Preferential priming of alloreactive T cells with indirect reactivity

Abstract

The relative contributions of the direct and indirect pathways in alloimmune responses have not been fully elucidated. We report a novel murine TCR transgenic system that can simultaneously track the CD4-direct (CD4-d), CD4-indirect (CD4-i) and CD8-direct (CD8-d) pathways after transplantation. Using this system, we have observed a profoundly greater proliferation of CD4-i T cells relative to CD4-d and CD8-d T cells after transplantation. Furthermore, a much larger proportion of CD4-i T cells attain an effector phenotype. We also analyzed endogenous, wild-type T cells using enzyme-linked immunospot analysis. In naïve mice, T cells with indirect reactivity were undetectable, but T cells with direct reactivity were abundant. However, 10 days after skin or heterotopic heart transplantation, CD4-i T cells comprised approximately 10% of the CD4+ response. Consistent with increased priming of the CD4-i pathway, we observed that the CD4-i T cells were further enriched in the effector cells migrating to the allograft and in memory-like T cells persisting after rejection. Thus, priming of the CD4-i pathway is favored after transplantation, allowing a rare population to rapidly become a major component of the CD4+ T-cell response in acute allograft rejection. The generalizability of this observation to other models remains to be determined.

Figure 1. Adoptive transfer of TCR-tg T cells allows for simultaneous tracking of three subtypes of alloreactive T cells

A) TCR-tg T cells representing the CD4-d (4C), CD4-i (TCR75) and CD8-d (2C) pathways of alloantigen recognition were purified by magnetic bead isolation and tested for purity by TCR-Vβ transgene expression and CD4 or CD8 expression using FACS analysis. B) Schematic of T cell purification, labeling with intravital dye (CFSE), and intravenous adoptive transfer into a syngeneic host. C) After transplantation, TCR-tg T cells could be identified amongst host T cells by the congenic cell surface markers, Ly5.1 and Thy1.1.

Figure 2. TCR-tg T cells have equivalent proliferative capabilities in vivo when Ag is abundant

CFSE-labeled TCR-tg T cells representing the CD4-d, CD4-i and CD8-d pathways of alloantigen recognition were co-adoptively transferred into syngeneic (B6) or semi-allogeneic F1 (BALB/c × B6) recipients. After 3 days, recipient splenocytes were harvested and their proliferation was assessed by CFSE dilution detected by FACS. Results are representative of two independent experiments with 2-3 mice per group.

Figure 3. CD4-i TCR-tg T cells are preferentially primed after transplantation

A) At various time-points after BALB/c heart transplantation, recipient splenocytes were isolated and the proliferation of adoptively transferred TCR-tg T cells was determined by CFSE dilution. Data is representative of at least 3 independent experiments, with at least 2-3 mice per experiment. B) CD44, CD62L and IFNγ expression by adoptively transferred CD4-d and CD4-i TCR-tg T cells were assessed by FACS three days (D3) following cardiac allograft transplantation. C) Proliferation of TCR-tg T cells following allogeneic skin transplantation at the draining axillary lymph nodes demonstrated by CFSE dilution. Data are representative of 3 independent experiments, 3 mice per experiment. D) Comparison of intracellular IFNγ production by CD4-d and CD4-i TCR-tg T cells 10 days after BALB/c skin transplantation. E) Equivalent migration of 4C and TCR75 TCR-tg T cells to spleen and lymph node. 2.5 × 10⁶ T cells from each TCR-tg were co-injected intravenously into B6 mice. 7 days later, spleen and lymph node were isolated and analyzed by FACS.

Figure 3. CD4-i TCR-tg T cells are preferentially primed after transplantation

A) At various time-points after BALB/c heart transplantation, recipient splenocytes were isolated and the proliferation of adoptively transferred TCR-tg T cells was determined by CFSE dilution. Data is representative of at least 3 independent experiments, with at least 2-3 mice per experiment. B) CD44, CD62L and IFNγ expression by adoptively transferred CD4-d and CD4-i TCR-tg T cells were assessed by FACS three days (D3) following cardiac allograft transplantation. C) Proliferation of TCR-tg T cells following allogeneic skin transplantation at the draining axillary lymph nodes demonstrated by CFSE dilution. Data are representative of 3 independent experiments, 3 mice per experiment. D) Comparison of intracellular IFNγ production by CD4-d and CD4-i TCR-tg T cells 10 days after BALB/c skin transplantation. E) Equivalent migration of 4C and TCR75 TCR-tg T cells to spleen and lymph node. 2.5 × 10⁶ T cells from each TCR-tg were co-injected intravenously into B6 mice. 7 days later, spleen and lymph node were isolated and analyzed by FACS.

Figure 3. CD4-i TCR-tg T cells are preferentially primed after transplantation

A) At various time-points after BALB/c heart transplantation, recipient splenocytes were isolated and the proliferation of adoptively transferred TCR-tg T cells was determined by CFSE dilution. Data is representative of at least 3 independent experiments, with at least 2-3 mice per experiment. B) CD44, CD62L and IFNγ expression by adoptively transferred CD4-d and CD4-i TCR-tg T cells were assessed by FACS three days (D3) following cardiac allograft transplantation. C) Proliferation of TCR-tg T cells following allogeneic skin transplantation at the draining axillary lymph nodes demonstrated by CFSE dilution. Data are representative of 3 independent experiments, 3 mice per experiment. D) Comparison of intracellular IFNγ production by CD4-d and CD4-i TCR-tg T cells 10 days after BALB/c skin transplantation. E) Equivalent migration of 4C and TCR75 TCR-tg T cells to spleen and lymph node. 2.5 × 10⁶ T cells from each TCR-tg were co-injected intravenously into B6 mice. 7 days later, spleen and lymph node were isolated and analyzed by FACS.

Figure 4. ELISPOT analysis of non-transgenic CD4-d and CD4-i T cells

After BALB/c → B6 skin transplantation, CD4+ T cells were purified from recipient axillary LN. CD4-d and CD4-i T cells were then identified by IL-2 and IFNγ ELISPOT assays by their response to direct or indirect alloantigen presentation. No TCR-tg T cells were utilized in these experiments. A) Naive B6 mouse (no transplant), IL-2; B) day (D) 10 after skin transplant, IL-2; C) D10 after skin transplant, IFNγ; D) D60 after skin transplant, IFNγ. Data are representative of two independent experiments with 2-3 mice per data point. E, F) IFNγ ELISPOT of draining lymph node CD4+ T cells 10 days after skin transplant with 3^rd party controls.

Figure 4. ELISPOT analysis of non-transgenic CD4-d and CD4-i T cells

After BALB/c → B6 skin transplantation, CD4+ T cells were purified from recipient axillary LN. CD4-d and CD4-i T cells were then identified by IL-2 and IFNγ ELISPOT assays by their response to direct or indirect alloantigen presentation. No TCR-tg T cells were utilized in these experiments. A) Naive B6 mouse (no transplant), IL-2; B) day (D) 10 after skin transplant, IL-2; C) D10 after skin transplant, IFNγ; D) D60 after skin transplant, IFNγ. Data are representative of two independent experiments with 2-3 mice per data point. E, F) IFNγ ELISPOT of draining lymph node CD4+ T cells 10 days after skin transplant with 3^rd party controls.

Figure 5. CD4-i T cells are enriched in graft infiltrating lymphocytes

On postoperative day 7 after BALB/c → B6 heart transplantation, cardiac allografts were excised and CD4+ T cells were isolated from the graft infiltrating lymphocytes (GIL) using magnetic beads. Purified CD4+ T cells from GIL and spleen were tested for direct and indirect alloreactivity by IFNγ ELISPOT assay. Data represent 3 heart transplant recipients.

Figure 6. Differential priming of CD4-d and CD4-i pathways in fresh vs. healed grafts

Kaplan-Meier survival plot of BALB/c skin transplants on B6 Rag1^-/- hosts. A) Two million CD4-i TCR-tg T cells were adoptively transferred at the time of skin transplantation (fresh skin) or 100 days after skin transplantation (healed skin) (p = NS). B) Similarly, two million CD4-d TCR-tg T cells were adoptively transferred into recipients of fresh skin or healed skin transplants. 10⁷ BALB/c DCs were injected at the time of CD4-d TCR-tg T cell transfer in recipients with healed BALB/c allografts.