Scavenger receptor A (SR-A)^−/− mice mount normal primary and memory CD8⁺ T-cell (T_CD8+) immune responses upon viral challenge. SR-A^−/− (filled bars) and wild-type (open bars) mice were immunized with recombinant vaccinia virus expressing ovalbumin (rVACV-OVA) (a, c) or influenza A/PR8 (b, d) to measure primary (a, b) and memory (c, d) T_CD8+ responses. The ex vivo effector function of VACV or influenza-specific T_CD8+ via interferon (IFN)-γ production in response to numerous viral peptides was examined 7 days post immunization for primary responses or 30 days post immunization for memory responses. The total number of viral-specific T_CD8+ was measured. NS, not significant (P > 0·05). NP, nucleoprotein; PA, acid polymerase.

Scavenger receptor A (SR-A) is a receptor for glycoprotein 96 (gp96). Wild-type and SR-A^−/− bone marrow-derived dendritic cells (BMDCs) were examined for their ability to bind fluorescently labelled gp96 (a, b, d, e) or calreticulin (CRT) (c). (a) Histograms of background fluorescence (dashed line), binding to SR-A^−/− (solid grey line) or wild-type BMDCs (solid black line). (b) The mean fluorescence intensity (MFI) of these data. (c) Similar data after pulsing with CRT. **P < 0·05. Fluorescence microscopy was used to measure gp96 binding to wild-type (d) or SR-A^−/− (e) BMDCs in the presence or absence of fucoidin, a competitive inhibitor of SR-A binding.

Scavenger receptor A (SR-A)^−/− bone marrow-derived dendritic cells (BMDCs) are efficient in the uptake of glycoprotein 96 (gp96) and the processing and presentation of gp96-bound peptides to CD8⁺ T cells (T_CD8+). 5-(and 6-)carboxyfluorescein diacetate–succinimidyl ester (CFDA-SE)-labelled OT-1.SJL splenocytes were adoptively transferred into naïve recipient mice. Wild-type (a–d) and SR-A^−/− (e–h) BMDCs were pulsed with 10 μg of gp96 (a, c, e, g) or gp96 ovalbumin (OVA)_257–264 (b, d, f, h) at 4° (a, b, e, f) or 37° (c, d, g, h) for 30 min and transferred intravenously into recipient wild-type mice. Proliferation of adoptively transferred OT-1.SJL T_CD8+ was determined via CFDA-SE dye dilution 72 hr post immunization. The proportion of adoptively transferred cells proliferating was not significantly different (P > 0·05) between panels (b) and (f), or (d) and (h). Mice were immunized with wild-type or SR-A^−/− BMDCs pulsed with gp96-OVA_257–264 (i) in the absence (closed bars) or presence (open bars) of fucoidin. Seven days post immunization, CFDA-SE-labelled B6.SJL targets pulsed with relevant or irrelevant peptide were adoptively transferred into wild-type or SR-A^−/− mice. Effector function was measured as the percentage of targets killed. **P < 0·05; *P < 0·06; NS, not significant (P > 0·05).

Scavenger receptor A (SR-A)^−/− mice do not express SR-A, have normal levels of major histocompatibility complex class I (MHC-I) and mount normal primary peptide–specific CD8⁺ T-cell (T_CD8+) immune responses. Macrophages from wild-type (WT) mice (a, b) or SR-A^−/− mice (c) were stained with anti-Cd11b antibody alone (a) or anti-Cd11b antibody in tandem with anti-SR-A antibody (b, c) to measure SR-A expression levels via flow cytometry. MHC-I cell surface expression on wild-type (dotted line) or SR-A^−/− (thin solid line) strains (d) by fluorescence-activated cell sorting (FACS) analysis was compared with background levels (shaded). Wild-type or SR-A^−/− mice were immunized with ovalbumin (OVA)_257–264 peptide + lipopolysaccharide (LPS) + anti-CD40 antibody and the ex vivo effector function of OVA-specific T_CD8+ was measured via interferon (IFN)-γ production by enzyme-linked immunosorbent assay (ELISA) in response to OVA_257–264 peptide 5 days post immunization (e). **P < 0·05; NS, not significant (P > 0·05). MFI, mean fluorescence intensity.

Scavenger receptor A (SR-A)^−/− mice are efficient in the cross presentation of exogenous antigen to CD8⁺ T cells (T_CD8+). 5-(and 6-) carboxyfluorescein diacetate–succinimidyl ester (CFDA-SE)-labelled OT-1.SJL (a–c) or F5.SJL (d–g) splenocytes were adoptively transferred into wild-type (a, b, d, e, f) or SR-A^−/− (c, g) recipient mice. Recipient mice were immunized with β₂-microglobulin (β₂m)^−/− cells loaded with ovalbumin (OVA) (a–c) or with adenovirus (Ad; Adeno) expressing nucleoprotein (NP) driven by the cytomegalovirus (CMV) (d), keratin 14 (K14) (e, g) or surfactant protein C (SPC) (f) promoters. Proliferation of adoptively transferred T_CD8+ was determined 72 hr post immunization. The proportion of adoptively transferred cells proliferating was not significantly different (P > 0·05) between panels (e) and (g). Effector activity (h, i) of NT60 NP-specific T_CD8+ in wild-type (closed bars) or SR-A^−/− (open bars) mice was measured via production of interferon (IFN)-γ after recipient mice had been immunized intradermally with Ad expressing NP as described above. Spleens were harvested 6 days post immunization and T_CD8+ grown for 7 days. The percentage (h) and number (i) of IFN-γ-producing T_CD8+ were measured using peptide pulsation. NS, not significant (P > 0·05).

Scavenger receptor A (SR-A)^−/− mice are able to respond to glycoprotein 96 (gp96) and calreticulin (CRT) coupled to peptide as efficiently as wild-type mice. 5-(and 6-)carboxyfluorescein diacetate–succinimidyl ester (CFDA-SE)-labelled OT-1.SJL (a–j) or unlabelled OT-1 (m) splenocytes were adoptively transferred into wild-type [a–e, m (filled bars)] or SR-A^−/− [f–j, m (open bars)] mice. Recipients were immunized intradermally with gp96 (a, f), or 10 μg (b, g), 2 μg (c, h), 0·4 μg (d, i), or 0·08 μg (e, j) of gp96 ovalbumin (OVA)_257–264 or as shown (m). Proliferation of adoptively transferred OT-1.SJL CD8⁺ T cells (T_CD8+) was determined 72 post immunization. The proportion of adoptively transferred cells proliferating was not significantly different (P > 0·05) between panels (b) and (g), or (c) and (h), or (d) and (i), or (e) and (j). Wild-type and SR-A^−/− recipient mice were immunized with CRT-OVA20 (k, l) in the presence of lipopolysaccharide (LPS) (k) or poly I:C (l) or with recombinant vaccinia virus expressing ovalbumin (rVACV-OVA), 10 μg of gp96, or 10 μg of gp96 OVA_257–264 (m). Four (k, l) or seven days (m) post immunization, CFDA-SE-labelled B6.SJL targets pulsed with relevant or irrelevant peptide were adoptively transferred into wild-type or SR-A^−/− recipient mice and effector activity measured by comparing the clearance of cells pulsed with relevant peptide to clearance of cells pulsed with the irrelevant peptide. NS, not significant (P > 0·05); NTD, N-terminal domain.