Defective T-cell help to B cells and germinal center formation following immunization, but normal antiviral responses in r1ΔT/r2n mice. (A) Serum antibody levels in mice injected with NP-Ficoll (left panel) or NP-OVA (right panel) are shown from WT versus r1ΔT/r2n (Cre⁺) mice. Data are shown as means ± SEM of 4 or 5 mice per genotype using serum dilutions in the linear range of detection. (B) Representative spleen sections of NP-OVA–immunized WT or Cre⁺ mice stained with B220-PE to identify B cells and PNA-FITC to identify germinal centers. Quantification of 4 WT and 7 Cre⁺ sections revealed a significant reduction in germinal center number (mean ± SEM of WT versus Cre⁺, 6.5 ± 0.6 versus 4.4 ± 0.6; P < .05) despite equivalent numbers of follicles (8.5 ± 1 versus 11.3 ± 1.2; P > .1). Germinal center size, measured by counting total green pixels and dividing by germinal center number, was also significantly reduced (2897 ± 513 versus 1463 ± 271; P < .01). Images were visualized using a Nikon Eclipse TE1000-S microscope equipped with a Nikon 4×/0.1 numerical aperture Plane objective (Nikon, Melville, NY). Images were captured using a Phonometrics CoolSnap ES camera (Roper Scientific, Ottobrunn, Germany) and Media Cybernetics ImagePro Express software version 5.1.1.14 (Media Cybernetics, Silver Spring, MD). Images were overlaid in Adobe Photoshop CS2 (Adobe, San Jose, CA); measurements were made using ImageJ software version 1.37 (National Institutes of Health, Bethesda, MD). (C) Flow cytometric tetramer staining of splenic CD8⁺ T cells (i), intracellular measurement of IFN-γ production (ii), and CTL assay (iii) conducted 7 days after intraperitoneal infection of MHV in WT or r1ΔT/r2n (Cre⁺) mice. Tetramer stains are of sham and infected animals of each genotype and are representative of 5 mice per genotype. The percent of tetramer-positive cells is shown in blue. CTL and ICS data are shown as the mean ± SEM of 3 to 5 mice per genotype (iv, v). Viral titer in WT and Cre⁺ mice after intracranial infection. Brain (iv) and liver (v) titers were measured at days 5 and 12 after infection. Data are shown as the mean ± SEM of 3 to 5 mice. Similar responses were observed in r1f/r2n mice.