Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naïve cells

Abstract

The rules that govern memory T cell differentiation are not well understood. This study shows that after antigenic stimulation naïve CD8+ T cells become committed to dividing at least seven times and differentiating into effector and memory cells. Once the parental naïve CD8+ T cell had been activated, this developmental process could not be interrupted and the daughter cells continued to divide and differentiate in the absence of further antigenic stimulation. These data indicate that initial antigen encounter triggers an instructive developmental program that does not require further antigenic stimulation and does not cease until memory CD8+ T cell formation.

Figure 1. Varying the antigen dose affects the magnitude of the CD8⁺ T cell response and the recruitment of naïve CD8+ T cells, but all recruited cells divide extensively

(a–c) CFSE-labeled naïve P14 CD8⁺ T cells (∼1–2×10⁶) were adoptively transferred into B6 mice and these chimeric mice were then infected with (a) high (3×10⁴ CFU) (b) intermediate (3×10³ CFU) or (c) low (100 CFU) doses of LM-GP33. On days 1 and 7 after infection the CFSE fluorescence of splenic P14 CD8⁺ T cells was analyzed by flow cytometry by staining with anti-CD8α and D^b-GP(33–41) tetramer. Histograms show the CFSE intensity of CD8⁺D^b-GP(33–41)⁺ T cells. Note that on day 7 after intermediate- and low-dose infections, the P14 CD8⁺ T cells are found in only two CFSE peaks: cells that had not divided and cells that had divided more than seven times and were CFSE^neg. The percentage of P14 CD8⁺ T cells not recruited into the immune response are shown in parentheses. The total number of activated CFSE^neg P14 CD8⁺ T cells in a representative experiment are in boxes. (d–i) CFSE-labeled P14 CD8+T cells (50,000) were cultured for 4 days in vitro with (d) 10,000 (e) 5000 (f) 1000 (g) 200 (h) 100 or (i) 0 GP(33–41)-pulsed splenocytes. The cells were stained with anti-CD8α and D^b-GP(33–41) tetramers and CFSE fluorescence analyzed was by flow cytometry. The percentage of cells that were not activated and did not proliferate are indicated.

Figure 2. CD8⁺ T cells become committed to differentiate fully into effector CTLs after initial activation

CFSE-labeled naïve P14 CD8⁺ T cells (2×10⁶) were adoptively transferred into B6 mice that were then infected with LM-GP33 (100 CFU). On (a) day 1 and (b) day 7 after infection splenic P14 CD8⁺ T cells were identified using fluorescent D^b-GP(33–41) tetramers and the expression of proteins associated with T cell activation were analyzed with anti-CD44, anti-CD43 (clone 1B11), anti-CD62L and anti–IFN-γ. The expression of CD44, CD43 and CD62L on the recruited (R, filled histograms) and nonrecruited (NR, solid-line histograms) P14 CD8⁺ T cells. IFN-γ production by P14 CD8⁺ T cells 1 and 7 days after infection was analyzed by flow cytometry after 5 h of GP(33–41) peptide stimulation and then CD8⁺ surface and IFN-γ intracellular staining. Dot-plots represent CD8⁺ T cells; note that only CFSE^neg cells produce IFN-γ on day 7 after infection.

Figure 3. Activated CD8⁺ T cells continue to divide in the absence of antigenic stimulation in vitro

(a,b) CFSE-labeled Thy1.1⁺ naïve P14 CD8⁺ T cells (50,000) were stimulated with GP(33–41) peptide–pulsed splenocytes (5000 from Thy1.2⁺ mice) for 4 days in vitro. (a) At 48 h and (b) 96 h, cells were removed from the cultures and stained with anti-Thy1.1 and D^b-GP(33–41) tetramers. Histograms were gated on Thy1.1⁺D^b-GP(33–41)⁺ T cells. After 48 h, CFSE-labeled (c) naïve or (d) memory Thy1.2⁺P14 CD8⁺ T cells were added to the cultures described above. After an additional 48 h, the cells were stained with anti-Thy1.2 and D^b-GP(33–41) tetramers. Histograms were gated on Thy1.2⁺D^b-GP(33–41)⁺ T cells.

Figure 4. Repeated exposure to antigen is not necessary for activated CD8⁺ T cells to divide several times and differentiate into effector CTLs in vivo

(a) Thy1.1⁺ and Thy1.2⁺ P14 CD8⁺ T cells were stimulated for 24 h with GP(33–41) peptide and stained with anti-CD8α + anti-CD69 and D^b-GP(33–41) tetramers. Histograms are gated on CD8⁺D^b-GP(33–41)⁺ T cells and show the amount of CD69 on stimulated Thy1.1⁺(solid line), Thy1.2⁺(dashed line) or unstimulated (filled histogram) P14 CD8⁺ T cells. After 24 h of GP(33–41) peptide stimulation the (b) Thy1.1⁺ or (c) Thy1.2⁺P14 CD8⁺ T cells were purified using CD8α magnetic beads and the purity analyzed by staining for CD8⁺ and D^b-GP(33–41)–specific T cells. More than 95% of the purified cells were CD8⁺. (d) The purified Thy1.1⁺–stimulated P14 CD8⁺ T cells were CFSE-labeled and injected into B6 mice. (e) The purified Thy1.2⁺-stimulated P14 CD8⁺ T cells were not CFSE-labeled but were injected into a second set of B6 mice along with the naïve CFSE-labeled Thy1.1⁺P14 CD8⁺ T cells that served as sensors for presence of residual antigen. (f) A third set of B6 mice received the sensor P14 CD8⁺ T cells alone. (d–g) Five to six days after transfer, the extent of proliferation and differentiation of the Thy1.1⁺-stimulated or sensor P14 CD8⁺ T cells was analyzed. Splenocytes from the recipient mice were stained with anti-Thy1.1 and D^b-GP(33–41) tetramers. The histograms, which show CFSE fluorescence, are gated on Thy1.1⁺D^b-GP(33–41)⁺ T cells. The percentage of CFSE^pos or CFSE^neg P14 CD8⁺ T cells is shown. (g) IFN-γ production in Thy1.1⁺ stimulated or sensor P14 CD8⁺ T cells was analyzed by flow cytometry after a 5-h incubation with (+) or without (–) GP(33–41) peptide. The splenocytes were stained for surface Thy1.1 and intracellular IFN-γ and dot-plots are gated on Thy1.1⁺ T cells. The percentage of Thy1.1⁺ T cells producing IFN-γ are indicated.

Figure 5. IL-2 is important for activated CD8⁺ T cells proliferating in the absence of continuous antigenic stimulation

Naïve CFSE-labeled P14 CD8⁺ T cells were stimulated with GP(33–41) peptide– pulsed cells for 4 days in culture, as described in Fig. 3. After the first 24 h of stimulation, either anti–IL-2 + anti-CD25 (open histogram) or IgG isotype control antibodies (filled histogram) were added to the cultures every 12 h over the next 3 days. Histograms are gated on CD8⁺D^b-GP(33–41)⁺ T cells.

Figure 6. Activated CD8⁺ T cells are programmed to differentiate into long-lived, functional memory CD8⁺ T cells

Naïve CFSE-labeled Thy1.1⁺ P14 CD8⁺ T cells (2×10⁶) were adoptively transferred into naïve B6 (Thy1.2⁺) mice, which were then infected with LM-GP33 (∼100 CFU). At days (a, e) 7 (b, f) 15 (c, g) 35 and (d, h) 100 the extent of proliferation and differentiation of the P14 CD8⁺ T cells was examined by staining splenocytes with anti-Thy1.1 and D^b-GP(33–41) tetramer. (a–d) Histograms are gated on Thy1.1⁺D^b-GP(33–41)-specific T cells. The CFSE intensity of nonrecruited P14 CD8⁺ T cells decreases with time due to intrinsic quenching. (e–h) Splenocytes were incubated for 5 h either with (+) or without (−) GP(33–41) peptide and then Thy1.1 surface and IFN-γ intracellular staining was done. The dot-plots are gated on Thy1.1⁺ T cells.(i)The absolute numbers of recruited (CFSE^neg) and nonrecruited (CFSE^pos) P14 CD8⁺ T cells in the spleen were quantified on days 7–100 after low-dose LM-GP33 infection. (j) Thy1.1⁺ P14 CD8⁺ T cells stimulated with GP(33–41) peptide for 24 h in vitro were purified to ∼95%, CFSE-labeled and transferred into naïve B6 (Thy1.2⁺) mice that were devoid of contaminating GP(33–41)-expressing APCs, as described in Fig. 4. The number of Thy1.1⁺ P14 CD8⁺T cells persisting in a functional state was assessed, as described in a, 43 days after transfer.

Figure 7. Memory CD8⁺ T cells generated in antigen-limiting conditions can confer protective immunity

Naïve CFSE-labeled P14 CD8⁺ T cells (2×10⁶) were adoptively transferred into naïve B6 mice that were then infected with LM-GP33 (∼100 CFU). (a) LM-GP33–immunized mice and (b) naïve B6 mice were intravenously challenged 90 days later with LCMV clone 13 (2×10⁶ PFU). Eight days after clone 13 infection the splenocytes were stained with anti-CD8α + D^b-GP(33–41) and D^b-NP(396–404). The dot-plots are gated on CD8⁺ T cells and the percentage of tetramers GP(33–41)- or NP(396–404)–specific CD8⁺ T cells is shown. (c) At days 5 and 8 after LCMV clone 13 infection viral titers were determined by plaque assay analysis of the sera of LM-GP33-immunized and naïve B6 mice. (d) LCMV viral titers in the spleen were determined by plaque assay analysis 8 days after infection.

Figure 8. Models for proliferation and differentiation of naïve CD8⁺ T cells

(a) CD8⁺ T cell proliferation is dependent on repeated encounters with antigen. Each cell that is stimulated by antigen divides and progressively differentiates into effector CTLs and memory CD8⁺ T cells with each successive cell division. According to this model, it is essential that each daughter cell be stimulated with antigen, otherwise CD8⁺ T cell division, and possibly differentiation, would be halted upon antigen removal. (b) Naïve CD8⁺ T cells are developmentally programmed to divide at least seven to ten times and to differentiate into effector CTLs and long-lived functional memory CD8⁺ T cells. Optimal antigenic stimulation of the parental cell triggers this developmental program and the CD8⁺ T cells become committed to proliferation and differentiation. Further antigenic stimulation of the daughter cells may increase the number of times the activated CD8⁺ T cells divide, but it is unnecessary for this developmental program to progress.