Rescue of CD8 T cell-mediated antimicrobial immunity with a nonspecific inflammatory stimulus

Abstract

Reconstitution of protective immunity by adoptive transfer of pathogen-specific T cells has been successful in patients with compromised cellular immunity. The in vivo effectiveness of in vitro-expanded CD8 CTLs is variable, however. For example, adoptively transferred Listeria monocytogenes-specific CD8 CTLs only confer protective immunity if challenge infection occurs within 48 hours of T cell infusion. Herein we show that transferred CTLs persist in lymphoid compartments for many weeks, but that their response to bacterial challenge decreases during the first week following transfer. While T cells transferred less than 48 hours before infection proliferate, those transferred 7 days before infection die. Remarkably, treatment of mice with anti-CD40 at the time of T cell infusion reprograms transferred T cells, allowing them to proliferate and confer protective immunity upon bacterial challenge 7 days later. Our study demonstrates, for the first time to our knowledge that CD40-mediated stimuli can influence CD8 T cell activation independent of concurrent antigen exposure. The ability to modulate long-term responsiveness of CD8 T cells with a transient, nonspecific inflammatory stimulus has importation implications for adoptive immunotherapy.

Figure 1

In vitro–expanded LLO_91-99–specific CD8 CTLs have an effector phenotype and retain a diverse TCR Vβ repertoire 7–14 days after the second in vitro stimulation with peptide-pulsed APCs. (a) CD8 CTL line stained with mAb for CD8α, CD62L, Thy1.1, CD44, CD25, and H2-K^d tetramers complexed with LLO_91-99. Left panel: Dot plot gated on live CD8 and Thy1.1 T cells. Middle and right panels: Dot plots gated on live CD8 T cells. Numbers in upper quadrants represent percentages of total CD8 T cells. (b) Percentage specific lysis by the LLO_91-99–specific CD8 CTL line in the presence of different concentrations of LLO_91-99 peptide was determined by ⁵¹Cr-release assay using P815 (H2^d) target cells. (c) TNF and IFN-γ production by CD8 CTL line was assessed by standard intracellular cytokine staining following in vitro stimulation. (d) CD8 CTL line was stained for TCR Vβ expression with TCR Vβ–specific antibodies. The percentage of LLO_91-99–specific CD8 CTLs stained with each of the TCR antibodies is indicated. Data are representative of two independent experiments.

Figure 2

CD8 CTLs maintain an effector phenotype and do not proliferate after transfer into naive, syngeneic recipients. (a–d) Three and seven days after transfer of 6 × 10⁶ Thy1.1 CD8 CTLs into Thy1.2 BALB/c recipients, splenocytes were stained for CD8α, Thy1.1, CD44, CD25, and with LLO_91-99 H2-K^d tetramers. (a) Dot plots are gated on live CD8 T cells, and the percentage of cells positive for Thy1.1 (y axis) and LLO_91-99 (x axis) is shown. (b) Absolute number of cells that stained positive for Thy1.1 and LLO_91-99 on days 3 and 7 after transfer; each point represents an individual mouse. (c) LLO_91-99–specific CD8 CTLs were labeled with CFSE prior to transfer, and the percentage of CFSE⁺ Thy1.1 CD8 T cells 3 days after transfer is indicated. (d) Activation-marker expression of LLO_91-99–specific T cells was determined 72 hours after transfer. Staining for Thy1.1 is shown on the x axis, and staining with LLO_91-99 H2-K^d tetramers and for CD62L, CD25, and CD44 is shown on the y axis. Dot plots are gated on live CD8 T cells. (a, c, and d) Dot plots are representative of three to four mice per group.

Figure 3

LLO_91-99–specific CD8 CTLs confer a high degree of protective immunity to wild-type L. monocytogenes (Lm) infection 30 minutes or 48 hours after T cell infusion (a). T cell recipients are not protected from a challenge with LLO_Ser92 L. monocytogenes 30 minutes after CTL infusion, demonstrating in vivo antigen specificity (b). CFUs per organ are shown on the y axis in log scale. Time interval between CTL transfer and infection is indicated. Control animals received PBS, while experimental animals received 6 × 10⁶ LLO_91-99–specific CD8 CTLs. *Lower limit of detection. Data are the mean and SD of two to three animals per group and are representative of two independent experiments.

Figure 4

Protective immunity conferred by transferred CTLs correlates with their expansion 72 hours after infection. (a) We infused 6 × 10⁶ T cells into recipients, followed by infection with wild-type L. monocytogenes or LLO_Ser92 L. monocytogenes 30 minutes or 48 hours after transfer. Seventy-two hours after infection, splenocytes were stained for CD8α, Thy1.1, and with LLO_91-99 H2-K^d tetramers. Dot plots are gated on live CD8 lymphocytes. Staining for Thy1.1 is shown on the y axis, tetramer staining on the x axis. Dot plots represent 4–6 animals per group. (b) CD8 CTLs were labeled with CFSE and transferred into recipient mice that were either infected or left uninfected. 72 hours after transfer CFSE fluorescence of Thy1.1⁺, LLO_91-99–specific T cells was determined. (c) Absolute number of transferred T cells, determined by Thy1.1 and H2-K^d tetramers, is plotted for various conditions. Lane 1, Lm_Ser92 infection 30 minutes after CTL transfer; lane 2, Lm infection 30 minutes after CTL transfer; lane 3, Lm infection 48 hours after CTL transfer; lane 4, Lm infection 7 days after CTL transfer; lane 5, no Lm infection, day 3 after CTL transfer; lane 6, no Lm infection, day 7 after CTL transfer; lane 7, Lm infection 7 days after CTL transfer. 5, 6, and 7 received 100 μg anti-CD40 antibody intraperitoneally 2 days and 1 day before CTL infusion and 12 hours after infection. Each point represents an individual mouse. *^,†Difference in absolute numbers of transferred T cells between conditions * and ^† achieves statistical significance (P < 0.05 by Student’s t test).

Figure 5

Protective immunity and expansion of transferred CTLs is lost 7 days after infusion. Loss of protection is prevented by anti-CD40 antibody (FGK45) treatment. (a) Recipients received 6 × 10⁶ LLO_91-99–specific CTLs and were infected with wild-type L. monocytogenes 7 days later. Seventy-two hours after infection bacteria were counted in liver and spleen. Left panel: no FGK45; right panel 100 μg FGK45 intraperitoneally 2 days and 1 day before CTL transfer and 12 hours after infection. Control animals received PBS. (b) Left panel: Forward scatter (FSC) for LLO_91-99–specific CTLs 7 days after transfer in the presence (gray histogram) or absence (black line) of FGK45. Right panel: mean FSC intensity for two to three mice per group. (c) Seventy-two hours after infection, splenocytes were stained for CD8α, Thy1.1, and with LLO_91-99 H2-K^d tetramers. Right upper quadrants show percentages of total CD8 lymphocytes. (d) Two weeks after transfer of CTLs and administration of FGK45, as described in a but without infection of the recipients, splenocytes were restimulated in vitro with LLO_91-99. Percentage of specific lysis in the presence of different concentrations of LLO_91-99 peptide was determined by standard ⁵¹Cr-release assay, using P815 (H2^d) target cells. Diamonds, plus in vivo FGK45; squares, without in vivo FGK45. Data in a are the mean and SD of two to three animals per group and are representative of two independent experiments. Dot plots in c are representative of five to six animals. Data in d are the mean and SD of two animals.

Figure 6

CD4 T cell memory does not restore the ability of infused LLO_91-99–specific CD8 CTLs to expand or to confer protective immunity 7 days after transfer. (a) We transferred 6 × 10⁶ LLO_91-99–specific CTLs into recipient mice and infected the mice with wild-type L. monocytogenes 7 days later. Thirty minutes prior to infection, 50 × 10⁶ splenocytes from a BALB/c mouse immunized more than 28 days earlier with LLO_Ser92 L. monocytogenes were infused into the CTL recipients. Seventy-two hours after infection, spleens and livers were cultured; bacterial counts are plotted on the y axis. (b) We transferred 6 × 10⁶ LLO_91-99–specific CTLs into recipient mice that had been infected with LLO_Ser92 L. monocytogenes more than 28 days earlier. Seven days after CTL infusion, recipients were infected with wild-type L. monocytogenes, and 72 hours later, splenocytes were stained for CD8α and Thy1.1 and with LLO_91-99 H2-K^d tetramers. Dot plots are gated on live CD8 T cells. Staining for Thy1.1 is shown on the y axis, and tetramer staining is shown on the x axis. Numbers in the upper right quadrants represent percentages of total CD8 T cells. Data are representative of two animals per group.

Figure 7

The timing of anti-CD40 antibody administration determines outcome of CTL response to in vivo infection. CTLs were infused into recipient mice and challenged with L. monocytogenes 7 days later. Bacterial counts in liver and spleen were determined 72 hours after infection. Animals received anti-CD40 antibody or an isotype control at the indicated time points. Experimental animals received 6 × 10⁶ CTLs 7 days before infection (lanes 1–4). Control animals received PBS intraperitoneally (lanes 5 and 6). Data are representative of three animals per group. Lane 1, CD40 mAb 1 day and 2 days before CTL transfer and 12 hours after infection; lane 2, CD40 mAb 1 day and 2 days before CTL transfer; lane 3, CD40 mAb 12 hours after infection; lane 4, isotype control 1 day and 2 days before CTL transfer and 12 hours after infection; lane 5, isotype control 1 day and 2 days before CTL transfer and 12 hours after infection; lane 6, CD40 mAb 1 day and 2 days before CTL transfer and 12 hours after infection. Lower limit of detection, 50.

Figure 8

In vivo CD40 stimulation promotes differentiation of memory T cells from infused effector T cells. (a) CTLs were infused into recipient mice that received anti-CD40 antibody 1 day and 2 days earlier. Recipients were infected 7 days later and also received another dose of anti-CD40 antibody 12 hours after the infection. Three weeks later, mice were rechallenged with 100,000 wild-type L. monocytogenes, and 72 hours later, splenocytes were stained for CD8α, Thy1.1, and CD62L, and with LLO_91-99 H2-K^d tetramers. Dot plots are gated on live CD8 T cells. Thy1.1 is shown on the y axis, and tetramer staining is shown on the x axis. Numbers in the upper quadrants represent percentage of total CD8 T cells. (b) Recipient mice were infected 30 minutes after CTL infusion without anti-CD40 antibody administration. These recipients were challenged 3 weeks later and analyzed as described in a. These plots are representative of three animals per group.