Two patients with rituximab associated low gammaglobulin levels and relapsed covid-19 infections treated with convalescent plasma.

Patients with haematological malignancies are considered to be a risk group for developing severe Coronavirus disease (Covid-19). Because of the limitations of therapeutic options, the development of new treatment strategies is mandatory, such as convalescent plasma (CP). Herein we report the use of CP therapy as an off-label indication in two lymphoma patients with relapsed COVID-19 in the setting of low gammaglobulin levels because of previous rituximab chemo-immunotherapy. Both were PCR positive for SARS-CoV-2 but had an absence of antibodies to the virus more than one month later of symptoms initiation. They developed important respiratory and neurological complications. After CP infusion, neutralising antibodies were detected and viral load dissapeared in both patients leading to clinical improvement with no more Covid-19 relapse.

Abatacept induced long-term non-progressive reduction in gamma-globulins and autoantibodies: dissociation from disease activity control.

OBJECTIVES: To evaluate long-term effects on gamma-globulins and autoantibodies of abatacept (ABA) versus tumor necrosis factor inhibitors (TNFi) in rheumatoid arthritis (RA) patients. METHOD: Eighteen RA patients undergoing abatacept (ABA-RA) and 18 age/sex-matched patients treated with TNFi (TNFi-RA) were compared regarding clinical data, total gamma-globulins (TGG), specific subtypes (IgG, IgM, IgA), free light chains (FLC), IgM/IgG rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP3), and anti-mutated citrullinated vimentin (anti-MCV), assessed before and every 6 months, up to 24 months. EXCLUSION CRITERIA: previous abatacept/rituximab or low TGG (< 0.7 g/dL). RESULTS: At baseline, female sex (78 vs. 78%), age (55 vs. 53 years), DAS28 (5.73 vs. 5.67), TGG (1.4 vs. 1.35 g/dL), IgG (1168 vs. 1079 mg/dL), IgM (107 vs. 113 mg/dL), IgA (333 vs. 322 mg/dL), kappa (342 vs. 249 mg/dL), lambda (170 vs. 150 mg/dL), IgM-RF (76 vs. 53 UI), IgG-RF (63 vs. 25 UI), anti-CCP3 (216 vs. 189 UI), and anti-MCV (202 vs. 102 UI) were comparable in ABA-RA and TNFi-RA (p > 0.05). Similar disease activity improvement was observed in both groups. In ABA-RA, significant decreases (p < 0.05) were observed in TGG (1.4 vs. 1.05 g/dL), IgG (1168 vs. 997), IgA (333 vs. 278 mg/dL), kappa (342 vs. 257 mg/dL), lambda (170 vs. 144 mg/dL), IgM-RF (76 vs. 37 UI), IgG-RF (65 vs. 24 UI), anti-CCP3 (216 vs. 183 UI), and anti-MCV (202 vs. 60 UI) at 6 months, without further decreases. In contrast, TNFi-RA showed no decrease in any of such parameters. ABA-RA also had more often transient IgG levels under the lower limit of normality (66.7% vs. 33.3%, p = 0.046). No severe infection occurred. DAS28, ESR, and CRP correlated significantly to gamma-globulins and FLC at baseline (p < 0.05), but these correlations were longitudinally lost in ABA-RA, but not in TNFi-RA. CONCLUSION: ABA, but not TNFi, induces a safe, persistent, long-term, and non-progressive reduction in gamma-globulins and autoantibodies, including anti-MCV. This pattern is dissociated from disease activity control.Key Points• ABA induces a long-term and non-progressive reduction in gamma-globulins and FLC, which occurs regardless of disease activity control.• ABA-induced reduction in gamma-globulins and FLC promotes a dissociation of such parameters and disease activity.• The same pattern of reduction is observed in autoantibodies: IgM-RF, IgG-RF, anti-CCP3, and anti-MCV.• Low transient IgG can be observed in RA patients treated with ABA, but does not correlate to infection.

Immune Complexes Impaired Glomerular Endothelial Cell Functions in Lupus Nephritis.

Lupus nephritis (LN) is one of the most common and severe complications of lupus. However, the mechanisms for renal damage have not been well elucidated. There are evidences show that glomerular endothelial cells (GECs) are damaged in LN. Immune complexes can deposit in subendothelial area and could affect GEC functions. In the present study, we used heat-aggregated gamma globulin (HAGG) to simulate immune complexes and investigated their effects on GEC functions. Our results revealed that HAGG impaired different aspect of the GEC functions. HAGG changed cell morphology, upregulated the expression of active caspase-3, inhibited angiogenesis, and increased NO production in GECs. These results provide new clues for the mechanisms of renal damage and the pathology of LN.

Antigen Complexed with a TLR9 Agonist Bolsters c-Myc and mTORC1 Activity in Germinal Center B Lymphocytes.

The germinal center (GC) is the anatomical site where humoral immunity evolves. B cells undergo cycles of proliferation and selection to produce high-affinity Abs against Ag. Direct linkage of a TLR9 agonist (CpG) to a T-dependent Ag increases the number of GC B cells. We used a T-dependent Ag complexed with CpG and a genetic model for ablating the TLR9 signaling adaptor molecule MyD88 specifically in B cells (B-MyD88- mice) together with transcriptomics to determine how this innate pathway positively regulates the GC. GC B cells from complex Ag-immunized B-MyD88- mice were defective in inducing gene expression signatures downstream of c-Myc and mTORC1. In agreement with the latter gene signature, ribosomal protein S6 phosphorylation was increased in GC B cells from wild-type mice compared with B-MyD88- mice. However, GC B cell expression of a c-Myc protein reporter was enhanced by CpG attached to Ag in both wild-type and B-MyD88- mice, indicating a B cell-extrinsic effect on c-Myc protein expression combined with a B cell-intrinsic enhancement of gene expression downstream of c-Myc. Both mTORC1 activity and c-Myc are directly induced by T cell help, indicating that TLR9 signaling in GC B cells either enhances their access to T cell help or directly influences these pathways to further enhance the effect of T cell help. Taken together, these findings indicate that TLR9 signaling in the GC could provide a surrogate prosurvival stimulus, "TLR help," thus lowering the threshold for selection and increasing the magnitude of the GC response.

Pivotal role of HIV and EBV replication in the long-term persistence of monoclonal gammopathy in patients on antiretroviral therapy.

A high prevalence of monoclonal gammopathy (MG) has been observed in HIV-infected patients. We explored the conditions associated with long-term persistence of serum monoclonal protein (M protein) in HIV-infected patients on antiretroviral therapy (ART). Of 21 patients with MG, M protein disappeared in 12 patients (58%) over 5 years of ART. Higher level of serum γ-globulin and higher percentages of circulating plasmablasts and plasma cells were observed in patients with persistent MG compared with patients with transient MG. MG persistence was associated with the cumulative time of detectable plasma HIV RNA after ART initiation, detection of Epstein-Barr virus (EBV) DNA in plasma, and a high level of EBV DNA in B cells. Poor control of HIV replication and detectable EBV replication in plasma were both associated with long-term MG persistence in patients on ART. In the case of viral control, MG associated with HIV infection is usually transient.

IL-21 receptor is critical for the development of memory B cell responses.

Development of long-term humoral immunity, characterized by the formation of long-lived plasma cells (PCs) in the bone marrow and memory B cells, is a critical component of protective immunity to pathogens, and as such it is the major goal of vaccination. However, the mechanisms involved in the generation of long-term humoral immunity remain poorly understood. In this study, we used IL-21R-deficient (IL-21R.KO) mice to examine the role of the IL-21 pathway in the development of the B cell memory response. Primary IgG serum Ab responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenylacetyl (NP) hapten conjugated to chicken γ globulin were delayed in IL-21R.KO mice, but reached normal titers within 3 to 4 wk of immunization. IL-21R.KO mice formed germinal centers and generated normal numbers of PCs in their bone marrow. Additionally, memory B cell formation was similar in wild-type and IL-21R.KO mice. However, NP-specific memory B cells and PCs failed to expand following secondary immunization of IL-21R.KO mice, and consequently, secondary IgG Ab responses to NP hapten conjugated to chicken γ globulin were significantly impaired. These results identify the IL-21 pathway as a critical component of the memory B cell response.

Successful unrelated umbilical cord blood cell transplantation without conditioning for a neonate with severe combined immunodeficiency.

A neonate was diagnosed as having SCID from his umbilical cord blood cells immediately after birth because his older brother had died of SCID eight months earlier. One locus-mismatched unrelated umbilical cord blood cell transplantation without conditioning was performed at the age of 30 days. The CD3-positive cells were detected on day 14 post-transplantation. There were no peri-transplantation complications. Four yr after transplantation, the boy is in excellent condition and T and NK cell engraftments are complete. His peripheral B cells with a common gamma chain were not detected by flow cytometry, and he still needs IgG replacement; however, his IgM and IgA levels have gradually increased, and the dosage of IVIG per body weight has gradually decreased. We speculate that the very few B cells that proliferate from transplanted cord blood cells produce gamma globulin. Unrelated cord blood cell transplantation, even though mismatched, without conditioning would be a treatment option for neonates with severe combined immunodeficiency.

Preferential localization of IgG memory B cells adjacent to contracted germinal centers.

It has long been presumed that after leaving the germinal centers (GCs), memory B cells colonize the marginal zone or join the recirculating pool. Here we demonstrate the preferential localization of nitrophenol-chicken gamma-globulin-induced CD38(+)IgG1(+) memory B cells adjacent to contracted GCs in the spleen. The memory B cells in this region proliferated after secondary immunization, a response that was abolished by depletion of CD4(+) T cells. We also found that these IgG1(+) memory B cells could present antigen on their surface, and that this activity was required for their activation. These results implicate this peri-GC region as an important site for survival and reactivation of memory B cells.

A role for lysosomal-associated protein transmembrane 5 in the negative regulation of surface B cell receptor levels and B cell activation.

Mechanisms by which cell surface levels of the BCR are regulated remain largely unknown. We found that B cells lacking the lysosomal-associated protein transmembrane 5 (LAPTM5) expressed higher levels of cell surface BCR than did wild-type (WT) B cells after Ag stimulation in vitro and in vivo. In addition, LAPTM5-deficient mice contained an increased frequency of Ag-specific B cells and produced greater amounts of Abs than did WT mice after immunization with a T-dependent Ag. Adoptive transfer of LAPTM5-deficient B cells with WT T cells into RAG1-deficient mice revealed that the increased surface BCR levels and the enhanced B cell activation and Ab production were due to a B cell intrinsic defect. As they aged, the LAPTM5-deficient mice had increased titers of serum IgM and autoantibodies and immune complex deposition in the kidney. Immunofluorescent and biochemical analysis revealed that LAPTM5 physically interacted with the BCR complex and promoted its degradation in the lysosomal compartment in mouse B cells. These results demonstrate a role for LAPTM5 in the negative regulation of cell surface BCR levels and B cell activation.

The pericyte and stromal cell marker CD248 (endosialin) is required for efficient lymph node expansion.

CD248 is a cell surface receptor that specifically identifies fibroblasts and pericytes during development and in association with cancer and inflammation. However, its function is poorly defined and its role in lymphoid organs not studied. Here, we used (4-hydroxy-3-nitrophenyl)acetyl chicken gamma-globulin immunisation and mice lacking CD248 to study whether CD248 modulates popliteal LN (pLN) expansion and subsequent immune responses. We have found that CD248 is required for complete pLN expansion but not for co-ordination of B and T cell compartmentalisation or antibody production following (4-hydroxy-3-nitrophenyl)acetyl chicken gamma-globulin immunisation. In vitro, we show that CD248 expression in human MG63 stromal cells and mouse embryonic fibroblasts leads to a pro-proliferative and pro-migratory phenotype. This correlates with a proliferating CD248(+) population observed in vivo during pLN expansion. Taken together, these data highlight a role for CD248 in secondary lymphoid organ remodelling during adaptive immune responses.

Immunizations of human lymphocytes in vitro with a T-dependent antigen towards human monoclonal antibody production.

Human monoclonal antibodies have a plethora of applications, justifying the time and effort towards development of techniques for their efficient production. Attempts at establishing efficient reproducible techniques for activation of lymphocytes in culture have met with moderate success. In this study, human lymphocytes from peripheral blood and bone marrow were immunized in vitro with a T-dependent antigen--bovine gamma globulin. Whole blood, bone marrow and separated mononuclear peripheral blood cells were studied for the potential antibody secretory capabilities. The culture conditions included supplementation with heat treated autologous serum, spent medium from U-266 myeloma cell culture, which is known to contain B cell differentiation factors, and varied antigenic concentrations along with exposure duration. Although there was no appreciable difference in response between whole peripheral blood and whole bone marrow, there is a much better response when compared to isolated cell cultures; especially when culture conditions include antigenic withdrawal and supplementation with conditioned medium. However, lower antigenic concentration was required for whole bone marrow cultures. With optimal in vitro conditions for antigenic stimulation standardized, several options are available for the immortalization of such activated cells to obtain stable human hybridomas of interest.

CD93 is required for maintenance of antibody secretion and persistence of plasma cells in the bone marrow niche.

Plasma cells represent the end stage of B-cell development and play a key role in providing an efficient antibody response, but they are also involved in numerous pathologies. Here we show that CD93, a receptor expressed during early B-cell development, is reinduced during plasma-cell differentiation. High CD93/CD138 expression was restricted to antibody-secreting cells both in T-dependent and T-independent responses as naive, memory, and germinal-center B cells remained CD93-negative. CD93 was expressed on (pre)plasmablasts/plasma cells, including long-lived plasma cells that showed decreased cell cycle activity, high levels of isotype-switched Ig secretion, and modification of the transcriptional network. T-independent and T-dependent stimuli led to re-expression of CD93 via 2 pathways, either before or after CD138 or Blimp-1 expression. Strikingly, while humoral immune responses initially proceeded normally, CD93-deficient mice were unable to maintain antibody secretion and bone-marrow plasma-cell numbers, demonstrating that CD93 is important for the maintenance of plasma cells in bone marrow niches.

ICOS, CD40, and lymphotoxin beta receptors signal sequentially and interdependently to initiate a germinal center reaction.

Germinal center (GC) responses to T-dependent Ags require effective collaboration between Th cells, activated B cells, and follicular dendritic cells within a highly organized microenvironment. Studies using gene-targeted mice have highlighted nonredundant molecules that are key for initiating and maintaining the GC niche, including the molecules of the ICOS, CD40, and lymphotoxin (LT) pathways. Signaling through ICOS has multiple consequences, including cytokine production, expression of CD40L on Th cells, and differentiation into CXCR5(+) follicular Th cells, all of which are important in the GC reaction. We have therefore taken advantage of ICOS(-/-) mice to dissect which downstream elements are required to initiate the formation of GC. In the context of a T-dependent immune response, we found that GC B cells from ICOS(-/-) mice express lower levels of LTalphabeta compared with wild-type GC B cells in vivo, and stimulation of ICOS on T cells induces LTalphabeta on B cells in vitro. Administration of agonistic anti-LTbeta receptor Ab was unable to restore the GC response in ICOS(-/-) mice, suggesting that additional input from another pathway is required for optimal GC generation. In contrast, treatment with agonistic anti-CD40 Ab in vivo recovered GC networks and restored LTalphabeta expression on GC B cells in ICOS(-/-) mice, and this effect was dependent on LTbeta receptor signaling. Collectively, these data demonstrate that ICOS activation is a prerequisite for the up-regulation of LTalphabeta on GC B cells in vivo and provide a model for cooperation between ICOS, CD40, and LT pathways in the context of the GC response.

SAP expression in T cells, not in B cells, is required for humoral immunity.

SAP (also named SH2D1A) is an intracellular adaptor molecule expressed in T cells, natural killer (NK) cells, and some B cells. The SAP gene is mutated in X-linked lymphoproliferative (XLP) disease, a human immunodeficiency characterized by a faulty immune response to Epstein-Barr virus infection. Previous reports documented severe defects in antibody production and germinal center (GC) formation in SAP-deficient humans and mice genetically engineered to lack SAP expression. However, in vitro studies and adoptive transfer experiments provided conflicting data as to whether this phenotype is caused by a functional defect resulting from SAP deficiency in T cells, B cells, or both. Here, we ascertained which cell types are responsible for this humoral immunity defect by using a conditional gene targeting approach. We also thoroughly examined the expression pattern of SAP in normal immune cells by using intracellular flow cytometry. The results showed that expression of SAP in T cells, but not in B cells or NK cells, is required and sufficient for SAP-dependent antibody production and GC formation. These data provide a critical insight into the mechanism by which SAP regulates humoral immunity. They also help elucidate the basis of a severe human immunodeficiency.

CD40 Ligand-activated, antigen-specific B cells are comparable to mature dendritic cells in presenting protein antigens and major histocompatibility complex class I- and class II-binding peptides.

Dendritic cells (DC) are increasingly exploited for cell-based immunotherapy. However, limitations in ex vivo DC growth and DC functional heterogeneity have motivated development of complementary antigen-presenting cell sources. Here, the ability of CD40 ligand (CD40L)-activated B cells to fulfil that role was investigated. We demonstrate for the first time that non-specific or antigen-specific murine B cells can be grown for extended periods of time by stimulation with CD40L. These cells rapidly up-regulate and maintain high levels of co-stimulatory molecules. In a head-to-head comparison with DC, CD40L-expanded B cells were comparable to DC in the presentation of peptides to CD4+ and CD8+ T cells. While DC were superior to antigen non-specific CD40L-activated B cells with regard to whole protein (NP-BSA) processing and presentation, CD40L-expanded B cells from NP-BSA-immunized mice were comparable to DC when presenting BSA or NP-BSA to primed primary T cells or when presenting NP linked to an unrelated carrier, CGG, to naïve T cells. Thus, the combination of CD40L activation, which supports B-cell growth and augments intracellular protein processing, and antigen uptake via the B-cell receptor, allows for efficient uptake, processing, and presentation of whole protein antigens in a fashion comparable to that observed with mature DC. Like DC, CD40L-activated B cells efficiently home to secondary lymphoid organs in vivo. This system represents a unique tool for studying primary antigen-specific B cells and the results suggest that the outgrowth of large numbers of highly activated B cells represents a viable and practical complement to DC for cell-based immunotherapy.

[Death of the ovarian follicular cells in mice with impaired oogenesis of the immune nature].

An impairment of the meiotic maturation of the oocytes has been shown in vitro for 2 types of immune damage of the ovaries in mice induced by xenogenic antiovarial antibodies and immunization with allogenic ovaria. Impairment of the oogenesis was followed by the follicular cell death, primarily by on the apoptic way, but under the immunization with allogenic ovary a necrotic way of their death was also activated.