Chimeric antigen receptor (CAR) T cell therapy was recently proposed as a treatment for adults with B-cell-mediated autoimmune diseases (ADs) refractory to conventional immunomodulatory therapy. We present a case series of eight children with severe/refractory AD (four systemic lupus erythematosus, three dermatomyositis, one systemic sclerosis) treated at Ospedale Pediatrico Bambino Gesù, Rome, and University Hospital Erlangen with a single infusion of 1 × 106 kg-1 point-of-care manufactured autologous CD19 CAR T cells (zorpocabtagene autoleucel), in a hospital exemption (HE) program. In Europe, the HE pathway offers the opportunity to treat patients with life-threatening or seriously debilitating disorders who lack valid therapeutic options, using an advanced therapy medicinal product (ATMP) authorized on a nonroutine, single-patient basis. In contrast to the 'compassionate use' pathway, the ATMP does not necessarily need to have undergone clinical trials or marketing authorization applications. Manufacturing was successful in all patients, yielding several drug product bags. Once infused after lymphodepletion, zorpocabtagene autoleucel cells expanded in vivo, promoting prompt B cell clearance. Grade 1 cytokine release syndrome was reported in six patients, and grade 1 immune effector cell-associated neurotoxicity syndrome was reported in one patient. Late-hematotoxicity was limited to grade 1 in two patients. All these adverse events were manageable and no severe infections occurred. With a median follow-up of 16.5 months (range = 9-24 months), all patients experienced a clinically substantial improvement/resolution of AD, as evidenced by reduction in disease activity scores and signs of reversal of organ damage. This improvement enabled sustained discontinuation of immunomodulators, even after B cell reconstitution. The activation of formal clinical trials enrolling children and adolescents is urgently needed to confirm these preliminary results and to assess the long-term safety of this approach.

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) enables hematopoietic reconstitution through donor stem cells matched at essential HLA loci. While it offers potential for a graft-versus-leukemia (GvL) effect through elimination of residual malignant cells, it also carries the risk of graft-versus-host disease (GvHD) due to the recognition of healthy tissues. We identified two groups of HLA-II restricted antigens with distinct behavior towards HLA-DM. DM-resistant antigens are presented when HLA-DM is expressed. In contrast, DM-sensitive antigens require inhibition of HLA-DM by HLA-DO. Because HLA-DO expression is confined to hematopoietic antigen-presenting cells, DM-sensitive antigens cannot be presented on non-hematopoietic tissues, even under inflammatory conditions. Since HLA-DP is frequently mismatched in unrelated donor transplants, we hypothesized that CD4+ T cells targeting DM-sensitive antigens in a mismatched HLA-DP allele could be an ideal way to achieve GvL effect without inducing GvHD. Methods: To identify T cells recognizing DM-sensitive antigens, isolated CD4⁺ T cells from an HLA-DP–mismatched donor were co-cultured with HeLa cells expressing invariant chain (li) and one of the five most common HLA-DP molecules in the Caucasian population (DPB1*01:01, *02:01, *03:01, 04:01, 04:02). Activated T cells were isolated based on expression of CD137 and clonally expanded. Reactivity of T-cell clones against HeLa + Ii ± HLA-DP ± HLA-DM, malignant hematopoietic and non-hematopoietic cell lines, EBV-infected B cells, and primary AML blasts was characterized, along with cytotoxicity and cytokine profiles. T-cell receptors (TCR) of T-cell clones with the most favorable characteristics were sequenced in order to generate TCR-engineered T cells. TCR reexpression was achieved by orthotopic T-cell receptor replacement (OTR) using CRISPR. Results: Of 105 T-cell clones from nine donors, 79 targeted DM-sensitive and 26 DM-resistant antigens, distinguished by their reactivity in presence of HLA-DM. T-cell clones directed against DM-sensitive antigens did not recognize non-hematopoietic cells, even under inflammatory conditions. In contrast, HLA-DO positive malignant hematopoietic cell lines and primary AML blasts were recognized by T-cell clones targeting DM-sensitive antigens, indicating potential leukemia-specificity. Testing against cell lines from various donors suggests that antigen recognition by T cells was independent of donor-specificity, but rather directed against the mismatched HLA-DP possibly complexed with a monomorphic peptide. Across all experimental conditions, the recognition profile (sensitive or resistant) remained consistent regardless of target cell concentration. Donor-dependent cytotoxicity for isolated T-cell clones was observed and mainly mediated by Granzyme A and B. Extended cytokine analysis revealed secretion of INF-γ, IL-5, IL-13, and IL-6, accompanied by lower levels of IL-4. After reexpression, TCR-engineered T cells demonstrated high functionality as shown by IFN-γ ELISA and activation marker analysis via flow cytometry. Conclusion: We show feasibility to identify T cells directed against DM-sensitive antigens presented in mismatched HLA-DP alleles recognizing malignant hematopoietic cells, which in future may contribute to the development of GvL directed T-cell therapies with reduced risk of GvHD.

BackgroundRecombinant immunotoxins (RITs) are fusion proteins of a targeting domain, such as an antibody fragment, and a truncated toxin, including Pseudomonas exotoxin A (PE) or diphtheria toxin (DT). Limiting their possible fusion partners, the targeting domain of DT is typically fused C-terminally and that of PE is fused N-terminally. Among other factors, the activity of immunotoxins depends on the target antigen and the target-specific intracellular trafficking. Because a novel anti-CD138-PE immunotoxin was inactive against multiple myeloma, we hypothesized that rational toxin design would improve trafficking and, thus, cytotoxicity.MethodsWe, therefore, generated several variants with distinct domain sequences including the catalytic (C) units of PE and DT, the furin-cleavage site of PE (fu), the transport (T) domain of DT, and the ER retention sequence KDEL. The lead candidate—a fusion of PE and DT—was combined with antibodies against CD138, CD22, glypican-3, or mesothelin.ResultsThis final toxin moiety was internalized 4-fold more efficiently over time on average compared to PE-based RITs, regardless of the target antigen or cell type. Improved internalization of DT over PE did not depend on a specific domain of DT. Instead, it was competed dose-dependently by poly-D lysine (PDL), indicating a more unspecific charge effect of DT over PE. The improved internalization frequently translated to enhanced cytotoxicity. Finally, combinatorial treatment with actinomycin D demonstrated synergistic effects with the lead PE‐DT-fusion toxin.ConclusionThis novel and target-dependently more potent toxin moiety may provide a framework for future immunotoxin design, possibly widening the range of target antigens, including the ongoing efforts to enhance mesothelin- or CD138-targeting immunotoxins against cancer or autoimmune diseases.

Introduction Chimeric antigen receptor (CAR) T-cell therapy has become a standard-of-care in oncology, yet standardized monitoring of circulating CAR T cells remains a major challenge due to diverse CAR constructs and limited availability of detection reagents. The antibody domain of the CAR commonly consists of the heavy and light chain connected through a linker, typically either 4x glycine and 1x serine (G4S) or a defined amino acid sequence (Whitlow/218). Here, we evaluated the novel monoclonal antibodies (mAbs) targeting the linker sequence as a universal tool for CAR detection.Methods Using flow cytometry, we compared anti-linker mAbs with conventional reagents, including anti-idiotype CD19.FMC63 mAb, CD19 and BCMA antigen-based detection reagents (Ag), and anti-F(ab ')2 mAb. Analyses were performed on commercial CD19- and BCMA-directed CAR T-cell products and an investigational Claudin-6 (CLDN6) CAR T-cell product. Performance was further assessed across diverse experimental platforms for clinical monitoring and in a murine model to evaluate sensitivity and translational relevance.Results Linker-mAbs detected all tested CAR constructs with high specificity and sensitivity, matching target-specific binding with conventional Ag reagents. Anti-linker mAbs demonstrated minimal background and low limits of quantification both comparable to Ag reagents. Longitudinal monitoring in lymphoma and myeloma patients revealed consistent CAR T-cell kinetics between anti-linker mAbs and Ag reagents. High performance of linker-based CAR-detection was demonstrated in high-dimensional, multi-parameter flow cytometry, in immunofluorescence imaging and in a murine model of anti-CD19 CAR T cells.Conclusion These findings establish anti-Whitlow/218 and anti-G4S mAbs as sensitive, specific, and universal reagents for CAR detection across multiple targets, constructs, and species, providing a standardized platform for harmonization of CAR T-cell monitoring in preclinical, clinical trial, and diagnostic settings.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and fibrotic remodeling of the skin and internal organs. Fibrotic tissue changes are considered hardly reversible with current therapies, suggesting that new strategies are required to modulate the disease-associated molecular and cellular phenotype to enable regeneration of affected tissues. Here, analyzing skin biopsy samples from patients with SSc who had received CD19-CAR T cell therapy as part of the CASTLE study or named patient use, we demonstrate structural regeneration of SSc skin structure, as evidenced by recovery of skin papillae. Consistent with these histological changes, cyclic in situ hybridization and imaging mass cytometry analyses suggested that fibroblast populations shifted towards a physiological state, both in terms of composition and function. Moreover, we describe signs of vascular repair and changes in epidermal cell function. These results suggest that B cell depletion using CD19-CAR T cell therapy may lead to skin tissue remodeling in SSc and highlight its potential for tissue regeneration in fibrotic diseases.

OBJECTIVES:Women with SLE are at increased risk of infertility due to disease activity and exposure to gonadotoxic therapies. Anti‑CD19 chimeric antigen receptor (CAR) T‑cell therapy has recently been shown to induce durable, drug‑free remission in refractory SLE; however, its effects on ovarian function remain unknown. We assessed longitudinal changes in reproductive endocrine markers to evaluate the impact of CAR T‑cell therapy on ovarian reserve and hypothalamic-pituitary-ovarian (HPO) axis function. METHODS:Women with severe, treatment‑refractory SLE receiving autologous anti‑CD19 CAR T‑cell therapy (Miltenyi Biomedicine) in conjunction with standard lymphodepletion at two tertiary care centres were studied. Anti‑Müllerian hormone (AMH), oestradiol (E2), progesterone, follicle‑stimulating hormone (FSH), luteinizing hormone (LH) and prolactin were measured at baseline and 12 months after therapy. Blood samples were obtained independent of menstrual cycle phase. Paired statistical analyses were performed. RESULTS:Thirteen women were included; median age at CAR T‑cell therapy was 27 years. AMH levels were stable (1.54 ng/ml [IQR 0.64-3.15] vs 2.14 ng/ml [0.63-3.26]; P = 0.886). E2 (243 pmol/l [186-346] vs 484 pmol/l [277-829]; P = 0.339) and progesterone (0.90 nmol/l [0.80-0.90] vs 1.80 nmol/l [0.90-3.40]; P = 0.054) showed substantial variability without significant longitudinal change. FSH increased modestly (6.60 IU/l [4.00-7.90] vs 7.20 IU/l [4.50-9.90]; P = 0.033) without a concomitant decline in AMH. LH (6.10 IU/l [5.10-12.90] vs 7.00 IU/l [5.00-12.90]; P = 0.946) and prolactin (10.0 µg/l [8.0-10.0] vs 8.0 µg/l [5.0-9.0]; P = 0.123) remained stable. CONCLUSION:Anti‑CD19 CAR T‑cell therapy, including standard lymphodepletion, showed no signal of impaired ovarian reserve, as assessed by AMH and hormone profiles. Observed hormonal changes, including FSH, should be interpreted with caution given study limitations.

Abstract Systemic lupus erythematosus (SLE) is driven by pathogenic B cells. Yet, why some patients receiving B cell depletion achieve durable remission, whilst others fail remains unclear. Here we use CD19-directed chimeric antigen receptor (CAR) T cell therapy as a mechanistic probe in 18 patients with refractory SLE 1,2 , with longitudinal follow-up extending up to 40 months. We show that durable, drug-free remission is defined not by the depth of B cell depletion alone, but by the elimination of the extrafollicular (EF) B cell differentiation trajectory – specifically, activated naïve B cell precursors and CD11c + T-bet + double-negative type 2 B cells. In long-term responders, B cell reconstitution recapitulated healthy ontogeny, while the EF pathway remained truncated, coinciding with collapse of the interferon-rich milieu and contraction of PD1 hi T peripheral helper cells. In contrast, in relapse, persistently elevated CXCL13, interferons and expanded PD1 hi T cells preceded the B cell return, and nascent B cells immediately followed the EF differentiation trajectory in the confirmed absence of germinal centers in the lymph node, shortly followed by clinical symptoms. These findings indicate that CAR-T cell therapy achieves remission by breaking a feed-forward loop between the systemic inflammatory environment and extrafollicular B cell differentiation.

Chimeric antigen receptor (CAR)-T cells are considered a powerful therapeutic tool to reset the immune system in patients with autoimmune diseases. Innovative trial designs are needed to allow feasible testing of the safety and efficacy of CAR-T cells in clinical studies. CASTLE (CAR-T cells in systemic B cell mediated autoimmune disease) is a phase 1/2a two-stage optimal design basket study that investigated the safety and efficacy of zorpocabtagene autoleucel (Zorpo-cel, also known as MB-CART19.1), an autologous CD19 CAR-T cell product, in patients with treatment-resistant systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and idiopathic inflammatory myopathies (IIM). The primary safety outcome was the rate of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The secondary clinical efficacy outcomes were remission of SLE according to DORIS criteria, no progression of interstitial lung disease in SSc and American College of Rheumatology (ACR) major/moderate response in IIM after 24 weeks. A total of 24 patients were enrolled (10 with SLE, 9 with SSc and 5 with IIM), all receiving a single infusion of Zorpo-cel after stopping immunosuppressive treatments and receiving standard lymphodepletion with cyclophosphamide and fludarabine. Primary and secondary endpoints of CASTLE were met. Regarding safety, no CRS higher than grade 2 and no ICANS occurred. Regarding efficacy, 22 of the 24 patients achieved predefined efficacy endpoints, with 9 out of 10 patients with SLE reaching DORIS remission, 9 out of 9 patients with SSc showing no disease progression, and 4 out of 5 patients with IIM reaching ACR major/moderate response. Furthermore, all patients remained free of glucocorticoids and any other immunosuppressive treatment over the entire observation period of 24 weeks. CASTLE suggests the feasibility, safety and efficacy of Zorpo-cel in three different autoimmune diseases and paves the way for conducting a pivotal study. ClinicalTrials.gov identifier: NCT06347718, EudraCT identifier: 2022-001366-35.

Background Autoimmune hemolytic anemia (AIHA), immune thrombocytopenia (ITP), and antiphospholipid antibody syndrome (APLAS) are B cell-driven autoimmune diseases defined by pathogenic autoantibodies. CD19-directed chimeric antigen receptor (CAR)-T cells have recently demonstrated the ability to reset dysregulated B cells and induce long-lasting remission in refractory systemic autoimmune diseases. Evidence for efficacy in severe, treatment-refractory AIHA, however, is limited. Methods We report the clinical course of a 47-year-old woman with life-threatening cold- and warm-agglutinin AIHA refractory to nine prior treatment lines, accompanied by ITP and APLAS. In an uncontrolled flare, she received a fludarabine/cyclophosphamide-containing lymphodepletion followed by autologous CD19-directed, 4-1BB-costimulated CAR-T cells (zorpocabtagene-autoleucel [Zorpo-cel], 1 × 106/kg) on the basis of compassionate use. Treatment efficacy and safety were assessed over an 11-month follow-up period. Findings Zorpo-cel showed a rapid and sustained B cell depletion. Transfusion independence was achieved by day 7, with hemoglobin normalization by day 25, including resolution of hemolysis markers. Cold-agglutinin titers decreased, and previously elevated antiphospholipid antibodies normalized without recurrence throughout 11 months of follow-up. ITP stabilized. No cytokine release syndrome or neurotoxicity occurred. Mild transaminase elevation and thrombocytopenia were observed, most likely correlating with pre-existing severe iron overload due to erythrocyte transfusions. Conclusion This case demonstrates that CD19-directed CAR-T cell therapy can induce rapid, durable remission of severe, refractory cold-agglutinin AIHA and simultaneously improve coexisting APLAS and ITP on a favorable toxicity profile. However, more data from controlled clinical trials are needed for final conclusions. Funding This work was funded by Deutsche Forschungsgemeinschaft and Deutsche Krebshilfe.

ABSTRACT:CD19-directed chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of relapsed/refractory B-cell non-Hodgkin lymphoma (B-NHL) and recently showed effects in autoimmune diseases, such as systemic lupus erythematosus (SLE). Despite high levels of inflammation, toxicity seemed to differ between patients with SLE and B-NHL. We therefore compared the CAR T-cell kinetics and treatment-related side effects to better define the toxicity profiles. In contrast with the similar CAR T-cell expansion, patients with SLE revealed a lower incidence and severity of cytokine-release syndrome, immune effector cell-associated neurotoxicity syndrome, and immune effector cell-associated hematotoxicity. Although the neutrophil nadir was lower in patients with SLE after therapy, the platelet counts remained close to normal and hematotoxicity was shorter in SLE than B-NHL. The reduced hematotoxicity correlated with lower acute-phase inflammation, better hematologic reserve before CAR T-cell therapy, and distinct serum cytokine profiles. Interestingly, CAR T-cell persistence was consistently shorter, and the reconstitution of conventional T and B cells was faster in SLE. In both cohorts, B-cell reconstitution correlated with functional CD4+ T-cell recovery, indicating a general biologic process of hematopoietic and immune system regeneration. In summary, similar lymphodepletion and CAR T-cell pharmacokinetics led to distinct toxicity, demonstrating that CAR T-cell therapy had a favorable side-effect profile in SLE, including faster recovery of the adaptive immune system.

CD19 chimeric antigen receptor (CD19 CAR) T cell therapy has been shown to induce stable drug-free remission in patients with refractory autoimmune disease. The management of potential relapses is currently unclear. Here we report on a 45-year-old woman with treatment-refractory Jo-1-associated anti-synthetase syndrome, who initially achieved disease remission after CD19 CAR T cell therapy but then experienced disease relapse after 9 months. After reinfusion of the same product, CAR T cells failed to expand and T cells targeting the CD19 CAR were detected. Despite full-dose lymphodepletion, no clinical response was observed. After bridging with anti-CD38 antibody daratumumab, which was efficacious with limited durability, plasma-cell-targeting B-cell maturation antigen (BCMA) CAR T cell therapy was performed. BCMA CAR T cells expanded, cleared plasma cells in lymphoid tissue, reduced autoantibody levels and re-induced stable drug-free remission. This case highlights the challenges in CAR T cell reinfusion, the potential of alternative targets and products, and suggests that the depletion of plasma cells may enhance therapeutic outcomes in patients who become treatment-refractory.

Systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and idiopathic inflammatory myositis (IIM) are autoimmune diseases managed with long-term immunosuppressive therapies. Hu19-CD828Z, a fully human anti-CD19 chimeric antigen receptor (CAR) with a CD28 costimulatory domain, is engineered to potently deplete B-cells. In this study, we manufactured Hu19-CD828Z CAR T-cells from peripheral blood of SLE, IIM, and SSc patients and healthy donors (HDs). CAR-mediated, CD19-specific activity of these cells was evaluated in vitro by assessing cytotoxicity, cytokine release, and proliferation assays in response to autologous CD19+ B-cells, the CD19+ NALM-6 B-cell line, or a CD19- U937 non-B-cell line as targets. The results demonstrated an increased proliferation of Hu19-CD828Z CAR T-cells and dose-dependent cytotoxicity against primary autologous and NALM-6 B-cells compared to non-transduced controls or co-cultures with non-B-cells. Notably, autoimmune-patient-derived CAR T-cells produced lower levels of inflammatory cytokines than healthy-donor-derived CAR T-cells in response to CD19+ B-cell targets. These data support the potential of Hu19-CD828Z and its therapeutic cell product KYV-101 as a therapeutic strategy to achieve deep B-cell depletion in SLE, IIM, and SSc patients, and highlights its promise for broader application in B-cell-driven autoimmune disorders.

Despite advances in targeted therapies, treatment of chronic lymphocytic leukemia (CLL) remains challenging, highlighting the urgent need for effective new therapeutic strategies. Although chimeric antigen receptor (CAR) T-cell therapy dramatically improved outcomes in acute lymphoblastic leukemia (ALL), its efficacy in CLL is limited. We hypothesize that this disparity results from pronounced CAR T-cell exhaustion and the immunosuppressive tumor microenvironment (TME) in CLL. We utilized an autologous 3D TME co-culture model to investigate the functionality of CAR T cells derived from CLL and ALL patients within physiologically relevant conditions. Our results revealed increased exhaustion levels and diminished cytotoxicity of CAR T cells from CLL patients compared to those from ALL patients. Importantly, combining CAR T-cell treatment with interleukin-10 (IL-10) or CXCR4 blockade effectively improved cytotoxicity against CLL cells, even in stromal-protected regions within the 3D model. These findings offer insights into CAR T-cell dysfunction in CLL and support novel TME-targeted combination strategies to improve clinical outcomes.

Background: Chronic graft-versus-host disease (cGvHD) remains a major cause of late morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Aberrant germinal center (GC) activity, driven by dysregulated T-B cell interactions, contributes to disease pathogenesis. PD-1+ CXCR5+ follicular helper T (TFH) cells orchestrate GC responses by promoting memory B cell and plasmablast formation, and elevated circulating (c) TFH frequencies have been linked to cGvHD severity. Here, we report the identification of a cytotoxic TFH subset, previously published by us and referred to as killer TFH or TFK cells, co-expressing canonical TFH markers with high levels of the cytotoxic effector molecules NKG-7 and granzyme B (GRZB). This dual helper–killer phenotype suggests a unique capacity to both disrupt GC homeostasis and mediate direct tissue injury on MHC-II-upregulating epithelia. Integrating these cells into the immunopathogenic framework of cGvHD offers a novel perspective on disease initiation and progression. Methods: In a prospective trial, peripheral blood from 80 allo-HSCT patients was collected on median day +113 (range +91 to +190). In 24 patients, additional samples were obtained at cGvHD onset (median day +187, range +98 to +410) and 4 and 8 weeks later (9 de novo, 15 quiescent/progressive cases). CD4+ T cell subsets, particularly cTFH cells, were analyzed by flow cytometry. Results: At a median of day +113 post allo-HSCT, patients without current or future GvHD exhibited significantly higher frequencies of PD-1+ CXCR5+ cTFH cells (median 7.98%, range 16.92%) compared to those who later developed cGvHD (median 3.81%, range 21.36%, p<0.002). In the latter group, cTFH frequencies were already reduced at day +113 and remained low following disease onset.Subtype analysis revealed that cTFH1 (CXCR3+CCR6-) and cTFH1/17 (CXCR3+CCR6+) frequencies at day +113 were markedly elevated in patients who never developed GvHD (cTFH1: median 4.38%, range 13.25%; cTFH1/17: median 1.18%, range 4.2%) compared to those with subsequent disease (cTFH1: median 2.53%, range 9.96%, p<0.03; cTFH1/17: median 0.31%, range 2.02%, p<0.0002). cTFH2 (CXCR3-CCR6-) and cTFH17 (CXCR3-CCR6+) frequencies declined from day +113 to disease onset (week 0) in untreated cGvHD patients.A prominent difference was also observed for cytotoxic NKG-7+ (anti-TIA-1) GRZB+ CD4+ T cells: Patients not developing any GvHD later on displayed significantly higher levels of NKG-7+ and NKG-7+ GRZB+ T cells at day +113 (NKG-7+: 24.56%, range 64.85%; NKG-7+ GRZB+: median 6.03%, range 23.00%, p<0.03) than patients who later developed cGvHD (NKG-7+: median 12.07%, range 51.98%; NKG-7+ GRZB+: median 0.99%, range 25.95%). A trend mirrored in the TFH compartment, where killer cTFH cells (NKG-7+ GRZB+) were most abundant in patients without developing any form of GvHD.Receiver operating characteristic (ROC) analysis assessed on day +113 identified several populations with strong prognostic potential subsequent for cGvHD: cTFH cells (AUC = 0.77; p = 0.0019), NKG-7+ GRZB+ CD4+ cells (AUC = 0.73; p = 0.0081), cTFH1 cells (AUC = 0.73; p = 0.0072), and cTFH1/17 cells (AUC = 0.84; p<0.0001). Conclusion: Our findings reveal that early post-transplant preservation of cTFH subsets, particularly cTFH1, cTFH1/17, and killer cTFH cells, can be found in patients without future or present cGvHD, whereas their decline in peripheral blood precedes disease onset. The identification of a killer TFH phenotype with high GRZB and NKG-7 expression adds a novel layer to the immunopathogenesis of cGvHD. Furthermore, the predictive value of the cytotoxic NKG-7+ GRZB+ CD4+T cells indicates a potential benefit for risk stratification and will be correlated further with clinical parameters.

ABSTRACT:Chronic graft-versus-host disease (cGVHD) is characterized by dysregulation of the adaptive immune system, including an aberrant B-cell homeostasis after allogeneic hematopoietic stem cell transplantation (allo-SCT). It is uncertain, however, whether this B-cell dysregulation is a result of manifest cGVHD or develops as a sign of aberrant B lymphopoiesis after allo-SCT before cGVHD becomes apparent. To gain insight into the development of B-cell dysregulation before the onset of cGVHD, we analyzed B-cell subpopulations by multiparameter flow cytometry on days 90, 180, and 356 after allo-SCT in a prospective study design. After completion of follow-up, patients were assigned retrospectively to 3 groups according to onset of GVHD: (1) no GVHD (n = 17); (2) acute GVHD (aGVHD) without subsequent cGVHD (n = 32); and (3) cGVHD (n = 59). Although CD21lowCD11c+ B cells were increased in all groups, the frequency of CD20-CD38hi plasmablasts was significantly elevated already 90 days after allo-SCT in patients who subsequently developed cGVHD, compared to patients without GVHD or with aGVHD only (median of CD19+ cells, 5.9% vs 2.2% vs 2.2%; P = .0016 and .0304, respectively). Detailed molecular analysis of expanded plasmablasts revealed a dominance of the immunoglobulin A isotype, with molecular evidence for recent generation in mucosal sites and markers for intestinal homing. A large fraction of the clonally expanded plasmablasts produced antibodies that bound to subgroups of commensals known to produce short-chain fatty acids. In summary, our data suggest that dysregulated intestinal antibody responses against commensals contribute to the pathophysiology of cGVHD.