Natural Resistance-Associated Macrophage Protein Is a Cellular Receptor for Sindbis Virus in Both Insect and Mammalian Hosts

SummaryAlphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection

Natural Resistance-associated Macrophage Protein (NRAMP) is a cellular receptor for Sindbis virus in both insect and mammalian hosts

Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of for this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter Natural Resistance-Associated Macrophage Protein (NRAMP), as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were non-permissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multi-pass membrane proteins for infection

A self-help program for memory CD8+ T cells: positive feedback via CD40-CD40L signaling as a critical determinant of secondary expansion.

The ability of memory CD8+ T cells to rapidly proliferate and acquire cytolytic activity is critical for protective immunity against intracellular pathogens. The signals that control this recall response remain unclear. We show that CD40L production by memory CD8+ T cells themselves is an essential catalyst for secondary expansion when systemic inflammation is limited. Secondary immunization accompanied by high levels of systemic inflammation results in CD8+ T cell secondary expansion independent of CD4+ T cells and CD40-CD40L signaling. Conversely, when the inflammatory response is limited, memory CD8+ T cell secondary expansion requires CD40L-producing cells, and memory CD8+ T cells can provide this signal. These results demonstrate that vaccination regimens differ in their dependence on CD40L-expressing CD8+ T cells for secondary expansion, and propose that CD40L-expression by CD8+ T cells is a fail-safe mechanism that can promote memory CD8+ T cell secondary expansion when inflammation is limited

Memory CD8+ T cell expansion following homologous boost is CD4+ T cell independent but CD40L dependent.

<p>Mice primed with Lm-OVA were boosted 21 days later with the indicated dose of Lm-OVA. 5 days post-boost, OVA_257–264-specific CD8+ T cells were enumerated by IFN-γ intracellular cytokine staining. (A) CD8+ splenocytes were transferred from Lm-OVA primed B6.SJL (CD45.1+) mice into C57BL/6 (CD45.2+) recipients. Recipient mice were then treated with Lm-OVA and the frequency OVA_257–264-specific CD8+ T cells was determined by intracellular IFN-γ staining. (B) Mice were depleted of CD4+ cells at the time of the secondary immunization. Absolute OVA_257–264-specific CD8+ T cells five days post homologous Lm-OVA prime-boost (mean±SEM, <i>n</i> = 5). (C-E) Mice were treated with MR1 (αCD40L) or control antibody during boost. (C) Frequency of OVA_257–264-specific CD8+ T cells within total CD8+ cells with and without CD40L blockade (mean±SEM, <i>n</i> = 5). (D) Absolute number of IFN-γ-producing OVA_257–264-specific CD8+ T cells (mean±SEM). (E) Fold expansion of OVA_257–264-specific CD8+ T cells with and without CD40L blockade (mean absolute IFN-γ+ OVA_257–264 -specific CD8+ T cells per group relative to mean of matched HBSS group). *, P<0.05, **P<0.01, Mann-Whitney. Data are representative of two independent experiments.</p

Antibiotic treatment following heterologous boost recapitulates dependence on CD40L for mCD8+ T cell expansion.

<p>Mice were immunized with 10⁶ pfu VV-OVA and boosted 21 days later with 5×10⁶ cfu Lm-QV in the presence of MR1 or control antibody treatment. 8 hours following boost, indicated groups were treated with ampicillin to accelerate bacterial clearance. OVA_257–264-specific memory CD8+ T cell responses were assessed 5 days post-boost using intracellular cytokine staining for IFN-γ (<i>n</i> = 5). (A) Representative intracellular cytokine staining (median shown, ±SEM). (B) Total OVA_257–264-specific (IFN-γ+) CD8+ T cells (mean, ±SEM). *, P<0.05, Mann-Whitney. (C) Serum cytokines 24 h post-boost in untreated versus ampicillin-treated animals (mean, ±SEM). Data are representative of three independent experiments.</p

CD40L is required for mCD8+ T cell expansion following homologous (but not heterologous) boost, in the absence of CD4+ T cells.

<p>Mice were primed with 10⁷ cfu Lm-QV or 10⁶ pfu VV-OVA and boosted 21 days later with 10⁵ cfu Lm-QV. Starting on day 20 (1 day pre-boost), mice were depleted of CD4+ T cells. MR1 (αCD40L) or control antibody was administered to indicated groups during boost. OVA_257–264-specific CD8+ T cell responses were assessed 5 days post-boost by intracellular cytokine staining. (A) Frequency of OVA_257–264-specific CD8+ T cells within total CD8+ cells (mean±SEM, <i>n</i> = 5). (B) Total IFN-γ+ OVA_257–264-specific or B8R_20–27-specific mCD8+ T cells per spleen (mean±SEM). (C) Fold expansion of OVA_257–264-specific or B8R_20–27-specific CD8+ T cells after homologous or heterologous boost. (mean absolute IFN-γ+ OVA_257–264 -specific CD8+ T cells per group relative to mean of matched HBSS group). Data are representative of two independent experiments.</p

Accelerated clearance of homologous vaccine vector limits inflammation and correlates with increased CD40L dependence.

<p>(A–B) Mice were primed with 10⁷ cfu Lm-OVA, 10⁶ pfu VV-OVA, or HBSS and boosted 21 days later with 10⁷ cfu Lm-QV (<i>n</i> = 5). (A) Spleens and livers were harvested at 24, 48, and 72 hours following boost, and cfu were enumerated by plating organ homogenates (mean, ±SEM). (B) Serum cytokine levels at 4 and 24 h following boost (mean, ±SEM). (C–E) Mice were primed with 10⁷ cfu Lm-OVA or 10⁶ pfu VV-OVA and boosted 21 days later with 5 µg DEC205-OVA alone, 5 µg DEC205-OVA with 10⁵ cfu Lm-OVA, or HBSS. αCD40L or control antibody was administered to the indicated groups during boost. OVA_257–264-specific memory CD8+ T cell responses were assessed 5 days post-boost using intracellular cytokine staining for IFN-γ (<i>n</i> = 5). (C) Representative plots of OVA_257–264-specific T cell enumeration (median shown, ±SEM). (D) Total OVA_257–264-specific (IFN-γ+) CD8+ T cells per animal (mean, ±SEM). *, P<0.05, **, P<0.01, ***, P<0.001, ANOVA. Data are representative of two independent experiments.</p

Rapid expression of CD40L by CD8+ T cells following restimulation.

<p>Mice were primed with 1×10⁷ cfu Lm-QV and spleens harvest 7 days later. Splenocytes were restimulated in vitro with B8R_20–27 for the indicated time and then stained for intracellular IFN-γ, TNF and CD40L. (A) Univariate analysis of intracellular IFN-γ, TNF or CD40L within CD8+ T cells after restimulation. (B) Multivariate analysis of IFN-γ, TNF and CD40L over time within CD8+ T cells. (C) Protein expression profile of B8R_20–27 –specific CD8+ T cells after restimulation. Pie slices correspond to color legend in panel B. Outer arcs indicate slices expressing the phenotype of the inner slices. Each point in panels A and B indicates a single animal (5 mice per group), panel C represents the median of data from panel B.</p

CD40L expressing mCD8+ T cells promote secondary expansion following homologous boost.

<p>(A) Experimental design: Donor (B6.SJL) mice and recipient (C57BL/6 and CD40L-deficient) mice were concurrently primed with 10⁷ cfu Lm-QV. 21 days later, CD8+ T cells were purified from B6.SJL spleens and transferred into B6 and B6.<i>Cd40L</i>-/- mice. 24 h post-transfer, recipients were boosted with 5×10⁶ cfu Lm-QV in the presence of αCD40L or control antibody. OVA_257–264-specific memory CD8+ T cell responses were assessed 5 days following boost using intracellular cytokine staining for IFN-γ (<i>n</i> = 5). (B) Total OVA_257–264-specific (IFN-γ+) CD45.1+CD8+ T cells per animal (mean, ±SEM). *, P<0.05, **, P<0.01, Mann-Whitney. (C) Fold expansion of donor CD45.1+ IFN-γ + OVA_257–264-specific CD8+ T cells. Data are representative of three independent experiments.</p