Prolonged graft survival in older recipient mice is determined by impaired effector T-cell but intact regulatory T-cell responses

Abstract

Elderly organ transplant recipients represent a fast growing segment of patients on the waiting list. We examined age-dependent CD4(+) T-cell functions in a wild-type (WT) and a transgenic mouse transplant model and analyzed the suppressive function of old regulatory T-cells. We found that splenocytes of naïve old B6 mice contained significantly higher frequencies of T-cells with an effector/memory phenotype (CD4(+)CD44(high)CD62L(low)). However, in-vitro proliferation (MLR) and IFNgamma-production (ELISPOT) were markedly reduced with increasing age. Likewise, skin graft rejection was significantly delayed in older recipients and fewer graft infiltrating CD4(+)T-cells were observed. Old CD4(+) T-cells demonstrated a significant impaired responsiveness as indicated by diminished proliferation and activation. In contrast, old alloantigen-specific CD4(+)CD25(+)FoxP3(+) T-cells demonstrated a dose-dependent well-preserved suppressor function. Next, we examined characteristics of 18-month old alloreactive T-cells in a transgenic adoptive transfer model. Adoptively transferred old T-cells proliferated significantly less in response to antigen. Skin graft rejection was significantly delayed in older recipients, and graft infiltrating cells were reduced. In summary, advanced recipient age was associated with delayed acute rejection and impaired CD4(+) T-cell function and proliferation while CD4(+)CD25(+)FoxP3(+) T-cells (Tregs) showed a well-preserved function.

Figure 1. Altered T-cell repertoire in aged naive mice.

Contraction of the T-cell repertoire in aged naive mice: 3 and 18mths naive B6 mice were sacrificed and splenocytes stained for CD4⁺T-cell subpopulations (n = 6). CD4⁺T-cells were reduced in frequency and total numbers in older animals (A). Effector/memory CD4⁺T-cells and early activated CD4⁺T-cells accumulated in older animals while frequencies of regulatory T-cells were age-independent (B).

Figure 2. Naive old mice demonstrate a poor functional response to antigen in vitro.

Naive old mice demonstrate a poor functional response to allo-antigen in vitro; (A) Splenocytes from naive B6 mice were co-cultured with bm12 antigen for 72hrs. Following 12hrs of incubation with 3H TdR (Thymidine) the proliferation of responder cells was determined by Thymidine incorporation. (B) Young and old B6 splenocytes were co-cultured in ELISPOT plates and resulting IFNγ spots counted. Both, T-cell proliferation (A) and alloreactive IFNγ -production (B) were significantly impaired in aged mice indicating a reduced functional T-cell response (n = 6).

Figure 3. Recipient age prolongs skin allograft survival in WT mice.

Recipient age prolongs skin allograft survival in WT mice (A), (p<0.005) and is associated with reduced intragraft CD4⁺ T-cell infiltrates (B) (day 7, 200×).

Figure 4. Impaired immune response in old WT recipients.

Old WT animals demonstrate an impaired immune response after transplantation: Both, T-cell proliferation (A) and alloreactive IFNγ -production (B) were significantly reduced in aged mice indicating a poor T-cell response upon re-stimulation in vivo (n = 6).

Figure 5. Impaired T-cell activation and proliferation in aged WT recipients.

Impaired T-cell activation and proliferation of splenocytes of aged WT transplant recipients. 3 and 18mths old WT recipients of bilateral bm12 skin grafts were sacrificed by day 7 and splenocytes were re-stimulated with PMA/Ionomycin for 4hrs. Following surface staining (CD4, Vα2.1 and Vβ8.1) cells were permeabilized and stained with PE-conjugated anti-IFNγ, IL-2 or IL-10 mAbs and isotype control mAbs. Early activated CD4⁺T-cells were reduced in frequency in older animals (A) while effector/memory CD4⁺T-cells and regulatory CD4⁺T-cells accumulated. (B) Despite a reduced functional in vitro and in vivo response as indicated by impaired IL-2 production expression of proinflammatory cytokines is elevated with age at day 7 after restimulation (C).

Figure 6. Regulatory T-cell function remains unaltered with age.

Regulatory T-cell function remains age-independent: CD4⁺CD25⁻ T-cells (responder cells) and regulatory T-cells (FoxP3⁺) were co-cultured at different ratios in the presence of stimulator cells. The proliferation of responder cells was determined by Thymidine incorporation. Young and old Tregs suppressed the proliferation of responder cells in a dose-dependent manner. Co-culture of old Tregs with young responders cells demonstrated a well-preserved suppressor function of old regulatory T-cells comparable to the suppressor function of young regulatory T-cells.

Figure 7. Impaired CD4⁺ T-cell expansion leads to prolonged graft survival.

Impaired proliferation of alloantigen-specific CD4⁺ T-cells leads to prolonged graft survival in old skin graft recipients. Nude B6 mice were engrafted with two bm12 skin transplants following adoptive transfer of young or old 2×10⁶ transgenic CD4⁺ ABM T-cells. In line with the reduced CD4⁺ T-cell response in old WT recipients skin graft survival was significantly prolonged following transfer of old alloreactive CD4⁺ T-cells (A). One week after transplantation spleen, dLN and ndLN were harvested and leukocytes were stained for CD4⁺, Vα2⁺ and Vβ8⁺ expression to assess proliferation of adoptively transferred T-cells (B). In parallel, skin grafts were stained for graft infiltrating CD4⁺ T-cells (C). At day 7, absolute numbers of adoptively transferred CD4⁺ T-cells were counted in different lymphatic compartments in order to determine T-cell proliferation. A significantly reduced proliferation of old tg CD4⁺ T-cells was accompanied by decreased graft infiltration, underscoring a delayed rejection response (B and C).

Figure 8. Reduced proliferation of old alloreactive effector/memory CD4⁺ T-cells.

Old alloreactive effector/memory CD4⁺ T-cells proliferate less in response to antigen. Following adoptive transfer of young or old 2×10⁶ transgenic CD4⁺ ABM T-cells nude B6 mice were engrafted with two bm12 skin transplants. One week later spleen (A) and dLN (B) were retrieved, leukocytes were stained for CD4, Vα2 and Vβ8 expression and the expression of different cell surface markers (n = 4). Relative numbers of CD69⁺ and CD25⁺CD4⁺ T-cells were reduced in recipients spleens after transfer of old alloreactive T-cells indicating an impaired activation and proliferation. Similarly, alloreactive effector/memory CD4⁺ T-cells were significantly reduced 7 days after restimulation in vivo (A). In contrast, enhanced early activation (CD69⁺) of old tg CD4⁺T-cells in draining lymph nodes did not lead to a stronger expansion of effector/memory T-cells most likely due to an impaired IL-2 production (B).