Opposing effects of TGF-beta and IL-15 cytokines control the number of short-lived effector CD8+ T cells

Abstract

An effective immune response against infectious agents involves massive expansion of CD8(+) T cells. Once the infection is cleared, the majority of these effector cells die through unknown mechanisms. How is expansion controlled to maximize pathogen clearance and minimize immunopathology? We found, after Listeria infection, plasma transforming growth factor beta (TGF-beta) titers increased concomitant with the expansion of effector CD8(+) T cells. Blocking TGF-beta signaling did not affect effector function of CD8(+) T cells. However, TGF-beta controlled effector cell number by lowering Bcl-2 amounts and selectively promoting the apoptosis of short-lived effector cells. TGF-beta-mediated apoptosis of this effector subpopulation occurred during clonal expansion and contraction, whereas interleukin-15 (IL-15) promoted their survival only during contraction. We demonstrate that the number of effector CD8(+) T cells is tightly controlled by multiple extrinsic signals throughout effector differentiation; this plasticity should be exploited during vaccine design and immunotherapy against tumors and autoimmune diseases.

Figure 1. TGF-β signaling may be important for controlling the number and function of effector CD8⁺ T cells

(A) Splenocytes from 6 week old mice under steady state conditions were gated on CD8⁺ T cells. Data shown is representative of at least 10 different sets of mice with similar profiles. (B) 5×10⁴ naïve OTI/RAG1^-/- cells were adoptively transferred into wild type B6 hosts and OTI cell expansion in response to LM-OVA infection was measured in a longitudinal experiment. Data is plotted as the fraction of OTI cells among 25,000 other peripheral blood lymphocytes (PBLs). Average and s.d. of 4 mice is shown. (C) Total TGF-β levels detected with ELISA after acidification of plasma collected from infected mice from part (B).

Figure 2. A 3-fold increase in the number of OTI-DNR compared to OTI cells at the peak of CD8⁺ T cell clonal expansion in response to LM-OVA

(A) 2.5×10⁴ each of naïve OTI/RAG1^-/- CD45.1.1 and OTI/DNR/RAG1^-/-CD45.1.2 cells were adoptively co-transferred into naïve hosts, followed by infection with LM-OVA one day later. The absolute number of each OTI and OTI-DNR population from each organ is plotted over time. For the blood measurements, the number of OTI cells per 1 mL of blood is plotted. Data is representative of two independent experiments with similar results. (B-C) Co-transfer and infection was performed as in part A, except the same group of mice were bled for a longitudinal experiment to compare the kinetics of OTI and OTI-DNR T cell accumulation among PBLs while measuring plasma TGF-β levels in the same infected mice. Average and s.d. is shown. Data is representative of two independent experiments with similar results and a total of 10-15 mice in each experiment.

Figure 3. Blockade of TGF-β signaling does not alter cytokine production and CTL activity of effector CD8⁺ T cells

(A) Adoptive co-transfer and LM-OVA infection was performed as described in Fig. 2A. Intracellular cytokine staining of spleen cells isolated 7 days p.i. Host (CD45.2.2), OTI (CD45.1.1), and OTI-DNR (CD45.1.2) cells were identified based on staining with CD8 and the corresponding congenic markers. Splenocytes were stimulated with or without SIINFEKL peptide. 1 Representative histogram is shown. (B) Average and s.d. of 4 different co-transfers. Data is representative of 3 independent experiments with similar results. (C) In vivo CTL assay performed with effector OTI and OTI-DNR cells isolated from the spleen of infected mice 7 days p.i. Representative raw data is shown on the left. Summary of two independent experiments is shown on the right, with average and s.d. of 4 recipients of each population. (D) Relative mRNA levels of cytolytic molecules from day 7 effector cells. Samples were first normalized to HPRT. Relative fold increase over naïve OTI cells is shown. Data is average and s.d. of 3 independent experiments.

Figure 3. Blockade of TGF-β signaling does not alter cytokine production and CTL activity of effector CD8⁺ T cells

(A) Adoptive co-transfer and LM-OVA infection was performed as described in Fig. 2A. Intracellular cytokine staining of spleen cells isolated 7 days p.i. Host (CD45.2.2), OTI (CD45.1.1), and OTI-DNR (CD45.1.2) cells were identified based on staining with CD8 and the corresponding congenic markers. Splenocytes were stimulated with or without SIINFEKL peptide. 1 Representative histogram is shown. (B) Average and s.d. of 4 different co-transfers. Data is representative of 3 independent experiments with similar results. (C) In vivo CTL assay performed with effector OTI and OTI-DNR cells isolated from the spleen of infected mice 7 days p.i. Representative raw data is shown on the left. Summary of two independent experiments is shown on the right, with average and s.d. of 4 recipients of each population. (D) Relative mRNA levels of cytolytic molecules from day 7 effector cells. Samples were first normalized to HPRT. Relative fold increase over naïve OTI cells is shown. Data is average and s.d. of 3 independent experiments.

Figure 4. TGF-β promotes apoptosis of effector CD8⁺ T cells during clonal expansion

(A) Histograms showing BrdU incorporation of clonally expanding OTI and OTI-DNR cells from co-transfer experiments. Splenocytes were first gated on CD8⁺ T cells and host, OTI, and OTI-DNR subpopulations were separated based on congenic markers. (B) Pair-wise comparison of each subpopulation in 4 different animals with varying degrees of BrdU incorporation. Data are representative of 3 independent experiments with similar results. (C) Dead versus apoptotic cells from day 5 (PBL) and day 7 (spleen) of infected mice were identified by AnnexinV and PI staining. OTI and OTI-DNR cells were then separated based on congenic markers amongst the PI-population. Average and s.d. of 3 (day 5) and 4 (day 7) mice per group is shown.

Figure 5. Selective apoptosis of SLECs by TGF-β

(A) Phenotypic comparisons between OTI and OTI-DNR effector CD8⁺ T cells. Histograms comparing the expression of each surface molecule on host CD8⁺ (solid gray), OTI (dashed line), and OTI-DNR (black line) T cells from co-transfer experiments. Data are representative of more than 4 independent experiments with similar results. (B) Expression pattern of indicated surface receptors was analyzed on OTI and OTI-DNR CD8⁺ T cells isolated from peripheral blood of LM-OVA infected mice at the indicated time points. Data is average and s.d. of 3-5 individual co-transfers and represents 1 out of 3 independent experiments with similar results. (C) Representative diagram of each effector sub-population separated based on CD127 and KLRG1 expression and the terminology used to describe each subset. (D) Representation of the difference seen in the percentages of OTI and OTI-DNR SLECs and MPECs isolated from the spleen of infected mice at the indicated time points. Time point -2 days p.i. refers to naïve cells prior to adoptive transfer. (E) Bar graphs (top) comparing the absolute number of total and each OTI and OTI-DNR sub-population from the spleen of infected mice at each indicated time point. Longitudinal graphs (bottom) comparing the contribution of each sub-population, on the same scale, to the overall increase in the number of OTI-DNR effector cells. Data is average and s.d. of 2-3 mice per time point and represents 1 out of 2 independent experiments with similar results.

Figure 5. Selective apoptosis of SLECs by TGF-β

(A) Phenotypic comparisons between OTI and OTI-DNR effector CD8⁺ T cells. Histograms comparing the expression of each surface molecule on host CD8⁺ (solid gray), OTI (dashed line), and OTI-DNR (black line) T cells from co-transfer experiments. Data are representative of more than 4 independent experiments with similar results. (B) Expression pattern of indicated surface receptors was analyzed on OTI and OTI-DNR CD8⁺ T cells isolated from peripheral blood of LM-OVA infected mice at the indicated time points. Data is average and s.d. of 3-5 individual co-transfers and represents 1 out of 3 independent experiments with similar results. (C) Representative diagram of each effector sub-population separated based on CD127 and KLRG1 expression and the terminology used to describe each subset. (D) Representation of the difference seen in the percentages of OTI and OTI-DNR SLECs and MPECs isolated from the spleen of infected mice at the indicated time points. Time point -2 days p.i. refers to naïve cells prior to adoptive transfer. (E) Bar graphs (top) comparing the absolute number of total and each OTI and OTI-DNR sub-population from the spleen of infected mice at each indicated time point. Longitudinal graphs (bottom) comparing the contribution of each sub-population, on the same scale, to the overall increase in the number of OTI-DNR effector cells. Data is average and s.d. of 2-3 mice per time point and represents 1 out of 2 independent experiments with similar results.

Figure 6. TGF-β promotes the apoptosis of effector CD8⁺ T cells both ex vivo and in vitro

(A) TGF-βRII expression on naïve and effector OTI and OTI-DNR sub-populations. After adoptive co-transfer and LM-OVA infection, OTI and OTI-DNR cells isolated from the spleen of infected mice were stained with CD127 and KLRG1 and either TGFβRII or the isotype control antibodies. MFI of TGF-βRII was determined by subtracting the MFI of background staining observed with isotype control, which varied from 3-6, from the MFI of TGF-βRII staining on the same sub-population of effector cells. (B) Experimental details of ex vivo treatment of OTI and OTI-DNR effector cells with TGF-β. (C) Upon experimental procedure described in part (B), OTI and OTI-DNR effector sub-populations were identified by the congenic marker and CD127/KLRG1 staining. The percent of live cells in each sub-population was determined from the forward and side scatter plots. The bar graph represents the fraction of live cells in TGF-β treated versus non-treated samples. Average and s.d. of 4 experimental wells is shown. Data is representative of 1 out of 2 independent experiments. (D) Experimental details of in vitro treatment of OTI and OTI-DNR effector cells with TGF-β. (E) Forward and side scatter plots and CFSE dilution of OTI and OTI-DNR cells after being stimulated for 48 hours with SIINFEKL-loaded APCs (top). Cells were washed and cultured in the absence or presence of 5ng/ml of TGF-β and 20ng/ml of IL-15, IL-7, or IL-2 for another 4 days. FACS plots represent total cell recovery and show all the events collected within 1 minute. AnnexinV+ (dead or apoptotic) and AnnexinV- (live) and the CFSE dilution of each fraction is shown. Data is representative of 1 out of 3 independent experiments with similar results.

Figure 7. TGF-β and IL-15 control the number of SLECs by exerting opposing effects on Bcl-2 levels in vivo

(A) Quantitative RT PCR comparing mRNA levels of Bcl-2 and T-bet in sorted naïve (prior to adoptive transfer) and effector (day 7 p.i.) OTI and OTI-DNR SLECs and MPECs. Samples were first normalized to HPRT. Fold change from naïve OTI cells is shown. Data is representative of 1 out of 3 independent experiments with similar results. (B) Intracellular staining of Bcl-2 and T-bet protein in day 7 effector OTI and OTI-DNR sub-populations from a co-transfer experiment. Data is representative of 1 out of 3 independent experiments with similar results. (C) 2.5×10⁴ each OTI and OTI-DNR cells were co-transferred into either IL-15^+/+ or IL-15^-/- hosts followed by LM-OVA infection. Fraction of total (left) and SLEC sub-population (right) of OTI and OTI-DNR cells were determined among total PBLs and converted to numbers of each cell type per 25,000 PBL. (D) Bar graphs comparing the calculated number of each OTI and OTI-DNR sub-population among 25,000 PBLs in IL-15^+/+ and IL-15^-/- hosts. Average and s.d. of 3 co-transfers into IL-15^+/+ and 7 co-transfers into IL-15^-/- mice is shown. (E) The average ratio of OTI-DNR/OTI SLECs in IL-15^+/+ versus IL-15^-/- hosts at each indicated time point post LM-OVA infection. (F) Representation of the difference seen in the percentage of each OTI and OTI-DNR sub-populations from the spleens of infected IL-15^+/+ and IL-15^-/- hosts at the indicated time points. (G) Comparison of intracellular Bcl-2 protein levels in each sub-population of OTI and OTI-DNR cells isolated from the spleen of IL-15^+/+ or IL-15^-/- hosts. (H) Absolute number of each OTI and OTI-DNR sub-population from the spleens of IL-15^+/+ and IL-15^-/- hosts. Data is average and s.d. of 2-3 co-transfers for each time point.

Figure 7. TGF-β and IL-15 control the number of SLECs by exerting opposing effects on Bcl-2 levels in vivo

(A) Quantitative RT PCR comparing mRNA levels of Bcl-2 and T-bet in sorted naïve (prior to adoptive transfer) and effector (day 7 p.i.) OTI and OTI-DNR SLECs and MPECs. Samples were first normalized to HPRT. Fold change from naïve OTI cells is shown. Data is representative of 1 out of 3 independent experiments with similar results. (B) Intracellular staining of Bcl-2 and T-bet protein in day 7 effector OTI and OTI-DNR sub-populations from a co-transfer experiment. Data is representative of 1 out of 3 independent experiments with similar results. (C) 2.5×10⁴ each OTI and OTI-DNR cells were co-transferred into either IL-15^+/+ or IL-15^-/- hosts followed by LM-OVA infection. Fraction of total (left) and SLEC sub-population (right) of OTI and OTI-DNR cells were determined among total PBLs and converted to numbers of each cell type per 25,000 PBL. (D) Bar graphs comparing the calculated number of each OTI and OTI-DNR sub-population among 25,000 PBLs in IL-15^+/+ and IL-15^-/- hosts. Average and s.d. of 3 co-transfers into IL-15^+/+ and 7 co-transfers into IL-15^-/- mice is shown. (E) The average ratio of OTI-DNR/OTI SLECs in IL-15^+/+ versus IL-15^-/- hosts at each indicated time point post LM-OVA infection. (F) Representation of the difference seen in the percentage of each OTI and OTI-DNR sub-populations from the spleens of infected IL-15^+/+ and IL-15^-/- hosts at the indicated time points. (G) Comparison of intracellular Bcl-2 protein levels in each sub-population of OTI and OTI-DNR cells isolated from the spleen of IL-15^+/+ or IL-15^-/- hosts. (H) Absolute number of each OTI and OTI-DNR sub-population from the spleens of IL-15^+/+ and IL-15^-/- hosts. Data is average and s.d. of 2-3 co-transfers for each time point.