However, anti-CD3Cactivated CD4+ T cells from mice demonstrated significantly decreased binding to immobilized ICAM-1CFc under physiologic flow conditions (Figure 4D). B cells and IgG antibody responses to TD antigen. mice. While there are data suggesting that B cells contribute to the impaired antibody response to TD antigens in DOCK8 deficiency (20), less is known about the role of T cells. We show that mice with selective DOCK8 deficiency in T cells mount poor IgG antibody responses to TD antigens, and have impaired GC formation and reduced numbers of GC B cells despite normal numbers of Tfh cells that are able to normally drive B cell differentiation in vitro. We demonstrate that activated DOCK8-deficient T cells have impaired LFA-1 activation and defective migration into GCs. Results DOCK8 expression in T cells is essential for a normal IgG antibody response to TD antigens. and mice, which lack DOCK8 only in T cells (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.134508DS1), and their controls were immunized in the hocks with the TD antigen TNP-KLH (2,4,6-trinitrophenylCkeyhole limpet hemocyanin). Serum anti-TNP IgG, but not IgM, antibody titers were significantly decreased in and mice (Figure 1A and Supplemental Figure 1B). Serum anti-TNP IgE antibody titers and IgE levels were not significantly different in mice and their controls (Supplemental Figure 1C). This may be Tartaric acid explained by known differences in the differentiation of B cells into IgE- versus IgG-secreting plasma cells (21). CD4+ T cells from TNP-KLHCimmunized mice proliferated and secreted IL-2 and IFN- normally in response to in vitro stimulation with KLH, demonstrating that they did not have a global activation defect (Supplemental Figure 1, D and E). Open in a separate window Figure 1 Impaired antibody responses to TD antigens in mice.(A) TNP-specific serum IgG levels on day 0 (unimmunized) and day 21 (immunized) from (left), mice (right), and controls after immunization in the bilateral hocks with TNP-KLH. (B) NP-specific serum IgG, TNP-specific serum IgG, and OVA-specific serum IgG measured on day 0 and day 21 after immunization of mice and controls with NP-OVA in the hocks, TNP-KLH i.p., or OVA i.p. A and B show data from 1 representative experiment of 2. = 4C5 mice/group. Data are presented as mean SEM. *< 0.05, **< 0.01, ***< 0.001 by ANOVA. The decreased IgG antibody response of mice to TNP-KLH was not specific for either antigen or route of immunization. mice had a decreased 4-hydroxy-3-nitrophenylacetyl hapten (anti-NP) IgG antibody response to hock immunization with NP-OVA in alum (Figure 1B), as well as decreased TNP- and OVA-specific IgG antibody responses to i.p. immunization with TNP-KLH and OVA in alum (Figure 1B). These findings demonstrate that DOCK8 expression in T cells is important for the IgG antibody response to TD antigen. DOCK8 expression in T cells is essential for normal GC formation and generation of GC B cells. GC development in draining LNs is first observed 2C3 days following immunization with TD antigen (12), and a mature GC forms by approximately 7 days (22). GC formation in the popliteal LN of mice was assessed by immunofluorescence microscopy on day 10 after immunization in the hock Tartaric acid with TNP-KLH in alum. GCs were significantly reduced in size in the LNs of mice compared with controls (Figure 2A). GCs are important for the formation of high-affinity IgG antibody (23). Levels of high-affinity antibodies against TNP were decreased in mice compared with controls 14 days after immunization with TNP-KLH (Figure 2B), suggesting that the smaller GCs formed in mice were less efficient in promoting antibody affinity maturation. Open in a separate window Figure 2 mice have a marked reduction in GC B cells after immunization with TD antigen.Draining LNs from and control mice immunized in the hocks with TNP-KLH were examined on day 2 for pre-GC B cells, on day 7 for GC B cells, and on day 10 for GC size. (A) Mouse monoclonal to LSD1/AOF2 Representative immunofluorescence photomicrograph of popliteal LNs (left). B cell follicles (IgD+) are in green, GCs (GL7+) in red, and the T cell zones (TCR+) in blue. Images are at 20 magnification. Quantification of GC size (right). = 4C5 mice/group in 2 pooled independent experiments. ANOVA; *< 0.05. (B) TNP-specific IgG affinity determined on day 14 after immunization. = 4 mice/group. Results are representative of 2 independent experiments. Tartaric acid (C) Representative flow cytometry plots, and percentages and numbers of.