Productive Germinal Center Responses Depend on the Nature of Stimuli Received by Anti-Insulin B Cells in Type 1 Diabetes-Prone Mice.

Islet autoantibodies, including those directed at insulin, predict type 1 diabetes (T1D) in mice and humans and signal immune tolerance breach by B lymphocytes. High-affinity insulin autoantibodies and T follicular helper cell involvement implicate germinal centers (GCs) in T1D. The VH125SD BCR transgenic model, in which 1-2% of peripheral B lymphocytes recognize insulin, enables direct study of insulin-binding B cells. Our prior studies showed that anti-insulin B cell receptor transgene site-directed to H chain locus mice fail to generate insulin Ab following T-dependent immunization, but it was unclear whether anti-insulin B cells were blocked for GC initiation, survival, or differentiation into Ab-secreting cells. Here, we show that insulin-binding B cells in T1D-prone anti-insulin B cell receptor transgene site-directed to H chain locus mice can spontaneously adopt a GC phenotype and undergo class switching to the IgG1 isotype, with little if any switching to IgG2b. T-dependent immunizations with insulin SRBC or insulin CFA drove anti-insulin B lymphocytes to adopt a GC phenotype, despite blunted insulin Ab production. Dual immunization against self (insulin) and foreign (4-hydroxy-3-nitrophenylacetyl hapten conjugated to keyhole limpet hemocyanin) Ags showed an anti-insulin (but not anti-4-hydroxy-3-nitrophenylacetyl) Ab block that tracked with increased expression of the apoptosis marker, activated caspase 3, in self-reactive GC B cells. Finally, T-independent immunization with insulin conjugated to Brucella abortus ring test Ag released immune tolerance to allow robust expansion of anti-insulin GC B cells and IgG-switched insulin Ab production. Overall, these data pinpoint GC survival and Ab-secreting cell differentiation as immune tolerance blocks that limit T-dependent, but not T-independent, stimulation of anti-insulin B cell responses.

High-Throughput Detection of Autoantigen-Specific B Cells Among Distinct Functional Subsets in Autoimmune Donors.

Antigen-specific B cells (ASBCs) can drive autoimmune disease by presenting autoantigen to cognate T cells to drive their activation, proliferation, and effector cell differentiation and/or by differentiating into autoantibody-secreting cells. Autoantibodies are frequently used to predict risk and diagnose several autoimmune diseases. ASBCs can drive type 1 diabetes even when immune tolerance mechanisms block their differentiation into antibody-secreting cells. Furthermore, anti-histidyl tRNA synthetase syndrome patients have expanded IgM+ Jo-1-binding B cells, which clinically diagnostic IgG Jo-1 autoantibodies may not fully reflect. Given the potential disconnect between the pathologic function of ASBCs and autoantibody secretion, direct study of ASBCs is a necessary step towards developing better therapies for autoimmune diseases, which often have no available cure. We therefore developed a high-throughput screening pipeline to 1) phenotypically identify specific B cell subsets, 2) expand them in vitro, 3) drive them to secrete BCRs as antibody, and 4) identify wells enriched for ASBCs through ELISA detection of antibody. We tested the capacity of several B cell subset(s) to differentiate into antibody-secreting cells following this robust stimulation. IgM+ and/or IgD+, CD27- memory, memory, switched memory, and BND B cells secreted B cell receptor (BCR) as antibody following in vitro stimulation, whereas few plasmablasts responded. Bimodal responses were observed across autoimmune donors for IgM+ CD21lo and IgM- CD21lo B cells, consistent with documented heterogeneity within the CD21lo subset. Using this approach, we detected insulin-binding B cell bias towards CD27- memory and CD27+ memory subsets in pre-symptomatic type 1 diabetes donors. We took advantage of routine detection of Jo-1-binding B cells in Jo-1+ anti-histidyl tRNA synthetase syndrome patients to show that Jo-1-binding B cells and total B cells expanded 20-30-fold using this culture system. Overall, these studies highlight technology that is amenable to small numbers of cryopreserved peripheral blood mononuclear cells that enables interrogation of phenotypic and repertoire attributes of ASBCs derived from autoimmune patients.

Adult Mosquitoes Infected with Bacteria Early in Life Have Stronger Antimicrobial Responses and More Hemocytes after Reinfection Later in Life.

The immunological strategies employed by insects to overcome infection vary with the type of infection and may change with experience. We investigated how a bacterial infection in the hemocoel of the African malaria mosquito, Anopheles gambiae, prepares the immune system to face a subsequent bacterial infection. For this, adult female mosquitoes were separated into three groups-unmanipulated, injured, or infected with Escherichia coli-and five days later all the mosquitoes were infected with a different strain of E. coli. We found that an injury or a bacterial infection early in life enhances the ability of mosquitoes to kill bacteria later in life. This protection results in higher mosquito survival and is associated with an increased hemocyte density, altered phagocytic activity by individual hemocytes, and the increased expression of nitric oxide synthase and perhaps prophenoloxidase 6. Protection from a second infection likely occurs because of heightened immune awareness due to an already existing infection instead of memory arising from an earlier, cured infection. This study highlights the dynamic nature of the mosquito immune response and how one infection prepares mosquitoes to survive a subsequent infection.

Metabolites Involved in Immune Evasion by Batrachochytrium dendrobatidis Include the Polyamine Spermidine.

Amphibians have been declining around the world for more than four decades. One recognized driver of these declines is the chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Amphibians have complex and varied immune defenses against B. dendrobatidis, but the fungus also has a number of counterdefenses. Previously, we identified two small molecules produced by the fungus that inhibit frog lymphocyte proliferation, methylthioadenosine (MTA) and kynurenine (KYN). Here, we report on the isolation and identification of the polyamine spermidine (SPD) as another significant immunomodulatory molecule produced by B. dendrobatidis SPD and its precursor, putrescine (PUT), are the major polyamines detected, and SPD is required for growth. The major pathway of biosynthesis is from ornithine through putrescine to spermidine. An alternative pathway from arginine to agmatine to putrescine appears to be absent. SPD is inhibitory at concentrations of ≥10 µM and is found at concentrations between 1 and 10 µM in active fungal supernatants. Although PUT is detected in the fungal supernatants, it is not inhibitory to lymphocytes even at concentrations as high as 100 µM. Two other related polyamines, norspermidine (NSP) and spermine (SPM), also inhibit amphibian lymphocyte proliferation, but a third polyamine, cadaverine (CAD), does not. A suboptimal (noninhibitory) concentration of MTA (10 µM), a by-product of spermidine synthesis, enhances the inhibition of SPD at 1 and 10 µM. We interpret these results to suggest that B. dendrobatidis produces an "armamentarium" of small molecules that, alone or in concert, may help it to evade clearance by the amphibian immune system.