IL-17A-producing γδ T cells and classical monocytes are associated with a rapid alloimmune response following vascularized composite allotransplantation in mice

Abstract

Background: Vascularized Composite Allotransplantation (VCA) is an important therapeutic option for patients that incur debilitating injuries to the face or limbs. The complexity and immunogenicity of tissue types within VCA grafts pose unique challenges and necessitate the use of intensive immunosuppression; however, graft rejection remains a challenge in VCA.

Methods: Deep proteomic profiling and high dimensional analysis with cytometry time of flight were used to define the cell types and effector mechanisms elicited by VCA in BALB/c (H-2Kd) > C57BL/6 (H-2Kb) limb recipients. Spleen and cervical draining lymph nodes were collected post-transplant days 1, 3, 5, and 7 (n =4-6 mice/group/day). We identified dynamic, coordinated signatures in T cell and monocyte populations associated with VCA allograft rejection.

Results: In comparison to syngeneic transplant recipients, allogeneic recipients exhibited significant alterations in the immune cell populations within secondary lymphoid tissues. These changes included very early expansion of double-negative TCRβ^- T cells, including IL-17A-producing γδ T cells, and patrolling monocytes. Subsequently, CD8+CD62L+ T cells and CD8+ effector/effector memory T cells (Teff/Tem), Ly6C^hiCCR2^hiCX3CR1^low classical monocytes, CD4+ Teff/Tem, and CD8+CD25^hiCCR7^low Teff/Tem were increased by day 5. CD8+CD25^hiCCR7^low Teff/Tem with the highest expression of IFN-γ, perforin, and granzyme B were enriched by day 7.

Conclusions: High dimensional proteomic analysis reveals multiple innate and Teff/Tem subsets in acute rejection following VCA. In particular, IL-17A-producing γδ T cells and classical monocytes may be particularly important in initiating the alloimmune response in VCA recipients.

Keywords: IFN-γ; IL-17; classical monocyte; granzyme; mouse; perforin; vascularized composite allotransplantation; γδ T cell.

Figure 1

Experimental design and high dimensional analysis on myeloid cells in the spleen. (A). Experimental design. This figure was created with BioRender.com. (B). UMAP visualization of eight myeloid cell clusters. (C). Heatmap showing that eight clusters were classified into cDC1, cDC2, pDC, classical monocytes, intermediated monocytes, patrolling monocytes, activated macrophages, and steady state macrophages according to the combination of representative surface markers. (D). Clusters 5, 6, and 7 are identified as classical monocytes, intermediated monocytes, and patrolling monocytes, respectively based on the variable expression of Ly6C, CCR2. (E). Pie charts showing the proportions of each cluster at three time points post-transplant. (F). Proportions of each cluster at three time points after transplant. The allogeneic group showed significant increases in patrolling monocytes on day 1 and classical monocytes on day 5 compared to the syngeneic group. **: P < 0.01 by Sidak’s multiple comparisons test.

Figure 2

High dimensional analysis on T cells in the spleen. (A) UMAP visualization of T cells classified into 18 clusters. (B) Left, heatmap showing the expressions of surface markers in Treg, CD4+/CD8+ Tnaïve, CD4+ and CD8+ Teff/Tem, CD8+CD62L+ T cells, and DN T cells. Right, heatmap demonstrating their expressions of cytokines, perforin, and granzyme B (C) Left, UMAP visualization of Granzyme B expression. Right, CD8+CD25^hiCCR7^low Teff/Tem (cluster 10) are distinguished from other effector T cell populations by increased Granzyme B expression.

Figure 3

Dynamic changes of T-cell clusters in the spleen following VCA. (A). Proportions of each cluster in each sample at four time points following transplant. (B). Bar graphs showing the proportions of the seven clusters at four time points post-transplant. Allogeneic recipients exhibited the early expansion of DN TCRβ- T cells (cluster 18) followed by CD8+ Teff/Tem (cluster 12) and CD8+CD62L+ T cells (cluster 9). Three distinct CD4+/CD8+ Teff/Tem clusters (clusters 6, 4, and 10) were increased on day 5 in allograft recipients, and the increase of CD8+CD25^hiCCR7^low Teff/Tem (cluster 10) in allograft recipients persisted through day 7 and was accompanied by marked expansion of CD8+CD25^lowCCR7^low Teff/Tem (cluster 15). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 by Sidak’s multiple comparisons test.

Figure 4

High dimensional analysis on T cells in the draining lymph nodes. (A). UMAP visualization of T cells classified into 18 clusters. (B). Left, heatmap showing the expressions of surface markers in Treg, CD4+/CD8+ Tnaïve, CD4+ and CD8+ Teff/Tem, CD8+CD62L+ T cells, and DN T cells. Right, heatmap demonstrating their expressions of cytokines, perforin, and granzyme B, (C). Proportions of each cluster in each sample at four time points post-transplant. The predominant population in the lymph nodes were CD8+CD25^lowCCR7^low Tem (cluster 10). (D). The allogeneic group showed significant increases in DN TCRβ- T cells (cluster 16) on day 1 and CD8+CD25^lowCCR7^low Tem (cluster 10) on day 7 compared to the syngeneic group. **: P < 0.01, ****: P < 0.0001 by Sidak’s multiple comparisons test. (E). Additional experiments presented that cluster 16 corresponded to TCRγδ+IL17A+, cluster 17 to TCRγδ-IL17A-, and cluster 18 to TCRγδ+IL17A- cells.

Figure 5

Summary of the cell populations associated with rejection in VCA allografts. In the very early post-transplant period, DN TCRβ- T cells, including IL-17A+ γδ T cells, were significantly elevated in the spleen and draining lymph nodes of allogeneic recipients, and patrolling monocytes were abundant in the allogeneic spleen. Subsequently, CD8+CD62L+ T cells and CD8+ Teff/Tem cells were significantly increased in the spleens of allogeneic recipients compared to syngeneic recipients, followed by significant increases in classical monocytes, CD4+ Teff/Tem cells, and CD8+ Teff/Tem cells in the allogeneic spleen. Thereafter, CD8+ Teff/Tem cells expressing the highest levels of IFN-γ, perforin, and granzyme B became significantly more prevalent in the allogeneic spleen than in the syngeneic spleen. This figure was created with BioRender.com.