Limiting the amount and duration of antigen exposure during priming increases memory T cell requirement for costimulation during recall

Abstract

Donor-reactive memory T cells (Tmem) can play an important role in mediating graft rejection after transplantation. Transplant recipients acquire donor-reactive Tmem not only through prior sensitization with alloantigens but also through previous exposure to environmental pathogens that are cross-reactive with allogeneic peptide-MHC complexes. Current dogma suggests that most, if not all, Tmem responses are independent of the requirement for CD28 and/or CD154/CD40-mediated costimulation to mount a recall response. However, heterogeneity among Tmem is increasingly being appreciated, and one important factor known to impact the function and phenotype of Ag-specific T cell responses is the amount/duration of Ag exposure. Importantly, the impact of Ag exposure on development of costimulation independence is currently unknown. In this study, we interrogated the effect of decreased Ag amount/duration during priming on the ability of donor-reactive Tmem to mediate costimulation blockade-resistant rejection during a recall response after transplantation in a murine model. Recipients possessing donor-reactive Tmem responses that were generated under conditions of reduced Ag exposure exhibited similar frequencies of Ag-specific T cells at day 30 postinfection, but, strikingly, failed to mediate costimulation blockade-resistant rejection after challenge with an OVA-expressing skin graft. Thus, these data demonstrate the amount/duration of Ag exposure is a critical factor in determining Tmem's relative requirement for costimulation during the recall response after transplantation.

Figure 1. Limiting the duration of infection and peptide presentation in LM-OVA infected mice with ampicillin treatment results in similar peak frequencies and numbers of OT-I T cells

Naïve B6 mice were adoptively transferred with 10⁴ OT-I T cells and infected with 10⁴ CFU of LM-OVA and either left untreated or treated with ampicillin as described in Materials and Methods. A) Mice were sacrificed and spleens were harvested at days 1, 2, 3, 4, and day 6 post-infection and plated as described in Materials and Methods for assessment of bacterial load. Results shown are cumulative from three independent experiments, with 2-3 mice per timepoint. B-C) Spleens harvested at the indicated timepoints were stained for K^b/SIINFEKL expression and enumerated using R-PE MESF beads. B, Representative example of K^b/SIINFEKL staining vs. isotype control at 48 h. C, Cumulative results from 3 mice/group/timepoint demonstrated that K^b/SIINFEKL expression in ampicillin-treated mice at days 2 and 3 post-infection was lower than untreated mice. D-F) Recipients of OT-I T cells were analyzed for the kinetics and magnitude of expansion at days 6, 10, 13, 21, and 30 post-LM-OVA infection. Data shown in C are representative results of the frequency of Thy1.1⁺CD8⁺ T cells from four independent experiments of 5 mice per group, per experiment. Data shown in D are a representative example of frequency of Thy1.1⁺ of CD8⁺ T cells at day 30 post-infection. Data shown in E are representative results of the frequency (left) and absolute number (right) of Thy1.1⁺CD8⁺ T cells detected at day 30 post-infection from three independent experiments of 5 mice per group, per experiment. There is no statistically significant difference in the absolute number of Thy1.1⁺ CD8⁺ memory T cells between groups (p=0.8413).

Figure 2. Limiting the duration of infection and peptide presentation results in long-term skin graft acceptance following CTLA-4 Ig and anti-CD154 treatment

LM-OVA infected, OT-I-transferred recipient mice received mOVA skin grafts and, where indicated, received CTLA-4 Ig alone, anti-CD154 alone, or a combination of the two on days 0, 2, 4, and 6 post transplantation as described in Materials and Methods. A) Untreated controls uniformly experienced graft rejection with accelerated kinetics as compared to naïve mice (MST 11 and 13.5 d for no amp vs amp, respectively), whereas B) costimulation blockade-treated animals receiving ampicillin treatment went on to long-term graft survival (MST 180 d, as compared to 13 d for mice that did not receive ampicillin treatment, p<0.001). Mice that rejected between days 179 and 196 are depicted as having rejected at day 180 post-transplant. C) Recipients experiencing limited antigen exposure demonstrated rejection with CTLA-4 Ig and anti-CD154 alone (MST 11 and 12.5 d, respectively) whereas naïve skin graft recipients under single pathway blockade experienced prolonged graft survival (MST 22 and 189 d, respectively). p=0.0013 for CD28 blockade and p=0.0003 for CD154 blockade as compared to untreated controls. Results shown are from four independent experiments, each experiment with five mice per group.

Figure 3. Effector T cells generated under limited antigen stimulation show similar cytokine profiles and decreased KLRG-1 expression

Mice infected with LM-OVA after OT-I transfer were either treated with ampicillin or left untreated and sacrificed 9 days post infection. Splenocytes were harvested for cytokine production assays and assessment of phenotypic expression markers. A-C) Splenocytes from LM-OVA-infected OT-I recipients were restimulated in vitro with SIINFEKL peptide for 4 hours, and analyzed for the presence of intracellular IFN-γ, TNF, and IL-2. Untreated and treated mice showed similar levels of production of IFN-γ, IL-2, and TNF production. Data shown in A are representatives of three independent experiments, where n=5 animals per group per experiment. Data in B and C summarize data for all three independent experiments. D) Thy1.1⁺ CD8⁺ T cells from untreated or ampicillin-treated mice were analyzed for surface expression of activation markers. Results indicated decreased KLRG-1 expression on Thy1.1⁺CD8⁺ T cells from ampicillin-treated recipients as compared to untreated controls (p<0.001). Results shown in D are cumulative mean fluorescence intensities of Thy1.1⁺ CD8⁺ T cells of two-four separate experiments, for a total of five-15 mice per group.

Figure 4. Memory CD8⁺ T cells generated under conditions of limited antigen results in similar levels of cytokine production and similar phenotypic profiles

Mice infected with LM-OVA after OT-I transfer were either treated with ampicillin or left untreated, sacrificed 30 days post infection and splenocytes were harvested for cytokine production assays and assessment of phenotypic expression markers. A-C) Splenocytes from LM-OVA-infected OT-I recipients were restimulated in vitro with SIINFEKL peptide for 4 hours, and analyzed for the presence of intracellular IFN-γ, TNF, and IL-2. Untreated and ampicillin-treated mice showed similar levels of production of IFN-γ, IL-2, and TNF. Data shown in A are representatives of two independent experiments, where n=5 animals per group per experiment. Data in B show summary data for both independent experiments. D) CD8⁺Thy1.1⁺ T cells from untreated or ampicillin-treated recipients were analyzed at day 30 post-infected for expression of CD62L, CD127, KLRG-1. CD28, and CD25. Mean fluorescence intensities of Thy1.1⁺ CD8⁺ T cells of two separate experiments are shown, for a total of five- 10 mice per group.

Figure 5. Memory T cells primed under conditions of limited antigen exposure exhibit diminished recall responses in the presence of costimulation blockade

A) Experimental design schematic showing that untreated or ampicillin-treated LM-OVA infected animals were grafted with an OVA-expressing skin graft at day 30 post-infected. Mice were then left untreated or treated with CTLA-4 Ig and anti-CD154 as described in Materials and Methods. Six days after transplantation, splenocytes and draining lymph nodes were harvested for cytokine production assays and assessment of phenotypic expression markers. B, C) Frequencies of Thy1.1⁺ CD8⁺ T cells were analyzed in spleen. Frequencies shown are %Thy1.1⁺ of total CD8⁺ T cells. *p<0.05 as compared to untreated controls receiving neither ampicillin nor costimulation blockade. C, right panel) Absolute numbers were calculated using TruCount analysis. *p<0.05 as compared to untreated controls receiving neither ampicillin nor costimulation blockade. Results shown are representative (B) and summarized data (C) from three independent experiments with a total of 8-10 mice per group. D-F) Splenocytes from indicated groups were harvested on day six post-transplant and restimulated in vitro to assess cytokine production. Representative flow plots for IFN-γ/ TNF production (D) and IFN-γ/ IL-2 production (E) are shown. F, Summary data from two independent experiments with a total of 8-9 mice per group are shown. Results indicated that whereas costimulation blockade treatment of untreated mice did not diminish cytokine responses upon recall (p=NS), costimulation blockade treatment of ampicillin-treated animals resulted in a significant decrease in cytokine producing cells (*p<0.01).