Volume 4, Issue 3, Pages 263-273 (March 1996) Disruption of CD40–CD40 Ligand Interactions Results in an Enhanced Susceptibility to Leishmania amazonensis Infection  Lynn Soong, Jian-Chao Xu, Iqbal S Grewal, Peter Kima, Jiaren Sun, B.Jack Longley, Nancy H Ruddle, Diane McMahon-Pratt, Richard A Flavell  Immunity  Volume 4, Issue 3, Pages 263-273 (March 1996) DOI: 10.1016/S1074-7613(00)80434-3

Figure 1 The Course of L. amazonensis Infection in Wild-Type and CD40L−/− Mice Mice (8 per group) were infected with 2 × 106 late-log phase parasites subcutaneously into the right hind footpad. The course of infection was monitored by measuring footpad swelling, which is expressed as a ratio of the infected right foot to that of the uninfected left foot. The mean ± SEM are shown from one of three similar experiments. Asterisk, indicates lesions with surface ulceration. Immunity 1996 4, 263-273DOI: (10.1016/S1074-7613(00)80434-3)

Figure 2 Histopathology of Infected Tissues from Wild-Type and CD40L−/− Mice during Infection with L. amazonensis Sections of foot or draining LN were prepared at indicated weeks after infection and stained with hematoxylin and eosin. (a) Foot section from wild-type mice at 4 weeks postinfection shows infected MΦs (arrows). Original magnification, 200×. (b) Foot section from CD40L−/− mice at 4 weeks postinfection shows heavily infected MΦs (arrow). Original magnification, 200×. (c) Demonstrates surface ulceration (arrow) and extensive necrosis in foot from wild-type mice at 12 weeks postinfection. Original magnification, 5×. (d) Neither ulceration nor necrosis were observed in infected foot from CD40L−/− mice at 12 weeks postinfection (as compared with [c]). Original magnification, 5×. (e) LN from wild-type mice at 12 weeks postinfection occasionally showed limited organisms in a multinucleated giant cell (arrow), and plasma cells (small arrow). Original magnification, 200×. (f) LN from CD40L−/− mice at 12 weeks postinfection shows heavily infected MΦs (arrow), and surrounding lymphocytes with barely visible cytoplasm. Original magnification, 200×. Immunity 1996 4, 263-273DOI: (10.1016/S1074-7613(00)80434-3)

Figure 3 Draining LN Cells from L. amazonensis–Infected CD40L−/− Mice Produce No Measurable Levels of IFNγ or LT–TNF Single cell suspensions (1 × 106/ml) were prepared from groups of 2–4 wild-type (closed symbols) or CD40L−/− mice (open symbols) at 8 weeks postinfection and were stimulated with indicated doses of promastigote lysates for 72 hr. Supernatants were harvested and assayed individually for IFNγ (A) or LT–TNF (B) activities using a specific two-site ELISA or a cytotoxicity assay, respectively. Values are expressed in mean ± SD. Immunity 1996 4, 263-273DOI: (10.1016/S1074-7613(00)80434-3)

Figure 4 In Vivo Expression of Lymphokine Transcripts in the Draining LN Cells from Wild-Type and CD40L−/− Mice LNs were harvested from groups of 2–3 wild-type or CD40L−/− mice at 8 weeks postinfection. The total RNA was isolated immediately. After reverse transcription from equivalent amounts of total RNA, a competitive PCR assay was performed using cDNAs and various concentrations of the polycompetitor construct (pQRS) containing sequences for IFNγ, IL-4, IL-2, and HPRT. In each reaction, lanes 1 and 7 contained 1.8 ng/ml of the plasmid pQRS and the following lanes contained serial 2-fold dilutions of pQRS. The amplification products from all samples (lower bands in each lane) were separated from amplified competitor sequences (upper bands) in ethidium bromide–stained agarose gels. The comparison between amplifed products from control plasmids and from samples is based upon the overall intensity. DNA MW makers (100 bp ladder, GIBCO) are shown from 100–600 bp. Immunity 1996 4, 263-273DOI: (10.1016/S1074-7613(00)80434-3)

Figure 5 P8-Immunized CD40L−/− Mice Are Not Protected against Challenge with L. amazonensis Groups of 8 wild-type (A) or CD40L−/− mice (B) were immunized by three intraperitoneal injections of purified P8 plus C. parvum (closed symbols). Control groups were immunized similarly with C. parvum (open symbols) alone. The mice were challenged 3 weeks after the last immunization with 105 late-log phase promastigotes of L. amazonensis in the right hind foot. The course of infection was monitored by measuring footpad swelling, as indicated in Figure 1. Asterisk, indicates lesions with surface ulceration. (C) At 8 and 11 weeks postinfection, 4 mice from each group were sacrificed. Lesion parasite burdens were determined using a limiting dilution assay and are expressed as numbers of parasites (in log scale) per gram of foot tissue. Values represent the mean from each group. Immunity 1996 4, 263-273DOI: (10.1016/S1074-7613(00)80434-3)

Figure 6 Impaired Nitrite Production in MΦs from Infected CD40L−/− Mice At 13 weeks postinfection, peritoneal MΦs from groups of two infected wild-type (closed bars) or CD40L−/− mice (open bars) were collected and cocultured in 24-well plates (5 × 105 cells/ml/well) with 7 × 105 draining LN cells from either wild-type or CD40L−/− mice in the presence of indicated concentrations of parasite lysates. Culture supernatants were collected after 72 hr. Nitrite concentration was measured using the Griess reaction and calculated by comparison to NaNO2 as a standard. Shown is one of two similar experiments. Immunity 1996 4, 263-273DOI: (10.1016/S1074-7613(00)80434-3)

Figure 7 MΦs from Naive CD40L−/− Mice Are Not Defective in Nitrite Production and B7-2 Expression Peritoneal MΦs from 3–4 naive wild-type (closed symbols) and CD40L−/− mice (open symbols) were collected and cultured for 48 hr in 96-well plate (1 × 105 cells/well) in the presence or absence of different concentrations and combinations of rIFNγ and LPS (A) or rIFNγ and rTNFα (B). The supernatants were collected and nitrite concentration was determined as indicated in Figure 6. There was no measurable level of nitrite in supernatants from cells culured in medium alone, and no significant level in cells cultured with 0.1–100 U/ml of rIFNγ alone (data not shown). Similar patterns of nitrite production were observed in thioglycollate-induced peritoneal MΦs and bone marrow–derived MΦs (data not shown). Peritoneal MΦs from wild-type (C) and CD40L−/− (D) mice were kept on ice or incubated with LPS for 18 hr. Cells were collected and stained as in Experimental Procedures. One-color profiles of PE and FITC staining were generated by gating on FITC–MAC-1+ cells. Thin and thick lines represent cells stained with the control or anti-B7-2 antibodies, respectively. Immunity 1996 4, 263-273DOI: (10.1016/S1074-7613(00)80434-3)