Inotuzumab Ozogamicin Murine Analog–Mediated B-Cell Depletion Reduces Anti-islet Allo- and Autoimmune Responses

B cells participate in the priming of the allo- and autoimmune responses, and their depletion can thus be advantageous for islet transplantation. Herein, we provide an extensive study of the effect of B-cell depletion in murine models of islet transplantation. Islet transplantation was performed in hyperglycemic B-cell–deficient(μMT) mice, in a purely alloimmune setting (BALB/c into hyperglycemic C57BL/6), in a purely autoimmune setting (NOD.SCID into hyperglycemic NOD), and in a mixed allo-/autoimmune setting (BALB/c into hyperglycemic NOD). Inotuzumab ozogamicin murine analog (anti-CD22 monoclonal antibody conjugated with calicheamicin [anti-CD22/cal]) efficiently depleted B cells in all three models of islet transplantation examined. Islet graft survival was significantly prolonged in B-cell–depleted mice compared with control groups in transplants of islets from BALB/c into C57BL/6 (mean survival time [MST]: 16.5 vs. 12.0 days; P = 0.004), from NOD.SCID into NOD (MST: 23.5 vs. 14.0 days; P = 0.03), and from BALB/c into NOD (MST: 12.0 vs. 5.5 days; P = 0.003). In the BALB/c into B-cell–deficient mice model, islet survival was prolonged as well (MST: μMT = 32.5 vs. WT = 14 days; P = 0.002). Pathology revealed reduced CD3+ cell islet infiltration and confirmed the absence of B cells in treated mice. Mechanistically, effector T cells were reduced in number, concomitant with a peripheral Th2 profile skewing and ex vivo recipient hyporesponsiveness toward donor-derived antigen as well as islet autoantigens. Finally, an anti-CD22/cal and CTLA4-Ig–based combination therapy displayed remarkable prolongation of graft survival in the stringent model of islet transplantation (BALB/c into NOD). Anti-CD22/cal–mediated B-cell depletion promotes the reduction of the anti-islet immune response in various models of islet transplantation

B Cell Depletion Reduces the Number of Autoreactive T Helper Cells and Prevents Glucose-6-Phosphate Isomerase-Induced Arthritis

The therapeutic benefit of B cell depletion in patients with rheumatoid arthritis has provided proof of concept that B cells are relevant for the pathogenesis of arthritis. It remains unknown which B cell effector functions contribute to the induction or chronification of arthritis. We studied the clinical and immunological effects of B cell depletion in glucose-6-phosphate isomerase-induced arthritis. We targeted CD22 to deplete B cells. Mice were depleted of B cells before or after immunization with glucose-6-phosphate isomerase (G6PI). The clinical and histological effects were studied. G6PI-specific antibody responses were measured by ELISA. G6PI-specific T helper (Th) cell responses were assayed by polychromatic flow cytometry. B cell depletion prior to G6PI-immunization prevented arthritis. B cell depletion after immunization ameliorated arthritis, whereas B cell depletion in arthritic mice was ineffective. Transfer of antibodies from arthritic mice into B cell depleted recipients did not reconstitute arthritis. B cell depleted mice harbored much fewer G6PI-specific Th cells than control animals. B cell depletion prevents but does not cure G6PI-induced arthritis. Arthritis prevention upon B cell depletion is associated with a drastic reduction in the number of G6PI-specific effector Th cells

B Cells Regulate Neutrophilia during Mycobacterium tuberculosis Infection and BCG Vaccination by Modulating the Interleukin-17 Response

We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization. © 2013 Kozakiewicz et al

SJL/J mice are susceptible to G6PI-induced arthritis.

<p>A) Age matched female DBA/1 and SJL/J mice were immunized with 400 µg G6PI in complete Freund's adjuvant and scored for clinical arthritis severity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024718#s2" target="_blank"><i>Materials and Methods</i></a>. B) Histological features of G6PI-induced arthritis in SJL/J mice. The overview (left, ×25) shows the severe destruction of the joint. Cartilage is completely missing, and parts of the underlying bone are infiltrated as well (center, ×100). The inflammatory infiltrate consist mainly of neutrophil granulocytes, but lymphocytes and plasma cells are clearly visible (right, ×400). The underlying pannus consists of activated fibroblasts and is covered by an enlarged synovial lining. In the synovial space, a putrid exudate with numerous granulocytes is found (all figures, H&E staining).</p

B cell depletion in CD22-cal treated mice.

<p>Mice were treated with CD22-cal at days −6 and −1 and were immunized with 400 µg G6PI in complete Freund's adjuvant. Control mice were immunized but otherwise left untreated. Lymph nodes (LN), spleens and bone marrow were isolated, counted and analyzed for the percentage of CD19⁺ B and CD3⁺ T lymphocytes. Shown are the total number cells harvested (left) or the total number of B- (middle) or T-lymphocytes (right) in the various organs. Data are representative for at least three mice per time point.</p

G6PI-specific Ig-levels in CD22-cal treated mice.

<p>Mice were treated with CD22-cal as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024718#pone-0024718-g002" target="_blank">Fig. 2</a>. Sera of mice were harvested at the indicated time points. Serially diluted sera (1/100, 1/400, 1/1600, 1/6.400, 1/25.600, 1/102.400, 1/409.600) were tested by ELISA for the presence of anti-G6PI-Ig's of the indicated isotypes. Shown are the optical densities, corrected for background. The data represent three indiviual mice per group and time point.</p

Serum transfer in CD22-cal treated mice.

<p>CD22-cal treated mice (day −6, day −1) or untreated mice were immunized with G6PI. At day 9 and 11, CD22-cal treated mice received 2 injections of pooled serum from either arthritic or naïve SJL/J mice. Shown is the clinical arthritis score from all mice per group (<i>n</i> = 8 per group).</p

G6PI-induced arthritis in CD22-cal treated mice.

<p>SJL/J mice were immunized with 400 µg G6PI in complete Freund's adjuvant and treated at the indicated time points with CD22-cal. Mice were scored for clinical signs of arthritis. A) Treatment with CD22-cal before (day −6, day −1) immunization or in early preclinical stage (day 3, day 11) delay the onset and reduces the incidence of arthritis, compared to untreated controls (Left). Furthermore, prophylactic CD22-cal treatment strongly reduces the clinical severity of arthritis which still develop disease; this effect was much lower in mice treated in the induction phase (right). B) H&E stained sections were scored for the extend of acute (infiltration of neutrophil granulocytes) and chronic (infiltration of mononuclear cells) inflammation as well as joint destruction (cartilage and bone erosions) in a semiquantitative manner. For each parameter a maximum score of 4 could be reached. Shown are the sums score of all paws of individual at day 36 after immunization. C) Clinical score of mice that were treated with CD22-cal as described above at days 15 and 20 after immunization.</p

IL-22 Is Required for Imiquimod-Induced Psoriasiform Skin Inflammation in Mice.

Psoriasis is a common chronic autoimmune skin disease of unknown cause that involves dysregulated interplay between immune cells and keratinocytes. IL-22 is a cytokine produced by the TH1, TH17, and TH22 subsets that are functionally implicated in the psoriatic pathology. We assessed the role of IL-22 in a mouse model where psoriasiform skin inflammation is triggered by topical application of the TLR7/8 agonist imiquimod. At the macroscopic level, scaly skin lesions induced by daily applications of imiquimod in wild-type mice were almost totally absent in IL-22-deficient mice or in mice treated with a blocking anti-IL-22 Ab. At the microscopic level, IL-22-deficient mice showed a dramatic decrease in the development of pustules and a partial decrease in acanthosis. At the molecular level, the absence or inhibition of IL-22 strongly decreased the expression of chemotactic factors such as CCL3 and CXCL3 and of biomarkers such as S100A8, S100A7, and keratin 14, which reflect the antimicrobial and hyperproliferative responses of keratinocytes. IL-22 also played a major role in neutrophil infiltration after imiquimod treatment. IL-23 was required for IL-22 production, and γδ TCR lymphocytes represented the major source of IL-22 in lymph nodes from imiquimod-treated mice. However, T cells were not absolutely required for IL-22 production because imiquimod-induced IL-22 expression in the skin is still preserved in Rag2(-/-) mice. Taken together, our data show that IL-22 is required for psoriasis-like lesions in the mouse imiquimod model and is produced by both T cells and innate immune cells