Helper Lineage Research Articles

Enhanced detection of antigen-specific T cells by a multiplexed AIM assay

Broadly applicable methods to identify and characterize antigen-specific CD4+ and CD8+ Tcells are key to immunology research, including studies of vaccine responses and immunity to infectious diseases. We developed a multiplexed activation-induced marker (AIM) assay that presents several advantages compared to single pairs of AIMs. The simultaneous measurement of four AIMs (CD69, 4-1BB, OX40, and CD40L) creates six AIM pairs that define CD4+ Tcell populations with partial and variable overlap. When combined in an AND/OR Boolean gating strategy for analysis, this approach enhances CD4+ Tcell detection compared to any single AIM pair, while CD8+ Tcells are dominated by CD69/4-1BB co-expression. Supervised and unsupervised clustering analyses show differential expression of the AIMs in defined T helper lineages and that multiplexing mitigates phenotypic biases. Paired and unpaired comparisons of responses to infections (HIV and cytomegalovirus [CMV]) and vaccination (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) validate the robustness and versatility of the method.

Pre- and Post-Treatment Immune Contexture Correlates with Long Term Response in Large B Cell Lymphoma Patients Treated with Axicabtagene Ciloleucel (axi-cel)

Background: Axi-cel is a US and EU-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for pts with relapsed/refractory large B cell lymphoma after 1 prior therapy as a result of ZUMA-7 (NCT. In ZUMA-1 (NCT02348216), the pivotal study which initially led to the approval of axi-cel in the 3L+ setting, the objective response rate was 83% (58% complete response rate; Locke et al. Lancet Oncol. 2019) and at 5 years follow up an OS rate of 42.6%, 5 years durable response and 5 years disease specific survival at 51% ( Neelapu et al. Blood 2023). T cell-related biology (Immunosign 21; Immunoscore®IC) measured pretreatment in the tumor microenvironment (TME) was associated with response to axi-cel ( Scholler et al., 2022). Increased density of activated PD-1+LAG-3+/−TIM-3−CD8+ T cells, measurable pretreatment, facilitates clinical response in pts post-axi-cel. This expanded analysis characterized the impact of axi-cel treatment on the TME immune contexture and examined associations between immune cell subsets and relapse. Methods: Baseline and post-infusion tumor biopsy samples were analyzed by multiplex immunohistochemistry ( Brightplex®). Four panels were developed and applied to assess T cell infiltration (CD3 CD8 FOXP3 TIM3 PD1 LAG3 TOX), regulatory T cell subtyping (CD3 CD8 GATA3 TBET RORg BCL6), T cell activation & exhaustion (CD3 CD8 TIM3 LAG3 PD1 GZMB KI67), macrophage (CD68 CD64 CD163 CD204 CD206 PDL1) and MDSC (CD3 CD11B CD68 CD14 CD15 LOX1 S100A9) subsets. Transcriptomic analysis was performed using nCounter® Pan Cancer panel. The association between T cell subtypes and macrophages cell subset density, and probability to relapse was evaluated. T test values were based on Brightplex analysis. Results: 34 tumor biopsies (16 at baseline, 18 post-infusion) from 26 pts were analyzed including 11 relapsed (6 CR/5 PR) and 15 durable response (15 CR). The baseline and post-infusion TME comprised all major macrophages, MDSC and T cell subsets, with diverse distribution across samples analyzed. Low proinflammatory M1 macrophage density was observed at baseline and post-infusion across patients. In relapsed patients, baseline tumors were enriched with a significantly higher proportion of protumoral M2 macrophage (p<0.0001). A shift of M1-M2 polarization was observed between baseline and post-infusion (Baseline 33%M1M2 / 65%M2, post infusion 76%M1M2 / 21%M2, p<0,0001) across the cohort. In contrast, in relapsed patients, M-MDSC cell density was significantly increased post treatment (p=0.0420). In addition, Innate lymphocyte cells subtype 2 (ILC2) cell density, previously described to be positively correlated with improved overall survival in CRC ( Huang et al. 2021 Cancers), was significantly decreased in relapsed patients post infusion (p=0.019).Post-infusion, durable response was associated with a significant increase in pan-Macrophages (p=0.0360) and cytotoxic T cell subset densities: CD8 naïve T cells (p=0.016); Activated Cytotoxic T cell (GZMB+)(p=0.0203); PD1+TIM3+LAG3- cytotoxic T cell (p=0.0117); CD8 regulatory T cell (FOXP3+)(p=0.012). Regarding T helper lineage, ongoing response was associated with a significant increase of: CD4 naïve T cells (p=0.021); T helper Th2 (p=0.020); cytotoxic T lymphocyte TC2 and TC1/TC2 cell densities (respectively p=0.0052 and 0.011).Additional characterization of the immune contexture and correlative analysis of cell subsets will be presented. Conclusion: These results suggest that baseline proportions of protumor M2 macrophages may predict disease relapse after axi-cel treatment. Following infusion, relapse is also associated with spatial enrichment of M-MDSC, whereas durable response is related to increases in T cell subpopulation densities inclusive of cytotoxic T cells, T helper, TC1/TC2 ratios and innate T-subpopulations. These findings suggest that axi-cel treatment drastically impacts tumor immune mobilization, infiltration and contexture, which correlates with long term response.

Cutting Edge: Bach2 Integrates Cytokine Signals to Arbitrate Differentiation Decisions between T Follicular Helper and Th17 Lineages.

CXCR5 is a hallmark of T follicular helper (Tfh) cells. The mechanism of CXCR5 induction, however, is still incompletely understood. In this study, we report that in mice with the absence of transcription factor Bach2, the Th17-inducing cytokines IL-6 and TGF-β together induced CXCR5 expression invitro. Mechanistically, IL-6/STAT3 drove Cxcr5 promoter activity via the upstream site 1 regulatory element, whereas TGF-β enhanced permissive histone modifications, and the STAT3 binding to the site 1 regulatory element was higher in the absence of Bach2. Subsequently, despite previous studies showing enhanced Th17 cell differentiation in the absence of Bach2 invitro, we found that invivo, the Bach2 deficiency led to an enhanced Tfh cell response at the expense of the Th17 cell response. These findings suggest that Bach2 helps integrate cytokine signals to arbitrate differentiation decisions between Tfh and Th17 lineages.

Autosomal recessive T-bet and RORgammaT deficiencies underlie mycobacterial susceptibility

Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by selective, inherited predisposition to clinical disease caused by weakly virulent mycobacteria, such as M.bovis BCG vaccines and environmental mycobacteria (EM). Twenty MSMD-causing genes have been identified. Their mutations affect IFN-γ-related immune circuits by impairing either the production of, the cellular response to IFN-γ, or both. T-bet and RORγT are transcription factors that govern the lineage commitment of two different T helper lineages, TH1 and TH17 cells, respectively, in mice. Surprisingly, both autosomal recessive (AR) T-bet and RORγT deficiencies in humans underlie mycobacterial susceptibility. Here we summarize the molecular, cellular, and clinical features of a recently identified patient with AR T-bet deficiency and three new patients with AR RORγT deficiency. Human AR T-bet deficiency underlies mycobacterial disease by preventing the development of innate (NK) and innate-like adaptive lymphocytes (iNKT, MAIT, and Vδ2+ γST cells) as well as IFN-γ produced by them. It also underlies peripheral eosinophilia and recurrent upper respiratory inflammation due to excessive production of TH2 cytokines, particularly IL-5 and IL-13, by CD4+ αβT cells. Although humoral immunity in inherited T-bet deficiency remains grossly intact, T-bet is required for the in vivo and in vitro development of a distinct subset of human B cells in humans resembling murine “age-associated B cells” that are associated with infection and autoimmunity. We also identified three new patients with AR RORγT deficiency that underlies both mycobacterial and fungal susceptibility. Inherited RORyT deficiency abolishes the development of iNKT and MAIT cells, two IFN-γ-producing innate-like adaptive T cell subsets, and impairs the production of IFN-γ by Vδ2+ γδT cells in response to mycobacterial stimulation. The production of IFN-γ by purely adaptive apT cells remains largely intact upon mycobacterium-specific stimulation. The development of TH17 cells and the production of TH17 cytokines are abolished in inherited RORyT deficiency, thereby underlying susceptibility to C.albicans. Collectively, impaired development of and defective production of IFN-γ by MAIT, V82+ γ8T cells, and iNKT cells are sufficient to cause mycobacterial susceptibility, whereas the contribution of purely adaptive apT cells to antimycobacterial immunity, at least against M.bovis BCG or EM, is probably inconsequential.Table 1(abstract: 257) ML-model Scores for IEI Test Cohort Baseline vs. Enhanced Model/Manual vs. Automated Term Extractioncase_dxscore_bn36_manualscore_bn47_update_manualscore_change_expertPrediction_Expert_BN36score_bn36_automaticscore_bn47_update_automaticscore_bn_change_HPOPrediction_HPO_BN36CVID0.991.000.0111.001.000.001SIgAD0.060.880.8210.140.690.551AT0.891.000.1110.020.110.090Hypogam0.541.000.4610.040.960.920Hypogam1.001.000.0011.001.000.001AT0.101.000.9011.001.000.001THIG0.020.690.6700.170.840.671CVID vs. Hypogam0.030.900.8701.001.000.001DGS0.541.000.4610.330.930.601DGS0.451.000.5510.380.990.611CVID-common variable immunodeficiency, SIgAD-selective IgA deficiency, AT-ataxia telangiectasia, Hypogam-hypogammaglobulinemia, THIG-transient hypogammaglobulinemia of infancy, DGS-Digeorge SyndromeTable 2(abstract: 257) Full feature set and frequency comparision by term extraction method.ConceptManualAutomated HPOBarnard Exact p_valueimm_dx_test0.70.30.06care_team0.60naMeds0.50naFatigue0.40.60.26iei_dz_mention0.40.4nasn_jiearing_loss0.30naAbscess0.20.10.34iei_gene_mention0.20naclin_care0.20naexam_findings0.20.7***0.021lymphoma0.20.2Napna_fewer_two0.20.40.26pna_greater_two0.20.11thrombocytopenia0.20.2naopportunistic infection0.20.2nahypocalcemia0.20.11Ataxia0.20narec_fever0.20naother_infect0.20.50.11FTT0.10.6***0.011Otorrhea0.10nalymphadenopathy0.10.40.076dysmorphic_feat0.10.1naWarts0.10naSepsis0.10nasinusitis_greater_four0.10nafe_def0.10.20.34lymphopenia0.10.30.16neutropenia0.10.20.34mycobact_dz0.10namycosis_superficial0.10nainv_herpes_dz0.10nagranuloma_skin0.10naaspergillosis0.10naChemo0.10nametabolics0.10.1napolyarthritis0.00naorganomegaly0.00.3naseptic_shock0.00nahyper-eos0.00.1nameningococcal_dz0.00naSle0.00.1nabronchiectasis0.00.1napulm_fibrosis0.00naAiha0.00naencephalitis0.00naheme_manifestation0.00.4na***denotes significant difference at a type I error rate of 0.05 [Display omitted]

IL-2 rescues impaired T cell function upon loss of Itk via metabolic reprogramming

Abstract IL-2 inducible T cell kinase (Itk) is a critical component of T cell receptor (TCR) signaling required for activation of PLCg and downstream transcription factors, that regulate expression of effector molecules. Loss of Itk impairs T-cell activation by modulating the strength of TCR signal. Itk −/−mice have defects in T cell development with decreased proliferation and impaired differentiation to multiple CD4 T helper lineages. Most severe defects are seen in differentiation of Th9 cells, which produce the cytokine, IL-9. Under Th9 differentiation conditions, Itk −/−cells initially appear similar to WT cells but at 72hrs fail to sustain long-term expression of effector molecules. Th9 differentiation could be rescued by inclusion of IL-2. Itk −/−CD4 T cells show decreased mTORC1 signaling and do not maintain expression of IRF4, c-Myc, pRb and multiple nutrient transporters, including the Glucose transporter 1 (GLUT1), transferrin receptor (CD71), and amino acid co-transporter (CD98), all of which were rescued by IL-2. IL-2 is not required initially but can be added as late as 48hrs into culture. Using SCENITH—a flow cytometry-based technique that profiles metabolism—we found that WT differentiated Th9 cells were primarily glycolytic. In contrast, Itk −/−Th9 cells were less synthetic and more dependent on mitochondrial respiration. Inclusion of IL-2 in Itk −/−cultures rescued metabolic profiles, comparable to WT cells. Our results suggest that metabolic flexibility is central to dictating the functional/effector fate of CD4 T cells and that the Tec kinase Itk and IL-2-mediated pathways are required for maintenance of metabolic integrity, providing novel insight into mechanisms by which IL-2 can rescue T cell functional defects. This work was supported by the Intramural Research Program of NIAID, NIH.

Human T follicular regulatory cells with helper and regulatory lineage origins.

Human T follicular regulatory cells with helper and regulatory lineage origins.

Id3 expression identifies CD4+ memory Th1 cells

Memory CD4+ T cells play a pivotal role in mediating long-term protective immunity, positioning them as an important target in vaccine development. However, multiple functionally distinct helper CD4+ T-cell subsets can arise in response to a single invading pathogen, complicating the identification of rare populations of memory precursor cells during the effector phase of infection and memory CD4+ T cells following pathogen clearance and the contraction phase of infection. Furthermore, current literature remains unclear regarding whether a single CD4+ memory T-cell lineage gives rise to secondary CD4+ T helper subsets or if there are unique memory precursor cells within each helper lineage. A majority of T follicular helper (Tfh) cells, which have established memory potential, express Id3, an inhibitor of E protein transcription factors, following acute viral infection. We show that expression of Id3 definitively identified a subset of cells within both the CD4+ Tfh and T helper 1 (Th1) lineages at memory time points that exhibited memory potential, with the capacity for significant re-expansion in response to secondary infection. Notably, we demonstrate that a subset of Th1 cells that survive into the memory phase were marked by Id3 expression and possessed the potential for enhanced expansion and generation of both Th1 and Tfh secondary effector cell populations in a secondary response to pathogen. Additionally, these cells exhibited enrichment of key molecules associated with memory potential when compared with Id3lo Th1 cells. Therefore, we propose that Id3 expression serves as an important marker to indicate multipotent potential in memory CD4+ T cells.

Immune tolerance of food is mediated by layers of CD4+ T cell dysfunction.

Gastrointestinal health depends on the adaptive immune system tolerating the foreign proteins in food1,2. This tolerance is paradoxical because the immune system normally attacks foreign substances by generating inflammation. Here we addressed this conundrum by using a sensitive cell enrichment method to show that polyclonal CD4+ T cells responded to food peptides, including a natural one from gliadin, by proliferating weakly in secondary lymphoid organs of the gut-liver axis owing to the action of regulatory T cells. A few food-specific T cells then differentiated into T follicular helper cells that promoted a weak antibody response. Most cells in the expanded population, however, lacked canonical T helper lineage markers and fell into five subsets dominated by naive-like or T follicular helper-like anergic cells with limited capacity to form inflammatory T helper 1 cells. Eventually, many of the T helper lineage-negative cells became regulatory T cells themselves through an interleukin-2-dependent mechanism. Our results indicate that exposure to food antigens causes cognate CD4+ naive T cells to form a complex set of noncanonical hyporesponsive T helper cell subsets that lack the inflammatory functions needed to cause gut pathology and yet have the potential to produce regulatory T cells that may suppress it.

The Tyrosine Kinase Tec Regulates Effector Th17 Differentiation, Pathogenicity, and Plasticity in T-Cell-Driven Intestinal Inflammation.

T helper (Th) 17 cells are not only key in controlling infections mediated by extracellular bacteria and fungi but are also triggering autoimmune responses. Th17 cells comprise heterogeneous subsets, some with pathogenic functions. They can cease to secrete their hallmark cytokine IL-17A and even convert to other T helper lineages, a process known as transdifferentiation relying on plasticity. Both pathogenicity and plasticity are tightly linked to IL-23 signaling. Here, we show that the protein tyrosine kinase Tec is highly induced in Th17 cells. Th17 differentiation was enhanced at low interleukin-6 (IL-6) concentrations in absence of Tec, which correlates with increased STAT3 phosphorylation and higher Il23r expression. Therefore, we uncovered a function for Tec in the IL-6 sensing via STAT3 by CD4+ T cells, defining Tec as a fine-tuning negative regulator of Th17 differentiation. Subsequently, by using the IL-17A fate mapping mouse combined with in vivo adoptive transfer models, we demonstrated that Tec not only restrained effector Th17 differentiation but also pathogenicity and plasticity in a T-cell intrinsic manner. Our data further suggest that Tec regulates inflammatory Th17-driven immune responses directly impacting disease severity in a T-cell-driven colitis model. Notably, consistent with the in vitro findings, elevated levels of the IL-23 receptor (IL-23R) were observed on intestinal pre- and postconversion Th17 cells isolated from diseased Tec−/− mice subjected to adoptive transfer colitis, highlighting a fundamental role of Tec in restraining IL-23R expression, likely via the IL-6-STAT3 signaling axis. Taken together, these findings identify Tec as a negative regulator of Th17 differentiation, pathogenicity, and plasticity, contributing to the mechanisms which help T cells to orchestrate optimal immune protection and to restrain immunopathology.

PD-1 through Asparaginyl Endopeptidase Regulates Foxp3 Stability in Induced Regulatory T Cells

CD4+ T cell differentiation into multiple T helper lineages is critical for optimal adaptive immune responses. Lineage instability has been attributed to Tregs and Th17 cells but Th1 cells are acclaimed for their functional stability. In this report, we investigated the intrinsic mechanism by which PD-1 signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+ iTregPDL1 cells). Tbet+iTregPDL1 cells were capable of preventing autoimmunity and alloimmunity in murine models of experimental colitis and experimental graft versus host disease (GvHD). PDL-1 binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells by specifically down regulating the endo-lysosomal protease asparaginase endopeptidase (AEP). AEP cleaved Foxp3 in control Tbet+ iTregs and iTreg cells resulting in Foxp3 degradation. Blocking AEP, by knockdown and selective inhibitor, enhanced Foxp3 stability and regulatory function in Tbet+ iTreg cells. In addition, Aep-/- iTregs significantly inhibited GvHD and maintained Foxp3. Of note, PD-1 mediated Foxp3 maintenance in Tbet+ Th1 cells was significantly operational in tumor infiltrating lymphocytes. Collectively, this report has identified a novel intrinsic function for PD-1 in converting Th1 cells into Tregs through an intrinsic proteolytic pathway. DisclosuresNo relevant conflicts of interest to declare.

MiR-141-3p and miR-200a-3p are involved in Th17 cell differentiation by negatively regulating RARB expression.

Among T helper (Th) lineages differentiated from naïve CD4+ T cells, interleukin (IL)-17-producing Th17 cells are highly correlated with the pathogenesis of autoimmune disorders. This study aimed to clarify the involvement of miR-141-3p and miR-200a-3p in Th17 cell differentiation as well as explore their potential target genes involved. For this purpose, human naïve CD4+ T cells were cultured under Th17 cell polarizing condition. The differentiation process was confirmed through measurement of IL-17 secretion using the ELISA method and assessment of Th17 cell-defining genes expression during the differentiation period. MiR-141-3p and miR-200a-3p downstream genes were identified via consensus and integration in silico approach and their expression pattern and alterations were evaluated by quantitative real-time PCR. Finally, direct interaction between both microRNAs (miRNAs) and their common predicted target sequences was approved by dual-luciferase reporter assay. Highly increased IL-17 secretion and Th17 lineage-specific genes expression confirmed Th17 cell differentiation. Our results have demonstrated that miR-141-3p and miR-200a-3p are Th17 cell-associated miRNAs and their expression level is upregulated significantly during Th17 cell induction. We have also found that retinoic acid receptor beta (RARB) gene, whose product has been reported as a negative regulator of Th17 cell generation, is a direct target of both miRNAs and its downregulation can affect the transcriptional level of JAK/STAT pathway genes. Overall, our results have identified two novel Th17 lineage-associated miRNAs and have provided evidence for the RARB-dependent mechanism of miR-141-3p and miR-200a-3p-induced Th17 cell differentiation and hence Th17-mediated autoimmunity.

Ca2+-dependent protein acyltransferase DHHC21 controls activation of CD4+ T cells.

Despite the recognized significance of reversible protein lipidation (S-acylation) for T cell receptor signal transduction, the enzymatic control of this post-translational modification in T cells remains poorly understood. Here, we demonstrate that DHHC21 (also known as ZDHHC21), a member of the DHHC family of mammalian protein acyltransferases, mediates T cell receptor-induced S-acylation of proximal T cell signaling proteins. Using Zdhhc21dep mice, which express a functionally deficient version of DHHC21, we show that DHHC21 is a Ca2+/calmodulin-dependent enzyme critical for activation of naïve CD4+ T cells in response to T cell receptor stimulation. We find that disruption of the Ca2+/calmodulin-binding domain of DHHC21 does not affect thymic T cell development but prevents differentiation of peripheral CD4+ T cells into Th1, Th2 and Th17 effector T helper lineages. Our findings identify DHHC21 as an essential component of the T cell receptor signaling machinery and define a new role for protein acyltransferases in regulation of T cell-mediated immunity.

Cutaneous T cells promote distinct cellular outcomes in human keratinocytes and fibroblasts

Abstract Human skin is a barrier tissue home to a highly specialized immune environment in which leukocytes coordinate with local epithelial and stromal tissue in response to physical, chemical, and biological stresses. T cells are abundant in the skin and are required for both host defense to pathogens and the subsequent repair of damaged tissue. Cutaneous lymphocytes include both resident and migratory fractions, which can be identified and isolated using expression of the cutaneous lymphocyte antigen (CLA). There are multiple subsets of CLA+ T cells, each having a distinct phenotype and functional capacity, however their precise roles in contributing to host-protective and tissue-repair responses remains incompletely understood. We assessed the function of eight different CD4+CLA+ cutaneous T cell subsets from the skin and blood, including traditional T helper lineages and a novel subset of CD4+CLA+CD103+ T cells with a tissue repair phenotype, by bulk RNAseq analysis of keratinocytes and fibroblasts following administration of activated T cell supernatants. Differential gene expression and pathway analysis reveal distinct cellular outcomes in keratinocytes and fibroblasts that depend strongly on T cell source and the activity of specific cytokines. Our findings define a diverse array of immune-induced states in the skin that are dependent on the unique functional capacity of various T cell subsets, thereby constructing a comprehensive tool to map how cutaneous T cells contribute to pathological disruption, tissue repair and return to homeostasis within human skin.

Recent advances in understanding the Th1/Th2 effector choice.

For over 35 years since Mosmann and Coffman proposed the seminal “type 1 T helper (Th1)/type 2 T helper (Th2)” hypothesis in 1986, the immunological community has appreciated that naïve CD4 T cells need to make important decisions upon their activation, namely to differentiate towards a Th1, Th2, Th17 (interleukin-17-producing T helper), follicular T helper (Tfh), or regulatory T cell (Treg) fate to orchestrate a variety of adaptive immune responses. The major molecular underpinnings of the Th1/Th2 effector fate choice had been initially characterized using excellent reductionist in vitro culture systems, through which the transcription factors T-bet and GATA3 were identified as the master regulators for the differentiation of Th1 and Th2 cells, respectively. However, Th1/Th2 cell differentiation and their cellular heterogeneity are usually determined by a combinatorial expression of multiple transcription factors, particularly in vivo, where dendritic cell (DC) and innate lymphoid cell (ILC) subsets can also influence T helper lineage choices. In addition, inflammatory cytokines that are capable of inducing Th17 cell differentiation are also found to be induced during typical Th1- or Th2-related immune responses, resulting in an alternative differentiation pathway, transiting from a Th17 cell phenotype towards Th1 or Th2 cells. In this review, we will discuss the recent advances in the field, focusing on some new players in the transcriptional network, contributions of DCs and ILCs, and alternative differentiation pathways towards understanding the Th1/Th2 effector choice in vivo.

DNA methylation modulates allograft survival and acute rejection after renal transplantation by regulating the mTOR pathway

Acute rejection (AR) can lead to allograft dysfunction following renal transplantation, despite immunosuppressive treatments. Accumulating evidence points out a role for epigenetic modification in immune responses. However, the mechanism and contribution of DNA methylation in allograft survival remain unclear. In this study, we followed up patients who successively experienced end‐stage renal disease, renal transplantation with allograft function or dysfunction, and hemodialysis. Peripheral blood mononuclear cells were collected at different time points for analysis of the DNA methylation. Epigenetic modifier analysis was also performed to explore its effect of methylation in a mouse model of AR. Compared with the allograft‐stable cohort, patients who experienced AR‐induced allograft dysfunction demonstrated more changes in methylation patterns. Pathway analysis revealed that the hypermethylated areas in the allograft dysfunction group were associated with genes related to the mechanistic target of rapamycin (mTOR) signaling pathway. Moreover, in the mouse AR model, treatment with the DNA methyltransferase inhibitor—decitabine regulated the Th1/2/17/regulatory T cell (Treg cell) immune response via its demethylating role in the suppressing the activity of the mTOR pathway, which ultimately ameliorated renal allograft‐related inflammatory injuries. These results revealed that changes in methylation accompany AR‐induced allograft dysfunction after renal transplantation. Epigenetics may provide new insights into predicting and improving allograft survival.

A circulating population of CD4+ cutaneous resident memory T cells is diminished in humans with fibrotic disease

Abstract Tissue-resident memory T cells (TRM) persist mainly in non-lymphoid tissues where they promote immune recall responses to recurrent insults. We previously defined a population of CD4+CD103+ TRM cells from human skin that become CD69neg and re-enter the circulation, but maintain expression of the skin-associated marker cutaneous lymphocyte antigen (CLA) and the capacity to return to the skin. Comparing blood- and skin-derived CLA+ memory populations, we found that the recirculating fraction of CD103+ TRM are clonally, transcriptionally, and phenotypically most similar to skin-resident CD103+ T cells. We now report that the circulating CD4+CLA+CD103+ T cells are functionally distinct from classically-defined CD4+ T helper lineages, highlighted by the co-production of IL-22 and IL-13. As these cytokines can promote tissue repair macrophage- and epithelial cell-mediated fibrosis and wound healing, we assessed the circulating skin TRM pool during systemic scleroderma (SSc), an autoimmune disorder highlighted by fibrosis of the skin and the underlying organs. Compared to healthy donors, SSc patients had a reduced frequency of circulating CD4+CLA+CD103+ T cells with diminished per-cell IL-13 production, both of which correlated with late-onset disease. Our ongoing work aims to understand how changes in the circulating TRM fraction might be a litmus for the onset or progression of SSc and similar skin diseases. Therefore, circulating CD4+CLA+CD103+ T cells represent a broadly available pool from which the isolation and study of skin TRM cell responses is possible both in health and disease.

THEMIS functions as a cytoplasmic adaptor and nuclear transcription factor in developing thymocytes and mature T cells

Abstract THEMIS functions in the cytoplasm as an adaptor molecule which dampens TCR signaling. During thymic development, THEMIS sets the affinity threshold for activation, enabling the selection of conventional T cells in response to low affinity antigens. Although these events occur in the cytoplasm, THEMIS possess a nuclear localization signal (NLS) and a significant amount of THEMIS resides in the nucleus. To investigate the importance of THEMIS in the nucleus, we deleted the Themis NLS to force THEMIS in the cytoplasm. Surprisingly, T cell development was impaired similar to the Themis null mice, suggesting that THEMIS may have important roles in the nucleus as well. Alternatively, we established Themis mutant mice, in which THEMIS localized exclusively in the nucleus or where THEMIS was constantly present in both compartments. Normal conventional T cells were absent in both mutant mice. Since lack of THEMIS or the constant presence of THEMIS in the cytoplasm or nucleus or both, all resulted in aberrant T cell development it indicated that the translocation of THEMIS to or/and from the nucleus is essential for its function. To examine the role of THEMIS in T cells in the periphery, we generated mutant mice where THEMIS is expressed normal during thymic development but altered post-thymically. Adoptive transfer of naïve CD4 T cells with altered THEMIS expression into RAG deficient host induced enhanced sever colitis and furthermore, we observed that forced localization of THEMIS in the nucleus redirects the polarization of CD4 T cells toward the pathogenic TH17 helper lineage. These findings identify a dual function for THEMIS as a cytoplasmic adaptor during TCR signaling and a nuclear factor directing gene expression in thymocytes and mature T cells.

Constitutive CD8 expression drives innate CD8+ T-cell differentiation via induction of iNKT2 cells.

Temporal down-regulation of the CD8 co-receptor after receiving positive-selection signals has been proposed to serve as an important determinant to segregate helper versus cytotoxic lineages by generating differences in the duration of TCR signaling between MHC-I and MHC-II selected thymocytes. By contrast, little is known about whether CD8 also modulates TCR signaling engaged by the non-classical MHC-I-like molecule, CD1d, during development of invariant natural killer T (iNKT) cells. Here, we show that constitutive transgenic CD8 expression resulted in enhanced differentiation of innate memory-like CD8+ thymocytes in both a cell-intrinsic and cell-extrinsic manner, the latter being accomplished by an increase in the IL-4-producing iNKT2 subset. Skewed iNKT2 differentiation requires cysteine residues in the intracellular domain of CD8α that are essential for transmitting cellular signaling. Collectively, these findings shed a new light on the relevance of CD8 down-regulation in shaping the balance of iNKT-cell subsets by modulating TCR signaling.

Differences in Tumor Microenvironment Dictate T Helper Lineage Polarization and Response to Immune Checkpoint Therapy

Immune checkpoint therapy (ICT) shows encouraging results in a subset of patients with metastatic castration-resistant prostate cancer (mCRPC) but still elicits a sub-optimal response among those with bone metastases. Analysis of patients' bone marrow samples revealed increased Th17 instead ofTh1 subsets after ICT. To further evaluate the different tumor microenvironments, we injected mice with prostate tumor cells either subcutaneously or intraosseously. ICT in the subcutaneous CRPC model significantly increases intra-tumoral Th1 subsets and improves survival. However, ICT fails to elicit an anti-tumor response in the bone CRPC model despite an increase in the intra-tumoral CD4 Tcells, which are polarized to Th17 rather than Th1 lineage. Mechanistically, tumors in the bone promote osteoclast-mediated bone resorption that releases TGF-β, which restrains Th1 lineage development. Blocking TGF-β along with ICT increases Th1 subsets and promotes clonal expansion of CD8 Tcells and subsequent regression of bone CRPC and improves survival.

Differential interferon-gamma production potential among naïve CD4+ T cells exists prior to antigen encounter.

Individual CD4+ T cells can become one of a number of helper (Th) lineages with distinct effector functions. However, whether biases in Th potential exist prior to antigen encounter is unknown. Studies have identified cell-intrinsic functional heterogeneity among naïve T cells that can be parsed based on the strength of T-cell receptor (TCR) interactions with self-peptide. Here, using CD5 levels as a surrogate for the strength of these basal TCR signals, we sought to identify pre-existing effector biases in the CD4+ T-cell lineage. We show that exvivo-activated CD5lo CD4+ T cells produce greater amounts of the Th1 cytokine interferon-gamma (IFNγ) than their CD5hi counterparts. In addition, a greater percentage of CD5lo effector CD4+ T cells produce IFNγ in both polyclonal and monoclonal CD4+ T-cell populations after antigen challenge invivo. These results suggest that differential IFNγ production potential exists among CD4+ T cells prior to activation and independent of TCR affinity for foreign antigen.