Tracking the total CD8 T cell response to infection reveals substantial discordance in magnitude and kinetics between inbred and outbred hosts

Abstract

Determining the magnitude and kinetics, together with the phenotypic and functional characteristics of responding CD8 T cells, is critical for understanding the regulation of adaptive immunity as well as in evaluating vaccine candidates. Recent technical advances have allowed tracking of some CD8 T cells responding to infection, and a body of information now exists describing phenotypic changes that occur in CD8 T cells of known Ag-specificity during their activation, expansion, and memory generation in inbred mice. In this study, we demonstrate that Ag but not inflammation-driven changes in expression of CD11a and CD8alpha can be used to distinguish naive from Ag-experienced (effector and memory) CD8 T cells after infection or vaccination. Interestingly and in contrast to inbred mice, tracking polyclonal CD8 T cell responses with this approach after bacterial and viral infections revealed substantial discordance in the magnitude and kinetics of CD8 T cell responses in outbred hosts. These data reveal limitations to the use of inbred mouse strains as preclinical models at vaccine development and suggest the same dose of infection or vaccination can lead to substantial differences in the magnitude and timing of Ag-specific CD8 expansion as well in differences in protective memory CD8 T cell numbers in outbred individuals. This concept has direct relevance to development of vaccines in outbred humans.

Figure 1. Phenotypic changes on antigen-specific effector and memory CD8 T cells after bacterial infection

Naïve B6 mice were infected with 5×10⁶ (~ 0.1 LD₅₀) of a recombinant strain of attenuated L. monocytogenes expressing Ova₂₅₇ epitope (Att LM-Ova) and CD8 T cells were analyzed in the blood at day 7 post infection. Blood from naïve (non-infected) mice was used as a control. A, C) Ova-specific CD8 T cells were detected by tetramer (K^bOva) staining. B, D) The expression of the indicated phenotypic markers was analyzed on all CD8 T cells in naïve mice and on K^bOva^negative or K^bOva^positive CD8 T cell populations in LM-infected (day 7 or day 50 post infection) mice. Representative mice are shown. The numbers inside the dot plots represent the percentage of cells positive for indicated molecules.

Figure 2. The role of inflammation in changes of CD11a and CD8α expression on CD8 T cells

A) Experimental design. Naïve B6 mice were treated with CpG (1826, 50 μg/mouse; i.p), Poly(I:C) (200 μg/mouse; i.p.), or infected with Att LM (5×10⁶) at day 0. B) At day 6 p.i. CD11a and CD8α expression on CD8 T cells was analyzed in the blood. Numbers represent the frequency of CD11a^high/CD8α^low/CD8 T cells in the blood. Representative profiles are shown. C) Frequency of CD11a^high/CD8α^low/CD8 T cells in the blood presented as mean + SD for three mice per group. D) Experimental design. Naïve OT-I Thy1.1 cells (4×10⁵/mouse) were transferred into groups of naïve B6 Thy1.2 mice, and 1 day later, the recipient mice were treated with CpG or Poly(I:C), or infected with Att LM or Att LM-Ova (5×10⁶). Additional group of non-infected or treated recipient mice served as controls. E) At day 6 p.i. CD11a and CD8α expression on gated OT-I (Thy1.1) CD8 T cells was analyzed in the blood and spleen of individual mice. Numbers represent the frequency of CD11a^high/CD8α^low/CD8 T cells. Representative profiles are shown. F) Naïve B6 mice were immunized with DCs coated with Ova peptide (1×10⁶/mouse i.v.) and CD8 T cells were analyzed in the blood at day 7 post immunization. Blood from naïve (non-immunized) mice was used as a control. Ova-specific CD8 T cells were detected by tetramer (K^bOva) staining. G) The expression of the CD11a and CD8α was analyzed on all CD8 T cells in naïve mice and on K^bOva^positive and K^bOva^negative CD8 T cell populations in DC-immunized mice. Representative mice are shown.

Figure 3. Functional memory CD8 T cells express high levels of CD11a and low levels of CD8α after bacterial infection

A) Experimental design. Naïve B6 Thy1.2 mice were infected with Att LM (5×10⁶ CFU/mouse) and on days 35 or 90 CD8 T cells were sorted according to their CD11a expression into CD11a^high and CD11a^low subpopulations. CD11a^low CD8 T cells sorted from naïve (non-infected) mice were used as controls. Sorted populations of cells were adoptively transferred into naïve B6 Thy1.1 mice before Att LM infection (8×10⁶). B) Post-sort purity of transferred CD11a^low or CD11a^high CD8 T cells from naïve and immune mice. C) Total number of Thy1.2 CD8 T cells in the spleen. Data are presented as mean + SD for three mice per group per time point.

Figure 4. The magnitude of polyclonal CD8 T cell expansion after bacterial or viral infections of inbred strains of mice

Naïve BALB/c and B6 mice were infected with different doses of Att LM (low – 1×10⁵ CFU; high – 5×10⁶ CFU/mouse) or with Armstrong strain of LCMV (2×10⁵ PFU/mouse i.p.). At the peak of the CD8 T cell expansion (day 7 after LM; day 8 after LCMV) the frequency of CD11a^high/CD8α^low/CD8 T cells was determined in the blood and spleen. A) The frequency of CD11a^high/CD8α^low/CD8 T cells in the blood and spleen of individual mice. B) Total number of CD11a^high/CD8α^low/CD8 T cells in the spleen of the individual mice at the peak of the CD8 T cell expansion.

Figure 5. CD11a upregulation and CD8α downregulation can mark antigen-experienced CD8 T cells in tissues

A) Experimental design. Naïve OT-I Thy1.1 cells (500 cells/mouse) were transferred into naïve B6 Thy1.2 mice, and 1 day later, the recipient mice were infected with Att LM-Ova (5×10⁶). B) At day 7 p.i. the status of CD11a and CD8α expression was determined on OT-I (Thy1.1) CD8 T cells in the spleen. C) The frequency of CD11a^high/CD8α^low cells among all CD8 T cells (CD8 T cell gate; left column) or on OT-I cells (OT-I gate; right column) was determined in the indicated tissues. Numbers represent the frequency of CD11a^high/CD8α^low in gated populations. Representative profiles are shown.

Figure 6. Kinetics of polyclonal CD8 T cell responses in immune mice after heterologous infection

Groups of naïve and LCMV-Arm immune B6 mice (day 200+ after 2×10⁵ PFU i.p. infection) were infected with Att LM (5×10⁶ CFU) on day 0. Kinetic analysis of CD11a^high/CD8α^low CD8 T cells in the blood after LCMV and/or LM infections. Data are presented as a mean +/− SD for three mice per group.

Figure 7. Substantial variability in the magnitude of primary LM-specific CD8 T cell responses and protection to LM re-challenge in individual outbred mice

A) The status of CD11a and CD8α expression on CD8 T cells in the blood of individual naïve B6 (inbred) and Swiss Webster (SW; outbred) mice was analyzed before (day 0) Att LM infection (1×10⁶ CFU/mouse i.v.). At the peak of the CD8 T cell expansion (day 7 as described for inbred strains of mice) the frequency of CD11a^high/CD8α^low/CD8 T cells was determined in the blood. Representative profiles of the individual mice are shown. B) The frequency of CD11a^high/CD8α^low/CD8 T in the blood of individual mice before and at day 7 p.i. Numbers inside the graph represent the ‘inside group variability’ that is calculated by dividing the highest with the lowest responders inside the group. The line represents the mean. C) In separate experiment outbred mice were infected with LM as described above and the frequency of CD11a^high/CD8α^low/CD8 T cells was determined in the blood at the memory stage of the CD8 T cell response (day 46). Mice were split into two groups (high and low) according to the frequency of CD11a^high/CD8α^low/CD8 T cells and re-challenged with high dose of virulent LM (vir LM; 5×10⁵ CFU/mouse; ~ 50 LD₅₀). Naïve mice were introduced into the experiment as controls. D) At day 3 post secondary infection bacterial numbers were determined in the spleen and liver. All naïve mice infected with the high dose of vir LM died by day 3. The levels of infection in individual mice are shown. Survival in all groups of mice is indicated. L.O.D – limit of detection.

Figure 8. Substantial variability in the magnitude and kinetics of primary virus-specific CD8 T cell responses in outbred mice

A) The frequency of CD11a^high/CD8α^low/CD8 T cells in the blood of individual B6 and Swiss Webster mice before (day 0) and at day 8 post LCMV-Arm (2×10⁵ PFU/mouse i.p.) infection. Numbers inside the graph represent the ‘inside group variability’ that is calculated by dividing the highest with the lowest responders inside the group. The line represents the mean. B) Kinetic analysis of CD11a^high/CD8α^low/CD8 T cells in the blood after LCMV-Arm infection. The responses of individual mice are shown. Vertical gray line represents the day of the peak of the anti-LCMV CD8 T cell response as determined in inbred B6 mice.