T Follicular Helper Research Articles

Clonal composition and persistence of antigen-specific circulating T follicular helper cells.

T follicular helper (TFH ) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4+ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC TFH cells. However, the antigen specificity and relationship of these circulating TFH (cTFH ) cells with other memory CD4+ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vβ sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cTFH and non-cTFH subsets, although with different frequencies and effector functions. Interestingly, cTFH and non-cTFH cells specific for C.alb or TT had a largely overlapping TCR Vβ repertoire while the repertoire of Flu-specific cTFH and non-cTFH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1+ cTFH cells had a "GC TFH -like" phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cTFH and non-cTFH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cTFH with non-cTFH cells and on the heterogeneity and persistence of antigen-specific human cTFH cells.

Clinicopathological findings, prognosis, and Epstein–Barr virus infection in rheumatoid arthritis patients with other iatrogenic immunodeficiency-associated T- and NK-cell lymphoproliferative disorders

BackgroundOther iatrogenic immunodeficiency-associated (OIIA) T- and natural killer (NK)-cell lymphoproliferative disorders (TNK-LPDs) are rare in patients with rheumatoid arthritis (RA).MethodsWe investigated the clinicopathological characteristics, Epstein–Barr virus (EBV) infection, genetic findings, therapeutic response, and prognostic factors in 21 RA patients with OIIA TNK-LPDs and compared these with those of 39 with OIIA B-cell LPDs (B-LPDs) and 22 with non-OIIA B-LPDs.ResultsImmunohistologically, 11 patients (52%) showed CD4+ T-LPDs, and 7 had a T follicular helper (TFH) phenotype. The other nine patients (43%) showed CD8+ T-LPDs, and the remaining one (5%) had features of CD3+ CD4− CD8− nasal type TNK-cell lymphoma. CD30+, p53+, and CMYC+ atypical lymphocytes were identified in seven (33%), eight (38%), and five (24%) patients, respectively. In situ hybridisation detected EBV-encoded RNA (EBER) + large atypical lymphocytes in five patients (24%). Nine of 17 patients (53%) showed clonal peaks of TCRγ by polymerase chain reaction. Withdrawal of MTX and biologic drugs was effective in 12 patients (57%), and 8 (38%) received chemotherapies. Two patients with TFH+ or EBV+ CD4+ CD30+ large cell peripheral T-cell lymphoma, one with CD8+ systemic anaplastic large cell lymphoma, and two with systemic EBV+ CD8+ T-cell lymphoma of childhood showed a lethal progressive clinical course within 13 months. Moreover, > 500 U/L LDH, large atypical lymphocytes, expression of CD30, p53, and CMYC, and EBER+ atypical lymphocytes were significantly poor prognostic factors for overall survival (p < 0.05). Median interval from RA onset to OIIA TNK-LPDs was 72 months, which was shorter than 166 months in OIIA B-LPDs (p = 0.003). EBV+ atypical and reactive lymphocytes were frequently found in 15 patients with OIIA TNK-LPDs (71%), in 27 with OIIA B-LPDs (69%), and only in 3 with non-OIIA B-LPDs (14%).ConclusionsOIIA TNK-LPDs occurred in early phase of RA, compared with OIIA B-LPDs, and occasionally showed a lethal progressive clinical course. Detection of OIIA TNK-LPD patients with poor prognostic factors is necessary. EBV infection in immunosuppressed patients due to persistent RA, MTX, and biologic drugs may play a role in forming the tumour microenvironment and lymphomagenesis of TNK-LPDs.

Primary cutaneous peripheral T-cell lymphomas with a T-follicular helper phenotype: an integrative clinical, pathological and molecular case series study.

Primary cutaneous peripheral T-cell lymphomas with a T-follicular helper phenotype (pcTFH-PTCL) are poorly characterized, and often compared to, but not corresponding with, mycosis fungoides (MF), Sézary syndrome, primary cutaneous CD4+ lymphoproliferative disorder, and skin manifestations of angioimmunoblastic T-cell lymphomas (AITL). We describe the clinicopathological features of pcTFH-PTCL in this original series of 23 patients, and also characterize these cases molecularly. Clinical and histopathological data of the selected patients were reviewed. Patient biopsy samples were also analysed by targeted next-generation sequencing. All patients (15 men, eight women; median age 66 years) presented with skin lesions, without systemic disease. Most were stage T3b, with nodular (n = 16), papular (n = 6) or plaque (atypical for MF, n = 1) lesions. Three (13%) developed systemic disease and died of lymphoma. Nine (39%) patients received more than one line of chemotherapy. Histologically, the lymphomas were CD4+ T-cell proliferations, usually dense and located in the deep dermis (n = 14, 61%), with the expression of at least two TFH markers (CD10, CXCL13, PD1, ICOS, BCL6), including three markers in 16 cases (70%). They were associated with a variable proportion of B cells. Eight patients were diagnosed with an associated B-cell lymphoproliferative disorder (LPD) on biopsy, including Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (n = 3), EBV+ LPD (n = 1) and monotypic plasma cell LPD (n = 4). Targeted sequencing showed four patients to have a mutated TET2-RHOAG17V association (as frequently seen in AITL) and another a TET2/DNMT3A/PLCG1/SETD2 mutational profile. The latter patient, one with a TET2-RHOA association, and one with no detected mutations, developed systemic disease and died. Five other patients showed isolated mutations in TET2 (n = 1), PLCG1 (n = 2), SETD2 (n = 1) or STAT5B (n = 1). Patients with pcTFH-PTCL have pathological and genetic features that overlap with those of systemic lymphoma of TFH derivation. Clinically, most remained confined to the skin, with only three patients showing systemic spread and death. Whether pcTFH-PTCL should be integrated as a new subgroup of TFH lymphomas in future classifications is still a matter of debate. What is already known about this topic? There is a group of cutaneous lymphomas that express T-follicular helper (TFH) markers that do not appear to correspond to existing World Health Organization diagnostic entities. These include mycosis fungoides, Sézary syndrome, or primary cutaneous CD4+ small/medium-sized T-cell lymphoproliferative disorder or cutaneous extensions of systemic peripheral T-cell lymphomas (PTCL) with TFH phenotype. What does this study add? This is the first large original series of patients with a diagnosis of primary cutaneous PTCL with a TFH phenotype (pcTFH-PTCL) to be molecularly characterized. pcTFH-PTCL may be a standalone group of cutaneous lymphomas with clinicopathological and molecular characteristics that overlap with those of systemic TFH lymphomas, such as angioimmunoblastic T-cell lymphoma, and does not belong to known diagnostic groups of cutaneous lymphoma. This has an impact on the treatment and follow-up of patients; the clinical behaviour needs to be better clarified in further studies to tailor patient management.

Decreased levels of T follicular helper (CD4+CXCR5+) cells and CD27+CD38+ and CD27+CD38− B cells in ankylosing spondylitis patients correlate with markers of inflammation

AbstractThe purpose of this study was to study CD4+CXCR5+ T follicular helper (TFH) cells, CD27+CD38+ plasmablasts and CD27+CD38− memory B cells, as well as disease‐related factors in patients with ankylosing spondylitis (AS) from northern Sweden. Peripheral blood mononuclear cells (PBMC) from 50 patients with AS (mean age 52 ± 9 years, 66% men, 100% HLA‐B27 positive) and 50 pairwise matched blood donor controls (mean age 54 ± 9 years, 66% men) were stained with antibodies for CD27, CD38, CD19, CD3, CD4 and CXCR5 markers and analysed by flow cytometry. Patients with AS were examined with spinal x‐ray for radiographic alterations (mSASSS), and plasma levels of C‐reactive protein, erythrocyte sedimentation rate, as well as selected proinflammatory and regulatory cytokines were determined. Physical mobility, function and disease activity were registered by BASMI, BASFI and ASDAS‐CRP, BASDAI, respectively. Comparing AS patients and controls pairwise, we observed a 56% reduction of TFH cells in PBMCs from AS patients (P = .000008). Furthermore, a 20%‐30% reduction in plasmablasts and B memory cells (P ≤ .002 and P ≤ .007, respectively) was observed. In female patients, negative correlations between ESR and TFH, plasmablasts and B memory cells were observed; Rs = −0.551, P ≤ .02; Rs = −0.476, P ≤ .05 and Rs = −0.522, P ≤ .03, respectively. In addition, positive correlations between the regulatory cytokine IL‐10 and the proportion of B cells, IL‐22, and the proportion of plasmablasts as well as a negative correlation between levels of the proinflammatory cytokine IL‐6 and TFH were detected. Our observations indicate a role of an aberrant humoral immune response related to inflammation in AS.

Long-Term Outcomes of Patients Aged 65 Years or Older with Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL-NOS) and T-Follicular Helper T-Cell Lymphoma (TFHTCL) Treated with Curative Intent CHOP(like) Chemotherapy

Introduction CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) remains the standard treatment for older adults with non-anaplastic large cell lymphoma (ALCL), nodal peripheral T-cell lymphomas (PTCLs). However, recent studies suggest that patients (pts) > 75 years (y) have similar outcomes with CHOP compared to those receiving palliative regimens (Ellin et al. Hematological Oncology 2017). In addition, with studies demonstrating sensitivity of PTCLs to epigenetic therapies, especially in the T-follicular helper (TFH) subtypes, there are emerging clinical trials evaluating these agents in newly diagnosed PTCL patients > 60 y (NCT02232516). We evaluated the long-term outcomes of pts aged 65 y or older with PTCLs, a group generally not transplant eligible, treated with systemic therapy with a focus on those treated with curative intent CHOP(like) chemotherapy. Methods The BC Cancer Lymphoid Cancer Database was reviewed and pts > 65 years with PTCL, not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL) as well as other T-Follicular Helper PTCL (collectively referred to as TFH T-cell lymphoma [TFHTCL]) diagnosed between 2000-2021 and who received front-line chemotherapy were included. Diagnoses were based on the World Health Organization (WHO) classifications of 2001, 2008, and 2017, depending on the era of diagnosis. Time to progression (TTP) was measured from the date of pathologic diagnosis to the date of relapse/progression, death due to treatment toxicity or lymphoma. Results 152 pts were initially identified, of which 5 were excluded: stage IA/IAE (n=3); CNS involvement only (n=2). Of the remaining 147 pts, 67 (46%) had PTCL-NOS and 80 (54%) had TFHTCL. The median age was 74 y (range 65-90) and the majority of pts had high risk features: International Prognostic Index (IPI) score of > 3 (n=113, 76%), stage III/IV disease (n=136, 93%) and PS of > 2 (n=85, 58%). Most pts (n=127, 86%) received multi-agent chemotherapy with curative intent. All pts received CHOP/CHOP-like chemotherapy (including five pts treated with CHP-brentuximab vedotin)Over half of these patients (n=66, 52%) received attenuated doses of chemotherapy starting from cycle 1, typically 50-75% dose intensity. Only two patients received consolidative autologous stem cell transplant. The remaining 20 patients received chemotherapy with palliative treatment intent, the majority (n=18, 90%) receiving cyclophosphamide alone or with the CVP (cyclophosphamide, vincristine, and prednisone) regimen. The median follow-up of all pts using the reverse Kaplan-Meier method was 9.2 y (range 0.9 to 12.5). The estimated 5-y TTP and overall survival (OS) for all patients were 21%, and 26%, respectively. Outcomes were similar in PTCL-NOS and TFHTCL (5-y TTP 17% vs 25%; p = 0.26). Curative intent chemotherapy was used more frequently in the 65-74 y vs > 75 y age group (70% vs 36%, p = 0.004). As expected, outcomes were superior in those treated with curative intent (Table 1): Median TTP of 11.3 months (m) vs 3.0 m; median OS of 24 m vs 4.5 m. Improvements in TTP and OS extended to those > 75 y (p < 0.001 for both). Considering the curative intent cohort, 91% of the relapse/progression events occurred within the first 3 y, and there was no significant difference in outcomes (5-y TTP/OS) with increasing age: 65-75 (19%/25%), >75 (34%/35%) (p = 0.08/0.6). Further, the outcomes of pts who received attenuated doses of CHOP-like chemotherapy were not inferior compared to full dose chemotherapy (5-y TTP/OS 32%/37% vs. 17%/21% p= 0.2/0.3). 5-y TTP/OS by the IPI ranged from 30%/52% for IPI of 1-2 to 12%/14% for IPI 4-5. The prognostic index for T-cell lymphoma (PIT) was more effective at identifying a lower risk group: 5-y TTP/ OS (PIT 1 40%/57%; PIT 2 21%/28%; PIT 3 21%/20% PIT 4 9%/10%, p = 0.002/0.001). Both models were prognostic in PTCL-NOS and TFHTCL. (Table 1) Conclusion With mature follow-up, ~ 30% of all older pts treated with curative intent CHOP-like chemotherapy remain progression-free and are alive at 5 y. Although results remain suboptimal, robust older patients can still be considered for curative intent CHOP chemotherapy with tailored doses to mitigate toxicity. Those with low risk IPI/PIT scores have a more favourable outcome with this approach, with a 5-y OS of > 50%, suggesting a benefit of subsequent therapies. In contrast, higher risk disease have poor outcomes and novel therapeutic approaches should be considered. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Transcriptome Reveals Comprehensive Immune Profiles of T Follicular Helper Cell Lymphoma

Background: Angioimmunoblastic T-cell lymphoma and other nodal lymphomas of T follicular helper (TFH) cell origin (TFH lymphoma) have a poor prognosis and are associated with immune dysregulation. However, comprehensive immune profiles of TFH lymphoma have not been fully investigated. The RHOA G17V mutation (G17V) is a disease-specific mutation found in 60-70% of TFH lymphoma and considered to gain a novel function through binding to VAV1 protein. Nevertheless, the prevalence of homozygous (Homo) and heterozygous (Hetero) G17V mutations and the difference in their oncogenicity due to its zygosity have not been elucidated. Here, we performed single-cell transcriptomic analysis to characterize tumor and immune cells of TFH lymphoma. Methods: We performed single-cell RNA sequencing (scRNA-seq) and T-cell/B-cell receptor sequencing (scTCR/BCR-seq) for 9 lymph nodes (LNs) and 16 peripheral blood (PB) from 14 TFH lymphoma patients (9 at initial diagnosis [ID] and 5 at relapsed/refractory diagnosis [RR]), as well as 7 homeostatic LNs (HLNs) from patients with solid cancers (Fig. 1). Whole-exome sequencing (WES) was also performed for genetic mutation analysis using bulk DNA extracted from tumor tissues and PB of the same cohort as scRNA-seq. scRNA-seq and scTCR/BCR-seq libraries were constructed using the Chromium system (10x Genomics). Publicly available scRNA-seq data from 5 healthy donors (HDs) were also integrated as control. scRNA-seq data analysis was performed by Seurat, Scater, and Batchelor for quality control, integration, and clustering; SingleR for annotation; Immunarch for TCR/BCR repertoire analysis; gene set variation analysis (GSVA) for pathway analysis, and CellPhoneDB for cell-cell interaction analysis. Genomon2 pipeline was used for calling somatic mutations in WES data. Subsequently, VarTrix was applied to estimate mutations detected by WES in scRNA-seq data. Results: In total, 68,738 cells from LNs and 98,891 cells from PB were analyzed. Unsupervised clustering and annotation by canonical markers identified 4 major cell types: CD4/CD8+ T, natural killer (NK), B, and myeloid cells. Tumor cells were identified by the expanded TCR clonality and the TFH-like gene expression signatures. Next, using scRNA-seq data, we reanalyzed a total of 393 somatic mutations discovered by WES and identified 15 mutations in more than 5 cells of scRNA-seq data, of which only RHOA G17V was identified in multiple samples. G17V-mutated cells were detected in 4 LN and 6 PB samples, mostly restricted to tumor cells. G17V-Homo and Hetero cells were identified based on frequency of mutant reads. Differentially expression gene (DEG) analysis and GSVA in G17V-mutated tumor cells of LNs revealed that the regulatory signature was significantly activated in Homo, whereas the proliferation signature was up-regulated in Hetero. Additionally, DEG analysis identified three tumor-specific gene candidates and the expression of cell surface protein PLS3 was validated by flow cytometry and immunohistochemistry. Subsequently, we performed sub-clustering and GSVA of immune cells and found that dysfunctional CD8 T cells were significantly increased in LNs and PB of patients compared to controls and exhibited oligoclonal expansion by TCR repertoire analysis. Notably, TREG, complement component 1q positive macrophages (C1Q+ Mφ), and LAMP3+ dendritic cells (DCs) were significantly increased in LNs from patients with RR diseases than HLNs. DEG analysis and GSVA revealed high expression of PD-L1 and regulatory signatures in LAMP3+ DCs. Moreover, genes related to immune suppression were significantly upregulated in TREG of patients compared to those of controls. Finally, cell-cell interaction analysis uncovered that tumor cells and TREG of RR patients strongly interacted with C1Q+ Mφ and LAMP3+ DCs via CXCR3-CXCL9 and CCR4-CCL17 interactions, respectively, indicating that these myeloid cells not only induced TREG into tumor tissues but also actively interacted with tumor cells. Conclusion: Tumor cells of TFH lymphoma acquired the immunosuppressive signature in the clonal evolution process from the G17V Hetero to Homo. Furthermore, comprehensive immune profiling of TFH lymphoma revealed expansion of cells involved in the immune escape. These results unveiled the enrichment of immune evasive phenotypes in TFH lymphoma and suggested their contribution to treatment resistance in RR patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Characterizing Mature T-Cell Lymphoma Patients' Outcomes By Race

Background Mature T-cell lymphomas (TCL) are a rare and aggressive group of non-Hodgkin lymphomas with significant geographic variability. Data on TCL patient outcomes by race are limited, and minority populations are often underrepresented in TCL clinical trials in North America and Europe. Herein, we describe outcomes in patients with newly diagnosed TCL from an urban US tertiary care center based on race. Methods We retrospectively identified 125 patients with non-cutaneous and non-leukemic mature TCL diagnosed between 1/1/2009-12/31/2021 from the electronic medical records at the Hospital of the University of Pennsylvania. In a univariate model stratified by self-reported race, we compared baseline patient characteristics at initial presentation, treatment patterns (including first line (1L) therapy, use of CNS prophylaxis and hematopoietic cell transplant (HCT), number of subsequent lines of treatment), and disease outcomes. Multivariate Cox proportional hazards models controlling for race, sex, age, ECOG score, LDH level, initial stage of disease, presence of B symptoms, hemoglobin, platelet count, IPI score, number of sites of extranodal involvement (ENI), histology, and 1L treatments (CHOP, CHOEP, other intensive treatment, radiation only, as well as palliative care), and receipt of HCT were implemented to further investigate the impact of these clinical parameters on overall survival (OS) and progression-free survival (PFS). P-values <0.05 were considered significant. Hazard ratio (HR) is reported with 95% CI in brackets. Results We collected and evaluated data from 94 White patients, 21 Black patients and 10 patients of other races. Patients of other races comprised less than 10% of our cohort and were omitted from the analysis due to inadequate statistical representation. Compared to White patients, Black patients demonstrated comparable baseline characteristics at initial presentation, except for lower incidence of TCL with T-follicular helper phenotype (p=0.013), poor performance status (ECOG ≥ 2; p=0.005), and ≥2 sites of ENI (p=0.002). Treatment patterns for Black and White patients, including 1L chemotherapy, radiation therapy, CNS prophylaxis, HCT, and subsequent lines of treatment were not significantly different. On univariate analysis, response rates to initial therapy, PFS, and OS were similar for Black and White patients (Table 1). On multivariate analysis, race did not significantly impact OS (HR 0.72 [0.29-1.80], p=0.480) or PFS (HR 0.50 [0.17-1.47], p=0.206), though worse outcomes were observed with other established risk factors, including ECOG ≥ 2 (OS HR 3.32 [1.43-7.73], p=0.005), elevated LDH (OS HR 3.25 [1.37-7.71], p=0.008; PFS HR 2.29 [1.00-5.21], p=0.0.049), and low platelet count (OS HR=2.84 [1.22-6.60], p=0.015; PFS HR 2.73 [1.13-6.62], p=0.026). However, advanced stage was associated with better OS (HR 0.22 [0.09 -0.58], p=0.002). For both races, HCT in 1st remission was associated with significantly improved PFS (HR 0.03 [0.03-0.33], p=0.004) but not OS (HR 0.57 [0.14-2.35] p=0.44) (Table 2). Conclusion In this cohort of patients with TCL treated at an urban US tertiary care center with demographics reflective of the national population, we did not find outcomes affected by race despite the higher incidence of poor risk baseline prognostic factors in Black patients. Treatment patterns, including intensive therapy, use of transplant, and number of subsequent therapies, were also not affected by race in our cohort. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CNS Relapse in T-Cell Lymphoma Index: A Risk Score to Predict Central Nervous System Relapse in Patients with T-Cell Lymphomas

Background: Central nervous system (CNS) relapse (CNSr) in patients with aggressive non-Hodgkin lymphoma is uncommon but carries a high morbidity and mortality. Data in T- and NK-cell lymphomas (TCL) are sparse, though the few small case series describing CNSr in TCL report an incidence up to 9% with median overall survival (mOS) less than 3 months (mo). We previously reported a large (n=75) case series of CNSr in TCL (Abstract #1382, ASH 2021). Herein, we developed a prediction score for CNSr in newly diagnosed patients with TCL. Methods: We retrospectively collected clinicopathologic and treatment data from 19 US academic centers from patients diagnosed with TCL between 1/1/09-1/1/19 who experienced CNSr (CNSr cohort). We combined these data with that from patients diagnosed with TCL without CNSr from a single institution (Penn cohort) to create a training set (T-set) enriched for patients with CNSr. Patients with leukemic or cutaneous TCL subtypes were excluded. We applied a LASSO Cox proportional hazards model to the T-set to create a predictive score for factors associated with CNSr and validated this score in an independent population-based validation set (V-set) from the Swedish Lymphoma Registry, which was not enriched for CNSr. Results: We evaluated data from 91 and 135 patients in the CNSr and Penn cohorts, respectively, which were combined to form the T-set and compared them to the V-set (n=745). Median age in the T-set was lower than in the V-set (60 vs 67; p<0.001). The most common TCL subtypes in the T-set and V-set were peripheral TCL, not otherwise specified (PTCL, NOS; 23% vs 34%, respectively; p=0.002), TCL with T-follicular helper phenotype (23% vs 14%, respectively; p=0.001), and ALK-negative anaplastic large cell lymphoma (18% vs 15%, respectively; p=0.352). CNS prophylaxis, defined as intrathecal therapy or high-dose methotrexate, was more common in the V-set than in the T-set (15% vs 9%, respectively; p=0.012). Rates of stem cell transplantation (SCT) as consolidation in first remission (CR1) did not differ significantly between the T-set and V-set (p=0.140). The median follow-up time was 37.6 mo in the T-set and 95.9 mo in the V-set. The median progression free survival (mPFS) and mOS after diagnosis were 9.8 mo and 33.2 mo in the T-set, respectively, and 8.5 mo and 17.9 mo in the V set, respectively. Within the T-set, mPFS and mOS were shorter for patients in the CNSr cohort vs the Penn cohort (6.0 mo vs 37.1 mo [p<0.001] and 17.3 mo vs 78.1 mo [p<0.001], respectively). In univariate analysis of the CNSr-enriched T-set, CNS prophylaxis, SCT in CR1, or frontline etoposide did not significantly decrease the risk of CNSr. Using several clinicopathologic characteristics, we fitted a LASSO Cox proportional hazards model to the T-set, which selected histology, LDH, stage, B-symptoms, and ≥2 sites of extranodal involvement (ENI) for a weighted risk score of CNSr (Fig. 1A). Variables most strongly associated with CNSr were PTCL, NOS (HR 2.64), enteropathy-associated TCL (HR 2.92), and ≥2 sites of ENI (HR 2.66). We stratified the T-set into tertiles based on weighted scores (x), which was low-risk if x≤0.14, intermediate-risk if 0.14<x ≤1.11, or high-risk if x>1.11. Cumulative incidence of CNSr in each respective group was 19.6%, 55.1%, and 83.7%. When the same cutoffs were applied to weighted scores in the V-set, we confirmed separation into distinct low-, intermediate-, and high-risk groups with cumulative incidences of CNSr of 1.9%, 4.9%, and 7.3%, respectively (Fig. 1B). As the V-set population was not enriched for CNSr, the relative incidence is expectedly lower compared to the T-set. Compared to the low-risk group in the V-set, risk of CNSr was significantly increased in the high-risk group (HR 5.72, p=0.002) and increased with a trend toward significance in the intermediate-risk group (HR 3.35, p=0.057). Conclusions: In a cohort of patients with TCL enriched for CNSr, we were able to characterize several risk factors associated with CNSr and developed a predictive CNSr in T-cell lymphoma Index (CITI) that could stratify patients into 3 distinct risk groups. PTCL, NOS, EATL, and ≥2 sites of ENI had the highest impact on risk of CNSr. The CITI score was validated in an independent population-based cohort. Although CNSr was uncommon even in high-risk patients (7.3%), our data will inform clinical decision making and may allow for identification of patients with TCL at high-risk of CNSr in future studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Investigating Biomarkers for Primary Immunodeficiency Disorders in Patients with Autoimmune Cytopenia

Introduction: Autoimmune cytopenias (AICs) occur in the setting of immune dysregulation and autoimmunity. AICs are known to precede the classical presentation and subsequent diagnosis of primary immunodeficiency disorders (PIDs) in some cases, with a PID spectrum involving B, T, and Treg dysregulation. Conversely, PID patients are also at a markedly increased risk of AICs and patients co-diagnosed with AICs and PIDs face higher mortality rates likely due to delayed diagnosis or improper/delayed therapy. Further, co-diagnosed patients are often refractory to standard treatment. Early screening and diagnosis of PIDs in the setting of AICs could lead to more targeted therapies and potentially better patient outcomes. We intend to investigate biomarkers that differentiate immune dysregulation among those with or without PID in the setting of AIC. Methods: A retrospective analysis comparing clinical history, including symptoms, presence of PID, genetic testing and diagnosis and treatment outcomes was performed on AIC patients in our hospital from 2016 to 2019. We also performed extensive immune phenotyping by flow cytometric analysis. Patients with expansion of CD19hi CD21lo B cells were further evaluated for Tbet+ CD11chi features for potential autoreactivity. Results: Our original cohort of 103 AIC patients included patients who were diagnosed with AIC only (n=52) or co-diagnosed with AIC-PID (n=51). From this cohort, a total of 43 were evaluated for further immune phenotyping, including 35 AIC-PID patients and 8 AIC only patients. Of the AIC-PID patients, 23 (65.7%) had a genetically coded PID, including pathogenic variants in CTLA4, NFKB1, PI3K or partial DiGeorge, while 12 (34.3%) were diagnosed with CVID/CID. Flow cytometry analysis revealed significantly increased T-follicular helper cell (Tfh) population in the AIC-PID group compared to AIC only (p=0.03) and healthy donor (HD) (p=0.004). We saw a similar expansion of CD19hi CD21lo B cells within the AIC-PID group. Conversely, patients with AIC-PID had decreased percentage of T-regulatory cells (Tregs) when compared to AIC only and HD groups. These differences were amplified within the genetically coded AIC-PID patients in Treg and Tfh populations and maintained in CD19hi CD21lo B cells. Further investigation of clinical history within the symptomatic, genetically coded AIC-PID group yielded 3 (33.3%) patients responsive to treatment and 6 (66.7%) patients unresponsive or partially responsive to therapy. Interestingly, nonresponsive patients showed an increased percentage of CD19hi CD21lo B cells. Conclusions: Our analysis demonstrates the potential utility of biomarkers, notably CD19hi CD21lo B cells and their immunological function for detecting underlying PIDs within the setting of AIC patients. Though this B cell population needs further assessment regarding Tbet and CD11c expression, with studies ongoing, these biomarkers may lead to a screening methodology to increase clinical visibility and detection as well as hasten diagnosis of underlying PID and potentially predict response to therapy, therefore increasing effective treatment outcomes for patients co-diagnosed with AIC PID.

The Addition of Romidepsin to CHOEP and High-Dose Chemotherapy Plus Stem Cell Transplantation Did Not Ameliorate the Outcome of Untreated Angioimmunoblastic T-Cell or Follicular T-Helper Lymphoma: Subgroup Analysis of Phase II FIL-PTCL13 Study

Introduction: In the phase Ib/II study PTCL13 study (NCT02223208) we demonstrated feasibility of Romidepsin 14 mg/mq in addition to CHOEP followed by high-dose chemotherapy and stem cell transplantation (SCT), but no benefit was observed in term of progression free survival (PFS), and the enrollment of the trial was stopped due to inefficacy of the experimental combination. Other studies showed Romidepsin activity in relapsed or refractory PTCLs, especially in angioimmunoblastic (AITL) subtype, expecially when combined with azacytidine. On this basis, we conducted a sub-analysis of the PTCL13 trial, with the aim to evaluate the role of adding Romidepsin to CHOEP followed by high dose chemotherapy and SCT in newly diagnosis AITL and T-helper follicular lymphoma patients. Methods: Patients aged 18-65 eligible to SCT, with advanced PTCL-NOS, AITL/T-helper follicular and ALK negative anaplastic large cell lymphoma, were eligible to PTCL13 study. Treatment plan consisted of 6 courses of Ro-CHOEP every 21 days (14 mg/ms Ro day 1 and 8), followed by cisplatin-cytarabine-dexamethasone (DHAP) with stem cell harvest and SCT. Patients in complete response (CR) after induction proceeded to autoSCT, while those in partial response (PR), with an available HLA-matched donor, proceeded to alloSCT upfront. Results: 89 patients were enrolled into the phase Ib and II part of the study; median age was 55 years (IQR 49;60); 78 (91%) had stage III-IV and 31 (36%) IPI risk >2. Pathological materials were collected at the time of diagnosis, and centrally reviewed by expert hemo-pathologist; subgroups were: 34 PTCL-NOS, 21 ALK negative and 31 AITL/THF. The clinical characteristics of the 31 AITL/THF patients were: median age 56 years (IQR 52.5-59.5), 63% male, 31% had an intermediate-high IPI score risk and 50% had a bone marrow involvement at diagnosis. The overall response (ORR) at the end of induction with 6 Romidepsin-CHOEP was achieved in 7 AITL/THF patients (22%), with 6 (19%) patients in complete remission (CR); at the end of treatment, the ORR was observed in 15 (47%) AITL/THF patients. ORR and CR results are superimposable to those of the whole population. At a median follow-up of 28 months, the 24-months PFS for AITL/THF was 43% (95% CI: 0.22-0.61) compared to 24-months PFS for ALK-negative 37% (95% CI: 0.16-0.59) and for PTCL-nos 33% (95% CI: 0.18-0.48), p 0.136; the 24-months OS for AITL/THF was 78% (95% CI: 0.57-0.89) compared to 24-months OS for ALK-negative 63% (95% CI: 0.37-0.81) and for PTCL-nos 61% (95% CI: 0.40-0.76), p 0.556. Conclusions: In the FIL-PTCL13 the primary endpoint was not met and the enrollment of the trial was stopped due to inefficacy of the experimental combination. With the limits of the small number and the unpowered analysis, the benefit of adding romidepsin to chemotherapy was not observed in the subgroup AITL/THF patients, even if the PFS and the OS of this subgroup seem better than the others.

Role of CXCR5+ CD8+ T cells in human immunodeficiency virus-1 infection

Human immunodeficiency virus (HIV) infection can be effectively suppressed by life-long administration of combination antiretroviral therapy (cART). However, the viral rebound can occur upon cART cessation due to the long-term presence of HIV reservoirs, posing a considerable barrier to drug-free viral remission. Memory CD4+ T cell subsets, especially T follicular helper (T FH ) cells that reside in B-cell follicles within lymphoid tissues, are regarded as the predominant cellular compartment of the HIV reservoir. Substantial evidence indicates that HIV-specific CD8+ T cell-mediated cellular immunity can sustain long-term disease-free and transmission-free HIV control in elite controllers. However, most HIV cure strategies that rely on expanded HIV-specific CD8+ T cells for virus control are likely to fail due to cellular exhaustion and T FH reservoir-specialized anatomical structures that isolate HIV-specific CD8+ T cell entry into B-cell follicles. Loss of stem-like memory properties is a key feature of exhaustion. Recent studies have found that CXC chemokine receptor type 5 (CXCR5)-expressing HIV-specific CD8+ T cells are memory-like CD8+ T cells that can migrate into B-cell follicles to execute inhibition of viral replication. Furthermore, these unique CD8+ T cells can respond to immune checkpoint blockade (ICB) therapy. In this review, we discuss the functions of these CD8+ T cells as well as the translation of findings into viable HIV treatment and cure strategies.

New definitions of human lymphoid and follicular cell entities in lymphatic tissue by machine learning

Histological sections of the lymphatic system are usually the basis of static (2D) morphological investigations. Here, we performed a dynamic (4D) analysis of human reactive lymphoid tissue using confocal fluorescent laser microscopy in combination with machine learning. Based on tracks for T-cells (CD3), B-cells (CD20), follicular T-helper cells (PD1) and optical flow of follicular dendritic cells (CD35), we put forward the first quantitative analysis of movement-related and morphological parameters within human lymphoid tissue. We identified correlations of follicular dendritic cell movement and the behavior of lymphocytes in the microenvironment. In addition, we investigated the value of movement and/or morphological parameters for a precise definition of cell types (CD clusters). CD-clusters could be determined based on movement and/or morphology. Differentiating between CD3- and CD20 positive cells is most challenging and long term-movement characteristics are indispensable. We propose morphological and movement-related prototypes of cell entities applying machine learning models. Finally, we define beyond CD clusters new subgroups within lymphocyte entities based on long term movement characteristics. In conclusion, we showed that the combination of 4D imaging and machine learning is able to define characteristics of lymphocytes not visible in 2D histology.

The Transcription Factor YY-1 Is an Essential Regulator of T Follicular Helper Cell Differentiation.

T follicular helper (TFH) cells are a specialized subset of CD4 T cells that deliver critical help signals to B cells for the production of high-affinity Abs. Understanding the genetic program regulating TFH differentiation is critical if one wants to manipulate TFH cells during vaccination. A large number of transcription factor (TFs) involved in the regulation of TFH differentiation have been characterized. However, there are likely additional unknown TFs required for this process. To identify new TFs, we screened a large short hairpin RNA library targeting 353 TFs in mice using an in vivo RNA interference screen. Yin Yang 1 (YY-1) was identified as a novel positive regulator of TFH differentiation. Ablation of YY-1 severely impaired TFH differentiation following acute viral infection and protein immunization. We found that the zinc fingers of YY-1 are critical to support TFH differentiation. Thus, we discovered a novel TF involved in the regulation of TFH cells.

Classification and diagnostic evaluation of nodal T- and NK-cell lymphomas.

Nodal T- and NK-cell lymphomas are among the most frequent T-cell malignancies and most subtypes have aggressive clinical behavior. Evolving understanding of the biology and molecular characteristics of these lymphomas, as well as the development of new precision therapy approaches, underscores the importance of ongoing updates to the classification and diagnostic evaluation of this group of malignancies. Here, we discuss the classification of nodal T- and NK-cell lymphomas based on the 2022 International Consensus Classification of Mature Lymphoid Neoplasms (2022 ICC). Lymphomas of T-follicular helper cell origin are now grouped into a single entity, follicular helper T-cell lymphoma (TFH lymphoma), with three subtypes (angioimmunoblastic-type, follicular-type, and not otherwise specified), reflecting their common cellular origin and shared molecular and clinical characteristics. Classification of anaplastic large cell lymphoma (ALCL) remains essentially unchanged; DUSP22-rearranged cases are now considered a genetic subtype of ALK-negative ALCL. Primary nodal EBV-positive T-/NK-cell lymphoma is introduced as a new provisional entity; these cases were previously considered a variant of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS). PTCL, NOS remains a diagnosis of exclusion, with evolving molecular data indicating the presence of distinct subgroups, including PTCL-TBX21, PTCL-GATA3, and EBV-negative cytotoxic PTCLs. We also discuss diagnostic strategies to facilitate the 2022 ICC classification among nodal T- and NK-cell lymphomas and the distinction from nodal involvement by extranodal neoplasms.

The role of CXCR5+ CD4+T-follicular helper cells in idiopathic thrombocytopenic purpura

Background Primary immune thrombocytopenic purpura (ITP) is an autoimmune illness characterized by increased platelet breakdown in the peripheral blood as well as impaired platelet production and it is also characterized by platelet counts less than 100 × 109/L. T-follicular helper (TFH) cells have been linked to the pathogenesis of autoimmune conditions. Aim The study’s aim was to study the role of TFH cells and their function in ITP children. Patients and methods This research involved 25 ITP patients and 20 age-matched and sex-matched controls, with the frequency of circulating TFH cells measured by BD FACS Calibur Flowcytometer in both ITP patients and healthy controls. Results The results showed that the ratio of circulating CD4+ CXCR5+ PD1+ (TFH) in ITP (+) patients, that is, (ITP patients with antiplatelet antibody-positive results) was dramatically higher than those of ITP (−) patients, that is, (ITP patients with antiplatelet antibody-negative results) and it was discovered that the ratios of circulating CD4+ CXCR5+ PD1+ (TFH) were dramatically higher in ITP (+) patients than the control group. However, the ratio of circulating CD4+ CXCR5 +PD1+ (TFH) was not dramatically different between ITP (−) patients and the control group. Conclusion Patients with ITP have an increased number of TFH cells, which prove its critical role in B-cell proliferation, resulting in the development of autoantibodies that contribute to the pathophysiology of ITP.

High expression of the ANXA3 gene promotes immune infiltration and improves tumor prognosis in ovarian serous carcinoma using bioinformatics analyses.

Annexin A3 (ANXA3) expression change is related to tumor cell proliferation and might serve as a novel diagnostic and prognostic biomarker for cancer. However, its roles and mechanisms in ovarian serous cystadenocarcinoma (OV) have not yet been elucidated. This study aimed to investigate ANXA3 expression in OV, its association with immune infiltrates, and its prognostic roles in OV. The clinical data and gene expression profiles of 379 patients (189 with low ANXA3 expression and 190 with high ANXA3 level) with an OV diagnosis confirmed by histopathological examination were downloaded from The Cancer Genome Atlas database (https://portal.gdc.cancer.gov). The survival rate and expected survival time were used to measure disease prognosis. Survival curves were generated using the Kaplan-Meier method. Cox regression and a nomogram prediction model were used to analyze the relationship between ANXA3 and the survival rate. Logistic regression was used to analyze the relationship between clinicopathological features and ANXA3 expression. Protein-protein interactions among ANXA3 relevant proteins were established using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. The signaling pathways interacting with ANXA3 were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. High ANXA3 expression significantly correlated with lymph node infiltration (odds ratio =0.448, P=0.025) and overall favorable survival (hazard ratio =0.69, P=0.011). The Federation International of Gynecology and Obstetrics stages, primary therapy outcome, age, and residual tumor might serve as independent prognostic factors, whereas the ANXA3 levels could not independently predict OV prognosis. ANXA3 expression negatively and statistically (P<0.05) correlated with lymphatic invasion in Th17 cells, T follicular helper (TFH) cells, and T effector memory cells. The GO/KEGG pathway enrichment analysis confirmed the involvement of three signaling pathways in controlling the interaction of extracellular vesicles with ANXA3. High ANXA3 expression may contribute to tumor inhibition and a favorable prognosis to a certain extent by promoting the infiltration of TFH cells and Th17 lymphocytes or acting on extracellular vesicles inducing a stronger T-cell-mediated immunity against tumor cells.

Termination of CD40L co-stimulation promotes human B cell differentiation into antibody-secreting cells.

Human naïve B cells are notoriously difficult to differentiate into antibody-secreting cells (ASCs) in vitro while maintaining sufficient cell numbers to evaluate the differentiation process. B cells require T follicular helper (TFH ) cell-derived signals like CD40L and IL-21 during germinal center (GC) responses to undergo differentiation into ASCs. Cognate interactions between B and TFH cells are transient; after TFH contact, B cells cycle between GC light and dark zones where TFH contact is present and absent, respectively. Here, we elucidated that the efficacy of naïve B cells in ACS differentiation is dramatically enhanced by the release of CD40L stimulation. Multiparameter phospho-flow and transcription factor (TF)-flow cytometry revealed that termination of CD40L stimulation downmodulates NF-κB and STAT3 signaling. Furthermore, the termination of CD40 signaling downmodulates C-MYC, while promoting ASC TFs BLIMP1 and XBP-1s. Reduced levels of C-MYC in the differentiating B cells are later associated with crucial downmodulation of the B cell signature TF PAX5 specifically upon the termination of CD40 signaling, resulting in the differentiation of BLIMP1 high expressing cells into ASCs. The data presented here are the first steps to provide further insights how the transient nature of CD40 signaling is in fact needed for efficient human naïve B cell differentiation to ASCs.

A Bcl6 Intronic Element Regulates T Follicular Helper Cell Differentiation.

In response to an intracellular infectious agent, the immune system produces a specific cellular response as well as a T cell-dependent Ab response. Precursor T cells differentiate into effector T cells, including Th1 cells, and T follicular helper (TFH) cells. The latter cooperate with B cells to form germinal centers and induce the formation of Ab-forming plasmacytes. One major focal point for control of T cell differentiation is the transcription factor BCL6. In this study, we demonstrated that the Bcl6 gene is regulated by FOXO1-binding, cis-acting sequences located in a highly conserved region of the first Bcl6 intron. In both mouse and human T cells, deletion of the tandem FOXO1 binding sites increased the expression of BCL6 and enhanced the proportion of TFH cells. These results reveal a fundamental control point for cellular versus humoral immunity.

The Role of T Cells in Systemic Sclerosis: An Update

Systemic sclerosis (SSc) is a chronic disease characterized by microvasculopathy, autoantibodies (autoAbs), and fibrosis. The pathogenesis of the disease is incompletely understood. Microvasculopathy and autoAbs appear very early in the disease process. AutoAbs, such as those directed against DNA topoisomerase I (Topo I), are disease specific and associated with disease manifestations, and indicate activation of the adaptive immune system. B cells are involved in fibrosis in SSc. T cells are also involved in disease pathogenesis. T cells show signs of antigen-induced activation; T cells of TH2 type are increased and produce profibrotic cytokines interleukin (IL)-4, IL-13, and IL-31; CD4+ cytotoxic T lymphocytes are increased in skin lesions, and cause fibrosis and endothelial cell apoptosis; circulating T follicular helper (TFH) cells are increased in SSc produce IL-21 and promote plasmablast antibody production. On the other hand, regulatory T cells are impaired in SSc. These findings provide strong circumstantial evidence for T cell implication in SSc pathogenesis and encourage new T cell-directed therapeutic strategies for the disease.

A RORγt+ cell instructs gut microbiota-specific Treg cell differentiation.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFβ activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.