Requirements for bone marrow-derived antigen-presenting cells in priming cytotoxic T cell responses to intracellular pathogens

Abstract

Bone marrow (BM)-derived antigen-presenting cells (APCs) are potent stimulators of T cell immune responses. We investigated the requirements for antigen presentation by these cells in priming cytotoxic T lymphocyte (CTL) responses to intracellular bacterial and viral pathogens. [Parent-->F(1)] radiation BM chimeras were constructed using C57BL/6 donors and (C57BL/6 x BALB/c)F(1) recipients. Infection of chimeric mice with either Listeria monocytogenes or vaccinia virus expressing the nucleoprotein (NP) antigen from lymphocytic choriomeningitis virus (LCMV) primed H2-D(b)-restricted, but not H2-K(d)-restricted CTL responses, demonstrating the requirement for BM-derived APCs for successful priming of CTL responses to these pathogens. Surprisingly, this did not hold true for chimeric mice infected with LCMV itself. LCMV-infected animals developed strong CTL responses specific for both H2-D(b)- and H2-L(d)-restricted NP epitopes. These findings indicate that in vivo priming of CTL responses to LCMV is remarkably insensitive to deficiencies in antigen presentation by professional BM-derived APCs.

Figure 1

BM MHC haplotype determines CTL priming after Listeria infection. Three [B6→CB6] and three [CB6→CB6] chimeras were infected with 500 CFU recombinant Listeria. Immune splenocytes were stimulated in vitro with CB6 splenocytes coated with H2-K^d–restricted Listeria LLO_(91–99) (A and D) or p60_(217–225) (B and E) epitopes or with the H2-D^b–restricted NP_(396–404) epitope from the recombinantly expressed NP of LCMV. CTL activity was assayed 6 d later using P815 (H2^d) (A and B, and D and E) or EL4 (H2^b) (C and F) target cells coated with PBS (open symbols) or with 20 nM of the indicated peptide (filled symbols). Symbol types correspond to responder cells from individual mice. Similar results were obtained in four additional experiments.

Figure 2

Exogenous CB6 APCs can prime CTL responses to an H2-K^d–restricted Listeria epitope in [B6→CB6] chimeras. DCs isolated from spleens of CB6 mice were pulsed with 1 μM LLO_(91–99) and used to immunize two naive [B6→CB6] chimeras intravenously. After 3 wk, splenocytes from immunized chimeras were stimulated in vitro with LLO_(91–99) (left) or p60_(217–225) (right) peptides. Responders were assayed after 6 d in culture for lysis of P815 target cells coated with PBS (open symbols) or 50 nM of the corresponding peptide (filled symbols).

Figure 3

CTL priming during infection with LCMV is not dictated by BM MHC haplotype. Three [B6→CB6] (A–C) and two [CB6→CB6] (D and E) chimeras were infected intravenously with 10⁵ PFU LCMV-Armstrong. On day 9 after infection, ex vivo CTL activity was measured directly using P815-D^b target cells pulsed with PBS or with 1 μM of the indicated NP peptide. NP_(396–404) is presented by H2-D^b and NP_(118–126) is presented by H2-L^d. The results in this figure are representative of three experiments. A similar response pattern was observed in animals receiving an LCMV inocula of only 200 PFU LCMV-Armstrong per animal.

Figure 4

Recombinant vaccinia virus infection elicits CTL responses only to epitopes presented by BM-derived APCs. Two [B6→CB6] (A and B) and two [CB6→CB6] (C and D) chimeric mice were infected intravenously with 5 × 10⁶ PFU of VacNP. 6 d after infection, CTL activity in spleens of infected mice was quantitated using P815-D^b target cells pulsed with 1 μM of the indicated NP peptides as described in the legend to Fig. 2. Similar results were obtained in two additional experiments, and were seen in animals infected with 7.1 × 10⁶ PFU of VacNP.

Figure 5

Representation of H2-K^d–expressing APCs is similar in chimeric animals infected with recombinant vaccinia virus or LCMV. Splenocytes from VacNP- or LCMV-infected [B6→CB6] chimeras were harvested on day 6 or 9 after infection, respectively. Cells were stained with antibodies to CD3 and H2-K^d and analyzed by FACS^®.