H2-M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses

Abstract

Major histocompatibility complex (MHC) class Ib molecules have been implicated in CD8(+) T cell-mediated defenses against intracellular bacterial infection, but the relative importance of MHC class Ib-restricted T cells in antimicrobial immunity is unknown. In this report, we use MHC tetramers to characterize T cell responses restricted by H2-M3, an MHC class Ib molecule that selectively presents N-formyl peptides. We find that sizeable H2-M3-restricted T cell responses, occurring earlier than MHC class Ia-restricted T cell responses, are mounted after primary infection with the intracellular bacterium Listeria monocytogenes. These H2-M3-restricted T cells are cytolytic and produce interferon gamma. However, after a second L. monocytogenes infection, H2-M3-restricted memory T cell responses are minor in comparison to the much larger MHC class Ia-restricted responses. This first direct characterization of an MHC class Ib-restricted T cell response indicates that CD8(+) T cells responding to L. monocytogenes infection can be divided into two groups: H2-M3-restricted responses, which provide rapid and quantitatively substantial effector function during primary infections but contribute relatively little to memory responses, and MHC class Ia-restricted responses, which expand later during primary infection but form memory T cells that respond rapidly and dramatically in response to subsequent infections by the same pathogen.

Figure 1

Expression and peptide-dependent refolding of recombinant H2-M3. (A) H2-M3 cDNA was modified by PCR (as described in Materials and Methods), cloned into the pET3a vector, and expressed in E. coli strain BL21(DE3). Samples taken before and 1 and 3 h after IPTG induction were subjected to SDS-PAGE, followed by staining with Coomassie blue. The number of hours of IPTG induction before collection of each sample is indicated above the gel. (B) The H2-M3 construct containing the biotinylation sequence was further mutagenized by PCR to replace four C/G nucleotides in the first seven codons of the 5′ coding region with A/T nucleotides (see Materials and Methods). These changes greatly increased H2-M3 expression in E. coli after IPTG induction. Samples taken before and after IPTG induction were treated as in A. H2-M3 and β2m protein were refolded in the presence of the Listeria-derived f-MIGWIIA peptide (C) or the mitochondrial COI self-peptide (D) as described in Materials and Methods, concentrated, and subjected to size-exclusion chromatography on a Superdex 200HR column. Absorbance at 280 nm was measured and plotted. (E) Refolding of H2-M3/β2m complexes was also attempted in the absence of exogenous formyl peptide.

Figure 3

The magnitude of f-MIGWIIA–specific CTL responses to L. monocytogenes infection is different among mouse strains. Female age-matched C57BL/6, C3H/HeJ, and BALB/c mice were infected with 2,000 L. monocytogenes, and splenocytes were obtained on the indicated day after primary infection, enriched for CD8⁺ T cells, and stained with antibodies to CD8 and CD62L as well as one of the PE-conjugated tetramers. Two mice per strain were analyzed each day. (A) The dot plots show H2-M3/f-MIGWIIA (left panels) and H2-M3/COI self (right panels) tetramer stainings for one mouse per strain on day 7 after infection. Staining for CD62L (l-selectin) is shown on the x-axis, and tetramer staining on the y-axis. Dot plots are gated on CD8⁺ lymphocytes. Percentages in the upper left quadrants represent the percentage of CD8⁺ CD62L^low (activated) cells that are tetramer positive. (B) The average number of H2-M3/f-MIGWIIA and H2-M3/COI (self) tetramer–positive cells per spleen is shown for two mice of each strain on days 5, 7, and 9 after infection. SD are indicated.

Figure 2

H2-M3/f-MIGWIIA tetramers stain CTL lines and clones specific for the f-MIGWIIA peptide. f-MIGWIIA– and LLO_91–99 specific CTL lines and clones were generated by in vitro peptide restimulation of splenocytes from L. monocytogenes–infected mice as described in Materials and Methods. Cells were stained with anti-CD8, anti-CD62L, PE-conjugated H2-M3/f-MIGWIIA or H2-K^d/LLO_91–99 tetramers, and propidium iodide to identify dead cells. Histograms show tetramer staining gated on live CD8⁺ CTLs from f-MIGWIIA–specific C57BL/6 (A) and BALB/c (B) derived CTL lines, a BALB/c f-MIGWIIA–specific CTL clone (C), and a BALB/c LLO_91–99 specific CTL line (D).

Figure 4

H2-M3–restricted T cell responses peak earlier than H2-K^d–restricted responses during primary L. monocytogenes infection. Female, age-matched CB6/F1 mice were infected with 2,000 L. monocytogenes. Splenocytes were taken from uninfected mice (day 0) and infected mice on the indicated day after infection. CD8⁺-enriched splenocytes were stained with anti-CD8, anti-CD62L, and one of three PE-conjugated tetramers: H2-M3/f-MIGWIIA, H2-M3/COI self, or H2-K^d/LLO_91–99. Dot plots are gated on CD8⁺ lymphocytes, with CD62L staining on the x-axis and tetramer staining on the y-axis. The percentage of CD62L^low (activated), tetramer-staining cells is indicated in the upper left quadrant. Dot plots for each day are from one representative mouse. Four log phases are shown.

Figure 5

H2-M3–restricted T cells undergo little expansion during recall L. monocytogenes infection compared with H2-K^d–restricted T cells. Female, age-matched CB6/F1 mice were infected with 2,000 L. monocytogenes. After 48 d, the mice were reinfected with 100,000 bacteria. Day 0 represents a mouse not reinfected with L. monocytogenes, and is the same as day 48 from Fig. 4. Splenocytes were taken on the indicated days and were stained and analyzed as described in the legend to Fig. 4.

Figure 6

H2-M3–restricted T cell responses differ from H2-K^d–restricted T cells during both primary infection and reinfection with L. monocytogenes. The total number of CD8⁺ CD62L^low (activated), tetramer-positive cells (for H2-M3/f-MIGWIIA, H2-M3/COI self, and H2-K^d/LLO_91–99 tetramers) was quantified from mice at each time point during primary and recall studies. Absolute numbers (y-axis) are shown for primary infection (A) and recall infection (B) with days after infection marked on the x-axis. Averages and SD are shown for three mice per day except primary days 0 (uninfected), 20, and 48 (recall day 0), at which times there were two mice. Note that y-axes have different scales for primary and recall infections.

Figure 7

H2-M3–restricted T cells detected 5 d after primary infection are effective killers and secrete IFN-γ. (A) Splenocytes from a CB6/F1 mouse infected with 2,000 L. monocytogenes 5 d previously were enriched for CD8⁺ T cells followed by staining with anti-CD8, anti-CD62L, and PE-conjugated H2-M3/f-MIGWIIA tetramers. Tetramer-positive (left panel) and tetramer-negative (right panel) cells were sorted separately from the CD8⁺CD62L^low staining population and placed in a CTL assay with peptide-coated p815 target cells. The peptide used and percent specific lysis are shown on the x-axis and y-axis, respectively. The E/T ratios were ∼1:1 for the tetramer-positive cells and 10:1 for the tetramer-negative population. (B) Unsorted splenocytes from both immunized mice were placed in an ELISPOT assay for detection of IFN-γ–secreting cells. The number of IFN-γ–secreting cells detected is shown as the average of two mice (y-axis), with SD indicated. Four replicate wells were counted for each mouse. The stimulating peptide is indicated on the x-axis.