Genetic and Functional Characterization of the Mouse Trl3 Locus in Defense against Tuberculosis

Animals

Inbred, pathogen free, C57BL/6J (B6) and DBA/2J (D2) male mice were purchased from the Trudeau Institute Animal Breeding Facility. Mice were free of common viral pathogens as determined by routine testing performed by the Research Animal Diagnostic and Investigative Laboratory, University of Missouri (Columbia, MO). Trl3 (Chr.7) and Trl4 (Chr.19) congenic mouse strains were generated using a speed-congenic (marker-assisted) approach (22). In this protocol, successive F₁ backcross males were genotyped to identify individuals with the least residual donor genomic DNA, and were selected for further backcrossing. In these mice, the chromosome 7 (Trl3) or chromosome 19 (Trl4) segments from B6 strain was introgressed onto the genetic background of the D2 strain. For Trl3, the BXD19 recombinant inbred strain was used as the initial donor for the proximal portion of chromosome 7 (D7Mit178-D7Mit193) (see Fig. 1⇓A), whereas for Trl4 the BXD9 strain was the initial donor of the distal portion of chromosome 19 (D19Mit69-D19Mit137) (see Fig. 1⇓B). Once at the N4 generation, heterozygotes were intercrossed to generate the homozygote congenic lines and also to produce the double congenic line. Genotyping was conducted as previously described (20), using tail genomic DNA and sequence-specific oligonucleotide primers (see supplemental Table S1).⁴ All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of the Trudeau Institute.

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FIGURE 1.

Physical position of the chromosome 7 (Trl3) and chromosome 19 (Trl4) segments from C57BL/6J strain introgressed onto the genetic background of the DBA/2J strain. The locations of the DBA/2 (D2) (open line) and C57BL/6J (B6) (solid line) segments introgressed in D2.B6-Chr7 (A) and D2.B6-Chr19 (B) are shown, as delimited by markers D7Mit178-D7Mit193 for chromosome 7 and D19Mit69-D19Mit137 for chromosome 19. The schematic representation of the recombinant chromosomes are shown below the LOD score traces defining the Trl3 and Trl4 loci as previously mapped by whole genome scanning in informative (B6 X D2)F₂ mice (21 ). The positions of informative markers, and the length of chromosomes are given in centiMorgan, and are drawn to scale.

Infection with M. tuberculosis

The M. tuberculosis strain H37Rv was obtained from the Trudeau Mycobacterial Culture Collection as a frozen (−70°C) log phase stock dispersed in Proskauer and Beck medium (Difco) containing 0.01% Tween 80. For each experiment, a vial was thawed, subjected to 5-s ultrasound to break up aggregates, and diluted appropriately in PBS containing 0.01% Tween 80. Mice, 8 to 10 wk of age, were inoculated with 10² CFU by the aerosol route in a Middlebrook airborne infection apparatus (Tri Instruments). Bacilli were enumerated in the lungs of infected mice at 30 and 70 days of infection by preparing lung homogenates in PBS containing 0.05% Tween 80 and by plating 10-fold serial dilutions of the homogenates on Middlebrook enriched 7H11 agar (Difco). CFU counts were performed after 3–4 wk of incubation at 37°C, and the data are presented as log₁₀ of total CFU count per lung.

Enumeration of CD4 T cell by flow cytometry and ELISPOT

Mice were euthanized, and their lungs were perfused with PBS containing 10 U/ml heparin to remove intravascular leukocytes. The lungs were then perfused with an enzyme mixture consisting of 150 U/ml collagenase, 0.2 U/ml elastase (Roche Applied Sciences), and 1 mg/ ml DNase (Sigma-Aldrich) in RPMI 1640. The lungs were removed, diced into small fragments, and subjected to further enzyme digestion before being mechanically disrupted to form a single cell suspension (23). Total lung cells were suspended in RPMI 1640-FCS in two 5-ml tubes at 1 × 10⁷/ml, and incubated with brefeldin A (10 μg/ml) for 5 h at 37°C (Epicentre Technologies). They were then stained for flow cytometry with FITC anti-CD3, R-PE anti-CD4, and peridinin chlorophyll protein anti-CD8 mAbs. After fixation overnight in 0.5% paraformaldehyde they were stained for intracellular IFN-γ with allophycocyanin anti-IFN-γ mAb, as previously described (23), and analyzed by FACSCalibur flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences).

Changes in the total number of Ag-specific T cells in the lungs capable of making IFN-γ in response to M. tuberculosis Ags were determined using the ELISPOT assay (Mouse IFN-γ ELISPOT set; BD Biosciences), according to the manufacturer’s instructions and using total lung cells from a pool of mice (n = 4) (23, 24). The M. tuberculosis Ag preparation used to stimulate IFN-γ production was a sonicated extract of a M. tuberculosis culture (23) and an ESAT-6 (1–20) peptide (New England Peptide) (25), which is an early-secreted M. tuberculosis protein.

Immunocytochemistry

Lungs were fixed by an intratracheal infusion of 10% formalin followed by 24-h immersion (at 20°C) in the same fixative. The lungs were then dehydrated in serial baths of 70% and 100% ethanol, embedded in paraffin, and sectioned. Immunocytochemistry to detect the NO synthase (NOS)2 enzyme and staining for acid-fast bacteria were performed as previously described (26). Briefly, lung sections were incubated with affinity-purified rabbit anti-mouse NOS2 Ig (primary Ab), biotinylated goat anti-rabbit Ig (secondary Ab), and avidin-coupled biotinylated HRP with diaminobenzidine as the substrate. Sections were then stained for acid-fast bacilli (27) and counterstained with methylene blue. Photomicrography was performed with a Nikon Microphot-Fx microscope fitted with a Spot RT Slider camera (Diagnostic Instruments) using Spot RT Software for image acquisition.

Transcriptional profiling with microarrays

Total lung RNA (three samples per experimental group; 36 in total) from uninfected controls and M. tuberculosis-infected (for 30 and 70 days) B6, and D2 controls as well as the chromosome 7 and chromosome 19 congenics (D2.B6-Chr7 and D2.B6-Chr19, respectively) were used for transcriptional profiling with Affymetrix oligonucleotides chips (Mouse Genome 430 2.0 array), according to the manufacturer’s recommendations. To minimize technical variability, RNA processing steps (RNA extraction, probe labeling, and microarray hybridization) were executed in parallel for all samples. The GeneSifter microarray data analysis system (VizX Labs; www.genesifter.net) was used to analyze data generated from comparisons between control (uninfected) and M. tuberculosis-infected (day 30 and day 70) groups for B6, D2, D2.B6-Chr7, and D2.B6-Chr19 mice individually. Data were normalized by the robust multiarray average algorithm, and differential expression was tested by using either pairwise, or two-way ANOVA analysis with independent t tests. Complete microarray data (accession no. E-MEXP-1942) has been deposited in the ArrayExpress database (www.ebi.ac.uk/microarray-as/ae/).

Construction of mouse lines congenic for the TB-resistance loci Trl3 and Trl4

Host resistance loci Trl3 and Trl4 were selected for investigation for the following reasons: 1) Trl3 affects both pulmonary bacterial replication and host survival time following M. tuberculosis infection in independent genome scans of (B6 X D2)F₂ mice (20, 21), 2) Trl4 shows the strongest genetic effect in these mice (21), and 3) there is strong additive interactions between Trl3 and Trl4 that explains a large fraction of the phenotypic variance in (B6 X D2)F₂ mice (21). To study the independent contribution of Trl3 and Trl4 to the differential susceptibility phenotype of B6 vs D2, and to initiate the identification of the cellular and biochemical pathways underlying both genetic effects, we have used a marker-assisted strategy to construct congenic lines in which the chromosome 7 (Trl3) and chromosome 19 (Trl4) segments of B6 resistant background were independently introgressed onto the D2 susceptible background. For chromosome 7, the proximal segment delineated by markers D7Mit178 to D7Mit193 (Fig. 1⇑A) was transferred to create D2.B6-Chr7, whereas for chromosome 19 the distal chromosomal segment delineated by markers D19Mit69 to D19Mit137 was transferred to create D2.B6.Chr19 (Fig. 1⇑B). To increase the efficiency of the breeding process, B6 chromosomal segments to be introgressed onto D2 were donated by independent BXD recombinant congenic strains (that already contain a 50:50 ratio of B6 vs D2 genome) BXD9 (Trl4) and BXD19 (Trl3). Heterozygotes produced by serial backcrossing were then intercrossed to generate homozygotes at each locus. Independent Trl3 and Trl4 congenic lines were then intercrossed to generate the Trl3:Trl4 double congenic line.

Effect of chromosomes 7 and 19 on susceptibility to pulmonary infection with M. tuberculosis

To evaluate the effect of the transferred chromosome 7 (Trl3) and chromosome 19 (Trl4) B6 segments on the susceptibility of D2 mice to infection with M. tuberculosis, D2.B6-Chr7 and D2.B6-Chr19 singly congenic lines together with C57BL/6 (B6) and DBA/2 (D2) controls were infected with 10² CFU of virulent M. tuberculosis H37Rv by the aerosol route. At day 30 and day 70 postinfection, pulmonary bacterial load was determined. Similar bacterial loads were measured in the lungs of the four mouse strains at day 30 (Fig. 2⇓), a time point of the infection where the innate difference between B6 and D2 in TB susceptibility is not yet detectable. In the subsequent genetically controlled phase of infection (day 70), increased pulmonary replication of M. tuberculosis had occurred in D2 mice, resulting in a 16-fold increase in CFU from day 30 to 70. By contrast, the infection was held stationary in lungs of B6 mice over the same period (Fig. 2⇓), in agreement with previous reports (21). In these experiments, the D2.B6-Chr19 line showed microbial pulmonary loads (days 30 and 70) very similar to those of D2 mice, whereas a significant 7- to 8-fold reduction in lung CFU was measured in D2.B6-Chr7 mice compared with D2 controls (Fig. 2⇓). The double congenic line for the B6-derived chromosomes 7 and 19 segments was also tested in the same setting, and showed CFU counts at day 70 that were undistinguishable from those seen in D2.B6-Chr7 mice (data not shown). Therefore, although transfer of the B6-derived Trl3-containing chromosome 7 segment significantly reduces pulmonary replication of M. tuberculosis in otherwise permissive D2 mice, transfer of chromosome 19 (Trl4) does not enhance resistance by itself and does not improve on the protective effect of chromosome 7.

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FIGURE 2.

Replication of M. tuberculosis in the lungs of B6, and D2 control mice strains, and D2.B6-Chr7 and D2.B6-Chr19 congenic mouse lines. Resistant B6 and susceptible D2 mice, as well as D2.B6-Chr7 and D2.B6-Chr19 congenic lines were infected via the aerosol route with 10² M. tuberculosis H37Rv, and the number of M. tuberculosis bacilli were enumerated in the lungs (log₁₀ CFU) 30 and 70 days postinfection. CFU counts for individual mice are shown with mean values for each group (horizontal line). p < 0.05, by unpaired Student’s t test for statistically significant differences in CFU counts detected between B6 and D2 (∗), D2 and D2.B6-Chr7 (∗∗), B6 and D2.B6-Chr7 (∗∗∗), and B6 and D2.B6-Chr19 (∗∗∗∗).

The effect of the transferred chromosomes 7 and 19 B6-derived segments on host survival from M. tuberculosis infection was next determined (Fig. 3⇓). Following aerosol infection, susceptible D2 mice showed a mean survival time (MST) of ∼100 days, with all animals succumbing to infection by day 107. In contrast, resistant B6 mice all survived beyond the 220-day observation period. The MST of the D2.B6-Chr19 line was 89 days, a value similar to that of D2 controls. In contrast, D2.B6-Chr7 mice displayed a significant increase in survival time over D2, revealing an MST of 155 days with animals surviving up to day 199. Therefore, the protective effect of B6-derived chromosome 7 (Trl3) initially detected by the reduction in pulmonary microbial replication caused an increase in host survival time. These two phenotypes are also known to be influenced by Trl3 in informative (B6 X D2)F₂ mice (20, 21). Conversely, the protective effect of B6 alleles at Trl4 noted in the same (B6 X D2)F₂ mice (21) is lost upon transfer of chromosome 19 onto the D2 background.

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FIGURE 3.

Survival of B6, and D2 control mice strains, and D2.B6-Chr7 and D2.B6-Chr19 congenic mouse lines following aerosol infection with M. tuberculosis. B6, D2, D2.B6-Chr7, and D2.B6-Chr19 mice were infected via the aerosol route with 10² M. tuberculosis H37Rv, and survival to infection was monitored during 220 days.

Effect of Trl3 and Trl4 on T cell-mediated immune response in M. tuberculosis-infected lungs

To initiate studies on the mechanistic basis of increased resistance noted in the D2.B6-Chr.7 congenics (Trl3), we investigated the effect of this locus on the type and extent of Th1 response during M. tuberculosis infection. In these studies, B6 and D2 control mice together with D2.B6-Chr7 and D2.B6-Chr19 congenics were infected with M. tuberculosis and the number of IFN-γ-producing CD4⁺ and CD8⁺ T cells were enumerated in the lungs, 10, 20, 30, and 50 days following aerosol infection (Fig. 4⇓). In B6-resistant controls, there was a very robust accumulation of IFN-γ-producing CD4⁺ T cells (Fig. 4⇓A) and to a lower extent CD8⁺ T cells (Fig. 4⇓B). This T cell influx started at day 20, and peaked at day 30, a time point at which B6 mice start to restrict pulmonary bacterial replication. This cellular response subsided by day 50 in B6 mice. By contrast, no such rapid T cell influx of IFN-γ-producing T cells was seen in susceptible D2 controls. Rather, there was a slow accumulation of both CD4⁺ and CD8⁺ T cells in infected lungs of D2 mice that peaked at day 50 and with cell numbers four times lower than peak numbers observed in control B6 at day 30 postinfection. Conversely, the D2.B6-Chr7 congenic mice displayed kinetics of IFN-γ⁺ CD4⁺ and CD8⁺ T cell accumulation in the lung that were indistinguishable from the numbers seen in D2 susceptible controls (Fig. 4⇓, A and B). As expected, D2.B6-Chr19 congenics showed T cells recruitment kinetics similar to D2 controls.

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FIGURE 4.

Kinetics of T cell response in the lungs of M. tuberculosis-infected B6 and D2 control mice strains, and D2.B6-Chr7 and D2.B6-Chr19 congenic mouse lines. The different control and congenic lines were infected with 10² M. tuberculosis H37Rv by the aerosol route and the number of IFN-γ-producing CD4⁺ (A) and CD8⁺ (B) T cells were enumerated in the lungs at 10, 20, 30, and 50 days postinfection. In parallel, the appearance of Ag-specific, IFN-γ-secreting T cells in the lungs (total cells) was evaluated by ELISPOT assay (C), using either an ESAT-6 peptide or crude M. tuberculosis sonicate as antigenic stimuli, and RPMI 1640 culture medium as unstimulated control. Results are from pooled lung cells obtained from four mice per experimental group. Individual experimental points are representative of measurements performed in triplicate.

The effect of Trl3 on the production of Ag-specific IFN-γ-secreting T cells in the lung was also investigated by ELISPOT assay, using either a crude M. tuberculosis sonicate or an ESAT-6 (1–20) peptide (a major M. tuberculosis Ag) as antigenic stimuli (Fig. 4⇑C). In agreement with results reported in Fig. 4⇑, A and B, for the number of IFN-γ⁺ CD4⁺ and CD8⁺ T cells in the lungs of infected mice, there was a robust response to both ESAT-6 and M. tuberculosis sonicate in the lungs of control B6 mice that peaked at day 30 and diminished by day 50. In contrast, susceptible D2 controls lacked the peak at day 30, but instead showed a more progressive response that peaked at day 50, the last time point investigated. The response of D2 mice to the M. tuberculosis sonicate was most robust (compared with ESAT-6), and at day 50 the number of IFN-γ-secreting T cells in response to the sonicated extract was similar to the number measured in B6 (Fig. 4⇑C). Again, the number of Ag-specific IFN-γ-secreting T cells detected in both chromosomes 7 and 19 congenic lines was similar to that detected in the D2 controls. Together these results suggest that transfer of B6-derived Trl3 alleles in D2.B6-Chr7 congenics increases resistance to pulmonary TB by a mechanism that is unlinked to the ability of B6 mice to rapidly recruit IFN-γ⁺ CD4⁺ and CD8⁺ T cells to the lung.

Effect of Trl3 on histopathology of M. tuberculosis infected lungs

The lung histopathology of D2 and B6 mice following aerosol challenge with M. tuberculosis (at the time D2 mice begin to succumb from infection (day 90 postinfection)) has been previously described (28). In the present study, lung lesions observed at day 70 postinfection in parental and congenic strains were similar in size, number, and cellular composition (Fig. 5⇓, A–D), although in some cases the alveoli in lesions of D2 and D2.B6-Chr19 mice were beginning to show presence of neutrophils. The striking difference between the lesions of susceptible and resistant mice, however, was in the level of infection of individual macrophages (Fig. 5⇓, E–H), with those in the lesions of D2 (Fig. 5⇓H) and D2.B6-Chr19 (Fig. 5⇓G) mice containing much larger numbers of acid-fast bacilli. This finding suggests that the higher pulmonary load of M. tuberculosis detected in D2 and D2.B6-Chr19 mice may be explained by a much higher level of intracellular replication in individual macrophage, as opposed to a larger number of infected macrophages.

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FIGURE 5.

Lung histopathology of M. tuberculosis-infected mice at day 70 of infection. B6 and D2 control mice together with the D2.B6-Chr7 and the D2.B6-Chr19 congenic lines were infected by the aerosol route with 10² CFU of M. tuberculosis, and 70 days later lungs were harvested, and processed for immunocytochemistry as described in Materials and Methods. Briefly, tissue sections were stained for acid-fast bacilli (red), whereas NOS2 was detected by immunocytochemistry (brown) sections were counterstained with methylene blue. In lung lesions of B6, D2.B6-Chr7, D2.B6-Chr19, and D2 mice, acid-fast bacilli were confined to large foamy macrophages that stained positively for NOS2. Macrophages of D2 and D2.B6-Chr19 mice contained many more acid-fast bacilli than macrophages of B6 and D2.B6-Chr7 mice. Low power (×50) of B6 lungs (A), low power (×50) of D2.B6-Chr7 lungs (B), low power (×50) of D2. B6-Chr19 lungs (C), low power (×50) of D2 lungs (D), high power (×667) of B6 lungs (E), high power (×667) of D2.B6-Chr7 lungs (F), high power (×667) of D2.B6-Chr19 lungs (G), and high power (×667) of D2 lungs (H).

Because expression of the bactericidal enzyme NOS2 is a good indicator of IFN-γ-triggered macrophage activation, NOS2 protein expression was also evaluated in these M. tuberculosis-infected lungs by immunocytochemistry at day 70 postinfection (Fig. 5⇑). Despite important interstrain differences in pulmonary bacterial loads measured at day 70, comparable levels of NOS2 staining was seen in macrophages of B6 and D2 mice and the two congenic strains (Fig. 5⇑). Additional transcriptional profiling studies also showed similar levels of IFN-γ and NOS2 mRNA expression in lungs from all strains following 30 (see supplemental Fig. S1A) and 70 (see supplemental Fig. S1B) days of M. tuberculosis infection.⁴ This demonstration suggests that neither the B6 vs D2 interstrain difference in susceptibility to M. tuberculosis, nor the enhanced resistance associated with transfer of B6 alleles at Trl3 in D2.B6-Chr7 congenics are linked to differences in activation of the NO-based anti-M. tuberculosis defense.

Effect of Trl3 on transcript profiles in the lungs of M. tuberculosis-infected mice

To gain insight into the host cell types, cellular and molecular pathways possibly involved in the differential permissiveness to pulmonary replication of M. tuberculosis, we conducted transcript profiling studies on M. tuberculosis-infected lungs from congenic and parental strains. We were particularly interested in identifying groups of transcripts associated with increased resistance to M. tuberculosis infection of B6 mice and the D2.B6-Chr7 line. That list consists in the overlap between the lists commonly expressed in response to infection between resistant B6 mice and the D2.B6-Chr7, but that show a significant difference in modulation when compared with infected susceptible D2 mice.

In these experiments, B6 and D2 mice, as well as the D2.B6-Chr19 and D2.B6-Chr7 congenic lines, were infected with M. tuberculosis. Lungs were harvested at days 30 and 70, and RNA was prepared. Three independent RNA samples from each group were converted to labeled cDNAs and hybridized to Affymetrix oligonucleotides arrays (Mouse Genome 430 2.0 array). Hybridization results were analyzed with the Genesifter analysis program to characterize changes in gene expression. The quality of the hybridization data for all the arrays was evaluated by examining gene expression intensity distribution following robust multiarray average normalization (see box plots in supplemental Fig. S2A).⁴ All experimental groups revealed a similar median expression value and a similar distribution of gene expression intensity, indicating homogeneity of the data set. A similar analysis of the three replicates for individual experimental group also showed homogeneity of the data within each group, with no outliers (data not shown).

In a first set of analyses, gene expression data obtained at days 30 and 70 postinfection were compared with that of day 0 (day 30 vs day 0; day 70 vs day 0) for each strain to establish M. tuberculosis-induced strain-specific ratios (pairwise analysis). These ratios were then used to determine gene expression profiles similarities in all mouse strains. Statistical analysis (p < 0.01 by t test; fold change doubled or more) revealed that infection with M. tuberculosis had a dramatic and potent effect on the profile of gene expression of all mouse strains. Interstrain comparison revealed that 1232 and 1876 genes were commonly regulated in all mouse strains in response to M. tuberculosis infection and this level at day 30 and 70, respectively. Of those, a total of 1136 transcripts were modulated in response to infection at both time points and in all mouse strains (see supplemental Fig. S2B).⁴ Additional analysis of the top 100 genes showing the greatest degree of M. tuberculosis-induced modulation, showed that 30 genes (of these 100) were commonly regulated in all mouse strains at both time points (see supplemental Table S2).⁴ This gene list represents a fingerprint of the anti-TB transcriptional response following aerosol infection and includes genes such as Ifnγ, Stat1, Irg1, or Iigp1 as well as chemokine ligands Cxcl9, Cxcl10, Ccl8 and chemokine receptor Cxcr6. In contrast, seven and six transcripts were specifically modulated for the day-30 and day-70 time points, respectively, revealing potential stage-specific aspects of host response to pulmonary TB (see supplemental Table S2).⁴

In a second stage, transcript profiles associated with differential susceptibility to M. tuberculosis in resistant B6 and D2.B6-Chr7 lines vs susceptible D2 mice ((B6 vs D2) and (D2.B6-Chr7 vs D2)) were investigated using a two-way ANOVA analysis of the day-30 data set. We focused our analysis on day 30 because it is at this time point that resistant B6 mice start to restrict pulmonary bacterial replication, and hence are likely to express genes contributing to protection. Likewise, B6, D2, and D2.B6-Chr7 show similar pulmonary microbial loads at day 30 (as opposed to day 70) (Fig. 2⇑), thereby minimizing the effect of bacterial burden on identification of quantitative differences in expression associated with resistance to infection. Gene lists were generated by comparing M. tuberculosis-induced changes in transcript profiles (expression at day 30 postinfection vs day 0) in both resistant strains vs susceptible strain (either B6 or D2.B6-Chr7 compared with D2). The comparison of B6 and D2.B6-Chr7 to susceptible D2 mice revealed a total of 1109 and 1611 genes showing a statistically significant difference in regulation in response to infection (p < 0.05 by t test; fold change twice as much or more), respectively, including a subset of 284 genes commonly regulated between B6 and D2.B6-Chr7. We have selected a subset of 12 such transcripts for validation by semiquantitative RT-PCR (see supplemental Fig. S3).⁴ These experiments showed a validation success rate of ∼60%. Most nonvalidated transcripts gave poor amplification results and were expressed at very low levels in lungs of all mouse strains tested. A gene ontology report on these 284 (221 annotated) genes revealed that 42 (19% of annotated genes) of them were associated with the immune system, representing the most abundant group of genes. By removing genes in duplicate and genes for which the expression ratio in D2.B6-Chr7 congenics did not follow the expression kinetic observed in B6 when compared with D2, 32 genes (Irf7, Pglyrp1, Bcl3, Cd22, and Coro1a are located on chromosome 7) were identified (Table I⇓). A few chemokine ligand genes are present, including Cxcl5, for which differential expression in B6 and D2 mice has been previously reported (29). Moreover, 16 of the 221 annotated genes commonly regulated in B6 and D2.B6-Chr7 mice map to chromosome 7 (Table II⇓), including seven (Rps9, Pglyrp1, Apoc1, Bcl3, Cd22, Saa1, and Saa2) that are located within the boundaries of the chromosome 7 congenic segment introgressed in D2.B6-Chr7 and that contain the Trl3 locus. These genes constitute strong positional candidates for Trl3, including the B cell-specific regulatory gene Cd22 that maps directly under the Trl3 linkage peak (Fig. 6⇓A). The intrinsic and inducible expression profiles for the seven genes were quantitatively similar in B6 and D2.B6-Chr7 when compared with D2 mice. More precisely, gene expression in B6 and D2.B6-Chr7 was similarly higher (Rps9, Pglyrp1, and Cd22) or lower (Apoc1, Bcl3, Saa1, and Saa2) than in D2 mice. A representative example is the case of genes Cd22 and Rps9, in which there was robust induction expression of these genes in response to M. tuberculosis in B6 and D2.B6-Chr7 mice at day 30, whereas susceptible D2 mice did not show induction of expression of these two transcripts (Fig. 6⇓B), even 70 days postinfection (data not shown). Therefore, the dramatic expression of Cd22 in response to M. tuberculosis infection in the lungs of resistant strains compared with susceptible D2 mice represents a candidate expression quantitative trait locus (QTL) for Trl3.

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FIGURE 6.

Chromosome 7 genes differentially expressed in the lungs of B6/D2.B6-Chr7 mice compared with D2 at day 30 postinfection. A, The approximate position of annotated genes on chromosome 7 (Mouse Ensembl available at www.ensembl.org/Mus_musculus/index.html) that show differential expression in the lungs of B6/D2.B6-Chr7 mice vs D2, 30 days (ANOVA two-by-two interaction) following aerosol infection with 10² CFU of M. tuberculosis is shown. The position of cytogenetically identifiable chromosomal bands is indicated and drawn to scale. The position of the B6 Chr.7 segment (D7Mit178-D7Mit193) introgressed in D2.B6-Chr7 is shown together with the approximate boundaries of the Trl3 locus. B, Expression levels of the Cd22 and Rps9 genes measured either before (D0) or day 30 postinfection (D30) in the lungs of B6, D2, and D2.B6-Chr7 lines. The mean signal intensity (log₂) obtained from three individual samples per experimental group are shown with error bar representing SE.