Transcriptional repressor Blimp-1 promotes CD8(+) T cell terminal differentiation and represses the acquisition of central memory T cell properties

Abstract

During acute infections, a small population of effector CD8(+) T cells evades terminal differentiation and survives as long-lived memory T cells. We demonstrate that the transcriptional repressor Blimp-1 enhanced the formation of terminally differentiated CD8(+) T cells during lymphocytic choriomeningitis virus (LCMV) infection, and Blimp-1 deficiency promoted the acquisition of memory cell properties by effector cells. Blimp-1 expression was preferentially increased in terminally differentiated effector and "effector memory" (Tem) CD8(+) T cells, and gradually decayed after infection as central memory (Tcm) cells developed. Blimp-1-deficient effector CD8(+) T cells showed some reduction in effector molecule expression, but primarily developed into memory precursor cells that survived better and more rapidly acquired several Tcm cell attributes, including CD62L and IL-2 expression and enhanced proliferative responses. These results reveal a critical role for Blimp-1 in controlling terminal differentiation and suppressing memory cell developmental potential in effector CD8(+) T cells during viral infection.

Figure 1. Blimp-1 is preferentially expressed in terminally differentiated LCMV-specific effector and memory CD8 T cells

(A) Relative Blimp-1 mRNA levels (±SEM) in naïve or LCMV-specific P14 CD8 T cells measured by qRT-PCR. n=8-9 independent samples/time point. (B) Naïve (CD44^lo CD62L^hi) or D^bGP_33-41-specific CD8 T cells in the spleen of Blimp-1:YFP BAC tg mice were analyzed and mean percentage (±SEM) and MFI of Blimp-1:YFP^hi cells is shown (n≥6 mice/time point). (C) Relative level of Blimp-1 mRNA (±SEM) in KLRG1^lo IL-7R^hi and KLRG1^hi IL-7R^lo P14 CD8 T cells in the spleen from days 8, 15 and 60 p.i. as measured by qRT-PCR. (D) Blimp-1 (or Grp94) protein levels in FACS-purified KLRG1^hi IL-7R^lo or KLRG1^lo IL-7R^hi P14 effector CD8 T cells from 8-10 days p.i. Fold change represents average over 3 independent experiments (±SEM). (E,F) Blimp-1:YFP expression was analyzed in P14 BAC tg CD8 T cells stained with antibodies to IL-7R and KLRG1 or CD27 and CD62L in the blood (E) or spleen (F) at days 10-40 p.i. n≥3 experiments each with 2-3 mice per time point. (E) Percentages of cells in each quadrant is shown. Histogram plots below each contour plot show the percent of Blimp-1:YFP^lo cells and MFI of YFP in each quadrant. (F) Line graph shows the percent (±SEM) of Blimp-1:YFP^hi cells in KLRG1^hi or KLRG1^lo D^bGP_33-41-specific CD8 T cells in the spleen.

Figure 2. Blimp-1 deficiency increases effector CD8 T cell survival and prevents terminal effector cell differentiation

(A) Representative plots of splenic D^bGP_33-41 and D^bNP_396-404 tetramer+ CD8 T cells at 8 and 60 days post LCMV infection in WT and Blimp-1 CKO mice (percent of tetramer+ cells CD8+ T cells ±SEM is indicated). (B) Blimp-1 CKO (white bars) or WT littermate controls (black bars) were infected with LCMV and numbers of LCMV-specific CD8 T cells in the spleen (±SEM) were enumerated by IFNγ intracellular cytokine staining (ICCS) after 5hr stimulation with NP_396-404, GP_33-41, and GP_276-286 peptides. Note: numbers based on tetramer staining are similar to those based on ICCS. n=4 experiments each with 2-3 animals per group per time point. (C, D, E) Combined number (±SEM) of D^bGP_33-41-and D^bNP_396-404 tetramer+ CD8 T cells in various tissues at day 8 (C), day 35 (D), and day 150 (E) p.i. in spleen (SPL), inguinal lymph nodes (LN), bone marrow (BM), liver (LV), and lung (LG). (F) Expression of IL-7R and KLRG1 (left plots) or CD62L and CD27 (right plots) in D^bGP_33-41 tetramer+ CD8 T cells at day 8 or day 60 p.i. Mean percentages (±SEM) of 5-12 mice per time point. (G) Percent (±SEM) of CD62L^hi D^bGP_33-41-specific CD8 T cells in the spleens of WT (black squares) or CKO (open squares) animals following LCMV infection.

Figure 3. Blimp-1 deficiency alters the expression of genes known to regulate effector and memory CD8 T cell differentiation

(A-C) qRT-PCR analysis of Bcl6 (A), Tbx21 (B), and Tcf7 (C) mRNA expression in WT and CKO effector CD8 T cells from day 8 p.i. or WT naïve cells (CD44^lo CD62L^hi), SLEC (KLRG1^hi IL-7R^lo) or MPEC (KLRG1^lo IL-7R^hi) from day 8 p.i., and memory CD8 T cells from day 150 p.i. Percentages (±SEM) are representative of 2-3 independent experiments each with 2-3 samples. (D) Percent (±SEM) Kit receptor positive D^bGP_33-41-tetramer+ CD8 T cells from WT (grey histogram) and CKO (black line) animals 8 days after LCMV infection.

Figure 4. Blimp-1 deficiency alters CTL differentiation

(A) Blimp-1 CKO or WT littermate controls were infected with LCMV and analyzed for IFNγ and IL-2 production at days 8 and 60 p.i. Representative plots showing frequency of GP_33/34-41- and NP_396-404-specific CD8 T cells producing IFNγ and IL-2 based on ICCS after 5hr stimulation with peptide (note, GP_33-41 peptide activates both D^bGP_33-41- and K^bGP_34-41-specific CD8 T cells; similar results observed for GP_276-286-specific CD8 T cells). Mean (±SEM) IFNγ MFI is indicated next to plots. Bar graphs to right show the percent of IFNγ+ cells that co-produce IL-2. Note, no difference in TNFα was noted between the two groups of animals. * p < 0.05. (B) Granzyme B protein levels (percent GzB+ ±SEM) of WT (grey histogram) and CKO (black line) D^bGP_33-41-specific effector CD8 T cells in the spleen 5 and 7 days after LCMV infection. (C) Serum LCMV viral titers in WT (black boxes) and CKO (white boxes) animals at various points after infection. n=5-8 mice per group per time point. (D) Phenotype of 2° effector CD8 T cells from LCMV immune WT and CKO mice reinfected with LCMV-cl. 13 7 days prior. Virus was cleared in the serum in both groups by day 5 p.i.

Figure 5. Blimp-1 expression correlates with cells of lower proliferative capacity

(A) Blimp-1:YFP+ (black bar) or Blimp-1:YFP- (white bar) Thy1.1+ P14 memory CD8 T cells from day 60 p.i. chimeric mice were FACS-sorted, and 25,000 cells were transferred into naïve Thy1.2+ B6 recipients that were subsequently infected with rLM33. The number (±SEM) of donor cells recovered from the spleen 7 days p.i. was calculated. * p < 0.05. (B) Blimp-1 CKO (GzB-cre⁺; Prdm1^flox/flox; black line) or WT littermate (GzB-cre^-; Prdm1^flox/flox; shaded) effector CD8 T cells from days 8-10 p.i. were CFSE-labeled, stimulated with GP_33-41 peptide, and analyzed for cell division 48 hours later. Data representative of 4 independent experiments each with 2-3 samples. (C) Day 7 Blimp-1 CKO (CD4-cre⁺; Prdm1^flox/flox) or WT littermate (CD4-cre⁺; Prdm1^+/+) P14 effector cells were purified from “chimeric” mice using FACS, and 25,000 cells were transferred into naive congenic recipients that were subsequently infected with rVV33. The number of secondary effector cells in the spleen (±SEM) was calculated 7 days post-rVV33 infection. * p < 0.05. (D) 50,000 D^bGP_33-41-specific CD8 T cells from WT (GzB-cre^-; Prdm1^flox/flox) or Blimp-1 CKO (GzB-cre⁺; Prdm1^flox/flox) mice infected 90 days previously with LCMV were transferred into naïve RAG1-/- recipients that were subsequently infected with rLM33. The number of secondary effector cells in the spleen (±SEM) was calculated 7 days post-rLM33 infection. NSD=no significant difference. (E) Purified WT (GzB-cre^-; Prdm1^flox/flox) or Blimp-1 CKO (GzB-cre⁺; Prdm1^flox/flox) effector CD8 T cells from day 10 p.i. or WT memory cells from day 90 p.i. were CFSE-labeled, transferred into γ-irradiated recipients, and analyzed for cell division 6 days later. Histograms gated on D^bGP_33-41-specific CD8 T cells. Similar results were found for NP_396-404-specific cells. Cell division numbers and PI, proliferation index (mean number of cell divisions), are indicated. Data are representative of 3 independent experiments each with 2-3 samples.