Projected Dose Optimization of Amino- and Hydroxypyrrolidine Purine PI3Kδ Immunomodulators.

The approvals of idelalisib and duvelisib have validated PI3Kδ inhibitors for the treatment for hematological malignancies driven by the PI3K/AKT pathway. Our program led to the identification of structurally distinct heterocycloalkyl purine inhibitors with excellent isoform and kinome selectivity; however, they had high projected human doses. Improved ligand contacts gave potency enhancements, while replacement of metabolic liabilities led to extended half-lives in preclinical species, affording PI3Kδ inhibitors with low once-daily predicted human doses. Treatment of C57BL/6-Foxp3-GDL reporter mice with 30 and 100 mg/kg/day of 3c (MSD-496486311) led to a 70% reduction in Foxp3-expressing regulatory T cells as observed through bioluminescence imaging with luciferin, consistent with the role of PI3K/AKT signaling in Treg cell proliferation. As a model for allergic rhinitis and asthma, treatment of ovalbumin-challenged Brown Norway rats with 0.3 to 30 mg/kg/day of 3c gave a dose-dependent reduction in pulmonary bronchoalveolar lavage inflammation eosinophil cell count.

CD22 is required for formation of memory B cell precursors within germinal centers.

CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens.

Targeting antigens through blood dendritic cell antigen 2 on plasmacytoid dendritic cells promotes immunologic tolerance.

The C-type lectin receptor blood dendritic cell Ag 2 (BDCA2) is expressed exclusively on human plasmacytoid DCs (pDCs) and plays a role in Ag capture, internalization, and presentation to T cells. We used transgenic mice that express human BDCA2 and anti-BDCA2 mAbs to deliver Ags directly to BDCA2 on pDCs in vivo. Targeting Ag to pDCs in this manner resulted in significant suppression of Ag-specific CD4(+) T cell and Ab responses upon secondary exposure to Ag in the presence of adjuvant. Suppression of Ab responses required both a decrease in effector CD4(+) T cells and preservation of Foxp3(+) regulatory T cells (Tregs). Reduction in Treg numbers following Ag delivery to BDCA2 restored both CD4(+) T cell activation and Ab responses, demonstrating that Tregs were required for the observed tolerance. Our results demonstrate that Ag delivery to pDCs through BDCA2 is an effective method to induce immunological tolerance, which may be useful for treating autoimmune diseases or to inhibit unwanted Ab responses.

Controlling immune responses by targeting antigens to dendritic cell subsets and B cells.

Delivering antigens in vivo by coupling them to mAbs specific for unique receptors on antigen-presenting cells (APCs) is a promising approach for modulating immune responses. Antigen delivery to receptors found on myeloid dendritic cell (DC) subsets, plasmacytoid DCs and B cells has shown them all to be viable targets to stimulate either the cellular or humoral arms of the immune system. It is now evident that antigen-targeting approaches can also be used to invoke antigen-specific inhibition of immune responses. The outcome of activation versus inhibition is determined by a combination of factors that include the choice of APC, the receptor that is targeted, whether to include an adjuvant and, if so, which adjuvant to employ. In addition to their use as a means to modulate immune responses, antigen-targeting systems are also a useful method to investigate the function of DC subsets and the early mechanistic events that underlie the initiation of both cellular and humoral immune responses. In this review, we focus on the literature surrounding the control of B-cell responses when antigen is delivered to various APC subsets.

Overexpression of TLR7 promotes cell-intrinsic expansion and autoantibody production by transitional T1 B cells.

Toll-like receptor (TLR), a ligand for single-stranded RNA, has been implicated in the development of pathogenic anti-RNA autoantibodies both in systemic lupus erythematous (SLE) patients and in murine models of lupus. It is still unclear, however, where and how TLR7-mediated interactions affect the development of autoreactive B cells. We found that overexpression of TLR7 in transgenic mice (TLR7.1Tg) leads to marked alterations of transitional (T1) B cells, associated with their expansion and proliferation within the splenic red pulp (RP). This phenotype was intrinsic to the T1 subset of B cells and occurred independently of type 1 IFN signals. Overexpression of RNase in TLR7.1Tg mice significantly limited the expansion and proliferation of T1 cells, indicating that endogenous RNA complexes are driving their activation. TLR7.1Tg T1 cells were hyper-responsive to anti-IgM and TLR7 ligand stimulation in vitro and produced high concentrations of class-switched IgG2b and IgG2c, including anti-RNA antibodies. Our results demonstrate that initial TLR7 stimulation of B cells occurs at the T1 stage of differentiation in the splenic RP and suggest that dysregulation of TLR7 expression in T1 cells can result in production of autoantibodies.

Targeting antigens to CD180 rapidly induces antigen-specific IgG, affinity maturation, and immunological memory.

Antigen (Ag) targeting is an efficient way to induce immune responses. Ag is usually coupled to an antibody (Ab) specific for a receptor expressed on dendritic cells (DCs), and then the Ag-anti-receptor is inoculated with an adjuvant. Here we report that targeting Ag to a receptor expressed on both B cells and DCs, the TLR orphan receptor CD180, in the absence of adjuvant rapidly induced IgG responses that were stronger than those induced by Ag in alum. Ag conjugated to anti-CD180 (Ag-αCD180) induced affinity maturation and Ab responses that were partially T cell independent, as Ag-specific IgGs were generated in CD40- and T cell-deficient mice. After preimmunization with Ag-αCD180 and boosting with soluble Ag, both WT and CD40 knockout (KO) mice rapidly produced Ag-specific IgG-forming cells, demonstrating that Ag-anti-CD180 induces immunological memory. The potent adjuvant effect of Ag-αCD180 required Ag to be coupled to anti-CD180 and the responsive B cells to express both CD180 and an Ag-specific B cell receptor. Surprisingly, CD180 Ag targeting also induced IgG Abs in BAFF-R KO mice lacking mature B cells and in mice deficient in interferon signaling. Targeting Ag to CD180 may be useful for therapeutic vaccination and for vaccinating the immune compromised.

STALing B cell responses with CD22.

Suppressing unwanted humoral immune responses without compromising the host's ability to respond to foreign pathogens is a primary goal for therapies aimed at ameliorating harmful autoantibody production. Global suppression of the immune system via lymphocyte depletion and/or immunosuppressive drugs can have off-target effects, a limitation to conventional therapies. In this issue of the JCI, Macauley and colleagues utilize a novel platform to inhibit antigen-specific antibody production that preserves the immune system's ability to respond to unrelated antigens.

Extrafollicular B cell activation by marginal zone dendritic cells drives T cell-dependent antibody responses.

Dendritic cells (DCs) are best known for their ability to activate naive T cells, and emerging evidence suggests that distinct DC subsets induce specialized T cell responses. However, little is known concerning the role of DC subsets in the initiation of B cell responses. We report that antigen (Ag) delivery to DC-inhibitory receptor 2 (DCIR2) found on marginal zone (MZ)-associated CD8α(-) DCs in mice leads to robust class-switched antibody (Ab) responses to a T cell-dependent (TD) Ag. DCIR2(+) DCs induced rapid up-regulation of multiple B cell activation markers and changes in chemokine receptor expression, resulting in accumulation of Ag-specific B cells within extrafollicular splenic bridging channels as early as 24 h after immunization. Ag-specific B cells primed by DCIR2(+) DCs were remarkably efficient at driving naive CD4 T cell proliferation, yet DCIR2-induced responses failed to form germinal centers or undergo affinity maturation of serum Ab unless toll-like receptor (TLR) 7 or TLR9 agonists were included at the time of immunization. These results demonstrate DCIR2(+) DCs have a unique capacity to initiate extrafollicular B cell responses to TD Ag, and thus define a novel division of labor among splenic DC subsets for B cell activation during humoral immune responses.

CD28-B7 interaction modulates short- and long-lived plasma cell function.

The interaction of CD28, which is constitutively expressed on T cells, with B7.1/B7.2 expressed on APCs is critical for T cell activation. CD28 is also expressed on murine and human plasma cells but its function on these cells remains unclear. There are two types of plasma cells: short-lived ones that appear in the secondary lymphoid tissue shortly after Ag exposure, and long-lived plasma cells that mainly reside in the bone marrow. We demonstrate that CD28-deficient murine short- and long-lived plasma cells produce significantly higher levels of Abs than do their wild-type counterparts. This was owing to both increased frequencies of plasma cells as well as increased Ab production per plasma cell. Plasma cells also express the ligand for CD28, B7.1, and B7.2. Surprisingly, deficiency of B7.1 and B7.2 in B cells also led to higher Ab levels, analogous to Cd28(-/-) plasma cells. Collectively, our results suggest that the CD28-B7 interaction operates as a key modulator of plasma cell function.

The RIG-I-like receptor LGP2 controls CD8(+) T cell survival and fitness.

The RIG-I-like receptors (RLRs) signal innate immune defenses upon RNA virus infection, but their roles in adaptive immunity have not been clearly defined. Here, we showed that the RLR LGP2 was not essential for induction of innate immune defenses, but rather was required for controlling antigen-specific CD8(+) T cell survival and fitness during peripheral T cell-number expansion in response to virus infection. Adoptive transfer and biochemical studies demonstrated that T cell-receptor signaling induced LGP2 expression wherein LGP2 operated to regulate death-receptor signaling and imparted sensitivity to CD95-mediated cell death. Thus, LGP2 promotes an essential prosurvival signal in response to antigen stimulation to confer CD8(+) T cell-number expansion and effector functions against divergent RNA viruses, including West Nile virus and lymphocytic choriomeningitis virus.

B-cell selection and the development of autoantibodies.

The clearest evidence that B cells play an important role in human autoimmunity is that immunotherapies that deplete B cells are very effective treatments for many autoimmune diseases. All people, healthy or ill, have autoreactive B cells, but not at the same frequency. A number of genes influence the level of these autoreactive B cells and whether they are eliminated or not during development at a central checkpoint in the bone marrow (BM) or at a later checkpoint in peripheral lymphoid tissues. These genes include those encoding proteins that regulate signaling through the B-cell receptor complex such as Btk and PTPN22, proteins that regulate innate signaling via Toll-like receptors (TLRs) such as MyD88 and interleukin-1 receptor-associated kinase 4, as well as the gene encoding the activation-induced deaminase (AID) essential for B cells to undergo class switch recombination and somatic hypermutation. Recent studies have revealed that TLR signaling elements and AID function not only in peripheral B cells to help mediate effective antibody responses to foreign antigens, but also in the BM to help remove autoreactive B-lineage cells at a very early point in B-cell development. Newly arising B cells that leave the BM and enter the blood and splenic red pulp can express both AID and TLR signaling elements like TLR7, and thus are fully equipped to respond rapidly to antigens (including autoantigens), to isotype class switch, and to undergo somatic hypermutation. These red pulp B cells may thus be an important source of autoantibody-producing cells arising particularly in extrafollicular sites, and indeed may be as significant a source of autoantibody-producing cells as B cells arising from germinal centers.

Immune complex-mediated enhancement of secondary antibody responses.

Immunologic memory is a hallmark of the vertebrate immune system. The first antigenic exposure leads to a slow and modest immune response, whereas repeated exposure, even many years later, leads to a rapid and exaggerated response that is two to three orders of magnitude greater than the primary. In the case of humoral immunity, the increased efficacy of recall responses is due to the production of amplified levels of Ag-specific Ab, as well as the accelerated kinetics of their production. Current thinking suggests that this is due to selective activation of long-lived, Ag-specific memory B cells. A downside of restricting secondary responses solely to memory cells is that the repertoire of the memory B cell pool remains static while pathogens continue to evolve. In this study, we propose that during secondary responses, naive Ag-specific B cells participate alongside memory cells. We show that immune complexes formed in vivo between the Ag and pre-existing Abs from the primary response activate these naive B cells, inducing them to respond with accelerated kinetics and increased magnitude. Thus, the continued recruitment of new B cell clones after each antigenic exposure enables the immune system to stay abreast of rapidly changing pathogens.

Perfusion fixation preserves enhanced yellow fluorescent protein and other cellular markers in lymphoid tissues.

Fluorescent proteins are increasingly being used to analyze cellular gene expression and to facilitate tracking of cell lineages in vivo. One of these, enhanced yellow fluorescent protein (EYFP) has several properties such as intense fluorescence and little to no toxicity in cells, which makes it an excellent molecule to label proteins and cells of interest. In live cells, visualization of EYFP has been highly successful; however, detection of EYFP in lymphoid tissue sections, particularly in combination with other markers of interest has been difficult. This is because of the enhanced solubility of EYFP in the absence of fixation. When extended fixation protocols are employed, EYFP is preserved but detection of other cellular antigens becomes problematic due to over fixation. Here we demonstrate that EYFP-expressing T and B cells can be efficiently visualized in lymphoid tissue sections without compromising the ability to detect other cellular markers.

Interleukin-10 plays an early role in generating virus-specific T cell anergy.

BACKGROUND: Infection of mice with the Armstrong strain of lymphocytic choriomeningitis virus (LCMVARM) leads to a robust immune response and efficient viral clearance. This is in contrast to infection with the variant strain LCMVClone13, which causes functional inactivation of effector T cells and viral persistence. The mechanism by which LCMVClone13 suppresses the antiviral immune response and persists in its host is unknown. RESULTS: Here we demonstrate that infection with LCMVClone13, but not with LCMVARM, resulted in a steady increase in the serum levels of the immuno-inhibitory cytokine, IL-10. Blockade of IL-10 using neutralizing monoclonal antibody injections in LCMVClone13-infected mice led to dramatically enhanced effector T cell responses at 8 days post-infection. Even though IL-10 blockade resulted in decreased viral titers, the generation and maintenance of memory T cells was still compromised. The functional inactivation of CD8+ T cells in IL-10-blocked, chronically infected mice 30 days post-infection was incomplete as potent CTL (cytotoxic T lymphocytes) could be generated by in vitro re-stimulation. IL-10 knockout mice showed a similar pattern of antiviral CD8 T cell responses: early antiviral T cells were dramatically increased and viral levels were decreased; however, CD8 T cells in IL-10 knockout mice were also eventually anergized and these mice became persistently infected. CONCLUSION: Our data suggest that IL-10 plays an early role in LCMVClone13-induced tolerance, although other factors collaborate with IL-10 to induce virus-specific tolerance.

Identification of memory B cells using a novel transgenic mouse model.

Memory B cells help to protect the host from invading pathogens by maintaining persistent levels of Ag-specific serum Ab and generating rapid Ab responses upon re-exposure to Ag. Unambiguous identification of memory B cells has been a major obstacle to furthering our knowledge concerning both the development of B cell memory and secondary Ab responses due to an absence of specific cell surface markers. Germinal centers (GCs) are thought to be the major site of Ig hypermutation and Ag-driven selection of memory B cells. To develop a model that would identify GC-derived memory B cells, we generated transgenic mice that expressed cre recombinase in a GC-specific fashion. Interbreeding these mice with the cre-reporter strain, ROSA26R, produced progeny in which beta-galactosidase (beta-gal) was permanently expressed in B cells of the GC-memory pathway. Analysis following immunization with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma globulin showed that long-lived beta-gal+ B cells exclusively contained somatically mutated lambda1 V regions and were capable of producing Ag-specific Ab-forming cell (AFC) responses that were >100-fold higher than those afforded by beta-gal- B cells following adoptive transfer to naive hosts. Secondary challenge of immune mice showed that only approximately 20% of secondary AFCs expressed beta-gal. Interestingly, we found that somatic hypermutation of rearranged lambda1 V regions within secondary AFCs showed a strong correlation with beta-gal expression, suggesting that nonmutated B cells contribute significantly to secondary Ab responses. This model should provide useful insights into memory B cell development, maintenance, and differentiation following immunization or pathogenic infection.