Initial T cell receptor transgenic cell precursor frequency dictates critical aspects of the CD8(+) T cell response to infection

Abstract

Adoptive-transfer experiments with relatively large input numbers ( approximately 10(6)) of T cell receptor-transgenic (TCR-tg) T cells are widely used to model endogenous T cell responses to infection or immunization. We show that input numbers of naive TCR-tg T cells sufficient to squelch the endogenous response to the same epitope substantially alter the kinetics, proliferative expansion, phenotype, and efficiency of memory generation by the TCR-tg T cells in response to infection. Thus, responses from nonphysiologic input numbers of TCR-tg T cells fail to accurately mimic the endogenous T cell response. Importantly, seeding as few as approximately 10-50 TCR-tg T cells, which constitute a fraction of the endogenous repertoire, allowed vigorous proliferation and analysis of TCR-tg cells after infection in a scenario representing normal physiology for any individual TCR. These data strongly suggest that modeling the endogenous T cell response with TCR-tg cells will require every effort to approximate the endogenous precursor frequency.

Figure 1. Magnitude of CD8 T cell expansion at d7 p.i. does not correlate with input numbers of OT-1 cells

(A) Naïve B6 Thy1.2 mice received the indicated numbers of naïve Thy1.1 OT-1 cells and were infected with Att LM-Ova (~7×10⁶ CFU/mouse) (B) Ova257-specific CD8 T cells detected by ICS for IFN-γ on day 7 p.i. in representative mice seeded with the indicated numbers of OT-1 cells. Numbers in the left column represent frequencies of all Ova257-specific CD8 T cells in the spleen. Numbers in the right column represent the frequencies of Thy1.1⁺ OT-1 cells among all Ova-specific IFN-γ producing cells. (C) Total numbers (mean +/− SD) of Thy1.1⁺ (OT-1) and Thy1.1⁻ (endogenous – Endo) Ova257-specific CD8⁺ T cells per spleen (n=2–3 mice per group). LOD – limit of detection.

Figure 2. Initial precursor frequency alters the functional and phenotypic characteristics of CD8 T cells early after infection

(A) Purified naïve OT-1 Thy1.1 cells at the indicated numbers were transferred into B6 Thy1.2 mice and one day later the recipient mice were immunized with Att LM-Ova. Phenotypic (CD127) and functional (grB and IL-2) status of Ova257-specific CD8 T cells from representative mice on day 7 p.i. Grey lines represent the isotype control staining that was indistinguishable between groups. Percent of Thy1.1⁺ (OT-1) and/or Thy1.1⁻ (Endo) Ova257-specific CD8 T cells that expressed the indicated molecules or produced IL-2 after Ag stimulation is indicated. (B) Percent (mean + SD for 2–3 mice per group) of Ova257-specific CD8 T cells expressing TNF, grB, CD43 (1B11)^hi, IL-2, CD127, and CD62L at day 7 and day 36 (CD127 and CD62L) post challenge. (C) Expression of CD127 and CD62L on gated OT-1 Thy1.1⁺ CD8 T cells detected in the pooled blood samples from the indicated groups at d36 p.i.

Figure 3. Input CD8 T cell precursor frequency dictates the kinetics and efficiency of the CD8 T cell response

Purified naïve OT-1 Thy1.1 cells at the indicated numbers were transferred into B6 Thy1.2 mice and one day later mice were immunized with Att LM-Ova. Expansion of OT-1 Thy1.1 cells was followed in the blood at indicated days p.i.. (A) Detection of OT-1 cells in the blood of representative mice on day 7 p.i. Numbers represent the frequency of OT-1 cells among PBL. (B) Kinetic analysis of OT-1 response in the blood. Responses of individual mice in each group are shown. (C) Frequency of OT-1 cells detected at the peak (top, day indicated inside bars) of expansion or at a memory (bottom, day 74 p.i.) time point. Numbers inside panels indicate the fold-difference in frequency of OT-1 cells between the highest and lowest input groups.

Figure 4. Precursor frequency controls the degree and kinetics of CD127 and CD62L modulation in responding CD8 T cells

(A) Representative CD127 and CD62L expression on OT-1 cells from the blood at day 7 p.i.. Shaded histograms represent isotype control staining. Numbers represent frequency of cells that are positive for indicated markers. (B) Kinetic analysis of CD127 and CD62L expression on OT-1 cells at indicated days p.i.. Pooled PBL from each group were analyzed. (C) Expression of CD127 on OT-1 cells from the indicated groups of mice at the peak of the response (day indicated inside the bars). (D) Degree of contraction of OT-1 cells at day 74 p.i. is normalized from frequencies of OT-1 cells in the blood (calculated as 100%) at day 7 (upper panel) or day of the peak of the response (lower panel). Individual mice are shown.

Figure 5. OT-I TCR-tg T cells at low input numbers can be followed in vivo and behave as an endogenous CD8 T cells after infection

(A) Blood from an OT-1 Thy1.1 mouse was used as a source of naïve TCR-tg cells that were transferred into naïve B6 Thy1.2 mice one day before Att LM-Ova infection. (B) Frequency of OT-1 (Vβ5⁺ CD8⁺) in the blood of the donor mouse used for calculation of OT-I numbers transferred. (C) Detection of OT-1 cells in the blood of the recipient mice on day 6 p.i. Numbers represent the frequency of OT-1 cells among PBL. Individual mice are shown. (D) Frequency of OT-1 cells out of CD8 cells in the blood. (E) Ova257-specific CD8 T cells detected in the spleen by ICS for IFN-γ on day 7 p.i. in representative mice that received the indicated numbers of OT-1 cells. Numbers in the left column represent frequencies of all Ova257-specific CD8 T cells in the spleen. Numbers in the right column represent the frequencies of Thy1.1⁺ OT-1 CD8 T cells among all IFN-γ producing cells. (F) Total numbers (mean +/− SD) of Thy1.1⁺ (OT-1) and Thy1.1⁻ (endogenous – Endo) Ova257-specific CD8 T cells per spleen (n=3–4 mice per group). Numbers inside the panel indicate that 1 out of 4 analyzed mice had detectable OT-1 response in the lowest input group. (G) Representative profiles of CD127 and CD62L expression on gated endogenous (Endo) Ova257-specific or OT-1 CD8 T cells in the spleen at day 7 p.i. Profiles are from mice that received 70 OT-1 cells, indicated by the box in panel F. Shaded histograms represent isotype control staining. Numbers represent the frequency of Ova257-specific CD8 T cells that are positive for CD127 or CD62L.

Figure 6. Initial precursor frequency alters the kinetics and phenotype of P14 TCR-tg T cells responding to LCMV

(A) Purified naïve P14 Thy1.1 cells at the indicated numbers were transferred into B6 Thy1.2 mice and one day later the recipient mice were infected with LCMV. Expansion of P14 Thy1.1⁺ cells was followed in the blood at indicated days p.i.. (B) Kinetic analysis of P14 responses in the blood. Responses of individual mice from each group are shown. (C) CD127 and CD62L expression on gated P14 cells from pooled blood samples from each group at day 8 p.i. Shaded histograms represent isotype control staining. Numbers represent the frequency of P14 cells that are positive for CD127 or CD62L.

Figure 7. P14 TCR-tg cells at low input numbers mimic the endogenous CD8 T cell response after LCMV infection

(A) Blood from a P14 Thy1.1⁺ mouse was used as the source of the indicated numbers of naïve TCR-tg cells, which were transferred into naïve B6 Thy1.2 mice one day before LCMV infection. (B) Detection of P14 cells in the blood of recipient mice on day 8 p.i. Numbers represent the frequency of P14 cells among PBL. Individual mice are shown. (C) Frequency of P14 TCR-tg T cells of all CD8⁺ cells in the blood. (D) GP33-specific splenic CD8⁺ T cells detected by ICS for IFN-γ on day 7 p.i. in representative mice that received the indicated numbers of P14 cells before infection. Numbers in the left column represent frequencies of all GP33-specific CD8⁺ T cells in the spleen. Numbers in the right column represent the frequencies of Thy1.1⁺ P14 CD8⁺ T cells among all IFN-γ producing cells. (E) Total numbers (mean +/− SD) of Thy1.1⁺ (P14) and Thy1.1⁻ (endogenous – Endo) GP33-41-specific CD8⁺ T cells per spleen (n=3 mice per group). (F) Representative profiles of CD127 and CD62L expression on gated endogenous (Endo) GP33-specific or P14 CD8⁺ T cells in the spleen at day 8 p.i.. Profiles are from mice that received 6000 P14 cells, indicated by the box in panel E. Shaded histograms represent isotype control staining. Numbers represent the frequency of GP33-specific CD8⁺ T cells that are positive for CD127 or CD62L.