term tissue-resident Tm (Trm) cells in the target tissues of cGVHD20, but how those Trm cells are maintained remains unclear.

Stemness potential of CD8+ Tm cells has been reported21,22. CD8+ Tsm are characterized by expression of TCF1, Ly108, and CXCR5, with transcription factors (i.e., TCF1 and BCL6) and other factors (i.e., IFN-I, IL-27, ST2, and Bach2)23–25. TCF1 plays a fundamental role in maintaining immune response against infections and cancer by sustaining T cell capacity in proliferation, regeneration, and differentiation23,24,26. The TCF1-BCL6 axis counteract type I interferon to repress exhaustion and maintain T cell stemness27. CD69 is transiently expressed by early activated T cells but is maintained on tissue-resident T memory effector cells in association with markers such as P2RX7 and CD10328–30. Four subsets characterized by expression of Ly108 versus CD69 have been identified in a pathway leading to CD8+ exhausted Tm (Tex) in tissues of chronic infection and tumors. These include progenitor-like Ly108+CD69+ (Texprog1) and Ly108+CD69− (Texprog2), intermediate Ly108−CD69− (Texint), and terminally differentiated Ly108−CD69+ (Texterm) cells31. CD4+ TCF1+ T memory progenitors play essential roles in sustaining acute inflammation in allograft rejection and colitis32,33, but their role in sustaining autoimmune-like cGVHD pathogenesis and the mechanisms of their maintenance and differentiation in the tissues remain largely unexplored.

To understand the mechanisms that perpetuate chronic inflammation, in this study, we characterize CD4+ Tm subsets in cGVHD target tissues via combining transcriptomics, epigenomics, flow cytometry analysis, and adoptive transfer experiments. We identify four CD4+ Tm subsets: Ly108+CD69− stem-like T memory (Tsm), Ly108+CD69+ tissue-resident progenitor-like T memory (Trmp), Ly108−CD69+ tissue-resident T memory (Trm), and Ly108−CD69− intermediate T (Tint). Trm cells are the key pathogenic effector cells, and the Tsm are required to maintain enough Trm to mediate tissue inflammation. The Tsm, Trmp, and Trm cells are also observed in the liver tissues of cGVHD patients. Thus, Tsm play a critical role in perpetuating chronic inflammation, and the specific targeting of Tsm cells may represent an attractive approach in treating chronic inflammatory diseases.

Results
Four Tm cell subsets in cGVHD target tissues
BALB/c recipients engrafted with C57BL/6 donor cells developed cGVHD 30–60 days after HCT with infiltration and fibrosis in aGVHD overlapping targets such as the liver and lung and in salivary and lacrimal glands as prototypic cGVHD targets9. cGVHD and controls with no cGVHD were produced by injecting T cell-depleted bone marrow (TCD-BM) cells with or without splenic T cells from C57BL/6 donors. On day 60 after HCT, donor-type CD4+ T cells in the liver were sorted for scRNA-seq analysis. With unsupervised hierarchical clustering, we identified 7 clusters (C0-6) among CD4+ T cells (Fig. 1a). As shown in Fig. 1a, b and Supplementary Fig. 1a, b, C0 contained stem-like memory T cells (Tsm) with high expression of stemness markers25 (i.e., Tcf7, Slamf6, Id3, Lef1) and some migration markers (Cd62l and S1pr1). C1 contained naïve T cells (Tn) with high expression of stemness-related markers and resting markers (i.e., Cd62l, S1pr1 and Klf2). C2 contained intermediate T (Tint) cells34 with low expression of TCR signaling markers (i.e., Batf, Lat and Zap70) and high expression of NK markers [i.e., Klrk1 (NKG2D), Klrc1 (NKG2A) and Klrd1 (CD94)]. C3 contained tissue-resident memory T progenitors (Trmp) with high expression of stemness markers and tissue-residency markers (i.e., Bhlhe40, P2rx7). C4 contained terminally differentiated tissue-resident memory T cells (Trm) with high expression of tissue-resident markers and proinflammatory Ifng/Gzm and low expression of Tcf7. In addition, progenitor signatures were present in C0 Tsm, C1 Tn, and C3 Trmp (Fig. 1c), and strong proinflammatory signaling was present in C3 Trmp and especially in C4 Trm (Fig. 1d). The recently identified tissue-resident T helper cells (Trh)35 were mainly observed in C3 and C4, which had high expression of Pdcd1 (PD1), Icos, Maf and Il21 with low expression of Cxcr5 (Supplementary Fig. 1c). C5 and C6 T cells did not express typical markers related to Trm or Tsm and were not considered in the current studies. As compared to No-GVHD recipients, cGVHD recipients had a much lower proportion of C1 Tn, a slightly lower proportion of C0 Tsm, and higher proportions of C2 Tint, C3 Trmp, and C4 Trm (Fig. 1e). These results suggest that Tm subsets in cGVHD target tissues include Tsm, Trmp, Trm, and Tint subsets.

Alloreactive memory T cells were identified as CD44hiCD62L− in the tissues more than 30 days after HCT, and the tissue resident Tm cells were identified with expression of CD69 as described in our and others’ previous publications20,32,36. We used flow cytometry analysis of CD69 (tissue-residency marker) versus TCF1 or Ly108 (a surrogate for TCF1 expression)31,37 to validate the donor-type CD62L−CD44+CD4+ Tm subsets in cGVHD target tissues, of which >95% were derived from the injected CD4+ T cells and <5% from the de novo-generated CD4+ T cells that contained few Trm cells (Fig. 1f and Supplementary Fig. 1d). The Ly108+CD69− Tsm showed the highest expression of stemness-related markers (TCF1, Ly108). The Ly108+CD69+ Trmp showed lower expression of stemness-related markers but high expression of tissue-residency-related markers and exhaustion markers (CXCR6, PD1, TOX, TIGIT)38. The Ly108−CD69+ Trm and Ly108−CD69− Tint subsets showed low expression of TCF1 and ICOS (Fig. 1g). The Tint subset showed uniquely high expression of the NK-related-marker NKG2D (Fig. 1g). Trm cells showed high expression of terminal differentiation and effector markers (i.e., T-bet, CD38 and CD39) with higher percentages of IFN-γ+GM-CSF+ cells that are proinflammatory and pathogenic39 (Fig. 1g, h).

The presence of the Tm subsets in the tissues was validated with an assay of 5-min intravenous aCD4 mAbs to label cells in the circulation40. Most (≥85%) of Trmp and Trm but only a small portion (~35%) of Tsm were unlabeled tissue resident cells (Supplementary Fig. 1e). We have reported that CD4+ Tm cells in the cGVHD target tissues liver and lung formed lymphoid-like structures (TLS) consisting of T, B, DC and other cells20,35. We observed the four CD4+ Tm subsets in the TLS (Supplementary Fig. 1f). cGVHD recipients had higher percentages of Trmp, Trm and Tint but lower percentages of Tsm as compared with No-GVHD controls, but the yields of all 4 Tm subsets were higher in cGVHD recipients than in controls (Fig. 1i). The four CD4+ Tm subsets were also observed in the MHC-haploidentical model of C57BL/6 donor to (C57BL/6 x BALB/c) F1 recipients and MHC-matched model of C57BL/6 donor to C3H.SW recipients (Supplementary Fig. 2a–d). These results suggest that while Ly108/TCF1 expression reflects stemness and CD69, CD39 expression reflects the terminal differentiation of Tm, the terminally differentiated Ly108−CD69+/CD39+ CD4+ Tm subset in the GVHD target tissues is not anergic/exhausted. Instead, they contain highly pathogenic T effectors with high production of IFN-γ and GM-CSF.

Transcriptomic and epigenetic remodeling of the four Tm subsets
We performed combined RNA-Seq with ATAC-Seq to identify the transcriptomic and epigenomic relationships among four CD4+ Tm subsets. Principal-component analysis (PCA) confirmed that each of the four subsets was distinct (Fig. 2a). The sample distance heatmap showed that Tsm had a closer relationship with Trmp than with Trm or Tint, while Trm had a closer relationship with Tint than with other subsets (Fig. 2b). The heatmap and KEGG pathways of differentially expressed genes (DEG) among Tm subsets were grouped into six clusters (Fig. 2c, d and Supplementary Data 1). Tsm and Trmp both had higher expression of Cluster 2 DEGs that regulate DNA metabolic and mitotic cell cycle processes, while Trmp had higher expression of Cluster 4 DEGs that regulate inflammatory immune responses. Trm had higher expression of Cluster 3 DEGs that regulate lymphocyte activation and Cluster 6 DEGs related to defense responses. Tint had higher expression of Cluster 1 DEGs that regulate cell adhesion, Cluster 5 DEGs that regulate cell migration, and Cluster 6 DEGs. Compared to the Trmp subset, the Tsm subset showed higher expression of S1pr1, Nav1, E2f2 and Cdkn3 with lower expression of Cd244a (2B4), Il2, Slc7a8 and Gzma. In comparison to Trm subset, Tint subset showed higher expression of Nav1, S1pr1 and Itgax with lower expression of Il17rb, Deptor, Il9r and Ccl6 (Fig. 2e). These results support the stemness features of Tsm and Trmp and the circulatory potential of Tsm and Tint.

ATAC-seq analysis showed differences in chromatin accessibility in distal intergenic regions (31.59%), 1st introns (18.43%), other introns (26.94%), and promoter regions (15.96%) (Supplementary Fig. 3a). The Tsm and Trmp subsets showed high similarity in chromatin accessibility, as did the Trm and Tint subsets (Fig. 2f). Enriched open chromatin regions (OCRs) per Tm subset were analyzed by heatmap (Supplementary Fig. 3b) and opening or closing chromatin regions were compared at the transition stages of Tm cell subsets. More opening and closing chromatin regions were observed during the transition from Trmp to Trm than during the transitions from Tsm to Trmp or from Tint to Trm (Supplementary Fig. 3c). We used HOMER to compare the enrichment of TF binding sequences in chromatin-accessible regions. As compared with Tsm, TF binding motifs CTCF and CTCFL were enriched, but KLF1 was depleted in Trmp (Fig. 2g, the first panel). As compared with Trmp, TF binding motifs Gata and X.box were enriched, but BATF, Nur77 and Tcf7 were depleted in Trm (the second panel). As compared with Tsm, TF binding motifs Gata were enriched, but Tcf7 and BATF were depleted in Tint (the third panel). As compared with Trm, TF binding motif TCFL2 was enriched but BATF and X.box were depleted in Tint (the final panel). These results suggest that stemness-related TF binding sites closed sequentially from Tsm to Trmp and from Trmp to Trm and Tint, while differentiation and function-related TF binding sites opened sequentially across the four Tm subsets in the same order.

Specific TF binding sites were selected for comparison. Consistently, the stemness-related loci Slamf6, Tcf7 and Cxcr5, and exhaustion-related loci Tox and Eomes were more accessible in Tsm and Trmp than in Trm or Tint. The cell migration related loci Klf2 and S1pr1 were more accessible in Tsm and Tint than in Trmp and Trm. The T cell activation-related loci Tbx21 (T-bet), Bhlhe40, Prdm1 (Blimp1), Ifng and Gzmb were more accessible in Trm and Tint than in Tsm or Trmp. The NK cell-related genes loci Klrk1 (NKG2D) and Klrd1 (CD94) were more accessible in Tint than in other subsets (Fig. 2h and Supplementary Fig. 3d). Taken together, these results have unraveled the key transcriptomic and epigenetic changes (i.e., Tcf7 and Bhlhe40) during Tsm cell activation and differentiation.

T cell lineage development pathways and clonal expansion among Tm subsets
To unravel the differentiation pathways among the Tm subsets in the cGVHD target tissues, we performed pseudotime single-cell trajectory analysis, using combined scTCR-CDR3-Seq with scRNA-Seq. The results show a predicted T cell differentiation pathway starting from Cluster 0 Tsm to Cluster 3 Trmp, and finally to Cluster 4 IFN-γ+/Gzm+ Trm or Cluster 2 Tint (Fig. 3a). The apparent direct pathway from Cluster 1 Tn to Cluster 0 Tsm represents an artifact reflecting the long delay from the initial alloantigen stimulation of injected donor T cells on day 0 to the analysis on day 60 after HCT. Tm from the livers of No-GVHD and cGVHD recipients showed qualitatively similar differentiation pathways (Fig. 3a). But clonal expansion of TRA and TRB was observed mainly in cGVHD target tissues (Fig. 3b and Supplementary Fig. 4a). Consistent with clonal expansion in cGVHD, the size of clonotypes of cGVHD Tm cells consisted of small, medium, and large, while the Non-GVHD Tm cells were with dominant small size (Supplementary Fig. 4b, c). Tm from the livers of No-GVHD or cGVHD recipients showed little clonal overlap (Fig. 3c). The clonality index of Tsm, Trmp, and Trm were similar and lower than Tint or Tn (Fig. 3d). Trm showed a much lower proliferation or apoptosis than Tsm and Trmp (Supplementary Fig. 4d, e). These results indicate that the clonality of Trm cells in cGVHD target tissues is decided by Tsm cells, and the strongly biased clonal expansion of Trm may result from accumulation of Tsm progeny.

Based on combined TRA and TRB, we found top 20 expanded clonotypes with two representatives shown (Supplementary Fig. 4f, g). Expanded clonaltypes that were shared across Tsm, Trmp, and Trm, with a few Tint clusters, represented ~1800 counts among the 5000 analyzed cells (Fig. 3e, f). The highly expanded clonotypes showed dominant TRBV19 and TRBJ1-5 preferences (Supplementary Fig. 4h), and they were associated with higher expression of proinflammatory genes (i.e., chemokines, granzymes B and K) (Fig. 3g). These results suggest that clonally biased Tsm give rise to clonally expanded proinflammatory Trm cells in the cGVHD tissues.

Alloreactive donor T-derived Tsm differentiate into Tm subsets that mediate cGVHD
To validate the suggestion from pseudotime single-cell trajectory analysis that Tsm could differentiate into Trmp and Trm in the GVHD target tissues, we evaluated the percentages and yields of the four Tm subsets among CD62L−CD44hi Tm cells in the spleen, liver, and lung on days 7, 14, 30, and 60 after HCT. On day 7, ≥80% of the Tm cells in all tissues were Ly108+ Tsm-like (Supplementary Fig. 5a). At 14–30 days after HCT, the kinetics of Tm subsets in the spleen diverged from those in the liver and lung. While the percentage of Tsm-like cells in the spleen remained >60% from days 7–60, the yields of all subsets decreased to <5×104 after day 14 (Supplementary Fig. 5a), likely reflecting lymphopenia caused by aGVHD. In the liver and lung, the percentages of Tsm-like cells decreased from days 7 to 30 and remained at 10–20% from 30 to 60 days after HCT, while the percentages of other subsets slowly increased (Supplementary Fig. 5a). The yields of Tsm, Trmp, and Trm cells in the liver and lung increased and generally reached a peak at day 30, coinciding with the onset of clinical cGVHD in our murine models9. The yields of these subsets then remained stable or gradually decreased but persisted at far higher numbers (>10–50×104) than those in the spleen (Supplementary Fig. 5a). These results suggest that Ly108+ Tsm cells may give rise to the other 3 subsets in GVHD target tissues over time.

To assess the precursor-product relationships among the Tm subsets from GVHD target tissues, we performed adoptive transfer experiments. As depicted in Fig. 4a and Supplementary Fig. 5b, sorted congenic CD45.1+CD62L−CD44+ Tsm, Trmp, and Trm subsets from the liver and lung of primary cGVHD recipients 30 days after HCT were injected into adoptive recipients with GVHD induced by CD45.2+ donor cells on day 14 after HCT. The adoptively transferred CD45.1+ Tm subsets were analyzed 14 days later. The yields of cells derived from the injected CD45.1+ Tsm in the liver and lung were higher than those derived from Trmp, and those from Trmp were higher than from Trm (Fig. 4b). Tsm and Trmp gave rise to all four subsets, while Trm maintained themselves, and Trmp may have a potential to become Tsm (Fig. 4c). The adoptively transferred Tsm and Trmp cells also expressed high levels of TOX (Fig. 4d), consistent with its role in T cell persistence41. These results indicate that Ly108+ Tsm and Trmp, especially Tsm, have strong progenitor capacity and give rise to Trm in the GVHD target tissues, indicating that Tsm play an essential role in maintaining the Tm pool in the GVHD target tissues.

Furthermore, we evaluated the pathogenic capacity of Tsm, Trmp, and Trm subset from GVHD target tissues liver and lung. Before tissue harvesting, the mice were treated with Treg Protector to block NAD-induced death of Trm cells42. The Tm subsets were sorted with purity ≥95% (Supplementary Fig. 5b). To avoid the influence of de novo developed T cells, the adoptive recipients were reconstituted with BM (2.5×106 cells) from Rag-1−/− C57BL/6 donors with CD8+ T cells (0.1×106) from WT C57BL/6 donors to facilitate complete donor chimerism. 15 days after HCT, sorted Tsm, Trmp, and Trm subsets (0.25×106 each) from primary day 30 recipients were respectively transferred into adoptive recipients, which were monitored for clinical GVHD development for up to day 70 after HCT.

At ~45 days after HCT (30 days after transfer), the adoptive recipients given Tsm or Trmp but not Trm cells started to show body-weight loss and clinical GVHD manifestations (Fig. 4e). Histopathology scores in Tsm recipients were significantly higher than in Trmp recipients (Fig. 4f), although clinical scores showed no significant difference (Fig. 4e). The histopathology scores in Tsm and Trmp recipients were significantly higher than in Trm recipients (Fig. 4f). The yields of CD4+ Tm cells in the liver and lung were higher in Tsm recipients than in Trmp recipients, while the yields in Trm recipients were very low (Fig. 4g). These results indicate that among Tm subsets, Tsm have the strongest capacity to produce pathogenic Trm cells and cause the most severe cGVHD pathology.

In addition, we tested whether donor-type CD8+ T cells play a role in GVHD via a CD4+ T-dependent mechanism. First, we compared the expansion of donor CD8+ T cells in the adoptive recipients with or without infusion of donor-type CD4+ Tm subset. Adoptive transfer of Tsm but not Trmp or Trm induced significant expansion of the injected CD8+ T cells in the liver and lung (Supplementary Fig. 5c). Second, we tested whether donor CD4+ T cells affected the induction of cGVHD by donor CD8+ T cells. In the absence of donor-type CD4+ T cells, donor CD8+ T cells did not induce cGVHD or show expansion of CD8+ Trm cells (Supplementary Fig. 6a–d). Therefore, cGVHD is primarily maintained by donor-type CD4+ Tm especially Tsm, although donor-type CD8+ T cells may contribute some synergistic effect.

Finally, we evaluated the role of CD4+ Tm cell pool in sustaining cGVHD pathogenesis. The cGVHD mice were treated with depleting aCD4 (clone GK1.5) mAbs. Accordingly, cGVHD mice were treated weekly with i.p. injection of aCD4 mAbs (500 µg/mouse) from days 29 to 50 after HCT. Amelioration of cGVHD was indicated by higher body weight and prolonged survival compared to IgG-treated controls (Fig. 4h). At ~14 days after the last aCD4 mAbs treatment, however, the recipients showed rapidly decreasing body weight and died with recurrent pathologic evidence of GVHD (Fig. 4h, i). Further analysis showed that aCD4 mAbs treatment could completely deplete CD4+ T cells in the blood and dramatically reduce the pathogenic CD4+ Tm subsets in the cGVHD target tissues, particularly the Tsm subset (Fig. 4j and Supplementary Fig. 6e). Mice with recurrence of cGVHD at day 25 after the last aCD4 mAbs treatment was associated with reappearance of CD4+ Tsm and their progeny in the tissues (Fig. 4j and Supplementary Fig. 6e). Continuous aCD4 mAbs treatment prevented recurrent cGVHD (Fig. 4k). These results indicate that aCD4 mAb-treatment can significantly deplete the four Tm subsets and ameliorate cGVHD. Recurrence of cGVHD after the end of aCD4 mAb treatment is associated with recovery of Tsm, Trmp, and Trm. Taken collectively, Tsm plays a significant role in maintaining the pool of pathogenic Tm.

Impairment of Tsm formation and amelioration of cGVHD by Tcf1 or Bcl6-deficiency in donor T cells
pySCENIC analysis of the scRNA-seq dataset showed that, among the top 50 regulons, Lef1, Tcf7 (TCF1), Bach2 and Klf3 had high regulon activity in Tn and Tsm, while Tbx21, Nfatc1 and Bhlhe40 had high regulon activity in Trmp, Trm and Tint (Fig. 5a). Therefore, we used Tcf7−/− donor T cells to evaluate the changes of Tsm and their role in generating or maintaining the Tm pool in aGVHD and cGVHD overlapping target tissues liver and lung. We also evaluated histopathological changes in the salivary and lacrimal glands as cGVHD prototypic target tissues9. As compared with donor WT-T cells, Tcf7-deficient donor T cells (Tcf7−/−-T) showed significantly lower cGVHD severity, as judged by higher body weight and survival, especially during cGVHD period of 30–60 days after HCT (Fig. 5b), and by histopathology and fibrosis in the target tissues (Fig. 5c and Supplementary Fig. 7a, b). On day 60 after HCT, recipients of Tcf7−/−-T cells showed much lower yields of all four subsets of CD4+ Tm cells in the liver and lung (Fig. 5d) and markedly lower percentages of Tsm and Trmp compared with recipients of WT-T cells, although the percentages of Trm (Fig. 5d) and IFN-γ+GM-CSF+ cells were higher than in recipients of WT-T cells (Supplementary Fig. 7c). These results indicates that Tcf7 deficiency in donor T cells reduces Tsm quantity, leading to reduction of Trm quantity and attenuation of cGVHD.

TCF1 is associated with induction of BCL6 expression, which supports the persistence of CD8+ Tm cells27, and BCL6 also plays an important role in maintenance of CD4+ T cell progenitors during chronic LCMV infection27. Thus, we also compared cGVHD severity and Tm subset changes in recipients given donor WT-T or Bcl6−/−-T cells, and the results show similar patterns observed in recipients given Tcf7−/−-T cells (Fig. 5e–g and Supplementary Fig. 7a, b, d). Taken collectively, these results indicate that TCF1 and BCL6 play important roles in generating or maintaining Tsm that maintain the pool of pathogenic Trm in the cGVHD target tissues.

Impairment of Tsm differentiation into Trm and amelioration of cGVHD by Bhlhe40-deficiency in donor T cells
Although previous studies have shown that formation and maintenance of CD4+ Trm in experimental colitis is regulated by HOBIT43, we observed scant expression of Hobit in Trm from cGVHD target tissues (Supplementary Fig. 1b). Instead, we observed the high regulon activity of Bhlhe40 in Trm (Fig. 5a). Thus, we assessed the role of BHLHE40 and HOBIT in Trm expansion in the cGVHD target tissues, using Bhlhe40−/− or Hobit−/− donor T cells. As compared to recipients given WT-T cells, recipients given Bhlhe40−/−-T cells showed marked reduction of cGVHD severity, as judged by the higher survival rate and body weight after HCT (Fig. 5h) and by histopathology and fibrosis on day 60 (Fig. 5i and Supplementary Fig. 7a, b). Compared to WT-T cells, Bhlhe40−/−-T cells showed much lower yields of Tm cells (Fig. 5j) and much lower percentages of Trm, Tint and IFN-γ+GM-CSF+ cells, although higher percentages of Tsm in the liver and lung (Fig. 5j and Supplementary Fig. 7e). In contrast, recipients given Hobit−/− donor T cells did not reduce cGVHD severity or reduce Trm or their production of IFN-γ or GM-CSF (Supplementary Fig. 7f–h). These results indicate that BHLHE40 but not HOBIT in donor T cells plays a critical role in generating or maintaining proinflammatory Trm cells in the cGVHD target tissues.

MHCII-TCR interactions maintain Tsm and augment their differentiation into Trm
Previous studies have shown that donor-type APCs are involved in the pathogenesis of cGVHD18,44. We observed that among Tm subsets in cGVHD target tissues clonal expansion was with Trm but not Tint subsets 60 days after HCT (Fig. 3), suggesting that differentiation and expansion of pathogenic Trm is MHCII-TCR interaction-dependent. Consistent with a previous report44, we observed that donor-type APCs in cGVHD upregulated expression of MHCII (Fig. 6a and Supplementary Fig. 8a). Thus, we evaluated the impact of donor and host MHCII on Tm subset expansion. As compared to results with WT-donor BM→WT-recipients, WT-donor BM→MHCII−/− recipients had modestly reduced severity of cGVHD, while MHCII−/−-donor BM→WT-recipients had markedly reduced severity of cGVHD, as indicated by body weight loss and histopathology (Supplementary Fig. 8b, c). We also explored the MHCII-TCR interaction in regulating the changes of the four Tm subsets in the GVHD target tissues of recipients with MHCII deficiency in donor- or host-type APCs during aGVHD on day 7 and during cGVHD on day 60 after HCT. Flow cytometry analysis of Tm subsets in target tissues liver and lung showed that, at day 7, Tm cells in the tissues were dominantly Tsm cells (>60%) with small percentage of Trmp, Trm, and Tint (<20%) (Fig. 6b, c and Supplementary Fig. 8d, e). As compared with WT-donor BM→WT-recipients, WT-donor BM→MHCII−/− recipients but not MHCII−/−-donor BM→WT-recipients had lower yields of Tsm although the percentage was relatively increased in the lung and liver (Fig. 6b, c and Supplementary Fig. 8d, e). In contrast, at day 60, Tsm among Tm were reduced to ~20%, and Trm and Tint increased to ~40%. As compared with WT-donor BM→WT-recipients, MHCII−/−-donor BM→WT-recipients but not WT-donor BM→MHCII−/− recipients had lower numbers of Trmp and Trm in both liver and lung tissues, although reduction of Tsm only occurred in the lung (Fig. 6b, c and Supplementary Fig. 8d, e). As compared with WT-donor BM→WT-recipients, the percentages of IFN-γ+GM-CSF+ Trm in the liver and lung of MHCII−/−-donor BM→WT-recipients did not differ, but they were lower in WT-donor BM→MHCII−/− recipients (Fig. 6d). These results indicate that recipient MHCII plays a key role in generating Tsm and their proinflammatory capacity during aGVHD. In contrast, donor MHCII plays a significant role in maintaining Tsm and their differentiation into Trm during cGVHD.

In addition, we compared the Tm subsets derived from the injected and de novo generated donor-type CD4+ T cells in WT recipients given WT or MHCII−/− BM. As compared to the injected CD4+ Tm, the de novo generated CD4+ Tm showed much lower Trm percentages and lower percentages of IFN-γ+GM-CSF+ Trm, but higher percentage of Tsm and Trmp (Supplementary Fig. 8f, g). As compared to de novo-generated CD4+ Tm from WT-BM, the de novo-generated CD4+ Tm from MHCII−/− BM also showed lower percentages of Trm and IFN-γ+GM-CSF+ Trm (Supplementary Fig. 8f, g). These results indicate that Tsm derived from the injected CD4+ T cells but not de novo-generated CD4+ T cells play a critical role in supporting the pathogenic Trm pool.

IFN-γ induces MHCII expression by antigen-presenting cells and other cells45, but the impact of IFN-γ on regulation of tissue Tm subsets in cGVHD remains unclear. Recipients given IFN-γR−/− donor BM cells plus WT-T cells showed lower severity of cGVHD as judged with higher body weight and percentage of survival and lower pathology score and fibrosis on day 60 after HCT (Fig. 6e, f), which was associated with lower percentages of donor-type CD11c+CD11b+ DCs as well as associated with lower yields of Tsm, Trmp, and Trm in the lungs and liver but not reduction in the percentages of IFN-γ+GM-CSF+ Trm cells (Fig. 6g–i). These results indicate that IFN-γR signaling regulates expansion of MHCII-expressing donor DCs that maintains Tsm and augments their differentiation into pathogenic Trm cells to sustain cGVHD.

Tsm, Trmp, and Trm are present in the lymphoid-like structures in the liver tissues of cGVHD patients
To determine the clinical and translational implications of our experimental results, we investigated whether Tsm, Trmp, Trm could be identified in the blood and affected tissues from patients with cGVHD (Supplementary Table 1 and 2). We first analyzed CD4+ Tm subsets with TCF1 and T-bet in the blood of patients with or without cGVHD. CD45RA−FAS+ T cells are identified as Tm in human blood46. Among CD4+ Tm, the percentages and numbers of TCF1hi T-betlo stem-like memory T (Tsm) were lower and the percentages and numbers of TCF1lo T-bethi Teff cells in the blood were higher in patients with cGVHD than in those without cGVHD (Fig. 7a). The TCF1hi T-betlo Tsm subset express stemness-related markers22 with high levels of CD27, TCF1 and CXCR5 (Fig. 7b, c), while the TCF1lo T-bethi Teff subset express high levels of CD11b, CXCR3 and T-bet (Fig. 7b, c). These results show that cGVHD patients have high numbers of T-bethi Teff and lower numbers of TCF1hi Tsm cells in the blood when compared to controls without cGVHD.

We used imaging mass cytometry (IMC)47 to visualize Tm subsets in the liver biopsy specimens from patients with cGVHD (Supplementary Table 2). Liver tissues of cGVHD patients were stained with a 24-antibody panel (Supplementary Fig. 9a, Supplementary Table 3). With the UMAP algorithm and mapping, we found tertiary lymphoid-like structures (TLS) that consisted of Tm, dendritic cells, macrophages and B cells (Fig. 7d and Supplementary Fig. 9a-c). We analyzed 2 TLS regions from each of four patients (Fig. 7d and Supplementary Fig. 9c). CD4+ Tm cells in the liver of cGVHD patients had few Foxp3+ but many IFN-γ+GM-CSF+ cells (Fig. 7e, f). The Tm cells included Ly108+CD69− Tsm, Ly108+CD69+ Trmp, and Ly108−CD69+ Trm subsets, and the number of Tsm per field was significantly lower than the number of Trmp, or Trm (Fig. 7g and Supplementary Fig. 7d). We used immunofluorescent staining to validate the identification of TCF+CD69− Tsm, TCF1+CD69+ Trmp, and TCF1−CD69+ Trm cells among CCR7−CD45RA−CD4+CD3+ cells (Fig. 7h, i). These results indicate the presence of Tm subsets including Tsm, Trmp and proinflammatory Trm in lymphoid-like structures in the liver of patients contributing active cGVHD.

Methods
Study design
This study is designed to dissect the mechanisms about how the pathogenic CD4+ Trm pool is maintained to sustain the pathogenesis of cGVHD. First, with murine models of cGVHD, we applied flow cytometry sorting, combined single-cell RNA-Seq with TCR-CDR3-Seq, combined bulk RNA-Seq with ATAC-Seq, and in vivo transfer experiments to analyze, characterize, and validate the Tm subsets (i.e., Tsm) in cGVHD target tissues. Second, we used specific gene-deficient donor T cells to study the key that regulating the maintenance and differentiation of Tsm cells in the cGVHD target tissues. Finally, we attempt to link our observations with murine models to the pathogenesis of cGVHD in patients by using imaging mass cytometry (IMC) to visualize the Tsm cell subsets in tertiary lymphoid structures in the liver tissues of patients with active cGVHD.

Human samples
White blood cells were isolated and cryopreserved over in liquid nitrogen. Cells were thawed and T cells subsets were examined by flow cytometry. Patient tissue samples were also collected and analyzed with imaging mass cytometry or immunofluorescence staining to visualize regions of immune cell infiltration. City of Hope National Medical Center Institutional Review Board (IRB15172) and the Fred Hutchinson Cancer Center approved the study. All recruited volunteers provided written informed consent. The patients gave informed consent allowing sections from biopsies to be used for research if the material was no longer needed for clinical purposes or for any required retention.

Mice
BALB/c (NCI#555, H-2Kd), C57BL/6 (NCI#556, H-2Kb), BDF-1(NCI#99, H-2Kb/d) mice were purchased from the National Cancer Institute. Congenic CD45.1+ (JAX#002014), MHCII−/− (JAX#003584), Nur77-GFP (JAX# 016617), CD4-cre (JAX# 017336), CD4−/− (JAX# 002663), Rag1−/− (JAX# 002216), IFNgR−/− (JAX# 003288), Tcf7f/f (JAX# 030909) or Bhlhe40−/− (JAX# 029732) C57BL/6 mice were purchased from the Jackson Laboratory (JAX). Hobit−/− C57BL/6 mice were kindly provided by Timothy O’Sullivan at UCLA, Bcl6f/f C57BL/6 mice were obtained from Dr Markus Muschen12,54. CD4-cre/Bcl6f/f or CD4-cre/Tcf7f/f C57BL/6, MHCII−/− BALB/c and C3H.SW (JAX#000438) mice were bred at City of Hope Animal Research Center (COH-ARC). CD4-cre/Bcl6f/f or CD4-cre/Tcf7f/f C57BL/6 were generated by crossing CD4-cre mice with Bcl6f/f or Tcf7f/f mice, respectively. MHCII−/− BALB/c mice were generated by backcrossing MHCII−/− C57BL/6 mice with BALB/c mice for more than 10 generations. All mice are maintained in a specific-pathogen-free room at COH-ARC. The experimental and control mice were housed in separate cages. The donor and host mice were generally male and 8-12 weeks old. The City of Hope Institutional Animal Care and Use Committee (IACUC) have approved all animal protocols.

Induction and evaluation of chronic GVHD
BALB/c cGVHD recipients (8~12 weeks old male, H2d) were exposed to 850 cGy total body irradiation (TBI) using a [137Cs] source 8–10 h prior to HCT and then received an intravenously (i.v.) injection of CD45.1+ or CD45.2+ C57BL/6 (8~12 weeks old male, H2b) donor spleen cells (1×106) or enriched Thy1.2+ spleen T cells (0.2×106) and CD45.2+ or CD45.1+ T cell-depleted BM cells (TCD-BM, 2.5×106). TCD-BM cells were depleted of T cells by incubation with biotin-conjugated anti-CD4 and anti-CD8 mAbs and anti-biotin Microbeads (Miltenyi Biotec#130-097-046) and then pass through an autoMACS Pro cell sorter (Miltenyi Biotec, Germany). Enrichment of splenic Thy1.2+ T cells was achieved using mouse anti-CD90.2 microbeads (Miltenyi Biotec#130-121-278). The purity of the Thy1.2+ T cell enrichment was >98%, while the purity after T cell depletion was >99%. The experimental and control animals were bred separately, evaluation and scoring of clinical chronic signs of GVHD and clinical cutaneous GVHD have been previously described12,55.
To establish MHC-haploidentical and minor mismatched cGVHD mouse models, B6D2F1 and C3H.sw recipient mice were irradiated with 1100 cGy and then engrafted with 5 M TCD-BM alone (No GVHD) or with 8×106 splenocytes added (cGVHD).

Isolation of mononuclear cells from lymph and non-lymph tissues
Mice were euthanized by CO₂ inhalation according to institutional guidelines and tissues were collected immediately after euthanasia. The spleens were meshed through a 70 μm nylon cell strainer (VWR, 10199-657) and cells were collected. Bone marrow cells were flushed from femurs with PBS and meshed through a 70 μm nylon cell strainer to collect the cells. Liver tissues were flushed with PBS and meshed through a 70 μm strainer and cells were collected and washed with PBS containing 2% BSA. Hepatic mononuclear cells were further enriched with a 40% and 70% Percoll gradient (Sigma-Aldrich# P1644). For isolation of MNC from lung and skin, tissues were cut into pieces and digested with collagenase VIII (Sigma-Aldrich# C2139) and DNase I (Fisher Scientific# NC0947151) in RPMI-1640 (Fisher Scientific# 21870092)/10% FBS (Fisher Scientific# A2720803) complete medium at 37°C for 45 and 90 min, respectively. MNC were then isolated with Lympholyte-M (Cedarlane# CL5035). Mouse PBMCs were also enriched with mouse Lympholyte-M. Isolated cells were washed and counted with a Cellometer Auto 2000 Cell Viability Counter (Nextcelom).

Flow cytometry and antibody usage
Isolated mononuclear cells were used to measure surface antigens, transcription factors and intracellular cytokines. For surface antigen measurement, mononuclear cells were blocked with anti-CD16/CD32 mAbs for 15 min and cells were stained with a surface antibody mixture at 4˚C for 15 min. Cells were then washed and evaluated with the use of BD fortessa or Cytek Aurora instrument. For transcription factors analysis, mononuclear cells were stained with surface antigens and then fixed for 30 min with commercial Foxp3/Transcription Factor Staining Buffer Set (Fisher Scientific# 552300). Cells were then washed with permeabilization buffer (Fisher Scientific# 833356) and stained with an intracellular TF antibody mixture. For analysis of intracellular cytokines, isolated mononuclear cells were stimulated for 4 h with PMA (Fisher Scientific# 125491) plus ionomycin (Sigma Aldrich# I0634), and protein transport inhibitors (Fisher Scientific# BDB554724), and cells were then stained with a surface antibody mixture. Cells were later fixed with a commercial fixation and permeabilization kit (Fisher Scientific# 554722) and stained with a mixture of antibodies against intracellular cytokines. For BrDU staining, BrDU (Fisher Scientific# 550891) was i.p. injected at 500 μg/mouse, and tissues were harvested 3 h later. BrdU measurement was applied with BD APC BrdU Flow Kit (Fisher Scientific# 552598). And Annexin V expressions were determined with Annexin V apoptosis detection kit (Biolegend# 88-8007-72).
The antibodies used to stain murine cells included anti-CD4 (RM4-5), anti-CD4 (GK1.5), anti-CD3 (17A2), anti-CD8 (53-6.7), anti-TCRβ (H57-597), anti-H-2Kb (AF6-88.5), anti-CD45.1 (A20), anti-CD45.2 (#104), anti-CD62L (MEL-14), anti-CD69 (H1.2F3), anti-PD1 (29F.1A12), anti-CD44 (IM7), anti-Ly108 (13-G3), anti-CXCR6 (SA051D1), anti-CD39 (Duha59), anti-CD38 (#90), anti-TIGIT (Vstm3), anti-Fas (15A7), anti-TCF1 (C63D9), anti-T-bet (eBio4B10(4B10)), anti-TOX (REA473), anti-ICOS (7E.17G9), anti-NKG2D (CX5), anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-CD11c (N418), anti-MHCII (M5/114.15.2), anti-CD45 (30-F11), anti-GM-CSF (MP1-22E9), anti-IFN-γ (XMG1.2), more details see Supplementary Table 4.
The antibodies used to stain human PBMCs included anti-human CD3/CD4/CD8a (Antibodies cocktail, Thermo Fisher Scientific), anti-human CD95 (DX2), anti-human CD45RA (HI100), anti-human CD29 (TS2/16), anti-human CXCR5 (MU5UBEE), anti-human CCR7 (4B12), anti-human CD27 (O323), anti-human CD56 (NCAM), anti-human CXCR3 (G025H7), anti-human CD11b (M1/70), anti-human CD197 (3D12), anti-human T-bet (4B10) and anti-human TCF1 (C63D9).

Intravascular labeling with anti-CD4 mAbs
Mice were intravenously injected with 2 µg aCD4 mAbs (Clone: RM-4-5) in 200 µl sterile PBS for 5 min and then euthanized to harvest tissues. Mononuclear cells were isolated from tissues for flow cytometry analysis. Lung circulating CD4+T cells are i.v. anti-CD4 (Clone: RM-4-5) positive, and lung tissue localized immune cells (Trm) are defined by i.v. anti-CD4 (Clone: RM-4-5) negative among H2Kb+TCRβ+CD4+ (Clone: GK1.5).

Immunofluorescent Staining
Mouse liver cryosections were fixed with 4% PFA, blocked by SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific) for 30 min and incubated overnight at 4°C in primary antibody cocktails including rat anti-mouse CD4 (biolegend, clone RM4-5), rabbit anti-mouse Ly108 (Cell signaling, clone E2P7A,), goat anti-mouse CD69 (R&D, clone: Polyclonal). To measure CD4+ Tm cells infiltration in the liver of cGVHD patients, slides were stained with rabbit anti-hCD3 mAbs (Fluidigm :3170019D), Alexa Flour 488 conjugated anti-hCD4 mAbs (R&D, FAB8165G), mouse anti-hCCR7 (R&D, Clone # 150503), goat anti-hCD69 mAbs (R&D, AF2359) and DAPI or Alexa Flour 488 conjugated anti-hCD4 mAbs (R&D, FAB8165G), mouse anti-hCD45RA mAbs (Fluidigm, Clone: HI100), goat anti-hCD69 mAbs (R&D, AF2359), rabbit anti-TCF1 mAbs (Cell Signaling Technology, C63D9) and DAPI. Slides were washed with PBS-TB (PBS supplemented with 0.05% Tween 20 and 1% BSA) three times and stained with secondary antibody for 60 min at room temperature. Images were later acquired on a Confocal Zeiss LSM700 or LSM880 microscope at 200X or 400X magnification.

Histopathology
Liver, lung, salivary gland and lacrimal gland from No GVHD or cGVHD mice were harvested and fixed in 10% neutral formalin and processed in paraffin blocks. The tissue-embedded paraffin blocks were sectioned at 5 µm and sections were stained with haematoxylin and eosin (HE) and Masson’s trichrome by the Pathology Solid Tumor Core at the City of Hope. Slides were examined at 100x or 200x magnification and visualized with Zeiss Observer II at the City of Hope Light Microscopy Core.

Cell sorting
For adoptive transfer experiments, primary cGVHD recipients on day 30 after HCT were i.p. injected with 50 ug Treg-protector (anti-ARTC2 nanobody, Biolegend) 30 min before euthanasia. Mononuclear cells from the liver and lung were mixed, and total CD4+ cells were enriched with anti-mouse CD4 beads (Miltenyi). Enriched cells were then stained with anti-H2kb mAb, anti-CD4 mAb, anti-TCRβ mAb, anti-CD45.1 mAb, anti-CD44 mAb, anti-CD62L mAb, anti-CD69 mAb and anti-Ly108 mAb cocktail. Four Tm cell subsets including CD69−Ly108+, CD69+Ly108+, CD69+Ly108− among injected H2kb+CD45.1+CD44+CD62L−TCRβ+CD4+ T cells were sorted for adoptive transfer.
For scRNA-seq/scTCR-seq, cGVHD mice and No GVHD mice were euthanized on day 60 after HCT, mononuclear cells were isolated from the liver tissues and stained with a surface antibody mixture: anti-H2kb mAb, anti-CD4 mAb and anti-TCRβ mAb. Sorted H2Kb+TCRβ+CD4+ T cells were used for single cell sequencing.
For bulk RNA sequencing and ATAC sequencing, the liver tissues from cGVHD mice were harvested on day 60 after HCT. Isolated mononuclear cells were stained with anti-H2kb, anti-CD4, anti-TCRβ, anti-CD44, anti-CD62L, anti-CD69 and anti-Ly108. Four Tm cell subsets including CD69−Ly108+, CD69+Ly108+, CD69+Ly108−, CD69−Ly108− among injected CD45.1+H2kb+CD44+CD62L−TCRβ+CD4+ T cells were sorted for Bulk RNA-Seq and ATAC-Seq.

Adoptive Transfer Experiments
Chronic GVHD was induced by transplanting CD45.2+ (or CD45.1+) TCD-BM cells (2.5×106) and splenocytes (1×106) from CD45.1+(or CD45.2+)-congenic C57BL/6 donors into lethally irradiated BALB/c recipients. 30 days after HCT, mononuclear cells from the liver and lung were isolated and stained with anti-H2Kb mAb, anti-CD45.1 mAb, anti-TCR mAb, anti-CD4 mAb, anti-CD69 mAb, anti-Ly108 mAb, anti-CD62L mAb, anti-CD44 mAb and then sorted by ARIA SORP. One million sorted Tsm, Trmp and Trm cells were transferred into mild GVHD adoptive recipients at 14 days after HCT with CD45.2+ C57BL/6 donor spleen cells (0.5×106) and T cell-depleted BM (TCD-BM) cells (2.5×106). 14 days after cell transfer, MNCs from the liver and lung were analyzed for percentage and yield of CD4+ T cells and four subsets of CD4+ Tm subsets.
To evaluate CD4+ Tm cells in regulation of cGVHD in secondary recipients, 0.25×106 Tsm, Trmp and Trm cells were adoptively transferred into BALB/c GVHD mice at day 15 after engraftment with 2.5×106 Rag1−/− BM and 0.1×106 CD8+ T cells from B6 donors.

Anti-CD4 mAbs treatment of cGVHD mice
cGVHD mice were treated with intraperitoneal (i.p.) anti-mouse CD4 mAbs (BioXCell, Clone GK1.5) weekly at 500 µg/mouse from day 29 to 50 after HCT or from day 29 to 99 after HCT. Control groups were given Rat IgG mAb purchased from Jackson ImmunoResearch Laboratories Inc.

10× Genomics single-cell RNA/TCR library preparation and sequencing
scRNA-seq library was generated using the Chromium Single Cell V(D)J Reagent Kits (10× Genomics) according to the manufacturer’s instructions and targeting 5,000 cells, and scTCR-seq library was generated using Chromium Single Cell V(D)J Enrichment Kit, mouse T and B Cell (10× Genomics), following all steps according to the manufacturer’s protocols. The cDNA library was sequenced on illumina HiSeq 2500 sequencing System platform at 26+8+101 (Read 1: 26 cycles, i7 Index: 8 cycles, Read 2: 101 cycles; Sequencing depth: 20,000/ read pairs per cell) for gene expression, and at 151+8+151 (Read 1: 151 cycles, i7 Index: 8 cycles, Read 2: 151 cycles; Sequencing depth: 5000/ read pairs per cell) for BCR and TCR sequencing. Sequencing reads were processed using the Cell Ranger pipeline with the default settings (10x Genomics).

scRNA/TCR-sequencing analysis
Analysis of mouse single cell RNA-seq data was performed using “Seurat”56. The datasets were subjected to quality control (elimination of cells with RNA features <200 and mitochondrial RNA >10%) and SCT normalization retaining the top 2000 variable genes, followed by integration using the Seurat workflow. The integrated data was scaled and subjected to principal component analysis (PCA), following by clustering using the top 12 PCs. UMAP was used to perform the dimensional reduction for visualization. Genes with differential expressions were identified by using the FindMarkers function in Seurat. Psuedo-time analysis was performed using the “Monocle3” package57. T cell stemness/progenitor signature were score via “AddModuleScore” with published genesets58. Single cell TCR-CDR3 data were processed by the 10x CellRanger pipeline, and cell barcodes were matched to the single cell RNA-seq data using R scripts. Visualization of single cell RNA-seq and TCR-CDR3-seq data was performed using “Seurat” and “scRepertoire”59. To explore key transcription factors in each cluster, 6000 cells were randomly selected, and active transcription factors were identified with the pySCENIC package60.

Bulk RNA-sequencing library preparation, sequencing and data analysis
RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific, Waltham Massachusetts, US), and RNA integrity was determined using Bioanalyzer (Agilent). Library construction of 280 ng total RNA for each sample was made using KAPA mRNA HyperPrep Kit (Illumina Platforms) (Kapa Biosystems, Wilmington, USA) using 10 cycles of PCR amplification. Libraries were purified using AxyPrep Mag PCR Clean-up kit (Thermo Fisher Scientific). Each library was quantified using a Qubit fluorometer (Life Technologies), and the size distribution was assessed by using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Sequencing was performed on NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA) instrument using NovaSeq Reagent Kits to generate 101 bp Paired-end reads sequencing. Quality control of RNA-Seq reads was performed using FastQC.
For data analysis, raw RNA-seq sequences were subjected to adapter trimmed with Trimmomatic, and poly(A) tails were removed with FASTP. The trimmed reads were aligned to mouse genome mm10 using STAR with the default settings. The expression level of RefSeq genes were counted using “Rsubread”. The raw count data were normalized by trimmed mean of M-values (TMM) method using the Bioconductor package “edgeR”. Differential expression analysis was conducted using the quasi-likelihood (QL) F-test implemented in “edgeR” to identify differentially expressed genes, with the cutoff of at least two groups having average RPKM>=1, FDR<=0.05 and fold change>=1.5. The gene set enrichment analysis was performed using ClusterProfiler to identify the affected Hallmark and KEGG pathways from MSigDB v7, using the pre-ranked gene list sorted by the -log10(p-value) with a sign determined by the fold change direction.

Bulk ATAC-sequencing library preparation, sequencing and data analysis
To prepare for the nuclei, we spun 50,000 cells at 300×g for 5 min, followed by a wash using 50 μL of cold 1x PBS and centrifugation at 300×g for 5 min. Cells were lysed using 50 μL cold lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin and 0.5% BSA). Immediately after lysis, 500 μL wash buffer was added, and nuclei were spun at 500×g for 10 min using a refrigerated centrifuge. To avoid losing cells during the nuclei prep, we used a fixed angle centrifuge and carefully pipetted away from the pellet after centrifugations. Nuclei tagmentation and adapter ligation by Tn5 was performed using the Nextera DNA Sample Preparation kit (Illumina), followed by purification with The Oligo Clean & Concentrator kit (ZYMO) according to the manufacturers’ instructions. Transposed DNA fragments were amplified using the NEBNext Q5 HotStart HiFi PCR Master Mix with regular forward and reverse barcoded primers. The numbers of additional amplification cycles were determined by quantitative-PCR using the NEBNext HiFi Master Mix, SYBR Green I (Applied Biosystems) and Custom Nextera Primers. The final product was prepared by using double-sided bead purification 0.5X and 1.8x volume AMPure XP beads (Beckman Colter Inc.) and quality checked on 2100 Bioanalyzer (Agilent). Sequencing was performed using a NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA) instrument for 2×101 bp sequencing with ~50 million reads per sample.

Data analysis
ATAC-seq data were aligned to mouse genome mm10 using BWA-MEM with the default settings. Peak calling was performed by using Genrich. Peaks were annotated by the R package “ChIPseeker”. K means clustering was generated using R package “pheatmap”. GO analysis of peaks in each cluster was conducted by “clusterProfiler”. Reads in peaks were scaled into RPKM values followed by log2 transformation with offset of 1 for fold change calculation. Fold changes >= 2 and p value <= 0.05 across conditions were considered significant. ATAC peak changes across sample groups were plotted using R package “ggalluvial”. Motifs enriched at the ATAC-seq peaks with 250bp flanking region were identified using HOMER. The output from HOMER was parsed using R package “monaLisa” and volcano plots were generated using “EnhancedVolcano”.

Imaging Mass Cytometry
For performing imaging Mass cytometry (IMC), liver tissue sections from cGVHD patients, fixed in formalin and embedded in paraffin (FFPE), were dewaxed using fresh xylene, rehydrated with a series of alcohol dilutions, and washed with distilled water. Antigen retrieval was performed in a Biocare Medical decloaking chamber at 95°C for 65 min in Tris-EDTA buffer (pH=9). The slides were cooled to room temperature, blocked with SuperBlock T20 (PBS) Blocking Buffer (Thermo Scientific) for 30 min, and incubated overnight at 4°C with primary antibody mixtures as described below. After incubation, the slides were washed with PBS-TB (PBS supplemented with 0.05% Tween 20 and 1% BSA) three times and stained with a nuclear intercalator solution containing iridium 193 and 191 isotopes for 10 min at room temperature. Finally, the slides were washed with distilled water and dried at room temperature before data acquisition using a Hyperion imaging mass cytometer (Fluidigm). The data were stored as MCD files and txt files, and the MCD viewer, Fiji ImageJ61 software and Noise2Void62 were used for image processing.

IMC analysis
Raw IMC data were collected and stored in the MCD and txt file. Single cells were segmented with Steinbock guided by the ImcSegmentationPipeline shared by Bodenmiller’s group63. Output files were then imported into R using Rstudio interface for further analysis. Image and cell-level quality control and batch effect correction were conducted. Cell type labeling was applied by Shiny application of R package cytomapper. Briefly, the following markers were used to define cell types: B cells (CD20), Macrophage (CD68+ HLA-DR+), DCs (CD68− CD11c+ HLA-DR+), Endothelial cells (CD31+), epithelial cells (E-cadherin+), CD4+ Tsm (CD3+ CD4+ CD45RO+ Ly108+ CD69−), CD4+ Trmp cells (CD3+ CD4+ CD45RO+ Ly108+ CD69+), CD4+ Trm cells (CD3+ CD4+ CD45RO+ Ly108− CD69+), Tint cells (CD3+ CD4+ CD45RO+ Ly108− CD69−). Gated cells were then downloaded as SingleCellExperiment objects stored in the metadata. Label cells were then trained in a random forest classifier. Single cell visualization and spatial visualization were later performed with imcRtools package63.

Statistical analysis
The results presented in this study were obtained from at least two replicate experiments. Data are presented as mean ± SEM. Survival was compared using the log-rank test. When data were normally distributed, Student’s unpaired t-test was used to compare two groups. For multiple comparisons of one independent variable, ordinary one-way ANOVA with Holm-Šídák test was used, while for two or more independent variables, two-way ANOVA with Holm-Šídák test was used. Prism 9 GraphPad Software was used for statistical analysis. Statistical significance was considered at P<0.05 (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors investigated the role of tissue-resident donor CD4+ T cells in chronic graft-versus-host disease (GVHD) using mouse MHC-mismatched allogeneic bone marrow transplant (BMT) models. They conducted sc-RNA-seq on CD4+ T cells from the liver 60 days post-transplant and identified seven CD4+ T clusters. In chronic GVHD, four memory T-cell clusters predominated: Tsm, Trmp, Tby, and Trm, while the proportion of Tsm was reduced, suggesting their differentiation toward Trm.\nPseudotime trajectory analysis revealed a differentiation path from Tsm to Trmp and then to Trm or Tby, confirmed by adoptive transfer experiments. Tsm and Trmp gave rise to all four subsets, while Trm maintained themselves. Tsm and Trmp cells expressed high Tox levels, contributing to their persistence, while Trm were the most functional, producing high amount of IFN-g and GM-CSF. sc-TCR-seq revieled that expanded clonal types that were shared across Tsm, Trmp, and Trm, with a few Tby clusters, and clonally bised Tsm give rise to clonally expanded proinflammatory Trm cells in the cGVHD\nThe authors confirmed that early Tm expansion required host MHCII, while later maintenance depended on donor MHCII. IFNgR knockout reduced Tm and ameliorated GVHD, likely by reducing MHCII expression on donor antigen-presenting cells. Deficiency in BCL6, TCF-1, or Bhlhe40 in donor T cells also reduced GVHD, with TCF-1 and BCL6 critical for differentiation and/or maintenance of Tsm while Bhlhe40 critical for differentiation of Tsm into Trmp, Trm, and Tby, and for GM-CSF and IFN-g production. Imaging mass cytometry confirmed the presence of Tsm, Trmp, and Trm in lymphoid-like structures in the livers of cGVHD patients.\nIt is interesting that authors precisely analysed tissue resident donor T cells using multiple comprehensive methods, such as scRNA-seq, scTCR-seq, ATAC-seq, flow cytometry, and adoptive transfer experiments. However, authors need to address the following points.\n1. The major weakness of this study is that the authors did not demonstrate that Tsm is truly required for the development of chronic GVHD. They showed that CD4 depletion using monoclonal antibodies ameliorated chronic GVHD, but this method depletes not only Tsm but also the entire CD4 population throughout the body. TCF-1 and BCL6 should be expressed in a broad range of donor T cells, including CD8 T cells. The authors need to find a more specific method to target Tsm, or they should demonstrate that the adoptive transfer of Tsm can develop chronic GVHD in secondary recipients.\n2. The authors annotated cluster 2 as bystander activated T cells (Tby) based on the expression of Tby-associated markers such as NKG2D and lower expression of TCR signaling markers. However, NKG2D is a marker of CD8 Tby, and it has not been tested if CD4+ Tby express it. It could be misleading to use this annotation, and this reviewer recommends using a less definitive term to describe this population.\n3. Figure 1h: Tint\" should be labeled as Tby.\n4. The authors concluded that TCF-1, BCL6, or Bhlhe40 deficiency in donor T cells, or IFNgR deficiency in donor BM cells, ameliorated chronic GVHD. However, they only demonstrated improved survival and mitigation of body-weight loss. Essentially, this [B6 into BALB/c] model is an acute GVHD model (Reddy P: BBMT 2008; 14:129), therefore these data may indicate that these deficiencies ameliorated acute GVHD, not chronic GVHD. The authors need to show more chronic GVHD-specific findings were ameliorated, such as organ fibrosis or tear secretion. How did they rule out the impact of TCF- 1, BCL6, or Bhlhe40 deficiencies on CD8+ T cells? The authors showed that CD4 T cells were required for the induction of GVHD in this model, but they did not test whether CD8+ T cells play a role in GVHD via a CD4-dependent mechanism.\n5. The authors used a single strain combination; B6 donor and BALB/c recipients. To generalize their findings, they should use at least one more model of chronic GVHD.\n6. Figure 4C showed that Trmp give rise to Tsm, indicating bidirectional differentiation between Tsm and Trmp. This is similar to the interconversion between TexProg1 and TexProg2 shown in CD8+ T cells (Beltra JC: Immunity 2020). It is better to add brief discussion about this point.\n7. Figure S6d: The authors need to clarify in the figure legend when this analysis was performed.\n8. Figure 6f: CD11b⁻ MHCII⁺ cells were expanded in the recipients of IFNgR-deficient BM cells. The authors should briefly discuss about these cells.\n9. Figure 7: The authors need to add more detailed background information about the patients, such as donor sources, MHC matching, time points of sample collection, ongoing immunosuppression at sample collection, history of acute GVHD, and other relevant details.\n10. The authors only used GVHD models. Therefore, they should narrow the title of this manuscript to reflect its focus on chronic GVHD, rather than chronic tissue inflammation.", "reviewer_2": "In the current manuscript, the authors identified 4 subsets of CD4 T cells mediating cGVHD, defined their transcriptional and epigenetic signatures, and determined their differentiation/lineage relationship. In addition, the transcription factors responsible for individual subsets were confirmed. The distinct function for donor vs host-derived APC was established. Importantly, major findings in mouse experiments were validated using samples from human patients. The findings are important to the broad field of transplantation and T-cell biology.\nHowever, several concerns and questions remain to be addressed.\n1. Some results are from the liver, some are from the lung and some are from pooled liver and lung samples. A direct comparison between the liver and lung should be provided, including the cell number and percentage of 4 CD4 subsets. In addition, a secondary lymphoid organ control (e.g., the spleen) should be included.\n2. What are the identity and function of Tby? MHCII KO experiments suggest that Tby are enriched in the absence of MHCII (Fig. 6). However, some other results indicate that Tby can share the same TCR with other subsets (Fig. 3 and Fig S3). How to explain the changes in Tby population in Bcl6KO and Bhlhe40 KO? Can aNKG2D alone lead to Tby activation? The expression of NK markers may not necessarily result in TCR-independent memory T cells. For example, memory CD8 T cells can express NKG2D. These memory T cells can be activated via either TCR or NKG2D.\n3. Because CD69+ TRM and TRMP carry P2RX7, it is highly possible that during tissue digestion and cell isolation, these TRM will receive a pro-apoptotic signal, which can also explain why CD69+ TRM results in less progeny after transfer. Please use Treg Protector to block P2RX7-induced signals and repeat the experiment.\nSome minor points.\n1. For Figure 1g, please provide statistics. Some of the differences are marginal. Without statistical analysis, it is difficult to conclude.\n2. Fig. 1h, what is Tint? Another subset or Tby?\n3. Fig. S1f, in the lymphoid structures of mouse samples, are B cells detectable?\n4. Fig 6C, the Trm FACS profiles do not represent the statistical analysis.\n5. Line 309, the statement about the “expression level” of transcription factors is not accurate. Fig. 5a is based on Regulon activity analysis, not the expression level of TFs.", "reviewer_3": "Kong et al. use single-cell transcriptomics and epigenomics to characterize memory CD4+ T cell subsets in tissues (primarily liver and lung) of mice with chronic graft-versus-host disease (GVHD). They identify four distinct subsets: two with stem-like properties, denoted as Tsc (stem-like) and Trmp (resident memory precursor), and two more differentiated populations, Trm (resident memory) and Tby (bystander activated).\nThey use TCR sequencing and RNA velocity to posit a differentiation path from Tsc to Trmp to Trm. Flow cytometry with CD69 and Ly108 helps parse these populations, and adoptive transfer experiments show that Tsc and Trmp can give rise to all four subsets, while Trm cells largely produce more Trm. Using genetic knockouts, they demonstrate that Tcf7 and Bcl6 are required to generate Tsm, while Bhlhe40 supports Trm establishment. Loss of these factors greatly reduces CD4+ T cell numbers and improves disease outcomes. Interestingly, the authors find that Hobit is not significantly involved in disease which seems to be distinct from other findings in experimental colitis. Finally, they show evidence that CD69/Ly108 T cell subsets are present in lymphoid-like structures in the tissues of patients with chronic GVHD.\nOverall, this work provides an in-depth assessment of the polyclonal CD4+ T cell memory compartment in target tissues of GVHD and sheds light on some of the developmental requirements for establishment of those populations, and the relationship between them. Enthusiasm is tempered by over/miss- interpretation of the data and the modest advance this study makes in the field in light of recent publications. That being said, these data are clear and rigorous, and provide a necessary piece to the puzzle of how chronic GVHD is mediated and what cells should be targeted therapeutically.\nSpecific Feedback and Recommendations:\n1. Recent work that the authors cite shows that resident progenitor populations give rise to pathogenetic effector T cells in GVHD (Sacirbegovic et al., 2023 PMID: 36720219). The current manuscript links the progenitor population (here called Trmp and Tsc) to Trm. A more in-depth discussion of the similarities and differences between this study and the published paper is needed. It appears they are studying some of the same cell populations, but are calling them different names.\n2. The finding that Bhlhe40 supports Trm establishment is consistent with similar findings in acute infection and tumor contexts (Li et al., 2019 PMID: 31533057). This should be discussed and this paper cited.\n3. It has also been shown that TCF1 expression in CD4+ T cells is needed to cause GVHD (Mahinbanu et al., 2023 PMID: 36901757). Please consider this paper in the discussion.\n4. Since the manuscript focuses on GVHD, please adjust the title to better reflect this (e.g., replace \"chronic tissue inflammation\" with \"chronic graft-versus-host disease\").\n5. The authors make the conclusion that there is a linear differentiation path from Tsc to Trmp to Trm, however this is not fully supported by the data. As is, it’s unclear from the RNA trajectory analysis that there is a direct linear differentiation path. The authors should discuss the genes influencing the transitions from cluster to cluster which may help clarify if these transitions exist. The epigenetic analysis supports that Tsc and Trmp are more stem-like and Trm and Tby are more differentiated. In our opinion, it does not support the direct transition from Tsc--> Trmp--> Trm. Taken together, the data still does not rule on the possibility that Tsc transition directly into Trm, or that Trmp are not direct progeny of Tsc, but rather are established independently and maintained separately. In the adoptive transfer studies, Trmp cells can give rise to Tsm; If there were a linear differentiation path, this would mean Trmp de-differentiate back into Tsm which seems unlikely. Please amend the interpretation of the data to reflect a more balanced discussion of possible differentiation paths.\n6. Please clarify how the bystander-activated CD4 subset (Tby) was defined. It is at odds with the literature where bystander- activated T cells express CD69 (PMID: 31827070). The lack of antigen specificity makes it difficult to discern if these are bystander (in the sense they are not alloantigen specific), and/or bystander-activated (activated by cytokines, not TCR).\n7. In Figure 5, there is an overall decrease in the number of CD4+ cells lacking Tcf1, bcl6 or bhlhe40. This makes it difficult to attribute disease outcomes to a specific CD4 population, rather than just a total decrease in CD4s. A quantification of numbers of Tm cell subset in Figures 5e-g would help clarify this and is data the authors likely already have.\n8. Are Figures 1h, 6c, 6g (and any others with GMCSF/IFNg) showing an in vitro stimulation condition? Please clarify.\n9. Conclusions that Bhlhe40-deficiency blocks Tsm differentiation into Trm is not fully supported. The data show a reduction in CD4+ cells lacking Bhlhe40, and skewed proportions, but not necessarily that Bhlhe40-KO Tsc fail to generate Trm. A stronger conclusion would require transferring Bhlhe40-KO Tsc into a new mouse and demonstrating failure to recover Trm.\n10. The authors refer to CD69 as a tissue-residency marker, but it is also a marker of activation. This dual role should be clarified.\n11. There are a few instances where \"Tby\" is referred to as \"Tint\" in the figures. Please ensure consistent terminology.\n12. In the first paragraph of the Results section, it would be helpful to include citations for this disease model and a brief description of its associated pathology.\n13. The sort purity (or sort gating, if purity is unavailable) should be shown for the adoptive transfer studies. Since cell fate relationships are being concluded from these studies, it's important to show no contamination between cell subsets.\n14. Please include the table of genes used to generate Fig. 2C\n15. The histology images are low resolution and difficult to interpret", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWERS’ COMMENTS\nBelow are our point-to-point responses to reviewers’ critiques. Our responses are in italic.\nChanges are also made accordingly in the main text, with highlights. In addition, we have\ncorrected some errors that we found in the original submission while preparing source files. We\nhope that you will find that the revised manuscript is markedly improved. Thank you so much!\nReviewer#1 (Remarks to the Author):\n1. The major weakness of this study is that the authors did not demonstrate that Tsm is truly\nrequired for the development of chronic GVHD. They showed that CD4 depletion using\nmonoclonal antibodies ameliorated chronic GVHD, but this method depletes not only Tsm but\nalso the entire CD4 population throughout the body. TCF-1 and BCL6 should be expressed in a\nbroad range of donor T cells, including CD8 T cells. The authors need to find a more specific\nmethod to target Tsm, or they should demonstrate that the adoptive transfer of Tsm can develop\nchronic GVHD in secondary recipients.\nResponse: Taking reviewer’s advice, we have designed an adoptive transfer experiment to\ndemonstrate that transfer of Tsm can develop chronic GVHD in secondary recipients (see Fig. 4\ne-g and Fig. S5b, c) (see Results on pages 12-13).\nChronic GVHD usually occurs ~30 days after HCT in murine models (John F DiPersio, et al. Dis\nModel Mech. 2011;4(3):318-33). To more clearly demonstrate the role of Tsm, Trmp, and Trm from\nthe liver and lung tissues in induction of chronic GVHD in adoptive recipients, we established\nadoptive BALB/c recipients by injecting Rag-1-/- C57BL/6 BM cells (2.5x106) and donor-type CD8+\nT cells (0.1x106) to facilitate donor stem cell engraftment and avoid influence of de novo generated\ndonor-type T cells. 15 days after initial HCT, the adoptive recipients were injected respectively\nwith sorted Tsm, Trmp, or Trm cells (0.25x106 each) from primary chronic GVHD recipients 30\ndays after HCT. We observed that the adoptive recipients given Tsm or Trmp but not Trm cells\nshowed significantly increased clinical GVHD score and bodyweight loss as compared with\ncontrol recipients injected with PBS (see Fig. 4e). Although we found no significant difference in\nclinical GVHD scores between the recipients given Tsm or Trmp, the histopathology scores of\nGVHD target tissues liver and lung of recipients given Tsm were significantly higher than that of\nTrmp cells, and the latter were significantly higher than those given Trm cells. Higher\nhistopathology scores were associated with the higher numbers of infiltrating Trm cells in the\ntissues (see Fig. 4f, g). The numbers of tissue infiltrating Trm cells generated by Tsm were\nmarkedly higher than those generated by Trmp (See Fig. 4b, c, g). These results indicate that\nwhile both Tsm and Trmp can maintain the Trm pool, the Tsm have stronger capacity than Trmp\nin generating the pathogenic Trm and maintain bigger Trm pool. This information has been\nincorporated into text (see result section on pages12-13)\n2. The authors annotated cluster 2 as bystander activated T cells (Tby) based on the expression\nof Tby-associated markers such as NKG2D and lower expression of TCR signaling markers.\nHowever, NKG2D is a marker of CD8 Tby, and it has not been tested if CD4+ Tby cells express\n1\nit. It could be misleading to use this annotation, and this reviewer recommends using a less\ndefinitive term to describe this population.\nResponse: We understand the reviewer’s concern. We have replaced the term “Tby” with\nintermediate T (Tint) for the CD108-CD69- Tm subset in the current report. The term “Tint” was\nproposed for Ly108-CD69-CD8+ Tm subset by Belta et al (JC Beltra, et al.Immunity.2020;52(5):\n825-841.e828,Immunity.2023;56(12):2699-2718.e11). We adopted the usage of Ly108 versus\nCD69 from Belta et al report to divide CD4+ Tm into four subsets, including Ly108+CD69- Tsm,\nLy108+CD69+ Trmp, Ly108-CD69+ Trm, and Ly108-CD69- Tint. We incorporated this concept\ninto our current studies (see Introduction on pages 4-5).\n3. Figure 1h: Tint\" should be labeled as Tby.\nResponse: We have kept “Tint” as explained above.\n4. The authors concluded that TCF-1, BCL6, or Bhlhe40 deficiency in donor T cells, or IFNgR\ndeficiency in donor BM cells, ameliorated chronic GVHD. However, they only demonstrated\nimproved survival and mitigation of bodyweight loss. Essentially, this [B6 into BALB/c] model is\nan acute GVHD model (Reddy P: BBMT 2008; 14:129), therefore these data may indicate that\nthese deficiencies ameliorated acute GVHD, not chronic GVHD. The authors need to show\nmore chronic GVHD-specific findings were ameliorated, such as organ fibrosis or tear secretion.\nHow did they rule out the impact of TCF-1, BCL6, or Bhlhe40 deficiencies on CD8+ T cells? The\nauthors showed that CD4 T cells were required for the induction of GVHD in this model, but they\ndid not test whether CD8+ T cells play a role in GVHD via a CD4-dependent mechanism.\nResponse: We agree that the model of B6 donor to BALB/c recipient tends to develop more\nsevere acute GVHD, as Reddy et al pointed out (Reddy P, et al: Biol Blood Marrow\nTransplant.2008;14:129-35). However, our studies showed that the model of B6 donor to BALB/c\nrecipient also developed chronic GVHD with appropriate dose of donor T cells that allowed the\nrecipient to survive for more than 30-60 days (Song, et al. Front Immunol.2021; 12:700857, Wu,\net al. J Immunol. 2013;191(1): 488-99). Chronic GVHD in murine models and in many humans is\na sequela of acute GVHD (Filipovich et al. Biol Blood Marrow Transplant.2005;11(12):945-956).\nThe information has been incorporated into introduction (see page 4).\nThe Salivary and lacrimal glands are the prototypical target organs of chronic GVHD while gut,\nliver, and lung can overlap between acute and chronic GVHD (Wu, et al.J Immunol. 2013;191(1):\n488-99). In the current studies, we have stressed that those differences were observed 30-60\ndays after HCT during possible chronic GVHD period. Agreeing with the reviewer’s suggestion,\nwe have also added fibrosis evaluation in the liver and lung tissues as well as addition of\nhistopathology of salivary and lacrimal glands of recipients given transplant with Tcf7, Bcl6,\nBhlhe40, or IFNgR deficiency in donor T cells to evaluate their impact on chronic GVHD (see Fig.\n5c, f, i; Fig. 6f and Fig. S7a, b). The information has been incorporated into the text.\nTo test whether “CD8+ T cells play a role in GVHD via a CD4-dependent mechanism”, first, we\ncompared the expansion of donor CD8+ T cells in the adoptive recipients with or without infusion\nof donor-type Tm subset, we found that adoptively transferred CD4+ Tsm cells could induce the\nexpansion of CD8+ Tm cells in the liver and lung (Fig. S5c). Second, we tested whether donor\nCD8+ T cells induced chronic GVHD with expansion of Trm cells. We found that in the absence\nof donor-type CD4+ T cells, donor CD8+ T cells induced little signs of chronic GVHD or\n2\nexpansion of CD8+ Trm cells (Fig. S6a-d). Therefore, donor-type CD4+ T cells are required for\ninduction of chronic GVHD mediated by donor CD8+ T cells, in other words, donor CD8+ T cells\nmay augment cGVHD in a CD4+ T-dependent manner. The information has been incorporated\ninto the text (see Results on page 13).\n5. The authors used a single strain combination; B6 donor and BALB/c recipients. To generalize\ntheir findings, they should use at least one more model of chronic GVHD.\nResponse: Besides the full MHC-mismatched model of B6 donor to BALB/c recipient, we have\nadded MHC-haploidentical and MHC-matched models (see Fig. S2 and Results on pages 7-8).\n6. Figure 4C showed that Trmp give rise to Tsm, indicating bidirectional differentiation between\nTsm and Trmp. This is similar to the interconversion between TexProg1 and TexProg2 shown in\nCD8+ T cells (Beltra JC: Immunity 2020). It is better to add brief discussion about this point.\nResponse: We added a brief discussion (see pages 19-20)\n7. Figure S6d: The authors need to clarify in the figure legend when this analysis was performed.\nResponse: The previous Figure S6d has been moved to Fig.6d with showing IFN-+GM-CSF+ Trm\ncells in both liver and lung. The analysis was performed on day 60 after HCT, as now explained\nin the legend for Fig. 6d.\n8. Figure 6f: CD11b MHCII cells were expanded in the recipients of IFNgR-deficient BM cells.\nThe authors should briefly discuss about these cells.\n⁻ ⁺\nResponse: We have reanalyzed the data and replaced “CD11b-MHCII+” with “CD11b+CD11c+”\nDCs. CD11b+CD11c+ DCs have strong capacity to capture and process antigens and stimulate T\ncell proliferation (E Daro, et al. J Immonol.2000;165(1):49–58). (see Fig. 6a and Fig. S8a).\n9. Figure 7: The authors need to add more detailed background information about the patients,\nsuch as donor sources, MHC matching, time points of sample collection, ongoing\nimmunosuppression at sample collection, history of acute GVHD, and other relevant details.\nResponse: Information has been added (see Supplemental Tables 2, 3).\n10. The authors only used GVHD models. Therefore, they should narrow the title of this\nmanuscript to reflect its focus on chronic GVHD, rather than chronic tissue inflammation.\nResponse: We have changed the title to “Chronic GVHD”.\nReviewer #2 (Remarks to the Author):\n1. Some results are from the liver, some are from the lung and some are from pooled liver and\nlung samples. A direct comparison between the liver and lung should be provided, including the\ncell number and percentage of 4 CD4 subsets. In addition, a secondary lymphoid organ control\n(e.g., the spleen) should be included.\nand Lung of chronic GVHD recipients. We started the project with scRNA-seq and scTCR-CDR3-\nseq analysis of Tm cells from the liver tissue and combined RNA-Seq with ATAC-Seq analysis of\nTm cells from the lung tissues of the same recipients due to limited cell yields from each organ\n(Fig. 1-3). Then, we performed Tm subset transfer by combining Tm cells from the liver and lung\n(Fig. 4). Thereafter, we compared both liver and lung. However, due to space limit, we put liver in\nthe figures and the lung in the supplemental figures.\nTaking reviewer’s suggestion, during revision, we have put the liver and lung profiles side by side\nin comparison in the Figures (Fig. 4b, c, f, g, i, j; Fig.5c, d, f, g, i, j and Fig. 6b, c, d, f, g, h) and\nsupplementary Figures (Fig. S4d, e; Fig. S5; Fig. S6; Fig. S7 and Fig. S8c-e). Due to space limit,\nwe put representative flow patterns in the supplement in some cases (i.e., Fig. S6e and Fig. S8d,\ne).\nChronic GVHD recipients have lymphopenia with tiny and fibrotic spleen, and it is not feasible to\nanalyze Tm subsets in the spleen of chronic GVHD recipients in most of experiments. However,\nwe had compared the Tm subset changes in the spleen and GVHD target tissues in the kinetic\nstudies shown in Fig. S5a and detailed information was in Results on page 11.\n2. What are the identity and function of Tby? MHCII KO experiments suggest that Tby are\nenriched in the absence of MHCII (Fig. 6). However, some other results indicate that Tby can\nshare the same TCR with other subsets (Fig. 3 and Fig S3). How to explain the changes in Tby\npopulation in Bcl6KO and Bhlhe40 KO? Can aNKG2D alone lead to Tby activation? The\nexpression of NK markers may not necessarily result in TCR-independent memory T cells. For\nexample, memory CD8 T cells can express NKG2D. These memory T cells can be activated via\neither TCR or NKG2D.\nResponse: The focus of the current studies is on MHCII/TCR-dependent Tsm, Trmp, and Trm.\nCharacterization of Tby is not the focus of current report. Taking the advice from Rev. 1, we\nhave changed Tby to be less definitive term “Intermediate”, the term was proposed by John\nWherry group (Beltra, et al. Immunity. 2020;52(5):825-841.e8.). We have pointed out that the\nrole of Tint in cGVHD pathogenesis remains unclear and subject to future studies (see\nDiscussion on pages 19-20).\n3. Because CD69+ TRM and TRMP carry P2RX7, it is highly possible that during tissue\ndigestion and cell isolation, these TRM will receive a pro-apoptotic signal, which can also\nexplain why CD69+ TRM results in less progeny after transfer. Please use Treg Protector to\nblock P2RX7-induced signals and repeat the experiment.\nResponse: In our new transfer experiments, we have added Treg Protector to reduce NAD\ninduced cell death before cell sorting. This info has been added to text (see page 12) and\nMaterials and Methods (see page 28). We still observed less expansion of Trm cells in the\nadoptive recipients (see Fig. 4g and Fig. S5b).\nSome minor points.\n1. For Figure 1g, please provide statistics. Some of the differences are marginal. Without\nstatistical analysis, it is difficult to conclude.\nexpression (see Fig. 1g)\n2. Fig. 1h, what is Tint? Another subset or Tby?\nResponse: We have replaced Tby with Tint, and the definition of Tint was described in text (see\npages 4-5).\n3. Fig. S1f, in the lymphoid structures of mouse samples, are B cells detectable?\nResponse: The text on page 19 cites our previous publication showing that B cells are present\nin lymphoid structures (Kong et al. Blood. 2022;140(25):2740-2753). We did not stain B cells in\nthe Fig. S1f because the focus in the current study was on Tm subsets.\n4. Fig 6C, the Trm FACS profiles do not represent the statistical analysis.\nResponse: Fig. 6d now shows the statistical analysis.\n5. Line 309, the statement about the “expression level” of transcription factors is not accurate.\nFig. 5a is based on Regulon activity analysis, not the expression level of TFs.\nResponse: We have replaced “expression level” with “regulon activity level” (see page 15).\nReviewer #3 (Remarks to the Author):\n1. Recent work that the authors cite shows that resident progenitor populations give rise to\npathogenetic effector T cells in GVHD (Sacirbegovic et al., 2023 PMID: 36720219). The current\nmanuscript links the progenitor population (here called Trmp and Tsm) to Trm. A more in-depth\ndiscussion of the similarities and differences between this study and the published paper is\nneeded. It appears they are studying some of the same cell populations but are calling them\ndifferent names.\nResponse: The studies by Sacirbegovic et al focus on the role of local TCF1+ progenitor-like Tm\ncells in perpetuating acute GVHD. The authors did not divide the TCF1+ progenitor-like Tm cells\ninto subsets. Our current studies divided the TCF+ progenitor-like Tm cells into Tsm and Trmp as\nwell as further characterize their features, differentiation pathways, and pathogenic capacities.\nThis information has been added to the Discussion (see pages 19-21).\n2. The finding that Bhlhe40 supports Trm establishment is consistent with similar findings in acute\ninfection and tumor contexts (Li et al., 2019 PMID: 31533057). This should be discussed and this\npaper cited.\nResponse: A brief discussion has been added (see page 21).\n3. It has also been shown that TCF1 expression in CD4+ T cells is needed to cause GVHD\n(Mahinbanu et al., 2023 PMID: 36901757). Please consider this paper in discussion.\nResponse: This publication is cited in discussion (see page 21 and reference #49).\n5\n4. Since the manuscript focuses on GVHD, please adjust the title to better reflect this (e.g.,\nreplace \"chronic tissue inflammation\" with \"chronic graft-versus-host disease\").\nResponse: We have replaced “chronic tissue inflammation” with “chronic graft-versus-host\ndisease”.\n5. The authors make the conclusion that there is a linear differentiation path from Tsc to Trmp to\nTrm, however this is not fully supported by the data. As is, it’s unclear from the RNA trajectory\nanalysis that there is a direct linear differentiation path. The authors should discuss the genes\ninfluencing the transitions from cluster to cluster which may help clarify if these transitions exist.\nThe epigenetic analysis supports that Tsc and Trmp are more stem-like and Trm and Tby are\nmore differentiated. In our opinion, it does not support the direct transition from Tsc--> Trmp-->\nTrm. Taken together, the data still does not rule on the possibility that Tsc transition directly into\nTrm, or that Trmp are not direct progeny of Tsc, but rather are established independently and\nmaintained separately. In the adoptive transfer studies, Trmp cells can give rise to Tsm; If there\nwere a linear differentiation path, this would mean Trmp de-differentiate back into Tsm which\nseems unlikely. Please amend the interpretation of the data to reflect a more balanced\ndiscussion of possible differentiation paths.\nResponse: We understand the reviewer’s concern and have added a more balanced discussion\n(see page 20).\n6. Please clarify how the bystander-activated CD4 subset (Tby) was defined. It is at odds with the\nliterature where bystander-activated T cells express CD69 (PMID: 31827070). The lack of antigen\nspecificity makes it difficult to discern if these are bystander (in the sense they are not alloantigen\nspecific), and/or bystander-activated (activated by cytokines, not TCR).\nResponse: We understand the reviewer’s concern and have replaced the term “Tby” with “Tint”.\n(see Introduction on pages 4-5 and Discussion on pages 19-20).\n7. In Figure 5, there is an overall decrease in the number of CD4+ cells lacking Tcf1, bcl6 or\nbhlhe40. This makes it difficult to attribute disease outcomes to a specific CD4 population,\nrather than just a total decrease in CD4s. A quantification of numbers of Tm cell subset in\nFigures 5e-g would help clarify this and is data the authors likely already have.\nResponse: A quantification of numbers of Tm cell subsets has been added to Fig. 5d, g, j.\n8. Are Figures 1h, 6c, 6g (and any others with GMCSF/IFNg) showing an in vitro stimulation\ncondition? Please clarify.\nResponse: All T cell intracellular staining of GM-CSF/IFN-g are from in vitro stimulation with PMA\n+ Ionomycin with addition of Golgi blocker. This information is added to Methods (see page 25).\n9. Conclusions that Bhlhe40-deficiency blocks Tsm differentiation into Trm is not fully supported.\nThe data show a reduction in CD4+ cells lacking Bhlhe40, and skewed proportions, but not\nnecessarily that Bhlhe40-KO Tsc fail to generate Trm. A stronger conclusion would require\ntransferring Bhlhe40-KO Tsc into a new mouse and demonstrating failure to recover Trm.\nResponse: We understand the reviewer’s concern and have toned down our statement by\nreplace “block” or “prevention” to “impair” or “reduction” (see abstract and text on page 15).\n6\n10. The authors refer to CD69 as a tissue-residency marker, but it is also a marker of activation.\nThis dual role should be clarified.\nResponse: CD69 is expressed by T cells at early activation stage and memory stage. The\nCD69+ effector memory T cells are tissue-resident T cells. We have clarified the concept (see\nIntroduction on page 4).\n11. There are a few instances where \"Tby\" is referred to as \"Tint\" in the figures. Please ensure\nconsistent terminology.\nResponse: “Tint” has been used throughout the revised manuscript.\n12. In the first paragraph of the Results section, it would be helpful to include citations for this\ndisease model and a brief description of its associated pathology.\nResponse: “Citations for this disease model and a brief description of its associated pathology” is\nadded at the beginning of Results section (see page 6).\n13. The sorting purity (or sort gating, if purity is unavailable) should be shown for the adoptive\ntransfer studies. Since cell fate relationships are being concluded from these studies, it's important\nto show no contamination between cell subsets.\nResponse: Sorting gating and purity are shown in (see Fig. S5b).\n14. Please include the table of genes used to generate Fig. 2C.\nResponse: A supplemental Table is added for the genes (See Supplementary Table 1).\n15. The histology images are low resolution and difficult to interpret.\nResponse: The IMC resolution is enhanced with denoised program (see methods section on page\n33). To validate the IMC data, we also added multiple color immunofluorescent staining data (see\nFig. 7h, I).\nIn addition, while preparing this resubmission, we discovered some errors in Figures 4, 5, and 6.\nIn the original version of Figure 4k, 5 recipients and 2 deaths were inadvertently omitted from\nthe IgG-treated group. These errors have been corrected in this revision.\nIn the original Figure 5b, 5e, 5h, and Figure 6e, KO and WT controls did not consistently\noriginate from the same experiments, and body weight was not monitored in all recipients.\nIn the revised Figure 5b, 5e and 5h and Figure 6e, KO and WT controls are from the same\nexperiments. The revised Figures 5b and 5e each show combined results from 2 experiments\neach with 5 KO recipients and 5 WT controls. The revised Figure 5h shows combined results\nfrom 2 experiments each with 10 KO recipients and 5 WT controls.\nThe revised Figure 6e shows combined results from 2 experiments, the first with 5 KO recipients\nand 5 WT controls and the second with 10 KO recipients and 5 WT controls. In the second\nexperiment, all WT controls died prematurely within days 10 after HCT. Because this cage\n7\neffect exaggerates the survival difference in the combined results, a separate third experiment\nwas done with 5 additional WT controls. We have added Supplementary Figure 8h showing the\ncombined results with the 15 KO recipients and the 10 WT controls from the first and third\nexperiments. The supplementary figure does not include a p-value for this comparison. We\nhave added a note in the source data to explain why the third experiment was done and why we\nhave added Supplementary Figure 8h in the revised manuscript.\nIn each of these experiments, body weight was monitored in all recipients. The revisions in\nthese figures do not change any of the conclusions from the original submission. The data\nshow that deficiency of TCF1, Bcl6, Bhlhe40 and IfngR1 in donor T cells each attenuate the\nseverity of acute GVHD. In these experiments, the more relevant data pertain to their effects on\nthe biology of chronic GVHD when evaluated at day 60 after HCT.\n8"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The major weaknesses of the previous version have been adequately addressed in the revision. The newly added adoptive transfer experiments substantially strengthen the overall conclusions, demonstrating that Tsm and Trmp, but not Trm, can induce chronic GVHD upon adoptive transfer. Other concerns have also been addressed adequately.", "reviewer_2": "No more concerns. This work provides convincing evidence to illustrate the differentiation pathway, function and transcriptional regulation of pathogenic CD4 T cells during cGVHD.", "reviewer_3": "Reviewer responses to authors indicated below (and attached as color coded word doc.)\n1. Recent work that the authors cite shows that resident progenitor populations give rise to pathogenetic effector T cells in GVHD (Sacirbegovic et al., 2023 PMID: 36720219). The current manuscript links the progenitor population (here called Trmp and Tsm) to Trm. A more in-depth discussion of the similarities and differences between this study and the published paper is needed. It appears they are studying some of the same cell populations but are calling them different names.", "reviewer_author_response": "A point-by-point response to the reviewers' comments are inserted below:\nRESPONSE TO REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe major weaknesses of the previous version have been adequately addressed in the\nrevision. The newly added adoptive transfer experiments substantially strengthen the\noverall conclusions, demonstrating that Tsm and Trmp, but not Trm, can induce chronic\nGVHD upon adoptive transfer. Other concerns have also been addressed adequately."}, "author_response": ""}]

Decision letter