B-cell Elimination Research Articles

Advances in Understanding and Managing Myasthenia Gravis: Current Trends and Future Directions.

Myasthenia gravis (MG) is an autoimmune illness characterized by autoantibodies against the acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), and an increasing number of extra postsynaptic proteins. Pathogenic autoantibodies reduce the number of functional AChRs in the neuromuscular junction's (NMJ)muscle end plate. The cause of the autoimmune response is unknown, but thymic abnormalities and immune regulatory deficiencies are significant. The disease's incidence is likely influenced by genetic predisposition, with sex hormones and exercise playing a role. MGcan affect any age, race, or gender and can be caused by any stressor, with infections being the most frequent cause. Treatment focuses on airway support and the triggering incident. MG is a rare autoimmune disease causing fatigue-inducing weakness in the axial, respiratory, leg, and bulbar muscles. Initially affecting the eyes, most MGpatients experience at least one worsening symptom during their illness. The disease is mainly caused by antibodies against the AChR, dependence on the immune system within cells, and engagement of the complement system. The complement system plays a significant role in MG, and complement inhibition can both prevent the onset and slow its development. Ocular MG affects around 15% of people, with most patients having blocking antibodies against the cholinergic receptor. There may be correlations between thymoma and other autoimmune conditions, especially thyroid illness. Treatment and management for MGinvolve removing autoantibodies from circulation or blocking effector mechanisms using techniques such as complement inhibition, plasmapheresis, and B-cell elimination.

Co-Transduced CD19/BCMA Dual-Targeting CAR-T Cells for the Treatment of Non-Hodgkin Lymphoma

CAR-T cells targeting CD19 have accomplished impressive responses in relapsed/refractory non-hodgkin lymphoma (NHL) patients, but disease recurrence is a concerning hurdle to overcome. This complication is mainly due to a selective pressure on tumor cells expressing CD19, which leads to loss of expression or downregulation to levels that cannot be detected by CAR-T cells. Simultaneous targeting of more than one antigen has been proposed as a compelling strategy to prevent tumor escape. CD19 is expressed along B-cell differentiation, and BCMA is expressed not only on plasmatic cells but also on mature B-cells. Thus, most NHL subtypes express both CD19 and BCMA. Here, we hypothesized that dual CD19 and BCMA targeting could reduce tumor antigen escape and enhance the antitumor efficacy of CAR-T cells. CAR constructs targeting CD19 (ARI0001) or BCMA (ARI0002h) were engineered in our institution and both have demonstrated safety and efficacy for treating patients with B-cell lymphoid neoplasms and multiple myeloma, respectively. Both CARs contain the 41BB co-stimulatory domain in tandem with the CD3z. We tested the antitumoral efficacy of three different dual-targeting CD19/BCMA strategies: 1) a bicistronic vector encoding both CAR constructs separated by a T2A cleavage peptide (ARI0001-T2A-ARI0002h); 2) co-transduced T cells with ARI0001 and ARI0002h lentiviral vectors to obtain a product with three CAR populations comprising both single-CARs and a double positive population; 3) pooled ARI0001 and ARI0002. Conventional functional assays such as cytokine secretion and specific lysis after short-term co-culture of CAR-T cells with NHL cell lines co-expressing CD19 and BCMA at constitutive levels revealed that all strategies were active but, did not discriminate between single- and dual-CARs groups. In order to mimic a potential relapse due to CD19 downregulation, we developed a CD19low density cell line of Burkitt lymphoma through its expression under a weak promoter. After 7-days of co-culture of CAR-T cells with RAMOS-CD19Low, only the co-transduction product maintained tumor elimination. Next, we analyzed the capacity of single versus dual CAR-T cells to kill malignant B cells and release cytokines in co-culture with NHL Patient Derived Lymphoma Spheroids. We tested samples from patients with 1) follicular lymphoma, 2) diffuse large B cell lymphoma (DLBCL), and 3) DLBCL patients who relapsed or had refractory disease post-CD19 CAR-T cell treatment. Under these more challenging conditions, differences among effector groups were maximized. ARI0001 and bicistronic CAR-T cells displayed similar effector functions in terms of killing capacity and cytokine production, especially when CD19 was expressed at high densities. ARI0002h presented antitumor efficacy but not as high as ARI0001. Co-transduction and pool products showed increased cytokine production and killing, being capable of B-cell elimination in all cases. A high disease burden of Burkitt lymphoma in vivo model revealed that co-transduction product significantly increased mice survival compared to ARI0001. This increase in survival was even more remarkable when the model was CD19low. No significant differences were observed between the co-transduction and pool groups. In summary, we propose a relatively simple preparation of a dual-CAR-T cells targeting CD19 and BCMA, which is particularly effective against NHL cell lines expressing low CD19 densities. Co-transduction is advantageous with respect to the pool since it only requires one manufacturing process, decreasing the cost of the therapy and increasing patients' access to these novel therapies. Our studies provide a strong rational to test CD19/BCMA dual-CAR-T cells in clinical trials for the treatment of NHL patients.

Comparison of Plasma Cytokine Levels before and after Treatment with Rituximab in Patients with Rheumatoid Arthritis and Systemic Lupus Erythematosus-Associated Polyautoimmunity

Introduction: Coexistence of more than one autoimmune disease (AD) in a single patient is known as polyautoimmunity, and may be seen in up to 35% of patients with ADs. The elimination of B-cells using Rituximab (RTX) improves clinical status in different ADs. The role of cytokine production by B-cells is unclear in systemic lupus erythematosus (SLE) and polyautoimmunity. Methods: As an exploratory study, plasma from 11 patients with either rheumatoid arthritis (RA) or SLE-associated polyautoimmunity was assessed prior and 6 months after therapy with RTX. Eight healthy individuals were used as controls. Cytokine levels were measured using ELISA (IFN-α and TGF-β1) or Cytometric Bead Array (TNF-α, IL-1β, IL-6, IL-8, IL-10, and IL-12p70). Results: Prior to RTX, IL-6 was only elevated in RA and IL-8 was elevated in both RA and SLE-associated polyautoimmunity, compared with controls. After RTX, significant decreases of IL-6 in RA and IL-8 in SLE-associated polyautoimmunity were observed. Levels of other cytokines measured were either similar (IFN-α, TGF β1) or below the detection limit (TNF-α, IL-1β, IL-10, IL-12p70) for both patients and controls. Conclusion:Our data highlight the importance of B-cell cytokine secretion in RA and SLE-associated polyautoimmunity, and suggest a differential role in each pathology. A significant increase of IL-8 prior to RTX in both groups, and a significant decrease after therapy only in SLE-associated polyautoimmunity support the potential of IL-8 as a therapeutic target. The heterogeneity of the polyautoimmunity patient population highlights the importance of the selection of specific subsets in future research.

Development of Donor-Specific Isohemagglutinins Following Pediatric ABO-Incompatible Heart Transplantation

Graft acceptance following pediatric ABO-incompatible heart transplantation has been associated with a deficiency of donor-specific isohemagglutinins (DSI) due to B-cell elimination. Recent observations suggest that some of these patients do produce DSI. The purpose of this study was to examine the pattern of, risk factors for development and clinical impact of DSI. All children who underwent an ABO-incompatible heart transplant (1996-2009) were included. Serial postheart transplantation DSI titers and clinical outcomes were reviewed. DSI were produced in 27% of the patients (n = 11/41). Anti-A production was significantly greater in "at risk" patients than Anti-B (39% vs. 8%; p = 0.04). Risk factors associated with the development of DSI included: older age at transplantation (HR: 1.15/month, p = 0.04), pretransplant Anti-B level ≥ 1:8 (HR: 9.61, p = 0.004) and HLA sensitization (HR: 2.80, p = 0.11). The presence of DSI did increase the risk of cellular rejection but not antibody-mediated rejection, allograft vasculopathy, graft loss or death. Although these antibodies do not result in any significant clinical consequences, their presence suggests that B-cell tolerance is not the sole mechanism of graft acceptance.

Autoreactive B-cell elimination by pathogenic IgG specific for the same antigen: implications for peripheral tolerance

Harmful pathogenic IgG auto-antibodies are produced against desmoglein 3 (Dsg3) in pemphigus vulgaris, an autoimmune blistering disease. Dsg3 is a cadherin-type cell adhesion molecule expressed in desmosomes of the skin and mucous membranes. In AK7-transgenic mice expressing non-pathogenic AK7 IgM against Dsg3, autoreactive transgenic B cells escape from the deletion or inactivation and exist in the periphery. However, when a pathogenic anti-Dsg3 IgG1 mAb (AK23) capable of inducing blisters was injected into AK7-transgenic mice, AK7 B cells were eliminated from the bone marrow (BM) and spleen only when Dsg3 was expressed in the periphery. In contrast, non-pathogenic IgG mAbs (AK7, AK9) failed to eliminate AK7 B cells. Interestingly, the AK23-mediated elimination of mature AK7 B cells in the spleen was significantly diminished in AK7-transgenic mice on a Rag2(-/-) background while BM B cells were still eliminated, suggesting the presence of T-cell-dependent and -independent mechanisms. T cell transfer studies into AK7-Rag2(-/-) mice revealed that autoreactive B-cell elimination in the periphery requires CD4(+) T cells from wild-type mice but not from gld (FasL mutant) mice. The B-cell elimination was impaired in both BM and periphery when Bcl2 was over-expressed in AK7 B cells. These findings suggest that autoreactive B cells exist unless they are harmful, but once harmful or dangerous events such as tissue destruction are sensed, the mature autoreactive B cells in the periphery are eliminated via a Fas-mediated process in a CD4(+) T cell-dependent manner.

Pharmacodynamics of Rituximab in Kidney Transplantation

The B-cell depleting anti-CD20 antibody rituximab has become a therapeutic alternative in renal transplantation. However, understanding of the pharmacodynamics is limited. We have therefore studied the effect of single-dose rituximab, in combination with conventional triple immunosuppressive therapy, on the B-cell population in peripheral blood as well as in tissues, in kidney transplant recipients. Forty-nine kidney recipients received single-dose rituximab. The prevalence of B cells was assessed in peripheral blood, kidney transplant tissue, and in lymph nodes. In 88%, complete depletion of B cells in peripheral blood was observed and, 15 months after treatment, B cells were still undetectable in the majority of patients. In kidney tissue, B cells were also completely eliminated. In contrast, the B cells were not eliminated in lymph nodes, although a reduction was observed. In conclusion, single-dose rituximab in kidney transplant recipients evokes a long-term elimination of B-cells in peripheral blood as well as within the kidney transplant.

Rituximab in childhood systemic lupus erythematosus refractory to conventional immunosuppression

Rituximab, a chimeric monoclonal antibody specific for human CD20, has recently been used for the treatment of autoimmune diseases. A 14-year-old patient with severe systemic lupus erythematosus (SLE) and class IV glomerulonephritis presented with immunologic and clinical resistance to conventional immunosuppressive therapy for 10 months after diagnosis. To induce remission of active SLE, treatment with 6 monthly rituximab at 375 mg/m(2), oral mycophenolate and prednisone was initiated followed by maintenance rituximab every 3 months. The SLEDAI decreased significantly from 31 at diagnosis to 14 after nine applications of rituximab. Extrarenal symptoms of SLE improved significantly. However, after induction therapy with rituximab the patient presented a reversible intrinsic acute renal insufficiency for a period of 3 weeks. The discontinuation of the daily medication (oral prednisone and mycophenolate) by the patient herself may explain the progression of active SLE associated with the reversible acute renal failure. Under intensive immunosuppressive therapy improvement of active disease manifestations and stabilization of plasma creatinine concentrations to normal values was observed. However, proteinuria remained elevated and improved only after a protracted period (median protein-to-creatinine ratio 5.2 g/g, range 0.8-11.2 g/g). Hematuria and urinary cell casts persisted. In conclusion, the extrarenal symptoms of the patient responded particularly well to rituximab. However, despite complete B-cell elimination, renal remission of SLE was not achieved. Thus, it may be possible that humoral and cellular immune mechanisms have a fundamental involvement in the pathogenesis of SLE nephritis.

Signaling thresholds and interclonal competition in preimmune B-cell selection.

The need to eliminate autoreactive B cells must be checked against the need for a diverse B-cell repertoire. Protein tyrosine phosphatases SHP1 and CD45 act antagonistically within B cells to set the threshold level of antigen-receptor engagement required for B-cell elimination. The fate of B cells binding weak autoantigens is also regulated by interclonal competition. In the presence of a normal diverse repertoire of competitor B cells, the autoantigen-binding cells are excluded from follicles in spleen and lymph nodes and undergo rapid cell death. In the absence of interclonal competition, the autoreactive cells enter the follicular microenvironment and survive. A model in which B cells compete for access to limiting follicular niches in order to survive is discussed.