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Long-term protective immunity to Salmonella typhimurium infection in mice is mediated by specific CD4 T cells and antibodies (11). However, innate immune responses provide some degree of resistance at early times after infection. The Ity locus influences the quality of this innate resistance: mouse strains that express the Ity^r allele are relatively resistant to S. typhimurium infection, whereas strains that express the Ity^s allele are susceptible (14). Recent studies indicate that a gene product encoded within the Ity locus, called natural-resistance-associated macrophage protein 1 (Nramp-1), is responsible for innate resistance (3, 6, 28). Nramp-1 is exclusively expressed by macrophages and is responsible for the enhanced bactericidal activity seen in macrophages treated with gamma interferon (IFN-) (29).

More recently, it has been shown that Nramp-1 affects antigen processing, possibly by modulating phagosomal pH and regulating the activity of proteases in the late endosomal compartment (7, 13). It has also been reported to be responsible for increased surface expression of major histocompatibility complex class II molecules and inflammatory cytokine production by macrophages stimulated by bacterial lipopolysaccharide (2). These findings suggest the possibility that the resistance to bacterial infection imparted by Nramp1 is due to effects of macrophages on CD4 T cells and the immune responses that they direct. Indeed, it has been reported that mice expressing the Ity^r allele generate stronger delayed-type hypersensitivity responses and more immunoglobulin G2a antibodies in response to S. typhimurium than mice expressing the Ity^s allele (22). In addition, splenocytes from infected, resistant mice have been shown to produce more IFN- than splenocytes from infected, susceptible mice (1, 24). However, it was also recently reported that production of IFN- and several other T-cell-derived cytokines did not differ in vivo in resistant and susceptible mice, at least over the first 5 days after infection (5, 21).

Adoptive transfer of T cells from TCR transgenic mice and construction of a recombinant S. typhimurium strain expressing chicken OVA. We directly assessed the effects of Nramp-1 on T-cell activation by using a model system in which a population of chicken ovalbumin (OVA)-specific T cells from T-cell antigen receptor (TCR) transgenic mice could be physically tracked in vivo during infection with an S. typhimurium strain that expresses OVA. Because OVA-specific T cells are too rare to detect directly in the repertoire of normal mice, it was necessary to transfer into normal BALB/c recipients a small number of CD4 T cells from DO11.10 TCR transgenic mice that uniformly express a TCR specific for an OVA peptide-class II major histocompatibility complex molecule (18). The TCR expressed by DO11.10 T cells is not expressed on other T cells in BALB/c mice and can be detected with the KJ1-26 anticlonotypic antibody (8). DO11.10 T cells (2.5 × 10⁶) were injected intravenously into unirradiated BALB/c (Ity^s) mice (purchased from the National Cancer Institute, Frederick, Md.) or BALB/c.DBA2 Ity^r [BALB/c (Ity^r)] congenic mice (15, 23) (kindly provided by Bruce Zwilling, Ohio State University) as previously described (20).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Expression of chicken OVA in S. typhimurium using expression vector pYA3149. A schematic representation of the pYA3149-OVA plasmid containing a Ptrc promoter, the aspartate -semialdehyde dehyrogenase gene, and the entire OVA cDNA sequence inserted between the EcoRI and BamHI restriction sites of pYA3149 is shown in panel A. The nucleotides present at the junction between the OVA cDNA and the pYA3149 vector are illustrated below the plasmid map. Underlined sequences are from the OVA cDNA. The asterisk represents the stop codon. An immunoblot developed with an anti-OVA rabbit polyclonal antiserum (1:500 dilution; Sigma, St. Louis, Mo.) is shown in panel B. The arrow indicates the OVA protein expressed in 106 S. typhimurium x4550 cells containing pYA3149-OVA.

Recombinant S. typhimurium infection in BALB/c (Ity^r) and BALB/c (Ity^s) mice. The infectivity of the recombinant S. typhimurium was tested in BALB/c (Ity^s) and BALB/c (Ity^r) mice that did or did not contain DO11.10 T cells. After subcutaneous inoculation, the number of bacteria in the draining lymph nodes increased rapidly (Fig. 2A) such that a stable level was achieved by day 3 postinfection. An identical pattern was observed in BALB/c (Ity^s) and BALB/c (Ity^r) mice whether or not DO11.10 T cells were present. The bacteria recovered at all time points retained expression of OVA (data not shown). Thus, BALB/c (Ity^r) mice did not clear the OVA-expressing S. typhimurium organisms from the lymph nodes faster than did BALB/c (Ity^s) mice and the presence of initially naive DO11.10 T cells did not alter the course of the infection.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   S. typhimurium infection in the draining lymph nodes and spleens of BALB/c (Ityr) and BALB/c (Itys) mice. Groups of BALB/c (Itys) (triangles) and BALB/c (Ityr) (circles) mice that did (filled symbols) or did not (open symbols) receive DO11.10 T cells were infected subcutaneously with 108 live S. typhimurium organisms expressing OVA. At the indicated times after infection, CFU were measured by plating serial dilutions of draining lymph node (axillary, brachial, and inguinal lymph nodes) and spleen cell suspensions on MacConkey agar. Panel A shows the number of CFU recovered from the draining lymph nodes, and panel B shows the number recovered from the spleen. Results from 6 to 10 individual mice in each group and at each time point were pooled, and the logarithm of the mean number of CFU (± the standard deviation) (SD) is shown.

DO11.10 T cells proliferate similarly in BALB/c (Ity^s) and BALB/c (Ity^r) mice during the early phase of infection with recombinant S. typhimurium expressing OVA. The clonal expansion of DO11.10 T cells was monitored by flow cytometry in the lymphoid tissues of BALB/c (Ity^s) or BALB/c (Ity^r) recipients infected with OVA-expressing recombinant S. typhimurium. As previously shown (4, 12, 20), CD4⁺ KJ1-26⁺ T cells could not be detected in lymph node cells of mice that did not receive DO11.10 T cells (Fig. 3A). In contrast, a small population of CD4⁺ KJ1-26⁺ T cells was present in lymph nodes of mice that received DO11.10 cells (Fig. 3B). The numbers of DO11.10 cells detected after transfer were equivalent in BALB/c (Ity^s) and BALB/c (Ity^r) mice (Fig. 3E). The number of CD4⁺ KJ1-26⁺ DO11.10 T cells in the draining lymph nodes increased dramatically after subcutaneous infection with OVA-expressing S. typhimurium (Fig. 3C and E), such that a maximal level was achieved on day 5. This increase in DO11.10 T cells did not occur in recipients that were infected with control S. typhimurium that did not express OVA (Fig. 3D and E), indicating the specificity of the response. Accumulation of DO11.10 cells was also observed in the spleens of mice infected with OVA-expressing S. typhimurium (data not shown). An identical increase in the number of DO11.10 T cells in the draining lymph nodes was observed in BALB/c (Ity^s) and BALB/c (Ity^r) recipients 3 and 5 days after subcutaneous infection with OVA-expressing S. typhimurium. Twelve days into the infection, a time at which BALB/c (Ity^r), but not BALB/c (Ity^s), mice had cleared the bacteria from the spleen, the number of DO11.10 T cells in the draining lymph nodes was significantly lower in susceptible BALB/c (Ity^s) recipients than in resistant BALB/c (Ity^r) recipients (Fig. 3E). This difference was due mainly to the fact that the total number of cells in the lymph nodes was much greater in BALB/c (Ity^r) recipients than in BALB/c (Ity^s) recipients; the percentages of DO11.10 T cells present in the lymph nodes of the two groups were actually similar at this late time point. These results show that antigen-specific T cells expand and survive equally well in susceptible and resistant recipients, at least until late times after infection.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Detection of transferred DO11.10 T cells in lymph nodes. Pooled brachial, axillary, and inguinal lymph node cell suspensions were stained with anti-CD4-phycoerythrin (PE) and biotinylated KJ1-26, which uniquely recognizes the DO11.10 TCR, followed by streptavidin-fluorescein isothiocyanate, and analyzed on a flow cytometer. The percentage of CD4+ KJ1-26+ lymphocytes is indicated on each contour plot. The stained lymph node cells were from normal BALB/c (Itys) mice (A), BALB/c (Itys) mice injected with 2.5 × 106 DO11.10 T cells (B), BALB/c (Itys) recipients of DO11.10 T cells 5 days after subcutaneous infection with 108 OVA-expressing S. typhimurium cells (C), and BALB/c (Itys) recipients of DO11.10 T cells 5 days after subcutaneous infection with 108 control S. typhimurium cells (D). For panel E, pooled brachial, axillary, and inguinal lymph nodes were harvested from four to six individual mice per group per time point following subcutaneous infection of BALB/c (Itys) (circles) or BALB/c (Ityr) (squares) adoptive-transfer recipients with 108 OVA-expressing (filled symbols) or control (open symbols) S. typhimurium cells. The percentage of CD4+ KJ1-26+ T cells was determined as described above, and the total number of CD4+ KJ1-26+ T cells was calculated by multiplying the percentage of CD4+ KJ1-26+ T cells by the total number of viable cells in the lymph nodes. The mean total number of CD4+ KJ1-26+ cells ± the standard deviation (SD) is shown.

IFN- production by DO11.10 cells recovered from BALB/c (Ity^s) and BALB/c (Ity^r) mice during infection with OVA-expressing S. typhimurium. Since IFN- has been shown to be critical for control of S. typhimurium infection (10), attention was focused on the capacity of DO11.10 T cells to produce this lymphokine during infection. Draining lymph node cells from BALB/c (Ity^s) or BALB/c (Ity^r) recipients of DO11.10 T cells were harvested 5 or 10 days after infection and cultured in vitro with OVA peptide. On day 5 after infection, the same amount of IFN- was produced in response to the OVA peptide by lymph node cells from BALB/c (Ity^s) and BALB/c (Ity^r) recipients of DO11.10 T cells (group 2 in Fig. 4A). In contrast, 10 days after infection, OVA-stimulated lymph node cells from BALB/c (Ity^r) recipients made much more IFN- than did BALB/c (Ity^s) recipients (group 2 in Fig. 4B). However, the day 10 cultures contained different numbers of input DO11.10 T cells (Fig. 3E), and thus, it was important to determine whether this alone accounted for the differences in IFN- production by BALB/c (Ity^s) and BALB/c (Ity^r) recipients. Most of the IFN- detected was dependent on DO11.10 T cells because IFN- was barely detected from antigen-stimulated cultures of lymph node cells from mice that were infected with OVA-expressing S. typhimurium but did not receive DO11.10 T cells (group 4 in Fig. 4A). This dependence made it possible to estimate the amount of IFN- produced by each DO11.10 T cell by dividing the total amount of IFN- detected by the number of DO11.10 cells present in the culture. This analysis showed that 5 days after infection with OVA-expressing S. typhimurium, DO11.10 T cells from BALB/c (Ity^s) and BALB/c (Ity^r) recipients produced similar amounts of IFN- (group 2 in Fig. 4C), whereas 10 days after infection, DO11.10 T cells from susceptible BALB/c (Ity^s) recipients infected with OVA-expressing S. typhimurium produced about sixfold less IFN- than did cells from BALB/c (Ity^r) recipients (group 2 in Fig. 4D). This difference was specific to IFN- because no significant differences in antigen-stimulated interleukin-2 (IL-2), IL-5, IL-6, or tumor necrosis factor alpha production were observed (data not shown). No IFN- was produced by lymph node cells from recipients of DO11.10 T cells that were not infected (group 1 in Fig. 4A and C) or were infected with non-OVA-expressing bacteria (group 3 in Fig. 4A), consistent with the idea that naive T cells must differentiate in vivo in response to antigenic stimulation before acquiring the capacity to produce IFN- (34). Finally, recall IFN- production by DO11.10 T cells was similar in BALB/c (Ity^s) and BALB/c (Ity^r) recipients that had been primed 2 weeks earlier with soluble OVA in complete Freund's adjuvant (data not shown), indicating that the reduced IFN- production observed in infected BALB/c (Ity^s) recipients was related to bacterial infection.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Differential IFN- production by DO11.10 T cells recovered from S. typhimurium-infected BALB/c (Itys) and BALB/c (Ityr) recipients. Draining lymph node cells were harvested from BALB/c (Itys) (striped bars) or BALB/c (Ityr) (solid bars) recipients of DO11.10 T cells that were not infected (group 1) or infected with OVA-expressing (group 2) or control (group 3) S. typhimurium 5 (A) or 10 (B) days after infection. Lymph node cells were also prepared from mice that were infected with OVA-expressing S. typhimurium but did not contain DO11.10 T cells (group 4). The percentage of CD4+ KJ1-26+ T cells present was measured in a sample from each group as shown in Fig. 3. Some of the remaining lymph node cells from each group (5 × 105/well) were cultured in vitro with 5 µM OVA peptide (amino acids 323 to 339) for 48 h. The amount of IFN- in the supernatant was measured by enzyme-linked immunosorbent assay using Pharmingen antibodies. The number of DO11.10 T cells added to each culture was calculated by multiplying the percentage of CD4+ KJ1-26+ T cells in each group by 5 × 105. The amount of IFN- detected was then divided by the number of DO11.10 T cells to obtain an estimate of the amount of IFN- produced by each DO11.10 T cell added to the culture. Data from three independent experiments were pooled, and each bar represents results from at least six mice. The error bars represent the standard error of the mean. PI, postinfection.