It has been demonstrated by us as well as others that eosinophils may alter their phenotype and function based on the local environment (7, 9, 10). Since costimulation plays a critical role in T cell cytokine production and environmental polarization (11, 12), we examined the possibility that eosinophils may lose their tolerogenic properties in the absence of CSB immunosuppression. Cytokine expression was examined in BALB/c (H2 d) lung allografts transplanted into fully MHC-mismatched C57BL/6 (B6) (H2 b) recipient mice. In the absence of immunosuppression, lung allografts had higher levels of Th1-polarizing cytokines IFN-γ and TNF-α than did CSB-treated accepting lung grafts (Figure 1A). Limited amounts of Th2-polarizing cytokines IL-4, IL-13, IL-33, and GM-CSF were evident in lung allografts treated with or without CSB immunosuppression (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.128241DS1). We next evaluated lung-resident eosinophils from both CSB-treated and nonimmunosuppressed grafts. Based on a previously identified eosinophil polarization phenotype (7), we noted higher levels of Th1-defining (or E1-defining) features, such as CXCL9, CXCL10, CXCL11, and iNOS in eosinophils isolated from rejecting compared with CSB-treated lung allografts (Figure 1A). In no group was Th2 (or E2) polarization of eosinophils detected (Supplemental Figure 1A). Flow cytometric characterization demonstrated an absence of MHC II and costimulatory molecules such as CD80, CD86, and CD40. However, there were high levels of recipient-derived MHC I (H2K b), PD-L1, and CD101 on lung-resident eosinophils in the absence of CSB (Figure 1B). Notably, we did not detect BALB/c-derived H2 d-MHC on graft-resident eosinophils, indicating a lack of "cross-dressing" or antigen swapping for donor derived-antigens (ref. 13 and Supplemental Figure 1B). Thus, while eosinophils from rejecting lungs resembled those from accepting lungs in some aspects, we did observe some differences. It is thus possible that in the absence of immunosuppression, eosinophils may contribute to graft rejection rather than acceptance, specifically because CD101 expression has been previously associated with an inflammatory eosinophil subtype (14). In order to evaluate this directly, we conditionally depleted eosinophils from B6 iPHIL mice, in which the diphtheria toxin (DT) receptor is expressed under the control of the eosinophil peroxidase promoter (Supplemental Figure 1C and ref. 15). DT-treated mice or saline-injected controls were challenged with a BALB/c lung allograft in the absence of immunosuppression. Surprisingly, mice depleted of eosinophils had higher grades of rejection (Figure 1C), increased numbers of lung-resident T lymphocytes, increased rates of T cell proliferation, and increased effector cell differentiation by day 4 after engraftment (Figure 1D). These patterns of T lymphocyte activation and infiltration were especially prominent for CD8 + T cells. CD4 + T cell proliferation did ...

Despite standardized postoperative care, some lung transplant patients suffer multiple episodes of acute and chronic rejection while others avoid graft problems for reasons that are poorly understood. Using an established model of C57BL/10 to C57BL/6 minor antigen mismatched single lung transplantation, we now demonstrate that the recipient microbiota contributes to variability in the alloimmune response. Specifically, mice from the Envigo facility in Frederick, Maryland contain nearly double the number of CD4 + Foxp3 + regulatory T cells (T regs ) than mice from the Jackson facility in Bar Harbor, Maine or the Envigo facility in Indianapolis, Indiana (18 vs 9 vs 7%). Lung graft recipients from the Maryland facility thus do not develop acute or chronic rejection. Treatment with broad-spectrum antibiotics decreases T regs and increases both acute and chronic graft rejection in otherwise tolerant strains of mice. Constitutive depletion of regulatory T cells, using Foxp3-driven expression of diphtheria toxin receptor, leads to the development of chronic rejection and supports the role of T regs in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through T reg -dependent mechanisms and challenges the practice of indiscriminate broad-spectrum antibiotic use in the perioperative period. K E Y W O R D Sanimal models: murine, basic (laboratory) research/science, bronchiolitis obliterans (BOS), immunosuppression/immune modulation, lung disease: immune/inflammatory, lung transplantation/pulmonology, rejection: acute, rejection: chronic

Ex vivo expansion followed by reinfusion of tumor-infiltrating leukocytes (TILs) has been used successfully for the treatment of multiple malignancies. Most protocols rely on the use of the cytokine IL-2 to expand TILs prior to reinfusion. In addition, TIL administration relies on systemic administration of IL-2 after reinfusion to support transferred cell survival. The use of IL-2, however, can be problematic because of its preferential expansion of regulatory T and myeloid cells as well as its systemic side effects. In this study, we describe the use of a novel IL-2 mutant retargeted to NKG2D rather than the high-affinity IL-2R for TIL-mediated immunotherapy in a murine model of malignant melanoma. We demonstrate that the NKG2D-retargeted IL-2 (called OMCPmutIL-2) preferentially expands TIL-resident CTLs, such as CD8+ T cells, NK cells, and γδT cells, whereas wild-type IL-2 provides a growth advantage for CD4+Foxp3+ T cells as well as myeloid cells. OMCPmutIL-2–expanded CTLs express higher levels of tumor-homing receptors, such as LFA-1, CD49a, and CXCR3, which correlate with TIL localization to the tumor bed after i.v. injection. Consistent with this, OMCPmutIL-2–expanded TILs provided superior tumor control compared with those expanded in wild-type IL-2. Our data demonstrate that adoptive transfer immunotherapy can be improved by rational retargeting of cytokine signaling to NKG2D-expressing CTLs rather than indiscriminate expansion of all TILs.

donors to 9.0yrs (8.5-9.6) with ≥40yo donors]. Conclusion:There is a disproportionate (3x) benefit in median survival by using a younger donor heart (12-24yo) in younger recipients (18-49yo) compared to ≥65yo recipients. The potential gain in number of total posttransplant years by an age-specific organ allocation should be considered in this supply-constrained climate of transplant "economics".

A growing arm of adoptive immunotherapy for cancer involves the isolation, expansion and reinfusion of autologous tumor infiltrating leukocytes or TILs. TILs are enriched for tumor-reactive cytotoxic cells that are rendered inactive or anergic by multiple immunosuppressive mechanisms operating in solid tumors. Their separation from the tumor microenvironment followed by ex vivo activation and expansion in the presence of T cell receptor stimulation and interleukin-2 (IL-2) has been described to reverse this dysfunction and control tumor growth in some patients. Unfortunately, a large number of patients achieve no response or clinical benefit suggesting that TIL immunotherapy could benefit from refinement and improvement. Part of the limitation involves the reliance on IL-2 to support T cell expansion both in vitro and in vivo as this cytokine can result in substantial expansion and activation of regulatory T cells (Tregs) that limit the efficacy of immunotherapy. We have recently described a novel re-targeted form of IL-2, which utilizes NKG2D rather than α chain of IL-2R to form the high affinity receptor complex for β and γ chain signal transduction. This redirected cytokine fusion protein consists of a cowpox virus encoded NKG2D ligand called orthopoxvirus major histocompatibility complex class I-like protein or OMCP along with non-IL-2R-α binding mutant form of IL-2 (OMCPmutIL-2). To evaluate the effect of NKG2D-redirected cytokine delivery on TIL expansion, we isolated TILs from progressively growing B16ova melanoma and expanded them with transient anti-CD3/CD28 stimulation in the presence of continuously replenished wild-type IL-2 or OMCPmutIL-2. Compared to wild-type IL-2 the use of OMCPmutIL-2 resulted in specific and preferential expansion of NK cells, CD8+ T cells as well as γδ T cells after 2 weeks of culture. Significantly higher expansion was observed in CD8+ T (p=0.037), antigen-specific CD8+ tetramer+ (p=0.0014), NK cells (p<0.001) and T cells (p=0.002), with decreased expansion of Tregs (p=0.045) and MDSCs (p=0.0122) indicating preferential expansion of more cytolytic subtypes of TILs by OMCPmut.IL-2. Human melanoma TILs demonstrated a similar trend with NK, NKT, CD8+T and T cells expanding significantly more in OMCPmut.IL-2 whereas MDSCs and Tregs expanded more in IL-2. We next evaluated tumor growth in C57BL/6 mice bearing established melanoma reconstituted with 5 × 106 TILs expanded in either wild-type IL-2 or OMCPmutIL-2. We found significant tumor control in the group which received OMCPmutIL-2 expanded TIL compared to IL-2 expanded ones (178.83±140.01vs 970.15±330.47mm3 on day 22 of growth). We conclude that the use of the NKG2D retargeted common γ-chain cytokine, called OMCPmutIL-2, facilitates expansion of multiple lineages of TIL-resident cytotoxic lymphocytes and improves adoptive transfer immunotherapy over wild-type IL-2. Our data thus suggests that retargeting stimulation away from the traditional IL-2R-α chain represents a rational approach to TIL immunotherapy. Citation Format: Anirban Banerjee, Yizhan Guo, Lea Paragas, Jacqueline Slobin, Bayan Mahgoub, Dongge Li, Sarah Hein, John Westwick, Eric Lazear, Alexander S. Krupnick. Targeted expansion of NKG2D-expressing tumor infiltrating leukocytes improves adoptive transfer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 878.