Comparison of microbial growth on primed extracorporeal membrane oxygenation circuits in varying environments using different priming solutions.

BACKGROUND: Extracorporeal Membrane Oxygenation (ECMO) is a life support device for patients with severe heart and/or lung failure. Emergency situations require immediate ECMO response. Primed circuits have become a routine practice, as it may take 30-60 min to assemble and prime. There remains a lack of data to support the sterility of primed and stored ECMO circuits. This bench study assessed the impact of storage environment and priming solution on specific microbial growth of primed ECMO circuits. METHODS: Twelve adult ECMO circuits were tested for sterility for 56 days between September-December 2020. Circuits were assembled and primed in a perfusion lab in Chicago, IL. Six were stored in a sterile environment and six in a non-sterile environment, with three circuits primed using normal saline (NaCl) and three with Plasmalyte-A for each environment. Samples were collected on days 0, 3, 7, 14, 28, 42, and 56 in anaerobic bottle cultures testing for potential pathogen growth, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. RESULTS: Samples obtained from the 12 primed ECMO circuits demonstrated no microbial growth of S. aureus, P. aeruginosa, and E. coli in the bottle cultures. Similarly, there was no difference in the circuit sterility based on the storage environment (sterile vs nonsterile) or priming solution (NaCl vs Plasmalyte-A). CONCLUSION: Our findings showed that ECMO circuits can be primed for 56 days without evidence of the specified bacterial growth. Furthermore, the storage conditions and the prime utilized did not affect the sterility of the primed ECMO circuits.

Recipient Myd88 Deficiency Promotes Spontaneous Resolution of Kidney Allograft Rejection.

The myeloid differentiation protein 88 (MyD88) adapter protein is an important mediator of kidney allograft rejection, yet the precise role of MyD88 signaling in directing the host immune response toward the development of kidney allograft rejection remains unclear. Using a stringent mouse model of allogeneic kidney transplantation, we demonstrated that acute allograft rejection occurred equally in MyD88-sufficient (wild-type [WT]) and MyD88(-/-) recipients. However, MyD88 deficiency resulted in spontaneous diminution of graft infiltrating effector cells, including CD11b(-)Gr-1(+) cells and activated CD8 T cells, as well as subsequent restoration of near-normal renal graft function, leading to long-term kidney allograft acceptance. Compared with T cells from WT recipients, T cells from MyD88(-/-) recipients failed to mount a robust recall response upon donor antigen restimulation in mixed lymphocyte cultures ex vivo. Notably, exogenous IL-6 restored the proliferation rate of T cells, particularly CD8 T cells, from MyD88(-/-) recipients to the proliferation rate of cells from WT recipients. Furthermore, MyD88(-/-) T cells exhibited diminished expression of chemokine receptors, specifically CCR4 and CXCR3, and the impaired ability to accumulate in the kidney allografts despite an otherwise MyD88-sufficient environment. These results provide a mechanism linking the lack of intrinsic MyD88 signaling in T cells to the effective control of the rejection response that results in spontaneous resolution of acute rejection and long-term graft protection.

Preemptive donor apoptotic cell infusions induce IFN-γ-producing myeloid-derived suppressor cells for cardiac allograft protection.

We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid-derived suppressor cells (MDSCs) in the graft protection provided by ECDI-SPs is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b(+) cells in the spleen that phenotypically resemble monocytic-like (CD11b(+)Ly6C(high)) and granulocytic-like (CD11b(+)Gr1(high)) MDSCs. Both populations suppress T cell proliferation in vitro and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b(+)Gr1(high) cells to produce a high level of IFN-γ and to exhibit an enhanced responsiveness to IFN-γ by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-γ completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-γ-dependent mechanisms. This form of preemptive donor apoptotic cell infusions has significant potential for the therapeutic manipulation of MDSCs for transplant tolerance induction.

Adoptive T-cell transfer combined with a single low dose of total body irradiation eradicates breast tumors.

The major objectives of tumor vaccination are to induce the regression of established tumors and to favor the establishment of long-lasting tumor-specific immunity, capable of protecting the host from relapse. Immunotherapeutic strategies such as the administration of tumor-associated antigenic peptides offer one means to boost preexisting antitumor CD8+ T cell immunity. Our recent work reveals that established breast tumors are rejected and tumor recurrence prevented when low-dose irradiation is combined with the adoptive transfer of Mammaglobin A epitope-specific CD8+ T cells.

Adoptive transfer of Mammaglobin-A epitope specific CD8 T cells combined with a single low dose of total body irradiation eradicates breast tumors.

Adoptive T cell therapy has proven to be beneficial in a number of tumor systems by targeting the relevant tumor antigen. The tumor antigen targeted in our model is Mammaglobin-A, expressed by approximately 80% of human breast tumors. Here we evaluated the use of adoptively transferred Mammaglobin-A specific CD8 T cells in combination with low dose irradiation to induce breast tumor rejection and prevent relapse. We show Mammaglobin-A specific CD8 T cells generated by DNA vaccination with all epitopes (Mammaglobin-A2.1, A2.2, A2.4 and A2.6) and full-length DNA in vivo resulted in heterogeneous T cell populations consisting of both effector and central memory CD8 T cell subsets. Adoptive transfer of spleen cells from all Mammaglobin-A2 immunized mice into tumor-bearing SCID/beige mice induced tumor regression but this anti-tumor response was not sustained long-term. Additionally, we demonstrate that only the adoptive transfer of Mammaglobin-A2 specific CD8 T cells in combination with a single low dose of irradiation prevents tumors from recurring. More importantly we show that this single dose of irradiation results in the down regulation of the macrophage scavenger receptor 1 on dendritic cells within the tumor and reduces lipid uptake by tumor resident dendritic cells potentially enabling the dendritic cells to present tumor antigen more efficiently and aid in tumor clearance. These data reveal the potential for adoptive transfer combined with a single low dose of total body irradiation as a suitable therapy for the treatment of established breast tumors and the prevention of tumor recurrence.

Ethylenecarbodiimide-fixed donor splenocyte infusions differentially target direct and indirect pathways of allorecognition for induction of transplant tolerance.

Strategic exposure to donor Ags prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pretransplant infusion of donor splenocytes treated with the chemical cross-linker ethylenecarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allospecificities. To target indirect allospecific T cells, ECDI-SPs induce upregulation of negative, but not positive, costimulatory molecules on recipient splenic CD11c(+) dendritic cells phagocytosing the injected ECDI-SPs. Indirect allospecific T cells activated by such CD11c(+) dendritic cells undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allospecific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4(+)CD25(+)Foxp3(+) T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SP infusions target host allogeneic responses via a multitude of mechanisms, including clonal depletion, anergy, and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.

Induction of transplantation tolerance by allogeneic donor-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells.

Several studies have shown that recipient-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are involved in transplantation tolerance. However, it is not clear whether allogeneic donor-derived Tregs are able to regulate T cell alloreactivity after solid organ allograft transplantation. Related studies in experimental bone marrow transplantation have shown that allogeneic donor-derived Tregs are capable of promoting early and long-term allogeneic hematopoietic engraftment, accompanied by tolerance to donor and recipient antigens. However, in these models, donor-derived Tregs are syngeneic with respect to the T responder cells. The role of Tregs in solid organ transplantation models where recipient-derived T responder and donor-derived Tregs are allogeneic has been scarcely studied. In order to determine whether allogeneic Tregs were able to regulate T cell alloreactivity, CD4(+)CD25(-) and CD8(+) T responder cells were cultured with stimulator dendritic cells in several responder-stimulator strain combinations (C57BL/6-->BALB/c, BALB/c-->C57BL/6 and C3H-->BALB/c) in the presence of responder-derived, stimulator-derived or 3rd-party-derived Tregs. Then, the frequency of IFN-gamma+ alloreactive T cells was determined by means of ELISPOT assay. The results of this study demonstrate that, regardless of the responder-stimulator strain combination, both responder-derived and stimulator-derived Tregs, but not 3rd-party-derived Tregs, significantly inhibited CD4(+) and CD8(+) T cell alloreactivity. The effect of allogeneic stimulator-derived Tregs was dependent on IL-10 and TGF-beta and reversed by exogenous IL-2. In vivo experiments in nu/nu recipients reconstituted with CD4(+)CD25(-) T responder and Tregs showed that recipient and donor-derived, but not 3rd-party-derived Tregs, significantly enhanced skin allograft survival. Importantly, T cells from both recipient-derived and donor-derived Treg-reconstituted nu/nu recipients exhibited donor-specific unresponsiveness in vitro. These results show that allogeneic donor-derived Tregs significantly inhibit T cell alloreactivity and suggest their potential use in the induction of transplantation tolerance.