Xid Mice Research Articles

OxPhos in adipose tissue macrophages regulated by BTK enhances their M2-like phenotype and confers a systemic immunometabolic benefit in obesity.

Bruton's tyrosine kinase (BTK) is a non-receptor bound kinase involved in pro-inflammatory signalling in activated macrophages, however, its role within adipose tissue macrophages remains unclear. We have demonstrated that BTK signalling regulates macrophage M2-like polarisation state by up-regulating subunits of mitochondrially encoded electron transport chain Complex I (ND4 and NDL4) and Complex IV (mt-CO1, mt-CO2 and mt-CO3) resulting in an enhanced rate of oxidative phosphorylation (OxPhos) in an NF-κB independent manner. Critically, BTK expression is elevated in adipose tissue macrophages from obese individuals with diabetes, while key mitochondrial genes (mtC01, mtC02 and mtC03) are decreased in inflammatory myeloid cells from obese individuals. Inhibition of BTK signalling either globally (Xid mice) or in myeloid cells (LysMCreBTK), or therapeutically (Acalabrutinib) protects HFD-fed mice from developing glycaemic dysregulation by improving signalling through the IRS1/Akt/GSK3β pathway. The beneficial effects of acalabrutinib treatment are lost in macrophage ablated mice. Inhibition of BTK signalling in myeloid cells but not B-cells, induced a phenotypic switch in adipose tissue macrophages from a pro-inflammatory M1-state to a pro-resolution M2-like phenotype, by shifting macrophage metabolism towards OxPhos. This reduces both local and systemic inflammation and protected mice from the immunometabolic consequences of obesity. Therefore, in BTK we have identified a macrophage specific, druggable target that can regulate adipose tissue polarisation and cellular metabolism that can confer systematic benefit in metabolic syndrome.

Mice with genetic and induced B-cell deficiency as a model for disseminated encephalitozoonosis

Encephalitozoon cuniculi, an intracellular pathogen, lives in a balanced relationship with immunocompetent individuals based on the activity of T lymphocytes. We previously highlighted the greater susceptibility of B-1 cell-deficient mice (XID mice) to encephalitozoonosis. This study aimed to develop a model of disseminated and severe encephalitozoonosis in mice with combined immunodeficiency to elucidate the role of B cells. To address this objective, cyclophosphamide (Cy)-treated BALB/c and XID mice were inoculated with E. cuniculi, followed by the evaluation of the immune response and histopathological lesions. Immunosuppressed BALB/c mice manifested no clinical signs with an increase in the populations of T lymphocytes and macrophages in the spleen. Immunosuppressed and infected XID mice revealed elevated T cells, macrophages populations, and pro-inflammatory cytokines levels (IFN-γ, TNF-α, and IL-6) with the presence of abdominal effusion and lesions in multiple organs. These clinical characteristics are associated with extensive and severe encephalitozoonosis. The symptoms and lesion size were reduced, whereas B-2 and CD4+ T cells populations were increased in the spleen by transferring B-2 cells adoptive to XID mice. Moreover, B-1 cells adoptive transfer upregulated the peritoneal populations of B-2 cells and macrophages but not T lymphocytes and decreased the symptoms. Herein, we speculated the consistency in the development of severe and disseminated encephalitozoonosis in mice with genetic deficiency of Bruton’s tyrosine kinase (Btk) associated with Cy immunosuppression develop with that of the models with T cell deficiency. Taken together, these data emphasized the crucial role of B cells in the protective immune response against encephalitozoonosis.

X-linked immunodeficient (XID) mice exhibit high susceptibility to Cryptococcus gattii infection

Cryptococcosis is an opportunistic disease caused by the fungus Cryptococcus neoformans and Cryptococcus gattii. It starts as a pulmonary infection that can spread to other organs, such as the brain, leading to the most serious occurrence of the disease, meningoencephalitis. The humoral response has already been described in limiting the progression of cryptococcosis where the B-1 cell seems to be responsible for producing natural IgM antibodies, crucial for combating fungal infections. The role of the B-1 cell in C. neoformans infection has been initially described, however the role of the humoral response of B-1 cells has not yet been evaluated during C. gattii infections. In the present study we tried to unravel this issue using XID mice, a murine model deficient in the Btk protein which compromises the development of B-1 lymphocytes. We use the XID mice compared to BALB/c mice that are sufficient for the B-1 population during C. gattii infection. Our model of chronic lung infection revealed that XID mice, unlike the sufficient group of B-1, had early mortality with significant weight loss, in addition to reduced levels of IgM and IgG specific to GXM isolated from the capsule of C. neoformans. In addition to this, we observed an increased fungal load in the blood and in the brain. We described an increase in the capsular size of C. gattii and the predominant presence of cytokines with a Th2 profile was also observed in these animals. Thus, the present study strongly points to a higher susceptibility of the XID mouse to C. gattii, which suggests that the presence of B-1 cells and anti-GXM antibodies is fundamental during the control of infection by C. gattii.

X-Linked Immunodeficient Mice With No Functional Bruton's Tyrosine Kinase Are Protected From Sepsis-Induced Multiple Organ Failure.

We previously reported the Bruton's tyrosine kinase (BTK) inhibitors ibrutinib and acalabrutinib improve outcomes in a mouse model of polymicrobial sepsis. Now we show that genetic deficiency of the BTK gene alone in Xid mice confers protection against cardiac, renal, and liver injury in polymicrobial sepsis and reduces hyperimmune stimulation (“cytokine storm”) induced by an overwhelming bacterial infection. Protection is due in part to enhanced bacterial phagocytosis in vivo, changes in lipid metabolism and decreased activation of NF-κB and the NLRP3 inflammasome. The inactivation of BTK leads to reduced innate immune cell recruitment and a phenotypic switch from M1 to M2 macrophages, aiding in the resolution of sepsis. We have also found that BTK expression in humans is increased in the blood of septic non-survivors, while lower expression is associated with survival from sepsis. Importantly no further reduction in organ damage, cytokine production, or changes in plasma metabolites is seen in Xid mice treated with the BTK inhibitor ibrutinib, demonstrating that the protective effects of BTK inhibitors in polymicrobial sepsis are mediated solely by inhibition of BTK and not by off-target effects of this class of drugs.

How to B(e)-1 Important Cell During Leishmania Infection.

B-1 cells are an innate-like population of B lymphocytes that are subdivided into B-1a and B-1b distinguished by the presence or absence of CD5, respectively. B-1 cells can act as regulatory B cells, are able to present antigen and produce IL-10. Leishmaniasis in humans is a complex of diseases caused by parasites of the genus Leishmania. More than 20 species can infect humans, with each species causing the development of different immunological responses in the host. Susceptibility is usually related to the production of anti-inflammatory cytokines while the production of Th1 cytokines is indicative of resistance. However, few studies have attempted to evaluate the role of B-1 cells during either the in vivo infection or in vitro interaction with Leishmania parasites. In vivo studies were performed using XID mice model, BALB/Xid mice have a mutation in the Bruton's tyrosine kinase, which is an important enzyme for developing B-1 and maturing B-2 lymphocytes leading to the presence of immature B-2 cells. Here, we compile these studies and assess the influence of B-1 cells on disease progression with different Leishmania species.

Abstract # 3226 Demyelination and remyelination following a gliotoxic injury in the brainstem of XID mice

The mechanisms by which the neurophysiological and inflammatory responses to brain injury contribute to memory impairments are not fully understood. Recently, we reported that the innate immune receptor, toll-like receptor 4 (TLR4) enhances AMPA receptor (AMPAR) currents and excitability in the dentate gyrus after fluid percussion brain injury (FPI) while limiting excitability in controls. Here, we examine the cellular mediators underlying TLR4 regulation of dentate excitability and its impact on memory performance. In ex vivo slices, astrocytic and microglial metabolic inhibitors selectively abolished TLR4 antagonist modulation of excitability in controls, but not in rats after FPI, demonstrating that glial signaling contributes to TLR4 regulation of excitability in controls. In glia-depleted neuronal cultures from naïve mice, TLR4 ligands bidirectionally modulated AMPAR charge transfer consistent with neuronal TLR4 regulation of excitability, as observed after brain injury. In vivo TLR4 antagonism reduced early post-injury increases in mediators of MyD88-dependent and independent TLR4 signaling without altering expression in controls. Blocking TNFα, a downstream effector of TLR4, mimicked effects of TLR4 antagonist and occluded TLR4 agonist modulation of excitability in slices from both control and FPI rats. Functionally, transiently blocking TLR4 in vivo improved impairments in working memory observed one week and one month after FPI, while the same treatment impaired memory function in uninjured controls. Together these data identify that distinct cellular signaling mechanisms converge on TNFα to mediate TLR4 modulation of network excitability in the uninjured and injured brain and demonstrate a role for TLR4 in regulation of working memory function.

Infection by Leishmania (Leishmania) infantum chagasi causes intestinal changes B-1 cells dependent.

Evaluating the histopathological and morphometric changes caused by Leishmania (Leishmania) infantum chagasi infection either in the presence or absence of B-1 cells. Wild-type Balb/c and XID mice were used. Half of XID mice received B-1 cells adoptive transfer (XID+B1). Five animals from each group were infected (Balb/c I, XID I and XID+B1 I), totalizing six groups (n=5). After 45days of infection, the ileum was collected for histological processing and analysis. After infection, the XID animals showed an increase in the thickness of the intestinal layers, in the depth and width of the crypt and in the villi width. However, the Balb/c I group showed a reduction in almost all these parameters, whereas the villi width was increased. The villi height decreased in the infected XID animals; however, it was increased in the XID+B1 I group. Leishmania (L) infantum chagasiinfection caused a decrease in the number of Paneth cells; however, their area was increased. Finally, goblet cells and enterocytes presented different change profiles among groups. This study showed that the parasite infection causes structural and histopathological alterations in the intestine. These changes might be influenced by the absence of B-1 cells.

B-1 Cells May Drive Macrophages Susceptibility to Trypanosoma cruzi Infection.

B-1 cells can directly and indirectly influence the immune response. These cells are known to be excellent producers of natural antibodies and can secrete a variety of immunomodulatory molecules. They are also able to differentiate into B-1 cell-derived phagocytes (B-1CDP). B-1 cells can modulate macrophages to become less effective, and B-1CDP cells are more susceptible in infection models. In this work, we investigated the microbicidal ability of these cells in Trypanosoma cruzi infection in vitro. The results show that macrophages from BALB/c mice are more susceptible to infection than macrophages from XID mice. The resistance observed in macrophages from XID mice was abolished in the presence of B-1 cells, and this event seems to be associated with IL-10 production by B-1 cells, which may have contributed to the decrease of NO production. Additionally, B-1CDP cells were more permissive to intracellular T. cruzi infection than peritoneal macrophages. These findings strongly suggest that B-1 cells and B-1CDP cells have a potential role in the persistence of the parasite in host cells.

Balb/C XID mice do not develop hypothalamic astrogliosis after a hypercaloric diet

Balb/C XID mice do not develop hypothalamic astrogliosis after a hypercaloric diet

Targeting of the Yersinia pestis F1 capsular antigen by innate-like B1b cells mediates a rapid protective response against bubonic plague

The generation of adaptive immunity by vaccination is usually a prolonged process that requires multiple dosing over several months. Hence, vaccines are administered for disease prevention a relatively long time prior to possible infection as opposed to post-exposure prophylaxis, which typically requires rapid intervention such as antibiotic therapy. The emergence of pathogens resistant to common antibiotic treatments has prompted the search for alternative therapeutic strategies. We previously demonstrated that vaccination of mice with the F1 capsular antigen of Yersinia pestis elicits specific and effective yet, unexpectedly, rapid anti-plague immunity. Here, we show by applying genetic and immunological approaches that the F1 antigen is targeted by peritoneal innate-like B1b cells that generate a prompt T-independent (TI) anti-F1 humoral response. The rapid F1-mediated defense response was diminished in Xid (Btkm) mice in which B1 cell numbers and activity are limited. Binding of fluorophore-labeled F1 to peritoneal B1b cells was detected as soon as 6 h post vaccination, emphasizing the high speed of this process. By assessing the ability to achieve rapid immunity with monomerized F1, we show that the natural polymeric structure of F1 is essential for (i) rapid association with peritoneal B1b cells, (ii) early induction of anti-F1 titers and (iii) rapid TI immunity in the mouse model of bubonic plague. These observations shed new light on the potential of novel as well as well-known protective antigens in generating rapid immunity and could be implemented in the rational design of future vaccines.

Folate Deficiency Facilitates Genomic Integration of Human Papillomavirus Type 16 DNA In Vivo in a Novel Mouse Model for Rapid Oncogenic Transformation of Human Keratinocytes

Epidemiologic and in vitro studies suggest independent linkages between poor folate and/or vitamin B-12 nutrition, genomic human papillomavirus (HPV) type 16 viral integration, and cancer. However, there is no direct evidence in vivo to support the causative role of poor folate nutrition in HPV16 integration into the cellular genome. We tested the hypothesis that folate deficiency enables the integration of HPV16 into the genome of HPV16-harboring keratinocytes, and could thereby influence earlier transformation of these cells to cancer in an animal model. HPV16-harboring human keratinocytes [(HPV16)BC-1-Ep/SL] were differentiated into 3-dimensional HPV16-organotypic rafts under either folate-replete or folate-deficient conditions in vitro. These were then subcutaneously implanted in severely immunocompromised female Beige Nude XID (Hsd: NIHS-LystbgFoxn1nuBtkxid) mice (4-6 wk old, 16-18 g) fed either a folate-replete diet (1200 nmol folate/kg diet) or a progressively folate-deficient diet (600 or 400 nmol folate/kg diet) for 2 mo prior to raft-implantation surgery, and indefinitely thereafter. The tumors that subsequently developed were characterized for onset, pattern of growth, morphology, HPV16 oncogene expression, and HPV16-genomic integration. All HPV16-organotypic rafts developed in either folate-replete or physiologic low-folate media in vitro and subsequently implanted in folate-replete mice eventually transformed into aggressive malignancies within weeks. When compared to HPV16-high folate-organotypic raft-derived tumors from mice fed either a 1200 or 600 nmol folate/kg diet, those raft-derived cancers that developed in mice fed a 400 nmol folate/kg diet expressed significantly more HPV16 E6 (1.8-fold more) and E7 (2.8-fold more) oncogenic proteins (P=0.001), and revealed significantly more HPV16-integration sites in genomic DNA (2-fold more), either directly into, or in the vicinity of, cellular genes (P<0.05). This unprecedented animal model for the consistent rapid transformation of differentiated (HPV16)BC-1-Ep/SL-derived organotypic raft-keratinocytes to cancer in Beige Nude XID mice confirms that dietary folate deficiency can profoundly influence and modulate events leading to HPV16-induced carcinogenesis, and facilitates genomic integration of HPV16 DNA in vivo.

B-1 cells upregulate CD8 T lymphocytes and increase proinflammatory cytokines serum levels in oral encephalitozoonosis

Microsporidia are intracellular pathogens that cause severe disease in immunocompromised humans and animals. We recently demonstrated that XID mice are more susceptible to Encephalitozoon cuniculi infection by intraperitoneal route, evidencing the role of B-1 cells in resistance against infection. The present study investigated the resistance and susceptibility against E. cuniculi oral infection, including the role of B-1 cells. BALB/c and BALB/c XID (B-1 cells deficient) mice were orally infected with E. cuniculi spores. No clinical symptoms were observed in infected animals; histopathology showed lymphoplasmocytic enteritis with degeneration of the apexes of the villi in all infected groups. Higher parasite burden was observed in infected BALB/c XID mice. In the spleen and peritoneum, all infected mice showed a decrease of lymphocytes, including CD8+ T cells, mostly in infected BALB/c XID mice. Adoptive transfer of B-1 cells (XID + B-1) was associated with a lower parasite burden. Pro-inflammatory cytokines (IFN-γ, TNF-α and IL-6) increased mostly in infected XID + B1 mice. Together, the present results showed that BALB/c XID mice infected by the oral route were more susceptible to encephalitozoonosis than BALB/c mice, demonstrating the B-1 cells importance in the control of the immune response against oral E. cuniculi infection.

Mediation of transitional B cell maturation in the absence of functional Bruton\u2019s tyrosine kinase

X-linked immune-deficient (Xid) mice, carrying a mutation in Bruton’s tyrosine kinase (Btk), have multiple B cell lineage differentiation defects. We now show that, while Xid mice showed only mild reduction in the frequency of the late transitional (T2) stage of peripheral B cells, the defect became severe when the Xid genotype was combined with either a CD40-null, a TCRbeta-null or an MHC class II (MHCII)-null genotype. Purified Xid T1 and T2 B cells survived poorly in vitro compared to wild-type (WT) cells. BAFF rescued WT but not Xid T1 and T2 B cells from death in culture, while CD40 ligation equivalently rescued both. Xid transitional B cells ex vivo showed low levels of the p100 protein substrate for non-canonical NF-kappaB signalling. In vitro, CD40 ligation induced equivalent activation of the canonical but not of the non-canonical NF-kappaB pathway in Xid and WT T1 and T2 B cells. CD40 ligation efficiently rescued p100-null T1 B cells from neglect-induced death in vitro. These data indicate that CD40-mediated signals, likely from CD4 T cells, can mediate peripheral transitional B cell maturation independent of Btk and the non-canonical NF-kappaB pathway, and thus contribute to the understanding of the complexities of peripheral B cell maturation.

Characterization of Immunodeficient Mouse Models

Leakiness in SCID mouse models refers to the occasionally productive VDJ rearrangement leading to clonal expansion of these limited B and T cell clones. The rate and extent of leakiness in immunodeficient models is a factor of age as well as housing environment and the background strain of the model. In the current study, serum IgG was used to determine the extent of leakiness in various immunodeficient mouse models; survival and lymphoma incidence was also analyzed. Male and female (n=23–26) C.B‐17‐SCID, NOD SCID, SCID Beige, SHrN®, Beige Nude XID and Nude mice were housed in flexible film isolators. Blood was collected and analyzed using dried blood spot technology at 12, 16, 20 and 24 weeks of age for IgG concentration analysis using multiplex fluorescent immunoassay. The leakiness threshold was set at 5 μg/mL of IgG for analysis. At 24 weeks of age, necropsies were performed to determine incidence of lymphoma. At 24 wk of age, the SHrN® model had the smallest percentage of leaky animals followed closely by the NOD SCID. Beige Nude XID mice display an age associated increase in serum IgG levels as well as an increasing percentage of animals above the 5 μg/mL threshold from 12–24 weeks of age. Nude mice also show increases in IgG from 12 to 24 weeks of age. The NOD SCID and SHrN® mice had the lowest survival rate at 24 weeks; however, survivability in all models was greater than or equal to 79%. There is a positive correlation between the percentage of leaky animals and survival in males and females across all models. The C.B17‐SCID shows low incidence of lymphoma at 24 weeks of age whereas the NOD SCID model has the highest incidence of lymphoma but when the hairless mutation is crossed onto the NOD SCID mouse to create the SHrN®, incidence of lymphoma is decreased. This is the first study reporting IgG concentration in multiple immunodeficient models to examine leakiness as well as 24 week survival and lymphoma incidence. Additional studies measuring immune cell activity and function will be useful to further characterize immunodeficient models; however, these data will be valuable information in determining the best model for xenograft research.Support or Funding InformationThis research was privately funded by Envigo.

B-1 cells modulate the murine macrophage response to Leishmania major infection.

AIMTo investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major (L. major) in vitro.METHODSPeritoneal macrophages obtained from BALB/c and BALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10 (IL-10) production was quantified in the cellular supernatants using an enzyme-linked immunosorbent assay. The levels of the lipid mediator prostaglandin E2 (PGE2) were determined using a PGE2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE2-neutralizing drugs inhibited PGE2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major.RESULTSWe report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major-infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE2 in supernatants of L. major-infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major-infected macrophages when the B-1 cells were isolated from IL-10 knockout mice.CONCLUSIONOur results show that elevated levels of PGE2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell cultures.

Exploring the Functional Relevance of BTK Beyond Chronic Lymphocytic Leukemia (CLL) Cells: BTK Expression in Non-Malignant Immune Cells of the Microenvironment Mediates CLL Development and Progression In Vivo

Background:Bruton's tyrosine kinase (BTK) is a critical component of the BCR pathway, but BTK and/or BTK-related enzymes such as ITK, Tec, BMX and RLK are also expressed in T, NK and myeloid cells, all of which are important sources of CLL-associated immune dysfunction. Whether BTK and/or BTK-related enzymes within non-malignant immune cells have any effects on CLL progression and immune function is not known. This is highly relevant translationally, as newer generations of BTK inhibitors selectively target BTK while sparing BTK-related enzymes in other cells. As the effects of constitutively active BCR and BTK targeting can be adequately modelled in the TCL-1 and TCL-1xXID mouse models (Woyach et al. Blood 2014), we used these preclinical tools to investigate if BTK expression in non-malignant immune cells modulates CLL progression in vivo. Our aims were: (1) to characterize differences in phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic WT and BTK-inactive mice; (2) to assess BTK-related PD-L1 expression; and (3) to evaluate the in vivo relevance of BTK in non-malignant immune cells to CLL progression.Methods:Single-cell suspension cells were obtained from spleens from CLL-free matched WT B6 and BTK-inactive XID mice (n=11 each) and leukemic TCL-1xXID and TCL-1 mice (n=10 each). Flow cytometry was used to characterize the phenotype of B, T, NK and myeloid cells and myeloid-derived suppressor cells (MDSCs), PD-L1 expression, and T-cell degranulation. For survival experiments, WT B6 (n=29) and XID (n=27) mice were injected with 5x106 splenocytes from leukemic TCL1 donors and monitored daily. For in vivo depletion of monocytes/ macrophages, WT and XID (both n=12) mice received clodronate i.p. every 3 days from day 1 after injection of 3x107 splenocytes from leukemic TCL1 donors, and were euthanized after 36 days of treatment. RT qPCR was conducted on BTK-deficient XLA cell lines.Results:To explore whether BTK expression in non-malignant immune cells affects CLL development in vivo, we first conducted adoptive transfer experiments into WT and BTK-inactive XID mice. While WT mice had a median survival of 52 days after CLL development, survival was almost twice as long (90 days) in XID mice, indicating that BTK inactivation in non-leukemic cells prolongs survival. To identify whether this could be explained by differences in immune cell populations, we next characterized phenotype and function of T, NK and myeloid cells in leukemia-free and leukemic BTK functional and BTK-inactive mice. This comparison revealed that CLL-induced T-cell changes were broadly recapitulated in BTK-inactive mice, while significantly more immature (p=0.0206) and mature (p=0.008) NK cells were maintained. In the myeloid compartment, BTK activity did not affect the overall quantity of CD11b+ myeloid cells, but led to significant qualitative CLL-associated changes: while mice with functional BTK exhibited a significant CLL-driven loss of neutrophils (p=0.0002) and expansion of F4/80+CD11b+ macrophages (p=0.0001), leukemic BTK-inactive mice largely maintained these populations, suggesting anti-tumor properties of these cells. We next depleted myeloid cells with presumed BTK-associated anti-tumor properties in CLL-engrafted mice using clodronate. After 36 days of treatment, differences in spleen weights, CLL load in spleen and blood, and percentage of proliferating CLL cells were lost between WT and XID mice (all p>0.05), indicating that monocytes/ macrophages in XID mice indeed have in vivo anti-tumor functions. Neutrophils were expanded in both WT (median 44.7% of CD11b+ cells) and XID mice (43.7% vs. 6.1% in non-depleted mice). Interestingly, PD-L1 expression as a surrogate marker for tumor-associated immune suppression was generally significantly increased on antigen-presenting cells from BTK-inactive leukemic mice (CLL cells p=0.0002; inflammatory monocytes p=0.0014; patrolling monocytes p=0.001; MDSCs p=0.0201). RT qPCR experiments using XLA cell lines with modified BTK expression revealed that absence of BTK was associated with significantly higher PD-L1 RNA expression (p=0.0075), indicating a direct mechanistic link between BTK and PD-L1.Conclusions: Our data indicate that BTK in myeloid and NK cells directly mediates CLL development and progression in vivo. Targeting BTK in those cells in addition to CLL cells might provide substantial clinical benefits. DisclosuresWoyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.

Ibrutinib Potentiated NK Cell-Mediated Cytotoxicity in Mouse Models of B-Cell Lymphomas

Background:Ibrutinib, a first-in-class, once-daily oral inhibitor of Bruton’s tyrosine kinase (BTK), is indicated by the US FDA for the treatment of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma, including patients with deletion 17p, patients with mantle cell lymphoma who have received at least 1 prior therapy, and patients with Waldenström’s macroglobulinemia. In addition to blocking B-cell activation via inhibition of BTK signaling, ibrutinib was reported to modulate immune function by driving TH1 response through IL-2-inducible T-cell kinase (Dubovsky, Blood 2013). Ibrutinib was also shown to reprogram macrophages toward a TH1 phenotype that fostered CD8+ T-cell cytotoxicity in pancreas ductal adenocarcinoma-bearing mice (Gunderson, Cancer Discov 2016). Both the direct and indirect effects on TH1 response may play a role in the therapeutic efficacy of ibrutinib in models of lymphoma and solid tumors (Sagiv-Barfi, PNAS 2015). Another essential component of the immune system with a role against cancer is natural killer (NK) cells involved in the recognition and elimination of tumor cells. In this study, we evaluated the effects of ibrutinib on NK cell-mediated cytotoxicity in mouse models of B-cell lymphoma.Methods:Experiments in TMD8 xenograft models were performed in CB17.SCID and NSG mice, and experiments in an A20 syngeneic model were performed in BALB/c mice. Mice were orally administered ibrutinib (once daily) for a total duration of 2 weeks starting from the time tumors reached a volume of 100-150 mm3. Tumor BTK occupancy was determined by a gel-based probe assay 4 hours after the last dose of ibrutinib, and was normalized to the total BTK level. NK cell-mediated cytotoxicity was also evaluated in mice carrying X-linked spontaneous mutation in BTK, Btk(xid) mice (009361; Jackson Laboratory). For the NK cell cytotoxicity assay, mouse splenocytes were added to PKH67-labeled YAC-1 cells at different effector-to-target ratios and incubated at 37°C for 4 hours. Propidium iodide was added and flow cytometry was performed to determine target cell viability. Human or mouse cytokines/chemokines were quantified using MILLIPLEX® MAP Kit (HCYTMAG-60K-PX38 and MCYTMAG-70K-PX32).Results:In TMD8 ABC-DLBCL models, tumor suppression after ibrutinib treatment was only observed in CB17.SCID mice but not in NSG mice despite tumor BTK occupancy of >90% in both models. As these 2 strains of mice differ in their NK cell profile (NSG mice lack mature T, B, and NK cells whereas CB17.SCID mice are severely deficient in T and B cells), we were interested in studying the immune modulation function of ibrutinib on NK cells. Interestingly, elevated NK cell cytotoxicity was seen in Btk(xid) mice compared to Btk wild-type mice, suggesting a potential role of BTK depletion in NK cell function. Treatment with ibrutinib increased NK cell-mediated cytotoxicity in both syngeneic A20 B-cell lymphoma and TMD8 xenograft models (Figure 1), but did not affect NK cell population (% of NKp46+ or CD3-CD49b+). A20 tumor-bearing mice had lower NK cell cytotoxicity compared to non-tumor-bearing mice, suggesting suppression of NK cell function by tumors. In addition, ibrutinib reduced tumor-derived cytokines IL-6 and IL-10, which are negative regulators of NK cells. IFN-gamma secreted by nontumor cells was increased in the sera of CB17.SCID but not in NSG mice after ibrutinib treatment, providing further evidence of the increased NK function with ibrutinib. The detailed mechanisms of modulation of NK cell function by ibrutinib is currently under investigation.Conclusions:We report herein that ibrutinib enhanced NK cell-mediated cytotoxicity in mouse models of B-cell lymphomas. In addition to its direct effect on BTK inhibition in tumor cells, these data provide further evidence of the immune modulation function of ibrutinib. The role of ibrutinib in NK cell activation as observed in the current study may further expand the potential application of ibrutinib therapy. [Display omitted] DisclosuresKuo:Pharmacyclics, LLC, an AbbVie Company: Employment, Other: Travel, Accommodations, and Expenses, Patents & Royalties: Pharmacyclics, LLC, an AbbVie Company; AbbVie: Equity Ownership. Hsieh:Pharmacyclics, LLC, an AbbVie Company: Employment. Whang:Pharmacyclics, LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Huang:Juno: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment; Five Prime: Equity Ownership; Merrimack: Equity Ownership. Sirisawad:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment. Chang:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment, Patents & Royalties: Pharmacyclics, LLC, an AbbVie Company.

B-1 cell decreases susceptibility to encephalitozoonosis in mice

Encephalitozoon cuniculi is an opportunist intracellular pathogen of mammals. The adaptive immune response is essential to eliminate E. cuniculi, but evidence is mounting that the response initiated by the innate immune response may ultimately define whether or not the parasite can survive. B-1 cells may act as antigen-presenting cells or differentiate into phagocytes, playing different roles in many infection models. However, the role of these cells in the dynamics of Encephalitozoon sp. infections is still unknown. To investigate the role of B-1 cells in E. cuniculi infection, BALB/c and BALB/c XID (B-1 cells deficient) mice were infected with E. cuniculi spores. Cytometric analyses of peritoneal cells showed that B-1 cells and macrophages increased significantly in infected BALB/c mice compared to uninfected controls. Despite the increase in the number of CD4+ and CD8+ lymphocytes in XID mice, these animals were more susceptible to infection as evidenced histologically with more prominent inflammatory lesions and parasite burden. Pro-inflammatory cytokines increased in both infected BALB/c and BALB/c XID mice. To confirm B-1 cells role in encephalitozoonosis, we adoptively transferred B-1 cells to BALB/c XID mice and this group showed few symptoms and microscopic lesions, associated with an increased in cytokines. Together, these results suggest that B-1 cells may increase the resistance of BALB/c mice to encephalitozoonosis, evidencing for the first time the important role of B-1 lymphocytes in the control of microsporidia infection.

Infection with Paracoccidioides brasiliensis induces B-1 cell migration and activation of regulatory T cells

Paracoccidioidomycosis is a systemic mycosis endemic to Latin America. The infection is initiated by inhalation of conidia into the lung and may develop as localized or disseminated disease depending on the depression of cellular immunity. In the present study, we observed that intratracheal infection with Paracoccidioides brasiliensis caused the percentage of resident B-1 cells to decrease. Using xid mice reconstituted with B-1 cells, we observed that B-1 cells migrate to the spleen and stimulate increases in the regulatory T cell subpopulation in response to P. brasiliensis infection. Collectively, these data demonstrate an active role for B-1 cells in susceptibility to paracoccidioidomycosis.

Abstract 553: Ibrutinib, a BTK inhibitor, impairs the generation and function of myeloid derived suppressor cells

Abstract Myeloid derived suppressor cells (MDSC) interfere with anti tumor immune responses. MDSC have also been shown to antagonize the effectiveness of immune based therapies including immune checkpoint blockade. As a result, MDSC have received attention as potential targets for immune based combination therapies. There has been limited success in the identification of clinically active agents with the ability to inhibit the function or generation of MDSC. Ibrutinib is an orally available irreversible inhibitor of Bruton's tyrosine kinase (BTK) that is FDA approved for the treatment of B cell malignancies. In addition to B cells, cells of the myeloid lineage including monocytes and macrophages express BTK, and treatment with ibrutinib has been shown to alter their function and differentiation. As a result, it was hypothesized that ibrutinib would interfere with the function or generation of MDSC in the setting of cancer. MDSC isolated from the spleens of multiple murine tumor models (EMT6, 4T1, and C26) as well as MDSC from patients with metastatic melanoma expressed BTK. Treatment with ibrutinib at doses ranging from 0.1-5 μM inhibited the phosphorylation of BTK in both murine and human MDSC. Ibrutinib treatment of murine and human MDSC resulted in a significant reduction in nitric oxide (NO) production (p&amp;lt; 0.05), but had only modest effects on MDSC levels of IDO and arginase. Ibrutinib was also able to inhibit murine MDSC migration in response to EMT6 cell line conditioned media and the chemokine CXCL12 (p&amp;lt; 0.05). In addition, ibrutinib inhibited human MDSC migration in response to GM CSF (p&amp;lt; 0.05). Ibrutinib reduced the expression of the myeloid adhesion molecules CD11a (p&amp;lt; 0.05) and CD49D (p&amp;lt; 0.01) by MDSC, which could explain the reduction in migration. Importantly, ibrutinib significantly reduced the ability of MDSC to suppress CD8+ T cell proliferation compared to DMSO (21.98% vs. 12.49% proliferation, p&amp;lt; 0.05). Daily treatment with ibrutinib effectively inhibited the in vitro generation of human MDSC from monocytes by promoting HLA DR expression (p&amp;lt; 0.05). Using the EMT6 mammary carcinoma model in vivo, ibrutinib treatment resulted in a significant reduction of MDSC in both the spleen and tumor (p&amp;lt; 0.05). Ibrutinib also reduced the frequency of splenic MDSC in wild type B16F10 tumor bearing mice, but not in BTK mutant XID mice. In addition, both murine and human MDSC did not express significant levels of alternative ibrutinib targets including ITK, Bmx, and Blk. These results suggest that inhibition of BTK is the primary driver behind the observed effects of ibrutinib on MDSC function and generation. Finally, the combination of ibrutinib and anti PDL1 therapy was significantly more effective than either agent alone (p&amp;lt; 0.01 and p&amp;lt; 0.05) producing complete tumor regression in 50% of EMT6 tumor being mice. The results support further investigation of ibrutinib in combination with immune based therapies for solid tumors. Citation Format: Andrew R. Stiff, Prashant Trikha, Robert Wesolowski, Kari Kendra, Sarvani Uppati, David Abood, Elizabeth McMichael, Megan Duggan, Amanda Campbell, Natarajan Muthusamy, Susheela Tridandapani, Michael Caliguiri, John C. Byrd, William E. Carson. Ibrutinib, a BTK inhibitor, impairs the generation and function of myeloid derived suppressor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 553.