High-frequency alloreactive T cells augment effector function of low-frequency CD8+ T-cell responses under CD28/CD154 blockade

Abstract

Background: Blockade of costimulatory molecules is a potent method of inducing long-term graft survival. We have previously addressed the issue of donor-reactive T-cell precursor frequency on relative costimulation dependence and found that the presence of a high precursor frequency of donor-reactive CD8 T cells resulted in costimulation blockade-resistant graft rejection, whereas the presence of a low-frequency donor-reactive population did not. To address the mechanisms by which high-frequency T cells obviated the requirement for costimulation, we asked whether a low-frequency population responding concomitantly with a high-frequency response also demonstrated costimulation independence.

Methods: A model system was established in which B6 mice containing a low frequency of anti-membrane bound chicken ovalbumin (mOVA) responders and a high frequency of anti-BALB/c responders received a skin graft from B6.mOVAxBALB/c F1 donors in the presence or absence of cytotoxic T-lymphocyte antigen-4 Ig/anti-CD154 costimulatory blockade.

Results: The results revealed that in the presence of costimulation blockade, high-frequency anti-BALB/c T cells augmented the effector activity of low-frequency anti-mOVA T cells, but it did not enhance the accumulation of anti-mOVA T cells capable of mediating graft rejection.

Conclusions: These results demonstrate that both antigen-specific and antigen-independent factors contribute to the relative costimulation independence of high-frequency T-cell responses.

Figure 1. B6 recipients of mOVAxBALB/c skin grafts experienced costimulation blockade-resistant rejection

A, Diagram of experimental design in which B, Splenocytes from B6 mice were CFSE-labeled and adoptively transferred into B6, mOVA, or BALB/c hosts. Recipients were sacrificed five days later and splenocytes were stained with anti-H-2K^b to identify responder B6 cells, anti-CD4 and anti-CD8, and analyzed for CFSE division. Data shown are gated on CD4⁺ or CD8⁺ T cells. C, mOVA, BALB/c, or mOVAxBALB/c splenocytes were analyzed by flow cytometry for expression of H-2K^d and the presence of K^b/SIINFEKL peptide:MHC complexes. B, OT-I T cells were stimulated in vitro with mOVA, BALB/c or mOVAxBALB/c stimulators, and the number of OT-I T cells were quantified by TruCount analysis five days later. C, Skin from mOVA, BALB/c or mOVA x BALB/c donors was placed onto B6 recipients, which were left untreated or were treated with CTLA-4 Ig and anti-CD154 on days 0, 2, 4, 6. Results indicated that while the mOVA graft was protected by CoB (MST >60 d), both BALB/c and mOVA x BALB/c grafts showed only modest prolongation under costimulation blockade. Data shown are representative of three independent experiments with 4–5 mice per group.

Figure 2. Stimulation by an mOVAxBALB/c skin graft did not augment the accumulation of OT-I T cells arising from low frequency precursors in the presence of costimulation blockade

Recipients of 10⁶ Thy1.1⁺ OT- I and OT-II T cells were transplanted with skin from mOVA, BALB/c or mOVA x BALB/c donors and left untreated or were treated with CTLA-4 Ig and anti-CD154 on days 0, 2, 4, 6. Mice were sacrificed at day 10 and draining LN were harvested. Results indicated that while frequencies and absolute numbers of CD8⁺ (A,B) and CD4⁺ (C,D) Thy1.1⁺ anti-mOVA T cells were augmented the mOVAxBALB/c graft recipients relative to the mOVA recipients, these populations were not augmented in the presence of costimulation blockade. Mice receiving a BALB/c allograft served as negative controls for frequency of OT-I T cells in the absence of antigenic stimulation. Data shown are representative of four independent experiments with 3–5 mice per group.

Figure 3. Stimulation by an mOVAxBALB/c skin graft increased cytokine production in OT-I T cells arising from low frequency precursors

Recipients of 10⁶ Thy1.1⁺ OT- I and OT-II T cells were transplanted and treated as indicated. Draining LN cells were restimulated in vitro with for 6 hours. Results indicated that frequencies (A,B) and absolute numbers (C) of IFN-γ and TNF -producing CD8⁺ Thy1.1⁺ anti-mOVA T cells were augmented the mOVAxBALB/c graft recipients relative to the mOVA recipients both in the presence and absence of costimulation blockade. Data shown are representative of three independent experiments with 3–5 mice per group.

Figure 4. Stimulation of OT-I T cells by an mOVAxBALB/c skin graft did not result in costimulation blockade-resistant rejection of a second mOVA skin graft

Recipients of 10⁶ Thy1.1⁺ OT- I and OT-II T cells were transplanted with skin from mOVA (A), BALB/c (B) or mOVA x BALB/c (C) donors with or without costimulation blockade on the left dorsal thorax. These recipients were then challenged with a simultaneous mOVA skin graft on the right dorsal thorax (D, E, and F) and monitored for graft rejection. Results indicated that even if primed in the presence of an mOVAxBALB/c skin graft (Figure 4C), OT-I effectors arising from low frequency precursors failed to mediate costimulation blockade resistant rejection of an mOVA skin graft (Figure 4E). Data shown are representative of three independent experiments with 5 mice per group.

Figure 5. High frequency anti-BALB/c T cells did not augment the ability of low frequency BALB.B-reactive T cells to mediate costimulation blockade-resistant rejection

A, Splenocytes from B6 mice were CFSE-labeled and adoptively transferred into B6 or BALB.B hosts. Recipients were sacrificed four days later and splenocytes were stained with anti-H-2K^b to identify responder B6 cells, anti-CD4 and anti-CD8, and analyzed for CFSE division. Data shown are gated on CD4+ or CD8+ T cells. B) B6 mice were grafted with either BALB/c (H-2^d) or BALB.B (H-2^b) skin grafts on the left side in the presence or absence of costimulation blockade. C) Mice also received a BALB.B skin graft on their right dorsal thorax. Despite the costimulation blockade-resistant rejection observed in the recipients of BALB/c grafts (MST 26d), a simultaneous BALB.B skin graft on the same recipient was not rejected (MST > 70 days).

Figure 6. Impact of adjacent BALB/c skin graft on CD4+ and CD8+ anti-OVA T cell responses

Recipients of 10⁶ Thy1.1⁺ OT- I and OT-II T cells were transplanted with skin from an mOVA donor on the right dorsal thorax in the presence or absence of costimulation blockade Groups of mice also received a simultaneous adjacent BALB/c skin graft on the left dorsal thorax. Results indicated that the absolute number of both CD4⁺ and CD8⁺ donor-reactive OVA-specific T cells were enhanced in the presence of adjacent BALB/c skin graft (Figure 6A, 6B). In contrast, however, there was no augmentation in the frequency of cytokine-producing cells in costimulation blockade-treated recipients in the presence of an adjacent BALB/c (Figure 6C, D). Data shown are representative of two independent experiments with 3 mice/group.