Bronchoalveolar Lavage Fluid Reflects a TH1-CD21low B-Cell Interaction in CVID-Related Interstitial Lung Disease

BackgroundAbout 20% of patients with common variable immunodeficiency (CVID) suffer from interstitial lung disease (ILD) as part of a systemic immune dysregulation. Current understanding suggests a role of B cells in the pathogenesis based on histology and increased levels of BAFF and IgM associated with active disease corroborated by several reports which demonstrate the successful use of rituximab in CVID-ILD. It is debated whether histological confirmation by biopsy or even video-assisted thoracoscopy is required and currently not investigated whether less invasive methods like a bronchoalveolar lavage (BAL) might provide an informative diagnostic tool.ObjectiveTo gain insight into potential immune mechanisms underlying granulomatous and lymphocytic interstitial lung disease (GLILD) and to define biomarkers for progressive ILD by characterizing the phenotype of B- and T-cell populations and cytokine profiles in BAL fluid (BALF) of CVID-ILD compared to sarcoidosis patients and healthy donors (HD).MethodsSixty-four CVID, six sarcoidosis, and 25 HD BALF samples were analyzed by flow cytometric profiling of B- and T-cells and for cytokines by ELISA and Multiplexing LASER Bead technology.ResultsBoth sarcoidosis and CVID-ILD are characterized by a predominantly T-cell mediated lymphocytosis in the BALF. There is an increase in T follicular helper (TFH)-like memory and decrease of regulatory T cells in CVID-ILD BALF. This TFH-like cell subset is clearly skewed toward TH1 cells in CVID-ILD. In contrast to sarcoidosis, CVID-ILD BALF contains a higher percentage of B cells comprising mostly CD21low B cells, but less class-switched memory B cells. BALF analysis showed increased levels of APRIL, CXCL10, and IL-17.ConclusionUnlike in sarcoidosis, B cells are expanded in BALF of CVID-ILD patients. This is associated with an expansion of TFH- and TPH-like cells and an increase in APRIL potentially supporting B-cell survival and differentiation and proinflammatory cytokines reflecting not only the previously described TH1 profile seen in CVID patients with secondary immune dysregulation. Thus, the analysis of BALF might be of diagnostic value not only in the diagnosis of CVID-ILD, but also in the evaluation of the activity of the disease and in determining potential treatment targets confirming the prominent role of B-cell targeted strategies.

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AB0026 DECREASE OF ANGIOGENIC T CELLS IN CONNECTIVE TISSUE DISEASE-ASSOCIATED INTERSTITIAL LUNG DISEASE

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2676 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 1046.3-1047

Author(s):

V. Pulito-Cueto ◽

S. Remuzgo Martinez ◽

F. Genre ◽

B. Atienza-Mateo ◽

V. M. Mora-Cuesta ◽

...

Keyword(s):

T Cells ◽

Interstitial Lung Disease ◽

Connective Tissue ◽

Lung Disease ◽

Cell Subset ◽

Vascular Damage ◽

Type I ◽

Research Support ◽

Angiogenic T Cells

Background:Interstitial lung disease (ILD) is one of the most significant complications of connective tissue diseases (CTD), leading to an increase of the morbidity and mortality in patients with CTD [1]. A specific T cell subset termed angiogenic T cells (TAng), that promote endothelial repair and revascularization, have been involved in the pathogenesis of CTD [2-4]. However, to the best of our knowledge, no information regarding the role of TAng in CTD-ILD+ is available.Objectives:To study, for the first time, the potential role of TAng related to vascular damage in CTD-ILD+.Methods:Peripheral venous blood was collected from 40 patients with CTD-ILD+ and three comparative groups: 44 CTD-ILD- patients, 21 idiopathic pulmonary fibrosis (IPF) patients and 20 healthy controls (HC). All subjects were recruited from the Rheumatology and Pneumology departments of Hospital Universitario Marqués de Valdecilla, Santander, Spain. Quantification of TAng was performed by flow cytometry. TAng were considered as triple-positive for CD3, CD31 and CXCR4.Results:Patients with CTD-ILD+ exhibited a significantly lower TAng frequency than CTD-ILD- patients (p<0.001). Similar results were obtained when patients with CTD-ILD+ were compared with HC (p=0.004) although no difference was observed between CTD-ILD+ and IPF. In addition, a significant increase of TAng frequency was shown in patients with CTD-ILD- in relation to IPF patients (p<0.001), while no difference was observed between CTD-ILD- and HC.Conclusion:Our results reveal a decrease of TAng frequency related to vascular damage in CTD-ILD+. Furthermore, we disclose that the presence of ILD is associated with lower TAng frequency.References:[1]Expert Rev Clin Immunol 2018;14(1):69-82.[2]Circulation 2007;116(15):1671-82.[3]Ann Rheum Dis 2015 74(5):921-7.[4]PLoS One 2017;12(8):e0183102.Acknowledgements:Personal funds, VP-C: PREVAL18/01 (IDIVAL); SR-M: RD16/0012/0009 (ISCIII-ERDF); LL-G: INNVAL20/06 (IDIVAL); RP-F: START PROJECT (FOREUM); RL-M: Miguel Servet type I CP16/00033 (ISCIII-ESF).Disclosure of Interests:Verónica Pulito-Cueto: None declared, Sara Remuzgo Martinez: None declared, Fernanda Genre: None declared, Belén Atienza-Mateo: None declared, Victor Manuel Mora-Cuesta: None declared, David Iturbe-Fernández: None declared, Leticia Lera-Gómez: None declared, Raquel Pérez-Fernández: None declared, Pilar Alonso Lecue: None declared, Javier Rodriguez Carrio: None declared, Diana Prieto-Peña: None declared, Virginia Portilla: None declared, Ricardo Blanco Speakers bureau: Abbvie, Pfizer, Roche, Bristol-Myers, Janssen and MSD, Consultant of: Abbvie, Pfizer, Roche, Bristol-Myers, Janssen and MSD, Grant/research support from: Abbvie, MSD and Roche, Alfonso Corrales: None declared, Jose Manuel Cifrián-Martínez: None declared, Raquel López-Mejías: None declared, Miguel A González-Gay Speakers bureau: Pfizer, Abbvie, MSD, Grant/research support from: Pfizer, Abbvie, MSD

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Rituximab Administration before Allogeneic HSCT Is Associated with Accelerated T Cell Reconstitution after Transplantation: Kinetic Evidence for a Graft-Versus-Leukemia Effect

Blood ◽

10.1182/blood.v124.21.3905.3905 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3905-3905

Author(s):

Sakura Hosoba ◽

Christopher R. Flowers ◽

Catherine J Wu ◽

Jens R. Wrammert ◽

Edmund K. Waller

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cd8 T Cells ◽

Cell Subset ◽

Post Transplant ◽

T Cell Reconstitution ◽

Relative Value ◽

History Of

Abstract Introduction: Rituximab (R) administration results in depletion of blood B cells and suppression of B cell reconstitution for several months after, with suggestions that T cell reconstitution may also be impaired. We hypothesized that pre-transplant R would be associated with delayed B and T cell reconstitution after allo-HSCT compared with non-R-treated allo-HSCT recipients. Methods: We conducted a retrospective analysis of 360 patients who underwent allo-HSCT using BM or G-CSF mobilized PB. Recipients of cord blood, T cell depleted grafts and 2nd allo-HSCT were excluded. Analysis of lymphocyte subsets in at least one blood at 1, 3, 6, 12, and 24 months post-allo-HSCT was available for 255 eligible patients. Data on lymphocyte recovery was censored after DLI or post-transplant R therapy. Post-HSCT lymphocyte recovery in 217 patients who never received R (no-R) was compared to 38 patients who had received R before allo-HSCT (+R) including 12 CLL, 19 NHL, and 7 B-cell ALL patients. +R patients received a median of 9 doses of R with the last dose of R at a median of 45 days pre-transplant. Results: Mean lymphocyte numbers in the blood at 1, 3, 6, 12, and 24 months were B-cells: 55 ± 465/µL, 82 ± 159/µL, 150 ± 243/µL, 255 ± 345/µL, and 384 ± 369/µL (normal range 79-835); and T-cells: 65 ± 987/µL, 831 ± 667/µL, 1058 ± 788/µL, 1291 ± 985/µL, and 1477 ± 1222/µL (normal range 675-3085). Lymphocyte reconstitution kinetics did not vary significantly based upon the intensity of the conditioning regimen or related vs. unrelated donors allowing aggregation of patients in the +R and no-R groups (Figure). B cell reconstitution in the +R patients was higher at 1 month post-allo-HSCT (relative value of 143% p=0.008) and lower at 3 months post-transplant (19.2%, p=0.069) compared to no-R patients. Blood B cells in the +R group rebounded by the 6th month post-allo-HSCT and remained higher than the no-R group through the 24th month post-HSCT (197% at the 6th month, p=0.037). Higher levels of B-cells at 1 month in the +R group was due to higher blood B-cells at 1 month post-HSCT among 12 CLL patients compared with no-R patients (423%, p<0.001; Figure), while B-cell counts in the remaining +R patients (B-cell NHL and B-cell ALL) were lower than the no-R patients at both 1 and 3 months. Reconstitution of CD4+ and CD8+ T cells among +R patients were similar to no-R patients in the first month post-allo-HSCT and then rebounded to higher levels than the no-R group of patients (relative value 194%, p=0.077 at the 24th month for CD4+ T cell subset, and 224%, p=0.020 for CD8+ T cell subset; Figure). CLL patients had a striking increase in blood levels of donor-derived CD4+ and CD8+ T cells at 3 months post-transplant concomitant with the disappearance of blood B cells compared with no-R patients (relative value of 178% and 372%, p=0.018 and p=0.003, respectively; Figure). Long term T cell reconstitution remained higher for +R patients compared with no-R patients, even when CLL patients were excluded (relative value of 203%, p=0.005 at 24 months post-HSCT; Figure). Conclusions: We observed higher levels of blood B cells and T cells ³ 6 months post-allo-HSCT in +R patients compared with no-R patients. B cell recovery at 6 months post-transplant is consistent with clearance of residual plasma R given the 1-2 months half-life of R, and the median of 1.5 months between the last dose of R and allo-HSCT. The increased blood CD8+ T cells in the blood of CLL patients at 3 months post-allo-HSCT associated with clearance of the B-cells seen 1 month post-HSCT is consistent with a donor T cell-mediated GVL effect. Pre-transplant R therapy does not appear to have any long-term deleterious effect on immune reconstitution, indicating that post-allo-HSCT vaccination at ≥6 months may be efficacious. Figure: Kinetics of lymphocyte reconstitution after allo-HSCT varied by history of pre-transplant R administration and primary disease. Panels show mean counts of each lymphocyte subset at 1, 3, 6, 12 and 24 months post-allo-HSCT for: (1) B cell, (2) T cell, (3) CD4+ and (4) CD8+ T cells. Solid lines with triangle show no-R group; dashed lines with circles shows subgroups of CLL and NHL/ALL +R patients. Asterisks show p values from t-test of the comparison between CLL +R or the NHL/ALL +R patients with no-R patients. *p<0.05; ** p<0.01; *** p<0.001. Figure:. Kinetics of lymphocyte reconstitution after allo-HSCT varied by history of pre-transplant R administration and primary disease. Panels show mean counts of each lymphocyte subset at 1, 3, 6, 12 and 24 months post-allo-HSCT for: (1) B cell, (2) T cell, (3) CD4+ and (4) CD8+ T cells. Solid lines with triangle show no-R group; dashed lines with circles shows subgroups of CLL and NHL/ALL +R patients. Asterisks show p values from t-test of the comparison between CLL +R or the NHL/ALL +R patients with no-R patients. *p<0.05; ** p<0.01; *** p<0.001. Disclosures No relevant conflicts of interest to declare.

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Marginal Zone B Cells Regulate RBC Alloimmunization Toward Distinct RBC Alloantigens

Blood ◽

10.1182/blood.v128.22.3847.3847 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3847-3847

Author(s):

Patricia E. Zerra ◽

Seema R. Patel ◽

Connie M. Arthur ◽

Kathryn R. Girard-Pierce ◽

Ashley Bennett ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cd4 T Cells ◽

Antibody Formation ◽

Marginal Zone ◽

Cd4 T Cell ◽

Rbc Transfusion

Abstract Background: While red blood cell (RBC) transfusion can be beneficial, exposure to allogeneic RBCs can result in the development of RBC alloantibodies that can make it difficult to obtain compatible RBCs for future transfusions. Aside from phenotype matching protocols, no strategy currently exists that is capable of preventing RBC alloimmunization following therapeutic transfusion. As RBC alloantigens represent diverse determinants capable of driving distinct immune pathways, common immunological nodes must be identified in order to successfully prevent RBC alloimmunization against a variety of different alloantigens. Recent results demonstrate that marginal zone (MZ) B cells mediate anti-KEL antibody formation in the complete absence of CD4 T cells. However, whether MZ B cells similarly regulate RBC alloantibody formation against other RBC alloantigens remains unknown. As a result, we examined the role of MZ B cells and CD4 T cells in the development of RBC alloantibodies following exposure to the HOD (hen egg lysozyme, ovalbumin and duffy) antigen. Methods: Each recipient was transfused with HOD or KEL RBCs following either MZ B cell or CD4 T cell depletion using a cocktail of MZ B cell (anti-CD11a and anti-CD49d) or anti-CD4 depleting antibody, 4 and 2 days prior to transfusion. Control groups received isotype control injections in parallel. MZ B cell deficient (CD19cre/+ X Notch2flx/flx) and CD4 T cell deficient (MHC class II knockout) recipients were also used to examine the role of MZ B cells and CD4 T cells, respectively. Serum collected on days 5 and 14 post-transfusion was evaluated for anti-HOD or anti-KEL antibodies by incubating HOD or KEL RBCs with serum, followed by detection of bound antibodies using anti-IgM and anti-IgG and subsequent flow cytometric analysis. Evaluation of antibody engagement and overall survival of HOD or KEL RBCs was accomplished by labeling RBCs with the lipophilic dye, DiI, prior to transfusion, followed by examination for bound antibody and RBC clearance on days 5 and 14 post-transfusion by flow cytometry. Results: Similar to the ability of MZ B cell depletion to reduce anti-KEL antibody formation following KEL RBC exposure, depletion of MZ B cells significantly reduced anti-HOD IgM and IgG antibodies following HOD RBC transfusion. In contrast, injection of recipients with isotype control antibodies in parallel failed to prevent alloantibody formation following HOD or KEL RBC transfusion. Similar results were obtained following HOD or KEL RBC transfusion into recipients genetically deficient in MZ B cells. In contrast, although MZ B cells were required for HOD and KEL RBC-alloantibody formation, manipulation of CD4 T cells differentially impacted the ability of each antigen to induce alloantibodies. While transfusion of HOD or KEL RBCs resulted in robust IgM alloantibodies in the absence of CD4 T cells, depletion or genetic elimination of CD4 T cells significantly inhibited anti-HOD IgG antibody formation, while failing to impact IgG anti-KEL antibody formation. Consistent with this, while manipulation of CD4 T cells protected HOD RBCs from antibody deposition and subsequent RBC clearance, this same approach failed to similarly protect KEL RBCs following transfusion. In contrast, depletion of MZ B cells not only prevented detectable alloantibody production, but also completely protected HOD or KEL RBCs from antibody deposition and subsequent RBC clearance. Conclusion: These results suggest that while MZ B cells mediate a robust IgM antibody response following either KEL or HOD antigen exposure, MZ B cells appear to possess the capacity to orchestrate unique downstream IgG responses through CD4 T cell dependent and independent pathways contingent on target alloantigen. As a result, while manipulation of CD4 T cells may prevent alloantibody formation against some antigens, targeting this immune population inadequately prevents RBC alloantibody formation against all RBC antigens. As chronic transfusion therapy exposes recipients to a wide variety of alloantigens, these results suggest that MZ B cells may represent a central initiating node that governs RBC alloimmunization against a variety of RBC alloantigens, and may therefore serve as a useful target in preventing alloantibody formation in chronically transfused individuals. Disclosures No relevant conflicts of interest to declare.

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Critical Role of T Cells in PF4/Heparin Antibody Production

Blood ◽

10.1182/blood.v124.21.1554.1554 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1554-1554

Author(s):

Yongwei Zheng ◽

Mei Yu ◽

Anand Padmanabhan ◽

Richard H. Aster ◽

Renren Wen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Antibody Production ◽

Cd4 T Cells ◽

Wild Type ◽

Specific Antibodies ◽

Deficient Mice

Abstract Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder that can cause arterial or venous thrombosis/thromboembolism, and platelet factor 4 (PF4)/ heparin-reactive antibodies are essential to the pathogenesis of HIT. Our recent studies have demonstrated that marginal zone (MZ) B cells play a major role in production of PF4/heparin-specific antibodies. However, the role of T cells in production of these pathogenic antibodies is not clear. Here we showed that PF4/heparin complex-induced production of PF4/heparin-specific antibodies was markedly impaired in mice, in which CD4 T cells were depleted by administration of GK1.5 anti-CD4 monoclonal antibody. As expected, the CD4 T cell-depleted mice responded normally to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, in agreement with the lack of CD4 T cells in these GK1.5-treated mice. Further, following adoptive transfer of a mixture of wild-type splenic B cells and splenocytes from B cell-deficient μMT mice, T and B cell-deficient Rag1 knockout mice responded to PF4/heparin complex challenge to produce PF4/heparin-specific antibodies. In contrast, Rag1-deficient mice that received a mixture of wild-type splenic B cells and splenocytes from Rag1-deficient mice barely produced PF4/heparin-specific antibodies upon PF4/heparin complex challenge. These data suggest that T cells are required for production of PF4/heparin-specific antibodies. Consistent with this concept, mice with B cells lacking CD40 molecule, a B cell costimulatory molecule that helps T cell-dependent B cell responses, displayed a marked reduction of PF4/heparin-specific antibody production following PF4/heparin complex challenge. Also as expected, mice with CD40-deficient B cells were able to respond to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, consistent with the lack of T-cell help in these mice. Taken together, these findings demonstrate that T cells play an essential role in production of PF4/heparin-specific antibodies by MZ B cells. Disclosures No relevant conflicts of interest to declare.

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Intratumoral T cells have a differential impact on FDG-PET parameters in follicular lymphoma

Blood Advances ◽

10.1182/bloodadvances.2020004051 ◽

2021 ◽

Vol 5 (12) ◽

pp. 2644-2649

Author(s):

Karthik Nath ◽

Soi-Cheng Law ◽

Muhammed B. Sabdia ◽

Jay Gunawardana ◽

Lilia M. de Long ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Follicular Lymphoma ◽

B Cell ◽

Glucose Uptake ◽

Fdg Pet ◽

Prognostic Impact ◽

The Impact

Data on the prognostic impact of pretherapy 18F-fluorodeoxyglucose–positron emission tomography (FDG-PET) in follicular lymphoma (FL) is conflicting. The predictive utility of pretherapy total metabolic tumor volume (TMTV) and maximum standardized uptake value (SUVmax) on outcome appears to vary between regimens. Chemoimmunotherapies vary in the extent of T-cell depletion they induce. The role of intratumoral T cells on pretherapy FDG-PET parameters is undefined. We assessed pretherapy FDG-PET parameters and quantified intratumoral T cells by multiple methodologies. Low intratumoral T cells associated with approximately sixfold higher TMTV, and FL nodes from patients with high TMTV showed increased malignant B-cell infiltration and fewer clonally expanded intratumoral CD8+ and CD4+ T-follicular helper cells than those with low TMTV. However, fluorescently labeled glucose uptake was higher in CD4+ and CD8+ T cells than intratumoral B cells. In patients with FDG-PET performed prior to excisional biopsy, SUVmax within the subsequently excised node associated with T cells but not B cells. In summary, TMTV best reflects the malignant B-cell burden in FL, whereas intratumoral T cells influence SUVmax. This may contribute to the contradictory results between the prognostic role of different FDG-PET parameters, particularly between short- and long-term T-cell–depleting chemoimmunotherapeutic regimens. The impact of glucose uptake in intratumoral T cells should be considered when interpreting pretherapy FDG-PET in FL.

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Interleukin 21 Correlates with T Cell and B Cell Subset Alterations in Systemic Lupus Erythematosus

The Journal of Rheumatology ◽

10.3899/jrheum.120468 ◽

2012 ◽

Vol 39 (9) ◽

pp. 1819-1828 ◽

Cited By ~ 70

Author(s):

BENJAMIN TERRIER ◽

NATHALIE COSTEDOAT-CHALUMEAU ◽

MARLÈNE GARRIDO ◽

GUILLAUME GERI ◽

MICHELLE ROSENZWAJG ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Lupus Erythematosus ◽

Cd4 T Cells ◽

Th17 Cells ◽

Cell Subset ◽

Tfh Cells ◽

Interleukin 21

Objective.Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by alterations of the B cell subset, global regulatory T cell (Treg) depletion, and an increase in Th17 cells. Interleukin 21 (IL-21) plays a critical role in T cell and B cell homeostasis. Our objective was to determine the implication of IL-21 and IL-21-producing CD4+ T cells in the pathogenesis of SLE.Methods.Twenty-five patients with SLE and 25 healthy donor controls were included. Analysis of CD4+ T cells producing IL-21, Th1, Th2, Th17, Treg, follicular helper T (TFH) cells, and B cells was performed in peripheral blood, and levels of cytokines were measured in culture supernatants.Results.Circulating CD4+ T cells producing IL-21 were markedly expanded in patients with SLE compared to controls and were correlated with increased Th17, decreased Treg, and increased memory B cells. CD4+ T cells producing IL-21 were composed of CXCR5+ and CXCR5–CD4+ T cell subsets. Both IL-21-producing CXCR5+CD4+ T cells and CXCR5–CD4+ T cells were increased in patients with SLE, the CXCR5–CD4+ subset correlating with Th17 cells and Treg, while the CXCR5+CD4+ subset was correlated with alterations of the B cell subset. The CXCR5+CD4+ subset comprised mainly circulating Bcl6+CXCR5+CD4+ TFH cells that were markedly expanded in patients with SLE and were correlated with increased circulating Bcl6+CXCR5+ germinal center B cells.Conclusion.These findings suggest that IL-21, produced by distinct cellular CD4+ T cells, correlates with alterations of T cell and B cell subsets in SLE, and that targeting IL-21 could provide beneficial effects on both T cell and B cell alterations.

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The CD8 T Cell-Epstein-Barr Virus-B Cell Trialogue: A Central Issue in Multiple Sclerosis Pathogenesis

Frontiers in Immunology ◽

10.3389/fimmu.2021.665718 ◽

2021 ◽

Vol 12 ◽

Author(s):

Caterina Veroni ◽

Francesca Aloisi

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cd8 T Cells ◽

Epstein Barr Virus ◽

Cd8 T Cell ◽

Barr Virus ◽

Epstein Barr

The cause and the pathogenic mechanisms leading to multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), are still under scrutiny. During the last decade, awareness has increased that multiple genetic and environmental factors act in concert to modulate MS risk. Likewise, the landscape of cells of the adaptive immune system that are believed to play a role in MS immunopathogenesis has expanded by including not only CD4 T helper cells but also cytotoxic CD8 T cells and B cells. Once the key cellular players are identified, the main challenge is to define precisely how they act and interact to induce neuroinflammation and the neurodegenerative cascade in MS. CD8 T cells have been implicated in MS pathogenesis since the 80’s when it was shown that CD8 T cells predominate in MS brain lesions. Interest in the role of CD8 T cells in MS was revived in 2000 and the years thereafter by studies showing that CNS-recruited CD8 T cells are clonally expanded and have a memory effector phenotype indicating in situ antigen-driven reactivation. The association of certain MHC class I alleles with MS genetic risk implicates CD8 T cells in disease pathogenesis. Moreover, experimental studies have highlighted the detrimental effects of CD8 T cell activation on neural cells. While the antigens responsible for T cell recruitment and activation in the CNS remain elusive, the high efficacy of B-cell depleting drugs in MS and a growing number of studies implicate B cells and Epstein-Barr virus (EBV), a B-lymphotropic herpesvirus that is strongly associated with MS, in the activation of pathogenic T cells. This article reviews the results of human studies that have contributed to elucidate the role of CD8 T cells in MS immunopathogenesis, and discusses them in light of current understanding of autoreactivity, B-cell and EBV involvement in MS, and mechanism of action of different MS treatments. Based on the available evidences, an immunopathological model of MS is proposed that entails a persistent EBV infection of CNS-infiltrating B cells as the target of a dysregulated cytotoxic CD8 T cell response causing CNS tissue damage.

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CLL B Cells Develop Resistance to Ibrutinib By Reinvigorating the IL-4R - IL-4 Axis Blocked By Bruton's Tyrosine Kinase Inhibitors Including Acalabrutinib and Zanubrutinib

Blood ◽

10.1182/blood-2019-127255 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 477-477

Author(s):

Shih-Shih Chen ◽

Constantine S. Tam ◽

Alan G. Ramsay ◽

Priyadarshini Ravichandran ◽

Natalia C. Couto-Francisco ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Subset ◽

Advisory Committees

Bruton's tyrosine kinases inhibitors (BTKis) represent major advances in CLL therapy. However resistance to this form of therapy is emerging, and such patients often progress more rapidly. Hence there is an important need for therapies that address resistance. Microenvironmental input like IL-4 is critical for CLL disease progression. Compared with normal B cells, CLL cells exhibit significantly higher levels of surface membrane (sm) IL-4 receptor (IL4-R) and contain increased amounts of pSTAT6, a downstream mediator of IL-4R signaling. IL-4 stimulation of CLL B cells suppresses smCXCR4 and increases smIgM, thus promotes CLL cell retention and expansion. In this study, we aimed to examine if smIL-4R expression, IL4R signaling, and IL-4-producing cells are altered in patients sensitive or resistant to BTKis. To do so, T and B cell subset changes were studied overtime in 12 acalabrutinib-treated CLL patients, 6 zanubrutinib-treated CLL patients, 30 ibrutinib-sensitive and 5 ibrutinib-resistant CLL patients, 4 of which exhibited BTK mutations. Consistent with only ibrutinib inhibiting T-cell kinase (ITK), T-cell subset analyses revealed no changes in Th1, Th2, Th17, Th9, and Th22 cells after zanubrutinib or acalabrutinib treatment. In contrast, a Th1-biased T-cell immunity was observed in patients responsive to ibrutinib. In patients progressing on ibrutinib, significantly reduced Th2 T cells were found during the resistant as well as sensitive periods. In an in vitro T-cell function assay using T cells collected before and after the treatment with each BTKi, only ibrutinib treated patients exhibited a reduced ability of T cells to support CLL B cell survival. We next studied changes in CLL B cells, including numbers of IL-4, -10 and -13 producing B cells after BTKi treatment. IL-13 producing CLL B cells were not changed. IL-10 producing CLL B cells were reduced in both ibrutinib sensitive and resistant patients, but not in zanubrutinib or acalabrutinib treated patients. Importantly, IL-4 producing CLL B cells were significantly decreased in patients treated with all 3 BTKi. Significantly reduced smIL-4R levels, impaired IL-4R signaling, decreased smIgM and increased smCXCR4 were also seen in patients treated with each BTKi. To understand the mechanism responsible for inhibition of IL-4 production in CLL cells treated with BTKis, we stimulated CLL cells through IgM, Toll-like receptor and CD40L, finding that only anti-IgM stimulation significantly increased IL-4 production and p-STAT6 induction. We then explored the function of IL-4. IL-4 enhanced CLL B cell survival in vitro and this action was blocked by all 3 BTKis. Moreover, adhesion of CLL B cells to smIL-4R expressing stromal cells was decreased by IL-4 and IL-4R neutralizing antibodies, especially in M-CLL cases. In in vivo studies transferring autologous T cells and CLL PBMCs into alymphoid mice, we found less CLL B cells in mouse spleens post ibrutinib than zanubrutinib or acalabrutinib treatment. This might be due to the suppressed Th2 cells found only in ibrutinib, while IL-4 producing B cells were reduced in all 3 BTKi treated mice. These results support the idea that IL-4 promotes CLL B cell adhesion and growth in tissues. Finally, we investigated the IL-4/IL-4R axis in ibrutinib-resistant patients. Although IL-4 producing T cells remain reduced during the sensitive and resistant phases, CLL B cell production of IL-4 and expression of and signaling through smIL-4R returned when patients developed ibrutinib-resistance. When comparing paired ibrutinib-sensitive and -resistant CLL B cells collected from 3 patients in a xenograft model that requires T cell help, we found ibrutinib-resistant CLL B cells grew in vivo with only minimal (~15%) numbers of autologous T cells compared to B cells collected from ibrutinib-sensitive phase; this suggested a reduced requirement for T-cell help for growth of ibrutinib-resistant CLL cells. In summary, we found IL-4 is a key survival factor in the CLL microenvironment that also improves leukemia cell adhesion to stromal cells expressing smIL-4R. IL-4 production and signaling can be stimulated in CLL B cells through the B-cell receptor, and are consistently blocked by BTKis. Moreover, the recovered ability of ibrutinib-resistant CLL B cells to produce and respond to IL-4 leads to disease progression, suggesting blocking the IL-4/IL-4R axis is a potential treatment for ibrutinib-resistant CLL patients. Disclosures Chen: Pharmacyclics: Research Funding; Beigene: Research Funding; Verastem: Research Funding; ArgenX: Research Funding. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Zhou:BeiGene: Employment. Barrientos:Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Rai:Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees.

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Role of Adaptive Regulatory T Cells in the Induction of Tolerance to FVIII

Blood ◽

10.1182/blood.v112.11.1027.1027 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1027-1027 ◽

Cited By ~ 2

Author(s):

Jonathan Skupsky ◽

Elika Farrokhi ◽

David W. Scott

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Regulatory T Cells ◽

Mouse Model ◽

B Cell ◽

Fluorescent Protein ◽

Tolerance Induction ◽

Coagulation Factor

Abstract Individuals who lack a given protein may never properly develop self tolerance. Thus, the administration of protein therapeutics can lead to undesirable immune responses. A classic example is that patients with Hemophilia A can develop an inhibitory immune response to therapeutic treatments with coagulation factor VIII (fVIII). Several years ago, we developed a B-cell delivered gene therapy based approach to prevent this response and a mouse model to study the mechanisms of the induction and maintenance of immunological tolerance to fVIII inhibitors. This model takes advantage of a knock-in mouse with the transcription factor, FoxP3, in frame with green fluorescent protein (GFP). FoxP3 is considered to be a marker for regulatory T cells (Tregs). The FoxP3-GFP knock-in allows us to follow existing (natural) Tregs and the induction of adaptive Tregs. We have backcrossed this mouse, which expresses GFP in all FoxP3+ Tregs, with the hemophilic mouse, which has an existing deletion in fVIII (E16), for 10 generations. Establishing a mouse that is transgenic for both the FoxP3-GFP fusion and a deletion in fVIII will enhance future clinically oriented experiments to understand how tolerogenic B cells interact with Tregs in a hemophilia system. Lymphocytes from these mice have now been characterized using flow cytometry and confocal microscopy, and analyzed during treatment with tolerogenic B cells. These cells express Treg markers that include, CD25, CTLA-4, GITR and reduced amounts of CD127. However, the population of fVIII antigen-specific cells is small in this mouse model. Thus, it is difficult to detect changes over the natural variation found between mice in a colony, although small changes have been detected. To explore the underlying role of Tregs in tolerance induction, we have bred an ovalbumin (OVA)-specific, T-cell receptor (TCR) transgenic mouse that contains the FoxP3-GFP fusion (FoxP3GFP/DO11.10 and FoxP3GFP/DO11.10/Rag2−/−). Using our B-cell delivered tolerance protocol, we can prevent immunization when an immunologically competent mouse is immunized with OVA in adjuvant. When FoxP3GFP/DO11.10 TCR transgenic mice are treated with OVA-Ig transduced B cells, we found a significant increase in antigen-specific Tregs (p<0.05). Finally, when the same treatment is performed on FoxP3GFP/DO11.10/Rag2−/−, which completely lack natural regulatory cells, we found that FoxP3 was expressed in 4% (p<0.001) of T-cells above background. These cells express markers typical of Tregs. These data strongly support the hypothesis that transduced B cell treatment induces a regulatory phenotype in the antigen-specific T cell population. To extend this system to the hemophilia model, fVIII C2 tetramers will be used to label and isolate antigen-specific T cells during tolerance induction. Additionally, this model opens up avenues of analysis to intra-vital microscopy which gives a true representation of interactions in a live organism. (Supported by NIH RO1 HL061883, NIH T32 HL007698, and a predoctoral fellowship from the American Heart Association.)

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The Role of B and T Lymphocytes in Recurrent Acquired Thrombotic Thrombocytopenic Purpura.

Blood ◽

10.1182/blood.v114.22.3521.3521 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3521-3521

Author(s):

Mariagabriella Mariani ◽

Andrea Cairo ◽

Roberta Palla ◽

Luca Andrea Lotta ◽

Andrea Rovati ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cellular Immunity ◽

T Cell Subsets ◽

Surface Markers ◽

Thrombocytopenic Purpura ◽

B Cell Subsets

Abstract Abstract 3521 Poster Board III-458 Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disease characterized by thrombocytopenia, microangiopathic haemolytic anemia and widespread microvascular thrombosis, resulting in multiorgan ischemia. Acquired TTP, which accounts for approximately 95% of cases, can be either associated to anti ADAMTS13 autoantibodies or secondary to a number of associated conditions (tumors, organ transplantation, use of drugs, pregnancy). There are several key questions that remain unanswered, including the importance of cellular immunity in immunomediated TTP, and the search for laboratory markers that predict disease relapse, an event that occurs in 20% to 50% of patients who survive the acute initial episode. Since alterations of peripheral B and T cell subsets in patients with autoimmune diseases (i.e. rheumatoid arthritis and systemic lupus erythematous) are well established, the aim of this study was to analyze the role of B and T cells in acquired TTP and during its recurrence. Methods 36 healthy controls and 36 consecutive patients affected by acquired TTP during remission (defined as the maintenance of normalization of clinical and laboratory data for at least 30 days after the last plasma therapy following the resolution of the last acute episode) were characterized by flow cytometry for the quantification of: - different peripheral B cell subsets, using labeled surface markers anti-CD19-PerCP, anti-IgD-PE, anti-IgM-FITC, anti-CD27-APC, anti-CD38-FITC; - different peripheral T cell subsets, using labeled surface markers anti-CD3-FITC, anti-CD4-PE, anti-CD8-APC, anti-CD25-FITC. For Treg cell quantification (only 17 patients were analyzed), anti-CD3-PerCP, anti-CD4-FITC, anti-CD25-PE and the intracellular marker FoxP3 were used. Patients were classified in two subgroups: those who developed at least two episodes of TTP (n=19, with recurrence) and those who experienced a single episode only and no relapse during at least one year of retrospective observational time (n=17). ADAMTS13 activity was measured by residual collagen binding assay (Gerritsen et al, Thromb Haemost 1999). The presence of anti-ADAMTS13 IgG was evaluated by Western blotting and ELISA assays, using recombinant ADAMTS13 protein as antigen and patients' plasma as a source of antibody. The presence of anti-ADAMTS13 IgA, IgM, IgG subclasses (IgG1, 2, 3, 4) were evaluated by ELISA assays. For continuous variables, differences between controls and patients and between patients with or without recurrence were evaluated by the t-test; for discrete variables, by the chi square test. P values smaller than 0.05 were considered statistically significant. Analyses were performed using the SPSS package version 17.0. Results 1) TTP patients had an increased number of CD19+ B cells (mean ± SD 13% ± 5) compared with the control group (10% ± 3, p=0.001). No difference was observed in T cells subsets. 2) The results of the characterization of the two groups of patients (with and without recurrence) are reported in the table. Patients with and without recurrence did not differ either in the amount of Treg FoxP3 or in the presence of IgA, IgM and IgG subclasses. Discussion The increased B cell numbers in acquired TTP indicates an enhanced activation of cellular immunity. Analysis of B cell subsets, particularly of memory B cells, and of T cells CD24+CD25+ during remission might provide information on the likelihood of recurrence in TTP. In conclusion, in recurrent TTP patients the higher amount of B cells might result in persistent autoantibodies production whilst the decreased level of T cells CD4+CD25+ may lead to a decreased inhibition of autoreactive T cells. These findings may explain the higher level of recurrence in these patients. Disclosures: Peyvandi: Archemix Corporation: Consultancy.

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