- IsoPlexis’ functional single-cell proteomics identified, for the first time, allogeneic polyfunctional effector memory T cells (TEM) triggered by complement and alloantibody-activated endothelial cells (ECs) that may be key drivers of alloimmune rejection.
- Advanced visualizations from the IsoSpeak software enable stratification of distinct functional cell subsets with unique cytokine signatures, revealing increased polyfunctional TEM with cellular heterogeneity promoted by alloimmune responses. Furthermore, IsoPlexis’ platform identified decreased polyfunctionality of allogeneic TEM in response to ECs with either anti-IL-15 blocking or IL-1 receptor blockade (IL-1Ra). IL-1Ra pretreatment inhibited the augmentative effect of PRA-activated ECs on CD4+ and CD8+ TEM proliferation, but had less effect on PRA-enhanced T cell polyfunctionality than anti-IL-15 treatment.
- The polyfunctional responses and functional kinetics of alloreactive TEM to complement and alloantibody-activated ECs were able to be correlated to the in vivo setting with IsoPlexis’ proteomic profiling, mechanistically linking alloantibody to T cell-mediated allograft rejection.
Altered Functions and Heterogenous Responders Triggered by Complement & Alloantibody- Activated ECs Could be Key Drivers of Alloimmune Rejection
Allograft rejection, the rejection of transplanted tissue, is typically categorized as cell-mediated or antibody-mediated. Even though there are two separate categories, the processes involved in cell-mediated and antibody-mediated rejection can occur in combination, causing mixed rejection of the allograft. In a recent study, conducted by Xie, et al. IsoPlexis’ single-cell proteomics was used to characterize the relationship between antibody-mediated deposition of complement membrane attack complexes (MACs) on IFN-γ-primed human endothelial cells (ECs) and T cell proliferation, tying together the antibody-mediated and cell-mediated processes involved in rejection. IsoPlexis’ walk-away functional immune landscaping uncovered the drivers of T cell polyfunctionality which were in turn driving mixed allograft rejection and identified agonists that suppressed the detrimental effect of IFN-γ-primed ECs treated with human alloantibodies called panel-reactive antibodies (PRA).
Human graft ECs are the only graft structural cells that express high levels of Class I and II MHC molecules, which are the main targets of donor specific antibodies (DSAs), the antibodies which are generated during antibody-mediated transplant rejection1. ECs are also involved in cell-mediated rejection: they release costimulatory signals that engage TEMs, which correlate with T cell-mediated rejection. DSAs are also directly identified by alloreactive T cells. ECs can stimulate alloreactive CD8+ TEM to rapidly produce various cytotoxic lymphocytes (CTL) which create effector molecules that can kill grafts, such as perforin, granzymes, and cytokines. ECs can also rapidly stimulate CD4+ TEM that produce cytokines activating effector cells, such as more CTLs, B cells, NK cells, and macrophages1. All of these effector cells can contribute to allograft rejection. The magnitude of T cell activation response depends on both the strength of the antigen and the costimulatory signal released by the EC: stronger signals produce more potent and complex effector cells which release complex combinations of effector molecules1. This provides a presumed mechanism through which DSA can increase T cell responses and contribute to mixed allograft rejection.
In the study by Xie, et al., researchers sought to characterize the individual T cell activations resulting from these processes using a human immune system mouse model of allograft vasculopathy. While the researchers had previously analyzed bulk CD4+ and CD8+ TEM populations and found increased T cell proliferation and overall cytokine production, they noted that the functional heterogeneity of immune cells prevented them from drawing conclusions about the effect on individual T cells within the population1. In order to do so, the study used IsoPlexis’ single-cell proteomics to resolve this functional heterogeneity and uncover the individual immune cell subpopulations driving mixed rejection.
IsoPlexis’ Single-Cell Proteomics Reveals Drivers of Frequency and Polyfunctionality Among Both CD4+ and CD8+ Proliferative TEM Cells Associated with Transplant Rejection
The study found that treating IFN-γ-primed human ECs with PRA significantly increased the stimulatory effects of ECs on isolated peripheral blood allogeneic CD4+ and CD8+ TEM proliferation. When ECs were pre-treated with an IL-1 receptor inhibitor, (IL-1Ra) the effect of PRA was markedly diminished. The use of anti-IL-15 blocking antibody significantly suppressed augmentation of CD8+, with a lesser effect on CD4+ TEM expansion. The researchers also profiled CD4+ and CD8+ TEM which were not co-cultured with ECs as controls and used IsoPlexis’ functional single-cell proteomics to analyze the controls and the treated cells. The control groups did not produce any cytokines, suggesting that co-culture with ECs was the factor driving cytokine production in the proliferated populations.
IsoPlexis’ platform identified several distinct subsets of polyfunctional allogeneic CD4+ and CD8+ TEM cells. The IsoSpeak software suite was used to generate advanced visualizations, such as t-SNE, as well as perform analyses such as calculating the percentage of polyfunctional cells. The control T cells, which were stimulated by IFN-γ-primed ECs showed low levels of polyfunctionality. In contrast, allogeneic CD4+ and CD8+ TEM cells were found to be highly polyfunctional and were significantly upregulated after simulation by PRA-activated ECs. While both control and PRA-activated groups contained non-secretors, the T cells stimulated by PRA-activated ECs showed higher levels of polyfunctionality.
Many T cells in the PRA-activated group secreted >3 cytokines, while the control T cells secreted less than 3. Additionally, the secretions of the T cells that were stimulated by PRA-activated ECs showed stronger signal intensity. Individual T cells secreted more IFN-γ and TFN-α after stimulation with PRA-treated ECs compared to controls. Additionally, CD4+ TEM cultured with PRA-activated ECs secreted additional cytokines, IL-4 and MIP-1β. Thus, the data suggest that PRA activation of ECs promotes the secretion of more cytokines with stronger signal intensities in CD4+ and CD8+ TEM cells.
Reducing Drivers of Polyfunctionality and Decreasing Markers of Transplant Rejection
IsoPlexis’ single-cell proteomics revealed that treating IFN-γ-primed human ECs with PRA significantly increased the stimulatory effects on CD4+ and CD8+ TEM proliferation and polyfunctionality, contributing to higher rates of allograft rejection. The study also found that treating the ECs with anti-IL-15 antibody reduced this increase in polyfunctionality, suggesting decreased rates of rejection. The use of anti-IL-15 blocking antibody significantly reduced CD8+, but not CD4+ TEM expansion. Additionally, when ECs were treated with IL-1Ra before co-culture with T cells, the stimulatory effects of PRA on CD4+ and CD8+ TEM proliferation but had less effect than anti-IL-15 on T cell polyfunctionality. IsoPlexis’ identification of these treatments enables the suppression of a potentially significant driver of mixed allograft rejection.
IsoPlexis’ highly multiplexed functional single-cell proteomics analysis of allogeneic TEM cells identified polyfunctional responder subsets that were upregulated by complement and alloantibody-activated ECs, driving alloimmune rejection. The significance of polyfunctional T cell subsets is supported by research not only in alloimmunity, but also in inflammation, cancer, infectious disease, and beyond. These cells which are capable of co-producing multiple cytokines can be key drivers of disease pathogenesis as well as anti-tumor response and are uniquely revealed by IsoPlexis’ functional proteomics. While methods such as single-cell RNA-seq provide insight into gene expression, the connection between gene expression and function is tenuous. Functional single-cell proteomics is needed to determine the “true protein secretory pattern from live cells,” including identifying the highly polyfunctional effector cell subsets that lend deep insights into in vivo biology. Xie, et al.’s findings that TEM cell polyfunctionality is increased by interactions with MAC-activated ECs elucidates the relationship between alloantibody and T cell-mediated allograft rejection.
1. Xie CB, et al. Complement-activated human endothelial cells stimulate increased polyfunctionality in alloreactive T cells. American Journal of Transplantation 2021.