Tissue-resident Memory Research Articles

A novel “prime and pull” strategy mediated by the combination of two dendritic cell-targeting designs induced protective lung tissue-resident memory T cells against H1N1 influenza virus challenge

Vaccination is still the most promising strategy for combating influenza virus pandemics. However, the highly variable characteristics of influenza virus make it difficult to develop antibody-based universal vaccines, until now. Lung tissue-resident memory T cells (TRM), which actively survey tissues for signs of infection and react rapidly to eliminate infected cells without the need for a systemic immune reaction, have recently drawn increasing attention towards the development of a universal influenza vaccine. We previously designed a sequential immunization strategy based on orally administered Salmonella vectored vaccine candidates. To further improve our vaccine design, in this study, we used two different dendritic cell (DC)-targeting strategies, including a single chain variable fragment (scFv) targeting the surface marker DC-CD11c and DC targeting peptide 3 (DCpep3). Oral immunization with Salmonella harboring plasmid pYL230 (S230), which displayed scFv-CD11c on the bacterial surface, induced dramatic production of spleen effector memory T cells (TEM). On the other hand, intranasal boost immunization using purified DCpep3-decorated 3M2e-ferritin nanoparticles in mice orally immunized twice with S230 (S230inDC) significantly stimulated the differentiation of lung CD11b+ DCs, increased intracellular IL-17 production in lung CD4+ T cells and elevated chemokine production in lung sections, such as CXCL13 and CXCL15, as determined by RNAseq and qRT‒PCR assays, resulting in significantly increased percentages of lung TRMs, which could provide efficient protection against influenza virus challenge. The dual DC targeting strategy, together with the sequential immunization approach described in this study, provides us with a novel “prime and pull” strategy for addressing the production of protective TRM cells in vaccine design.

Tissue-resident memory T cells exhibit phenotypically and functionally heterogeneous in human physiological and pathological nasal mucosa

Pathogens commonly enter mucosal barrier tissues and tissue-resident memory T cells (TRM) are essential for preventing mucosal lesions. However, the immunological properties of TRM cells in nasal mucosa are poorly known. In comparison with control tissues, decreasing CD103+ TRM cells were observed in Chronic rhinosinusitis with nasal polyps (CRSwNPs) and sinonasal inverted papilloma (SNIP), which presented high capability to produce effector cytokines. In CRSwNPs, we found that CD103+ TRM cells with higher cytokine and Granzyme B coexpressed high PD-1, CD103− TRM cells expressed higher IL-10. Homogenates isolated from CRSwNPs induced CD103 expression on peripheral T cells which could be inhibited by blocking TGF-β. The frequencies of CD103+ TRM cells in CRSwNPs were extremely negatively correlated with neutrophil infiltration. CD103+ TRM cells from Staphylococcus aureus positive CRSwNPs had a stronger response to SEB. Taken together, two phenotypically and functionally distinct subsets of TRM cells exist in nasal tissues and play critical roles in the progress of CRSwNPs and SNIPs.

Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions.

IPSC-derived hypoimmunogenic tissue resident memory T cells mediate robust anti-tumor activity against cervical cancer

Functionally rejuvenated human papilloma virus-specific cytotoxic T lymphocytes (HPV-rejTs) generated from induced pluripotent stem cells robustly suppress cervical cancer. However, autologous rejT generation is time consuming, leading to difficulty in treating patients with advanced cancer. Although use of allogeneic HPV-rejTs can obviate this, the major obstacle is rejection by the patient immune system. To overcome this, we develop HLA-A24&-E dual integrated HPV-rejTs after erasing HLA class I antigens. These rejTs effectively suppress recipient immune rejection while maintaining more robust cytotoxicity than original cytotoxic T lymphocytes. Single-cell RNA sequencing performed to gain deeper insights reveal that HPV-rejTs are highly enriched with tissue resident memory Tcells, which enhance cytotoxicity against cervical cancer through TGFβR signaling, with increased CD103 expression. Genes associated with the immunological synapse also are upregulated, suggesting that these features promote stronger activation of Tcell receptor (TCR) and increased TCR-mediated target cell death. We believe that our work will contribute to feasible "off-the-shelf" Tcell therapy with robust anti-cervical cancer effects.

Abstract A012: STING agonists drive intrinsic type I interferon responses in monocytes for optimal anti-tumor immunity

Abstract The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is triggered by the sensing of cytosolic DNA, triggering the production of type I interferon (IFN-I) and play a crucial role in host defense against pathogens. This pathway also induces an anti-tumor response when tumor-derived DNA is sensed. Small molecule agonists called STING agonists activate this pathway and have been shown to cause tumor regression in mice. However, it is unclear how immune cells respond to STING agonist-induced IFN-I. In our study, we observed that monocytes were abundantly recruited to tumors during STING agonist therapy, and the lack of CCR2+ Ly6Chi inflammatory monocytes in CCR2-knockout (KO) mice led to impaired responses to STING agonist therapy of B16F10 and MC-38 tumors. Single-cell RNA sequencing (scRNA-Seq) of CD45+ cells from lymph nodes and tumors of Ifnar1 mixed bone marrow chimeric mice in which half of the immune cells lacked IFN-I receptors revealed that inflammatory monocytes upregulated chemokine receptor ccr5 and genes associated with MHC-I restricted antigen presentation in response to IFN-I. Using mice with B16.OVA.Tomato red fluorescent tumors, we demonstrated that IFN-I-responsive monocytes captured fluorescent antigens in tumors and transported them to lymph nodes. Additionally, the administration of CCR5 antagonist, maraviroc, significantly reduced the number of fluorescent antigen-carrying monocytes in the lymph nodes of red fluorescent tumor-bearing mice treated with STING agonists, indicating the involvement of CCR5 in monocyte-mediated antigen transport. Furthermore, NicheNet analysis of scRNA-Seq data showed that IL-18 - IL-18R1 interaction was the top predicted interaction between inflammatory monocytes and CD8+ T cells or NK cells. Blocking IL-18 remarkedly reduced the therapeutic efficacy of STING agonist treatment. Moreover, IFN-I acted on monocytes to upregulate glucocorticoid-induced TNFR-related (GITRL) providing a pro-survival signal to T cells. Deletion of GITRL on CCR2+ monocytes led to impaired tumor control, and the absence of the cognate receptor GITR on T cells led to impaired anti-tumor effector T cell responses in the tumors treated with and tissue-resident memory T cell accumulation in the skins that rejected tumors with STING agonist therapy. Taken together, our data suggests that STING agonists induce IFN-I-responsive monocytes to promote anti-tumor responses through secreting IL-18, potentially through transporting antigens to lymph nodes, and through providing a pro-survival signal for T cell accumulation in the tumors during STING agonist therapy. Citation Format: Tianning Yu, Melanie Girard, Tania H Watts. STING agonists drive intrinsic type I interferon responses in monocytes for optimal anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A012.

Abstract B007: Phenotypic Signatures of Circulating Neoantigen-Reactive CD8+ T Cells in Patients with Metastatic Cancers

Abstract Background: Immunotherapies represent some of the most promising approaches to treat metastatic solid epithelial cancers, yet their response rates remain low. Identifying antitumor T cells, their antigenic specificities, and their cognate T cell receptors (TCRs) will provide crucial insights into the design of next-generation engineered cellular immunotherapies. Circulating T cells from the peripheral blood (PBL) can provide a rich and non-invasive source for identifying and studying antitumor T cells as an alternative to tumor-infiltrating lymphocytes (TIL). Yet, pre-immunotherapy pre-surgery antitumor T cell frequencies circulating in PBL of patients with metastatic cancer are often low, limiting the accurate definition of their phenotypic states. Methods: We employed single-cell phenotypic profiling of 36 experimentally defined neoantigen-specific T cell clones from 6 metastatic epithelial cancer patients to derive the transcriptional and cell surface protein signatures of pre-surgery PBL-resident antitumor CD8+ T cells (NeoTCRPBL). and compared T cell gene expression signatures neoantigen TCR clonotypes between the PBL and TIL compartments. We developed a NeoTCRPBL gene signature to assess its sensitivity and specificity in discovering new antitumor TCRs from PBL of prospective patients with different solid tumor types. Results: Circulating NeoTCRPBL T cells were clonally expanded, but low in frequency in the PBL (⇐0.001-0.005% per clone) necessitating prior-enrichment for studies. NeoTCRPBL T cells exhibited phenotypes distinct from common blood T cell subsets and bystander viral-reactive T cells displaying transcriptional programs of both dysfunctional as well as tissue-resident memory T cells. Within the same patient, intra-clonotype comparison of 24 TIL-and PBL-neoantigen-specific T cell clones revealed that relative to their TIL counterparts, circulating NeoTCRPBLT cells displayed less-dysfunctional immunotherapy-response associated progenitor phenotypic states. Combined analysis of 100 antitumor T cell clones revealed that circulating NeoTCRPBL T cells largely targeted the same clonal, subclonal neoantigens with comparable avidity as TIL (>79% shared), but their TCR-repertoire was only partially shared with TIL (47% shared). Finally, prediction and testing of 64 clonally expanded TCRs based on NeoTCRPBL gene expression signature-enrichment from prospective PBL samples discovered 20 neoantigen-TCR clonotypes suggesting that the NeoTCRPBL signature can successfully identify antitumor TCRs from very low circulating PBL frequencies(< 0.002%). Conclusions: Circulating PBL-resident antitumor T cells are low in frequency exhibiting distinct clonotypic repertoire and phenotypic states in patients with metastatic solid tumors. The NeoTCRPBL signature provides an alternative source for identifying antitumor T cells and their TCRs non-invasively from pre-surgery blood samples from cancer patients enabling immune monitoring and immunotherapies. Citation Format: Sri Krishna, Rami Yoseph, Sivasish Sindiri, Frank J Lowery, Paul F Robbins, Steven A Rosenberg. Phenotypic Signatures of Circulating Neoantigen-Reactive CD8+ T Cells in Patients with Metastatic Cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B007.

Divergent molecular networks program functionally distinct CD8+ skin-resident memory T cells.

Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.

Bone Marrow CD8 + Tissue-Resident Memory T Cell Promote B Cell Differentiation and Inhibit Platelet Production in Patients with Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease in which enhanced anti-platelet T cell activity and dysfunction of CD8 + T cells are involved(Audia et al., 2013; Semple & Freedman, 1991). Persistent secretion of antibodies by long-lived plasma cells and memory B cells in the bone marrow contributes to ITP pathogenesis (Roeser, Lazarus, & Mahevas, 2023). However, the role of memory T cells, especially tissue-resident memory T cells (TRM), in ITP remains underexplored. TRM develop in specific tissues and play crucial role in local immunity. TRM exert their effects through mechanisms including the induction of local humoral responses, contributing to autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Chang et al., 2021; Gu, Song, Roh, Jung, & Kim, 2021). Bone marrow contains one of the largest pools of memory CD8 + T cells. However, the involvement of bone marrow TRM in ITP pathogenesis remains poorly understood. In this study, we observed a significantly higher proportion of CD8 + TRM (CD8 +CD45RA -CD45RO +CD69 +) in the bone marrow of ITP patients compared to healthy controls. However, no significant differences were observed in CD4 + TRM, CD8 + TCM and CD8 + TEM between the two groups. CD8 + TRM from ITP patients exhibited increased levels of IFN-γ, NLRP3, IL-1β, TNF-α, and decreased levels of IL-10 compared to those from healthy controls. Furthermore, CD8 + TRM proportions were significantly higher in glucocorticoid responders than non-responders, suggesting a potential role of CD8 + TRMs in mediating glucocorticoid therapeutic effects. Transcriptome sequencing revealed upregulated genes, including IGLC2, ASAP2, HIST2H2AA4, IGLV2-11, C1QA, ATP5MC1, IL5RA, and CYC1, and downregulated genes, including CCR9, HSPE1P18, RGCC, and ITGAE, in TRM cells from ITP patients compared to controls. GO/KEGG analysis of the top 500 upregulated genes highlighted pathways associated with B cell-associated immunity. To explore the effect of TRM on B cells, bone marrow CD19 + B cells were co-cultured with autologous TRM or memory T cells without TRM (TM) in the presence of IgM, and the proportion of memory B cells (CD19 +CD27 +CD38 -) and plasma cells (CD19 -CD24 -CD38 +) were measured by flow cytometry. The supernatants were collected, and cytokines and IgG level were tested by ELISA. Results showed that TRMs from ITP patients promoted the differentiation of memory B cells and IgG antibody production compared with those from healthy controls. However, no significant difference was observed in the proportion of naive B cells and plasma cells between the two groups. TRM, but not TM augmented B cell differentiation into memory B cells or plasma cells both in ITP patients and healthy controls. Additionally, TRM-primed B cells showed a stronger effect on antibody production and resulted in a higher level of IgG in supernatant compared with TM primed B cells. In line with these findings, the levels of B cell activating factor (BAFF), a proliferation inducing ligand (APRIL), and sCD40L, which were critical cytokines for B cell survival and differentiation, were significantly higher in supernatants from ITP patients than those from healthy controls. Moreover, the levels of BAFF, APRIL and CD40L in co-culture system with TRM were significantly increased compared to the system with TM, providing insights into the impact of TRM on B cell differentiation and function. To further investigate the role of TRM in regulating platelet production in ITP patients, we cocultured TRM with CD34 +cells in the presence of thrombopoietin (TPO), stem cell factor (SCF) and interleukin (IL)-3. Results showed that TRM did not suppress the production of CD41 + megakaryocytes from CD34 + cells, but reduced megakaryocyte apoptosis and increased the percentage of polyploidy (≥4N) megakaryocytes compared with TM. Additionally, TRM inhibited reticulated platelet formation and platelet production. Notably, dexamethasone (DXM) modulation alleviated certain suppressive effects on platelet production. In summary, our findings reveal the involvement of impaired bone marrow CD8 + TRM in the pathogenesis of ITP through the promotion of B cell differentiation and inhibition of platelet production. These results shed new light on the potential of immune therapy targeting TRM in the management of ITP.

ST2/ Rorγt-Dependent Th1/Th2-like CD4 + Trm Cells Mediate Cutaneous Chronic Gvhd

Allogeneic hematopoietic cell transplantation (Allo-HCT) is a curative therapy for relapsed hematological malignances, but the same alloreactive T cells that eliminate malignant cells in the recipient also mediate graft-versus-host disease (GVHD), and in long-term Allo-HCT patients chronic GVHD (cGVHD) remains the major cause of morbidity and mortality. Th subsets (i.e., Th1, Th2, and Th17) have been associated with cGVHD in certain tissues, but the Th subsets involved in cutaneous cGVHD remain undefined. In the current studies, with a murine model of C57BL/6 donor to MHC-matched C3H.SW recipient, we observed that although IFN-γ + Th1 cells were dominant in the gut, liver, and skin during early acute GVHD, the skin showed a marked expansion of IFN-γ +IL-13 + Th1/Th2-like cells 30 days after HCT at the onset of cGVHD, while IFN-γ +IL-13 - Th1 cells remained dominant in the liver and intestines. The cutaneous Th1/Th2-like cells exhibited characteristics of Ly108 -CD69 + tissue resident memory T (Trm) cells. The expansion of cutaneous Th1/Th2-like cells was RORγt and ST2-dependent because T cells from RORγt -/- or ST2 -/- C57BL/6 donors did not show expansion of the cutaneous Th1/Th2-like cells or induce cutaneous cGVHD. These results indicate that 1) IFN-γ +/IL-13 + Th1/Th2-like CD4 + Trm cells likely play a critical role in the pathogenesis of cutaneous cGVHD; 2) the differentiation of IFN-γ +CD4 + Th1 cells into IFN-γ +IL-13 + Th1/Th2-like cells is RORγt and ST2-dependent. The molecular mechanisms that underpin this differentiation pathway are under investigation.

Clonal Expansion and Differentiation of Stem-like Memory T Cells to Tissue-Resident Memory T Cells Perpetuates Pathogenesis of Chronic Graft-Versus-Host Disease

We and others have shown that tissue-resident memory CD4 + T cells play a critical role in maintaining chronic GVHD pathogenesis. However, the cellular and molecular mechanisms remain largely unknown. Ly108 and (TCF1) expression have been shown to reflect the stemness of memory CD8 + T cells. In the current studies, using the markers Ly108 (TCF1) and CD69, we identified four subsets of memory CD4 + T (Tm) cells in the GVHD target tissues, including Ly108 + CD69 - and Ly108 +CD69 + stem-like Tm cells, as well as Ly108 - CD69 - and Ly108 - CD69 + differentiatedTm cells. Compared to other Tm subsets, the Ly108 - CD69 + subset expressed the highest levels of IFN-γ and GM-CSF without upregulating anergy/exhaustion markers, indicating that they represent a terminally differentiated, tissue resident, pathogenic CD4 + memory T (Trm) subset. The Ly108 + Tm subsets showed self-renewal capacity and differentiation into Ly108 - Tm subsets, as demonstrated by adoptive transfer experiments. Using single-cell RNA sequencing (scRNA-seq) in conjunction with scTCR-seq analysis, we observed that Ly108 +CD69 - Tm subset was clonally related to the expanded Ly108 - CD69 - and Ly108 - CD69 + Tm subsets, suggesting a clonal expansion and differentiation of Ly108 + CD69 - stem-like memory T (Tsm) cells. In addition, we found that IFN-γ primed donor-type antigen-presenting cells (APCs) play an essential role in optimizing the transition from CD4 + Tsm cells to CD4 + Trm cells in STAT3- and BCL6-dependent manner, as indicated by ATAC-Seq analysis. Our results have elucidated a novel pathway of clonal expansion and differentiation of Tsm cells to Trm cells in the GVHD target tissues. These observations provide cellular and molecular mechanisms that explain how chronic GVHD is perpetuated by memory T cells in local tissues. XK and BW contributed equally.

Distinct Minor Neoplastic Follicular T-Cell Subsets Dictate Lymphoma Fates through Functional Specialization

Background: The presence and role of follicular T-cell populations other than T follicular helper (Tfh) cells, such as T follicular regulatory (Tfr) and cytotoxic (Tfc) cells, are gaining increasing attention in certain pathological states. However, the ecosystem of follicular T cells in the tumor microenvironment (TME) has not been fully elucidated. In particular, the significance of minor follicular T-cell subsets in the neoplastic follicular environment remains elusive. Here, we aimed to reveal the landscape of follicular T-cell alterations in various cancers, with a particular emphasis on the follicular lymphoma (FL) TME. Methods: We analyzed single-cell RNA/TCR sequencing data of >500,000 human T cells from FL (obtained from four cohorts) and 25 other cancer types, as well as homeostatic and reactive lymph nodes (LNs), to construct a comprehensive single-T-cell atlas. We investigated differentially expressed genes, RNA velocity, and TCR clonality using this atlas. To determine the functions of neoplastic follicular regulatory (Tnfr) and cytotoxic (Tnfc) T cells, we performed in vitro cytokineproduction and co-culture assays, in combination with cell activation/suppression, cell division, and apoptosis assays, using human FL samples. With the PhenoCycler-Fusion system, we conducted multiplex digital spatial profiling (DSP) of 169 FL samples from two independent cohorts (now being extended to 242 FL samples from three cohorts) for >25 antibodies. We also performed single-cell spatial and protein expression profiling and prognostic analysis. Results: In FL, distinct minor neoplastic follicular T-cell subsets-Tnfr and CD4 (Tnfc4) and CD8 (Tnfc8) Tnfc cells-increased relative to homeostatic LNs. The TCR repertoire analysis revealed that Tnfr cells shared clonotypes with conventional effector regulatory T (Trg) and Tfh cells, whereas Tnfc4 and Tnfc8 cells shared clonotypes with Tfh cells and effector and exhausted (Tcex) cytotoxic CD8 T cells, respectively. In line with these findings, the RNA velocity survey suggested that Tnfr, Tnfc4, and Tnfc8 cells originated from Trg, Tfh, and naïve-like CD8 T cells, respectively. Tnfr and Tnfc cells expressed higher levels of effector genes, including those involved in cytokine release, chemokine response, migration, and PD-1 signaling, than their reactive LN counterparts. The pan-cancer survey revealed that Tfr and CD4 Tfc cells were exclusive to FL, whereas the prevalence and gene expression profiles of CD8 Tfc cells varied across cancers. Tnfr cells were marked by abundant expression of IL10 and IL21, whereas Tnfc cells displayed a unique phenotype, as they concomitantly expressed markers of effector Tfh (e.g., CXCL13, CXCR5, and PDCD1), naïve/stem (e.g., CCR7 and TCF7), central memory (e.g., CD27, CD28, and SELL), and tissue-resident memory (e.g., ITGAE) cells. Hierarchical clustering demonstrated that Tnfc8 cells had transcriptional profiles similar to those of melanoma TCF1 +PD-1 +CD8 + stem-like T cells. DSP of FL detected Tnfr and Tnfc cells frequently localized within and around neoplastic follicles, forming a cellular neighborhood that allowed them to interact closely. Tnfr cells were distributed predominantly near Tfh cells. The functional co-culture assays demonstrated that Tnfr cells suppressed Tfh-cell activation and division, thereby inhibiting Tfh-mediated malignant B-cell activation and survival. Tnfc8 cells showed a higher cell division capability than that of Tcex cells, suggesting that Tnfc8 cells function as a pool of CD8 T cells in neoplastic follicles. The prognostic analysis revealed that Tnfr and Tnfc cell proportions correlated with early disease relapse (i.e., POD24) and predicted a significantly longer time-to-relapse ( P <0.05 for Tnfr and <0.001 for Tnfc cells) in FL. In the multivariate analysis, the prognostic impact of these two cell subsets was independent of the FLIPI. The prognostic analysis findings were confirmed using a validation cohort. Conclusions: Our multi-omics approach identified the expansion of minor neoplastic follicular T-cell subsets that carry unique transcriptional and functional profiles and robust prognostic impacts. These findings deepen our understanding of the biological and immunological roles of non-Tfh follicular T cells in the lymphoma TME and highlights their clinical potential for patient risk stratification and future therapeutic interventions.

Diverse macrophage populations contribute to distinct manifestations of human cutaneous graft-versus-host disease.

Graft-versus-host disease (GvHD) is a major life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT), limiting the broad application of HSCT for haematological malignancies. Cutaneous GvHD is described as a post-transplant inflammatory reaction by skin-infiltrating donor T cells and remaining recipient tissue-resident memory T cells. Despite the major influence of lymphocytes on GvHD pathogenesis, the complex role of mononuclear phagocytes (MNPs) in tissues affected by GvHD is increasingly appreciated. To characterize the identity, origin and functions of MNPs in patients with acute cutaneous GvHD. Using single-cell RNA sequencing and multiplex tissue immunofluorescence, we identified an increased abundance of MNPs in skin and blood from 36 patients with acute cutaneous GvHD. In cases of sex-mismatched transplantation, we used expression of X-linked genes to detect rapid tissue adaptation of newly recruited donor MNPs resulting in similar transcriptional states of host- and donor-derived macrophages within GvHD skin lesions. We showed that cutaneous GvHD lesions harbour expanded CD163+ tissue-resident macrophage populations with anti-inflammatory and tissue-remodelling properties including interleukin-10 cytokine production. Cell-cell interaction analyses revealed putative signalling to strengthen regulatory T-cell responses. Notably, macrophage polarization in chronic cutaneous GvHD types was proinflammatory and drastically differed from acute GvHD, supporting the notion of distinct cellular players in different clinical GvHD subtypes. Overall, our data reveal a surprisingly dynamic role of MNPs after HSCT. Specific and time-resolved targeting to repolarize this cell subset may present a promising therapeutic strategy in combatting GvHD skin inflammation.

Regulatory role of RGMb in lung injury promoted by the combination of carbon ion irradiation and anti-PD-1 antibody through Erk1/2 and p38 MAPK pathways

The combination of carbon ion radiotherapy and anti-PD-1 antibody represents a new approach to treating thoracic tumors. However, the lung damage caused by this combination therapy may limit its use, and the potential mechanisms for this are worthy of investigation. The objective of this research was to examine the potential involvement of repulsive guidance molecule b (RGMb) in lung damage promoted by the utilization of carbon ion irradiation combined with an anti-PD-1 antibody. The C57BL/6 mice have been randomly separated into four distinct groups: control, anti-PD-1, whole thorax carbon ion irradiation, and irradiation in combination with anti-PD-1 treatment groups (combination group). Detection of pathological changes in lung tissue using HE staining. Detection of pulmonary fibrosis by Masson staining and the hydroxyproline assay. ELISA to detect TNF-α, TGF-β, IL-6, and IL-1β expression levels within lung homogenates. The expression of RGMb, p38 MAPK, and Erk1/2 pathways was detected using a fully automated digital Western blotting system WES (ProteinSimple, USA). Flow cytometry was employed to analyze tissue-resident memory T cells (TRM) within the lung. Subsequently, the siRNA gene was employed to induce the downregulation of RGMb in mice in order to validate the involvement of RGMb in radiation-immune lung injury. The present study observed a significant increase in both inflammatory and fibrotic indicators within the mice group's lung tissue that received the combination treatment. The combination group exhibited elevated levels of TGF-β, TNF-α, IL-6, and IL-1β in lung homogenates. Anti-PD-1 antibody and carbon ion irradiation, upregulated RGMb, phospho-p38 MAPK and phospho-Erk1/2. The results obtained from the flow cytometry analysis indicated that the combination group was significantly higher in the number of clonal expansion TRMs, which were predominantly characterized by the expression of CD8+CD103+CD69-TRMs. The downregulate of RGMb via siRNA in mice resulted in a decrease in phospho-p38 MAPK and phospho-Erk1/2. The combination group exhibited a reduction in TNF-α, TGF-β, IL-6, and IL-1β in their lung tissues, and the number of CD8+CD103+CD69-TRM was significantly reduced. The combination group exhibited a significant improvement in inflammatory and fibrotic indicators within the lung tissues. Anti-PD-1 antibody and carbon ion irradiation synergistically regulate RGMb, leading to strong clonal expansion of lung TRM through the p38 MAPK and Erk1/2 pathways. The present study offers valuable insights into the treatment of lung injury due to the combined administration of carbon ion radiotherapy and anti-PD-1 antibody therapy.

Both Humoral and Cellular Immunity Limit the Ability of Live Attenuated Influenza Vaccines to Promote T Cell Responses.

One potential advantage of live attenuated influenza vaccines (LAIVs) is their ability to establish both virus-specific Ab and tissue-resident memory T cells (TRM) in the respiratory mucosa. However, it is hypothesized that pre-existing immunity from past infections and/or immunizations prevents LAIV from boosting or generating de novo CD8+ T cell responses. To determine whether we can overcome this limitation, we generated a series of drifted influenza A/PR8 LAIVs with successive mutations in the hemagglutinin protein, allowing for increasing levels of escape from pre-existing Ab. We also inserted a CD8+ T cell epitope from the Sendai virus nucleoprotein (NP) to assess both generation of a de novo T cell response and boosting of pre-existing influenza-specific CD8+ T cells following LAIV immunization. Increasing the level of escape from Ab enabled boosting of pre-existing TRM, but we were unable to generate de novo Sendai virus NP+ CD8+ TRM following LAIV immunization in PR8 influenza-immune mice, even with LAIV strains that can fully escape pre-existing Ab. As these data suggested a role for cell-mediated immunity in limiting LAIV efficacy, we investigated several scenarios to assess the impact of pre-existing LAIV-specific TRM in the upper and lower respiratory tract. Ultimately, we found that deletion of the immunodominant influenza NP366-374 epitope allowed for sufficient escape from cellular immunity to establish de novo CD8+ TRM. When combined, these studies demonstrate that both pre-existing humoral and cellular immunity can limit the effectiveness of LAIV, which is an important consideration for future design of vaccine vectors against respiratory pathogens.

Human adipose tissue as a major reservoir of cytomegalovirus-reactive T cells.

Cytomegalovirus (CMV) is a common herpesvirus with a high prevalence worldwide. After the acute infection phase, CMV can remain latent in several tissues. CD8 T cells in the lungs and salivary glands mainly control its reactivation control. White adipose tissue (WAT) contains a significant population of memory T cells reactive to viral antigens, but CMV specificity has mainly been studied in mouse WAT. Therefore, we obtained blood, omental WAT (oWAT), subcutaneous WAT (sWAT), and liver samples from 11 obese donors to characterize the human WAT adaptive immune landscape from a phenotypic and immune receptor specificity perspective. We performed high-throughput sequencing of the T cell receptor (TCR) locus to analyze tissue and blood TCR repertoires of the 11 donors. The presence of TCRs specific to CMV epitopes was tested through ELISpot assays. Moreover, phenotypic characterization of T cells was carried out through flow cytometry. High-throughput sequencing analyses revealed that tissue TCR repertoires in oWAT, sWAT, and liver samples were less diverse and dominated by hyperexpanded clones when compared to blood samples. Additionally, we predicted the presence of TCRs specific to viral epitopes, particularly from CMV, which was confirmed by ELISpot assays. Remarkably, we found that oWAT has a higher proportion of CMV-reactive T cells than blood or sWAT. Finally, flow cytometry analyses indicated that most WAT-infiltrated lymphocytes were tissue-resident effector memory CD8 T cells. Overall, these findings postulate human oWAT as a major reservoir of CMV-specific T cells, presumably for latent viral reactivation control. This study enhances our understanding of the adaptive immune response in human WAT and highlights its potential role in antiviral defense.

MYC expression and fatty acid oxidation in EGFR-TKI acquired resistance

This report expands on our previous research, highlighting a unique inverse correlation between MYC expression in tumor cells and immune cells during the development of EGFR-TKI resistance. It is observed that MYC expression and fatty acid oxidation (FAO) metabolism in tissue-resident memory (TRM) CD8 + T cells are significantly impaired. These findings offer new insights into the mechanisms of TKI resistance. Although the study is preliminary, it suggests caution when interpreting the effectiveness of MYC inhibitors in reversing TKI resistance, especially when immune factors are not considered.

Virus-specific TRM cells of both donor and recipient origin reside in human kidney transplants.

Tissue-resident lymphocytes (TRLs) are critical for local protection against viral pathogens in peripheral tissue. However, it is unclear if TRLs perform a similar role in transplanted organs under chronic immunosuppressed conditions. The present study aimed to characterize the TRL compartment in human kidney transplant nephrectomies and examine its potential role in antiviral immunity. The TRL compartment of kidney transplants contained diverse innate(-like) and adaptive TRL populations expressing the canonical residency markers CD69, CD103, and CD49a. Chimerism of donor and recipient cells was present in 43% of kidney transplants and occurred in all TRL subpopulations. Paired single-cell transcriptome and T-cell receptor (TCR) sequencing showed that donor and recipient tissue-resident memory T (TRM) cells exhibit striking similarities in their transcriptomic profiles and share numerous TCR clonotypes predicted to target viral pathogens. Virus dextramer staining further confirmed that CD8 TRM cells of both donor and recipient origin cexpress TCRs with specificities against common viruses, including CMV, EBV, BK polyomavirus, and influenza A. Overall, the study results demonstrate that a diverse population of TRLs resides in kidney transplants and offer compelling evidence that TRM cells of both donor and recipient origin reside within this TRL population and may contribute to local protection against viral pathogens.

Dissecting the human leptomeninges at single-cell resolution

Emerging evidence shows that the meninges conduct essential immune surveillance and immune defense at the brain border, and the dysfunction of meningeal immunity contributes to aging and neurodegeneration. However, no study exists on the molecular properties of cell types within human leptomeninges. Here, we provide single nuclei profiling of dissected postmortem leptomeninges from aged individuals. We detect diverse cell types, including unique meningeal endothelial, mural, and fibroblast subtypes. For immune cells, we show that most T cells express CD8 and bear characteristics of tissue-resident memory T cells. We also identify distinct subtypes of border-associated macrophages (BAMs) that display differential gene expressions from microglia and express risk genes for Alzheimer’s Disease (AD), as nominated by genome-wide association studies (GWAS). We discover cell-type-specific differentially expressed genes in individuals with Alzheimer’s dementia, particularly in fibroblasts and BAMs. Indeed, when cultured, leptomeningeal cells display the signature of ex vivo AD fibroblasts upon amyloid-β treatment. We further explore ligand-receptor interactions within the leptomeningeal niche and computationally infer intercellular communications in AD. Thus, our study establishes a molecular map of human leptomeningeal cell types, providing significant insight into the border immune and fibrotic responses in AD.

Usefulness of Biopsy Specimens for Evaluating CD103+ Tumor-resident Memory T Cells in Esophageal Cancer.

CD103+ tissue-resident memory T cells (TRM) in tumor sites are associated with a favorable prognosis and predict the effectiveness of immune checkpoint inhibitors. The detection of CD103+ TRM infiltration in biopsy samples could be beneficial for patients without surgical indications. However, the usefulness of TRM detection in biopsy tissue and the difference in TRM status between biopsy and surgical specimens' post-neoadjuvant chemotherapy have not been elucidated. In the present study, we aimed to elucidate whether we can detect TRM in biopsy specimens and the impact of chemotherapy on TRM infiltration. Tissue sections were obtained from 46 patients with esophageal cancer who received neoadjuvant chemotherapy and underwent radical esophagectomy in 2017. Immunohistochemistry was performed using an anti-CD103 antibody for biopsy and surgical specimens. We examined the relationship between CD103 expression, clinicopathological features, and prognosis for each patient. TRM infiltration was detected in the biopsy specimens. CD103 expression in biopsy specimens correlated with that in surgical specimens. Although there was no statistical significance in clinicopathological findings between CD103high and CD103low, patients with CD103high biopsy specimens exhibited favorable prognosis. The number of CD103+ cells was increased by chemotherapy: though with no survival benefit. Regardless of surgical indication, we were able to determine the TRM status even in biopsy specimens. CD103 evaluation at biopsy may be more useful and practical than evaluation in surgical specimens, enabling prediction of prognosis and response to immune therapy.

PD-1 Impairs CD8+ T Cell Granzyme B Production in Aged Mice during Acute Viral Respiratory Infection.

CD8+ T cell dysfunction contributes to severe respiratory viral infection outcomes in older adults. CD8+ T cells are the primary cell type responsible for viral clearance. With increasing age, CD8+ T cell function declines in conjunction with an accumulation of cytotoxic tissue-resident memory (TRM) CD8+ T cells. We sought to elucidate the role of PD-1 signaling on aged CD8+ T cell function and accumulation of CD8+ TRM cells during acute viral respiratory tract infection, given the importance of PD-1 regulating CD8+ T cells during acute and chronic infections. PD-1 blockade or genetic ablation in aged mice yielded improved CD8+ T cell granzyme B production comparable to that in young mice during human metapneumovirus and influenza viral infections. Syngeneic transplant and adoptive transfer strategies revealed that improved granzyme B production in aged Pdcd1-/- CD8+ T cells was primarily cell intrinsic because aged wild-type CD8+ T cells did not have increased granzyme B production when transplanted into a young host. PD-1 signaling promoted accumulation of cytotoxic CD8+ TRM cells in aged mice. PD-1 blockade of aged mice during rechallenge infection resulted in improved clinical outcomes that paralleled reduced accumulation of CD8+ TRM cells. These findings suggest that PD-1 signaling impaired CD8+ T cell granzyme B production and contributed to CD8+ TRM cell accumulation in the aged lung. These findings have implications for future research investigating PD-1 checkpoint inhibitors as a potential therapeutic option for elderly patients with severe respiratory viral infections.