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Eosinophils are bone-marrow-derived granulocytes known for their ability to facilitate clearance of parasitic infections and their association with asthma and other inflammatory diseases. The purpose of this review is to discuss the currently available human observational and animal experimental data linking eosinophils to the immunologic response in solid organ transplantation. First, we present observational human studies that demonstrate a link between transplantation and eosinophils yet were unable to define the exact role of this cell population. Next, we describe published experimental models and demonstrate a defined mechanistic role of eosinophils in downregulating the alloimmune response to murine lung transplants. The overall summary of this data suggests that further studies are needed to define the role of eosinophils in multiple solid organ allografts and points to the possibility of manipulating this cell population to improve graft survival.

Long-term survival of lung transplants lags behind other solid organs due to early onset of a fibrotic form of chronic rejection known as chronic lung allograft dysfunction (CLAD). Preventing CLAD is difficult as multiple immunologic and physiologic insults contribute to its development. Targeting fibroblast activation, which is the final common pathway leading to CLAD, offers the opportunity to ameliorate fibrosis irrespective of the initiating insult. Thy-1 is a surface glycoprotein that controls fibroblast differentiation and activation.To study the role of Thy-1 in CLAD, we utilized the minor antigen mismatched C57BL/6 (B6More severe CLAD was evident in B6Our findings suggest that the loss of Thy-1 on fibroblasts is a previously unrecognized cause of CLAD and its replacement may offer therapeutic applications for amelioration of this disease post-transplantation in the setting of infectious stress responses.

Pathways regulating lung alloimmune responses differ from most other solid organs and remain poorly explored. Based on our recent work identifying the unique role of eosinophils in downregulating lung alloimmunity, we sought to define pathways contributing to eosinophil migration and homeostasis. Using a murine lung transplant model, we have uncovered that immunosuppression increases eosinophil infiltration into the allograft in an IL-5-dependent manner. IL-5 production depends on immunosuppression-mediated preservation of donor-derived group 2 innate lymphoid cells (ILC2). We further describe that ischemia reperfusion injury upregulates the expression of IL-33, which functions as the dominant and nonredundant mediator of IL-5 production by graft-resident ILC2. Our work thus identifies unique cellular mechanisms that contribute to lung allograft acceptance. Notably, ischemia reperfusion injury, widely considered to be solely deleterious to allograft survival, can also downregulate alloimmune responses by initiating unique pathways that promote IL-33/IL-5/eosinophil-mediated tolerance.

Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body1, and regulates inflammation, cell proliferation, and tissue regeneration2–4. How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as ‘good-bye’ signals to actively modulate outcomes in tissues. Apoptotic cells communicate with neighbouring cells by the regulated release of specific metabolites, and a cocktail of select apoptotic metabolites reduces disease severity in mouse models of inflammatory arthritis and lung transplant rejection.

Gluten network formed by oxidation of glutenin sulfhydryl groups is the determinant of dough rheological properties, while chemical reagents including oxidants and reductants are both used as dough rheology improvers under different circumstances. This study compares the impact of sodium metabisulfite (SMBS) and azodicarbonamide (ADA), as the representative reductive and oxidative dough improvers, at series of concentrations that offer or remove the same number of electrons form dough, respectively. The alveographic characterization, protein distribution and glutenin composition analysis, and free sulfhydryl measurement were performed on dough containing redox equivalent SMBS or ADA. Finally, at each optimal concentration, the dough protein network was analyzed with confocal microscopy. Results showed that SMBS increased the free sulfhydryl content, loosened the microstructure of gluten network, and thus enhanced dough extensibility. ADA reduced the free sulfhydryl content, compacted the dough microstructure, thus enhanced the tenacity and baking strength of dough. It is therefore proposed that the reductants reduce disulfide bonds in gluten network and renders the formation of one-dimensional gluten network while oxidants promote the disulfide linkage and formation of three-dimensional gluten network. This study offers a theoretic foundation of differentiating dough rheology improvers for their specified application.

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 (Tregs ) 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 Tregs 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 Tregs in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through Treg -dependent mechanisms and challenges the practice of indiscriminate broad-spectrum antibiotic use in the perioperative period.

T cell mediated immunotherapy has gained credible traction for cancer treatment. To date experimental and clinical strategies have relied on antibody-mediated co-stimulatory blockade, or the administration of high doses of naturally occurring or engineered cytokines to increase T cell activation. Cytokine therapy is by limited undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here we describe functionality of a unique chimeric cytokine by design, called OMCPmutIL-2, binds with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signals through the common γ-chain cytokine receptor, thus eliminating IL-2Ra mediated off-target side effects. In addition to precise activation of cytotoxic T cells due to redirected binding we describe that OMCPmutIL-2 results in superior activation of both human and murine CD8+ T cells by improving their survival, decreasing exhaustion, and improving memory cell generation. This functional improvement is the result altered signal transduction based on the reorganization of surface membrane lipid rafts resulting in JAK-mediated phosphorylation of the T cell receptor (TCR) rather than canonical STAT/AKT signaling intermediates activated by other common γ-chain cytokines. This altered signaling pathway increases CD8+ T cell response to low affinity antigens, activates Nuclear Factor of Activated T Cells (NFAT) transcription factors, and enhances mitochondrial biogenesis with improved T cell survival, cytotoxicity, and memory cell generation. OMCPmutIL-2 thus outperforms other common γ-chain cytokines as both a reagent for T cell expansion and in vivo CD8+ T cell-based immunotherapy. Supported by grant I01 BX002299

Factors affecting mortality after lung surgery remain poorly explored. We performed pulmonary lobar resections(removing □ 1/5 of the total pulmonary volume) from C57BL/6 mice and evaluated quantitative changes in various leucocytes in the remaining lung, blood and spleen. A nearly four-fold increase in eosinophils was evident by day-7 post resection with a gradual return to baseline by day 30. Evaluation of the bone marrow demonstrated no change in common myeloid, granulocyte-monocyte or eosinophil progenitors. However the number of early stage and mature eosinophils doubled after lobar resection suggesting that pulmonary surgery augmented maturation of eosinophils. Neutralization of IL-5 decreased overall eosinophil numbers but the relative differences between resting and post-resection mice remained. Further evaluation of bone marrow demonstrated an increase in IL-25, IL-6, IL-9, KC and MCP-1 after pulmonary resection. We next performed right pneumonectomies in saline or diphtheria toxin-treated iPHIL mice, where diphtheria toxin receptor expression is limited to eosinophils, and noted a 70% mortality in eosinophil sufficient vs. 20% mortality in eosinophil depleted mice (p=.005). Severe hypoxia was noted in eosinophils sufficient vs. deficient mice (pO2 of 54.3+/−5.7 vs. 83.6+/−11.63; p=.04) four hours prior to resection. Taken together out data demonstrate that pulmonary surgery leads to bone marrow-specific maturation of eosinophils and systemic eosinophilia which directly contributes to perioperative mortality. Thus defining factors which may disrupt this process offers the opportunity to improve outcomes after lung surgery. Supported by PO1 AI116501, RO1 AI145108-01

Gamma delta (γδ) T cells can effectively recognize and kill colorectal cancer (CRC) cells, thereby suppressing tumor progression via multiple mechanisms. They also have abilities to exert a protumor effect via secreting interleukin-17 (IL-17). γδ T cells have been selected as potential immunocytes for antitumor treatment because of their significant cytotoxic activity. Immunotherapy is another potential anti-CRC strategy after an operation, chemotherapy, and radiotherapy. γδ T cell-based immunotherapy for CRC shows fewer side effects and better toleration. This review will outline the immune functions and the mechanisms of γδ T cells in the growth and progression of CRC in recent years, and summarize the immunotherapies based on γδ T cells, thus providing a direction for future γδ T cells in CRC research.

Ex vivo expansion followed by reinfusion of tumor infiltrating leucocytes (TILs) has been utilized successfully for the treatment of multiple malignancies. Most protocols rely on the use of the cytokine interleukin-2 (IL-2) to expand TILs prior to reinfusion. In addition, TIL administration relies on systemic administration of IL-2 post-reinfusion to support transferred cell survival. The use of IL-2, however, can be problematic due to its preferential expansion of regulatory T and myeloid cells as well as its systemic side effects. Here we describe the use of a novel IL-2 mutant retargeted to NKG2D rather than the high affinity IL-2 receptor 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 cytotoxic lymphocytes, such as CD8(+) T cells, NK cells, and γδT cells while wild-type IL-2 provides a growth advantage for CD4(+)Foxp3(+) T cells as well as myeloid cells. OMCPmutIL-2 expanded cytotoxic lymphocytes express higher levels of tumor homing receptors such as LFA-1, CD49a and CXCR3 which correlate with TIL localization to the tumor bed after intravenous injection. Consistent with this OMCPmutIL-2 expanded TILs provided superior tumor control compared to 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 cytotoxic lymphocytes rather than indiscriminate expansion of all TILs.

Objective: 2019 novel coronavirus (2019-nCov) has become a global health emergency. However, the clinical presentations are not well characterized. The study aimed to describe clinical characteristics of 2019-nCov pneumonia with meta-analytic approach, and to identify risk factors for developing severe cases.Methods: The electronic databases of PubMed, Google Scholar and MedRxiv were searched from December 2019 to February 2020. Records were included if they reported clinical characteristics of 2019-nCov pneumonia. Studies using crowd sourcing data for mathematical modeling but not reporting clinical data were excluded. The study was reported according to the PRISMA guideline. Data were extracted by independent reviewers. Proportions and mean values were pooled across component studies by using the meta-analytic approach. Data were pooled with fixed or random-effects model as appropriate. Clinical characteristics such as age, gender, symptoms, treatment and mortality outcome were pooled across studies if appropriate. Risk factors for development of severe cases were reported.Results: A total of 13 studies involving 5,729 patients were included for quantitative analysis. The mean age of the study population was 50 years (95% CI: 47 to 53). The most common initial symptoms were cough (68.0%; 95% CI: 65.6 to 70.4%), followed by fever (56.5%; 95% CI: 53.9 to 58.9%), fatigue (42.5%; 95% CI: 39.9 to 45.1%) and anorexia (31.7%; 95% CI: 26.5 to 38.4%). The severe cases accounts for 22.5% of the whole population (95% CI: 21.4 to 23.6%). The overall mortality rate was 1.8% (95% CI: 1.5 to 2.2%), which was consistent with the real time epidemic tracking data. There was substantial heterogeneity across included studies (O = 0.84; p < 0.001). A number of comorbidities and symptoms such as hypertension, COPD, dyspnea, elevated C-reactive protein and procalcitonin were found to be associated with increased risk of developing severe cases.Conclusions: Our study described clinical characteristics of the 2019-nCov pneumonia in a systematic way. Multiple risk factors were identified for severe cases.

Natural killer (NK) cells play a critical role in controlling malignancies. Susceptibility or resistance to lung cancer, for example, specifically depends on NK cell function. Nevertheless, intrinsic factors that control NK cell-mediated clearance of lung cancer are unknown. Here we report that NK cells exposed to exogenous major histocompatibility class I (MHCI) provide a significant immunologic barrier to the growth and progression of malignancy. Clearance of lung cancer is facilitated by up-regulation of NKG2D, NKp46, and other activating receptors upon exposure to environmental MHCI. Surface expression of the inhibitory receptor Ly49C/I, on the other hand, is down-regulated upon exposure to tumor-bearing tissue. We thus demonstrate that NK cells exhibit dynamic plasticity in surface expression of both activating and inhibitory receptors based on the environmental context. Our data suggest that altering the activation state of NK cells may contribute to immunologic control of lung and possibly other cancers.

Eosinophils are rare granulocytes that belong to the innate arm of the immune system. This cell population is traditionally defined as a destructive and cytotoxic mediator in asthma and helminth infection. Limited data in transplantation have suggested that eosinophils play a similar role in potentiating deleterious organ inflammation and immunologic rejection. Contrary to this long-held notion, recent data have uncovered the possibility that eosinophils play an alternative role in immune homeostasis, defense against a wide range of pathogens, as well as downregulation of deleterious inflammation. Specifically, translational data from small animal models of lung transplantation have demonstrated a critical role for eosinophils in the downregulation of alloimmunity. These findings shed new light on the unique immunologic features of the lung allograft and demonstrate that environmental polarization may alter the phenotype and function of leukocyte populations previously thought to be static in nature. In this review, we provide an update on eosinophils in the homeostasis of the lung as well as other solid organs.

Despite the accepted notion that granulocytes play a universally destructive role in organ and tissue grafts, it has been recently described that eosinophils can facilitate immunosuppression-mediated acceptance of murine lung allografts. The mechanism of eosinophil-mediated tolerance, or their role in regulating alloimmune responses in the absence of immunosuppression, remains unknown. Using lung transplants in a fully MHC-mismatched BALB/c (H2(d)) to C57BL/6 (H2(b)) strain combination, we demonstrate that eosinophils downregulate T cell–mediated immune responses and play a tolerogenic role even in the absence of immunosuppression. We further show that such downregulation depends on PD-L1/PD-1–mediated synapse formation between eosinophils and T cells. We also demonstrate that eosinophils suppress T lymphocyte responses through the inhibition of T cell receptor/CD3 (TCR/CD3) subunit association and signal transduction in an inducible NOS–dependent manner. Increasing local eosinophil concentration, through administration of intratracheal eotaxin and IL-5, can ameliorate alloimmune responses in the lung allograft. Thus, our data indicate that eosinophil mobilization may be utilized as a novel means of lung allograft–specific immunosuppression.

Dough elasticity and bread quality depend on the gluten network formation which by nature is the oxidative crosslinking of glutenin. It is known for long, either in the kitchen or in the laboratory, sodium chloride (NaCl) enhances dough rheological properties while itself is not an oxidant. However, besides NaCl induced gluten aggregation, the molecular mechanism behind this phenomenon is largely unknown. Azodicarbonamide (ADA) and Vitamin C (Vc) improve dough qualities with different mechanisms. Here, response surface methodology (RSM) was used to study the synergistic effect of ADA, Vc and NaCl on the dough and subsequent bread qualities. Results show that NaCl dominates the dough alveographic characters in the presence of oxidative flour improvers, and fine tune of NaCl concentration profoundly impacts subsequent bread properties. Consistent with free sulfhydryl assay and glutenin gel electrophoresis analysis, simulation study reveals that NaCl concentration affects glutenin conformation and intermolecular interaction which in turn impacts their orientation adjustment that lead to sulfhydryl contact and crosslinking.

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 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.

Purpose Despite the growing experience in lung transplantation a significant number of patients rapidly succumb to chronic rejection in the form of bronchiolitis obliterans (OB). The reasons for such discrepancies are unknown but have been suspected to relate to individual variability in gut and lung microbiome. Methods To study the role of the microbiome in controlling lung immunology we transplanted C57BL/10 lungs to C57BL/6 recipients from either Jackson Lab, Maine or Envigo facilities in Maryland vs Indiana. Histological grading of acute and chronic rejection, in the form of OB was performed on days 7 and 21 respectively with flow cytometric analysis of the lungs in parallel. Results Recipients from the Maine facility rapidly rejected lung grafts with high ISHLT rejection grades (2.8±0.37) by day 7 while Maryland recipients were rejection-free (0.25±0.25). Delayed histologic evaluation on day 21 post-engraftment revealed evidence of OB in 40% of Indiana recipients while 0% in Maine or Maryland mice(Fig.1A). Flow cytometric analysis revealed a high prevalence of CD4+Foxp3+ Tregs in Maryland recipients (29.56±2.32%) but lower in Maine and Indiana mice while T cell IL-17 levels were also different between them. Systemic different TGF-β levels were also observed. Treatment of Maryland recipients with broad-spectrum antibiotics affected TGF-β levels, decreased the Treg proportion (14.24±1.47%, p Conclusion Our data demonstrate that mice from different origins mount different lung allograft-specific microbiome-related immune responses. Furthermore it suggests that microbiome-dependent TGF beta levels may contribute to the generation of protective Tregs and ameliorate both acute and chronic rejection. Such data puts into question the notion that TGF-β contributes to lung fibrosis and opens up therapeutic avenues of microbiome manipulation to augment the TGF-β -Treg axis.

Lungs allografts have worse long-term survival compared with other organ transplants. This is most likely due to their unique immunoregulation that may not respond to traditional immunosuppression. For example, local NO generation by inducible NOS (iNOS) is critical for lung allograft acceptance but associates with rejection of other solid organs. The source of NO in accepting lung allografts remains unknown. Here, we report that, unlike the case for other pulmonary processes in which myeloid cells control NO generation, recipient-derived eosinophils play a critical and nonredundant role in iNOS-mediated lung allograft acceptance. Depletion of eosinophils reduces NO levels to that of recipients with global deletion of iNOS and leads to a costimulatory blockade-resistant form of rejection. Furthermore, NO production by eosinophils depends on Th1 polarization by inflammatory mediators, such as IFN-γ and TNF-α. Neutralization of such mediators abrogates eosinophil suppressive capacity. Our data point to what we believe to be a unique and previously unrecognized role of eosinophil polarization in mediating allograft tolerance and put into perspective the use of high-dose eosinophil-ablating corticosteroids after lung transplantation.

Isolation, ex vivo expansion followed by reinfusion of activated autologous lymphocytes is a successfully utilized and promising clinical treatment for both solid and liquid tumors. Although it has been routine for most clinical and experimental laboratories to rely on interleukin-2 (IL-2) to support T cell expansion this cytokine can result in substantial expansion and induction of regulatory T cells (Tregs), AICD, exhaustion and terminal differentiation. IL-2 signals when the three subunits of IL-2 receptor, namely α, β and γ form a heterotrimeric protein. We have recently described a novel re-targeted form of IL-2 which utilizes NKG2D rather than IL-2Rα to form the high affinity receptor complex for IL-2 signaling. This cytokine (OIL-2), targets signaling solely to NKG2D-expressing cells but its role in expanding CD8+ T cells for adoptive T cell therapy is unknown In direct comparison to IL2, OIL-2 resulted in significantly improved expansion of CD8+ T cells (70-fold in vs 345-fold increase in 3 weeks; p In conclusion we demonstrate that our first-in class cytokine is more effective and offers a superior method for ex vivo T cell expansion. OIL-2 mediated CD8+ T cell stimulation occur via an altered signaling complex which causes T cell metabolic re-programming. As a result, OIL-2 expanded T cells persist and offers better tumor clearance and provide superior protection against malignancy over T cells expanded through alternative protocols. Citation Format: Anirban Banerjee, Yizhan Guo, Sarah Hein, Alexander S. Krupnick. A novel first-in-class common-gamma chain cytokine facilitates expansion of CD8+T cells and offers a superior cellular source for adoptive T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 939.

Eosinophils play a surprising role in downregulating lung graft-specific alloimmunity, even without immunosuppression. We demonstrate here, a mechanism used by eosinophils to downregulate alloimmune responses. To study the mechanism of eosinophil dependent downregulation of alloimmune responses, we used a model of diphtheria toxin (DT) dependent ablation of eosinophils in iPHIL, a mouse model expressing DT receptor on the eosinophil-peroxidase gene locus. Fully MHC-mismatched Balb/c (H2Kd) lungs were engrafted to iPHIL (H2Kb) recipients without immunosuppression. We modeled alloreactivity in vitro, with mixed leukocyte cultures, comprising Th1-polarized eosinophils as modulators of T cells from C57BL/6J or Nur77-GFP mice stimulated by either allogeneic dendritic cells or anti-CD3/CD28 agonistic antibodies. Ablation of eosinophils in non-immunosuppressed lung graft recipients leads to enhanced T cell activation and graft rejection. Similarly, in vitro, we observed a dose and contact dependent downregulation of T cell activation by eosinophils. While contact between eosinophils and T cells is mediated through the upregulation of PD-L1 on both wild-type and iNOS−/− eosinophils after TH-1 polarization, the inhibition of T cell activation is iNOS dependent and is directly linked to the iNOS dependent inhibition of the TCR signaling early gene, Nr4a1, monitored in Nur77-GFP mice. Thus, following contact with T cells, iNOS-expressing eosinophils downregulate lung graft alloimmunity through a direct effect on TCR engagement early genes, leading to the inhibition of T cell activation. This provides an explanation for the surprising observation of lung-graft specific alloimmune downregulation by eosinophils.

Background and objectivesDelayed patient admission to the intensive care unit (ICU) from the ED is common in China. Patients with severe sepsis or septic shock requiring ICU admission are in need of specialised monitoring and tailored treatment. Delayed admission to the ICU might be associated with adverse clinical outcomes for patients with sepsis.MethodsPatients with sepsis admitted to the ICU from the ED from January 2010 to April 2018 were retrospectively identified from a clinical data warehouse. The primary endpoint was in-hospital mortality. Length of stay in ED (EDLOS) was compared between survivors and non-survivors. A multivariable regression model was employed to adjust for potential confounding due to patient clinical condition.ResultsA total of 1997 patients, including 473 non-survivors and 1524 survivors, were included. The crude mortality rate for patients with EDLOS 24 hours (31.8%). After adjusting for PaO2/FiO2, serum creatinine, age, Sequential Organ Failure Assessment, body mass index, lactate, comorbidities and infection site, EDLOS continued to be independently associated with increased risk of hospital mortality. Compared with the group with EDLOS 24 hours (OR 1.79, 95% CI 1.27 to 2.52) showed a significantly increased risk of death.ConclusionsOur study shows that prolonged EDLOS is independently associated with increased risk of hospital mortality in patients with sepsis requiring ICU admission.

Infusion of activated autologous lymphocytes or CAR-T cells is a promising clinical treatment for both solid and liquid tumors. Traditional methods for ex vivo T cell expansion have relied on transient stimulation of the T cell receptor in the presence of the common γ-chain cytokines. Although it has been routine for most clinical and experimental laboratories to rely on interleukin-2 (IL2) to support T cell expansion, this cytokine can result in expansion of regulatory T cells (Tregs) and activation induced death of effector cells. Furthermore, prolonged exposure to IL2 may result in differentiation of CD8+ T cells toward an effector phenotype, a terminally differentiated state that provides only short-lived and highly limited protection from malignancy. Alternate gamma chain cytokines, such as IL7 and IL15, do not expand Tregs and may offer an advantage for the expansion of central memory CD8+ T cells, that are superior in mediating tumor regression. Nevertheless, optimal protocols for T cell expansion have yet to be defined. We have recently demonstrated that IL2 targeted to NKG2D-expressing cells using the viral decoy ligand known as orthopox major histocompatibility complex class I-like protein (or OMCP for short), offers a superior method for NK cell expansion and NK mediated immunotherapy (Nat Commun 2016 Sep 21;7. doi: 10.1038/ncomms12878). The possibility of using this redirected cytokine (called OMCP-IL2 for short) for ex vivo CD8+ T cell expansion has not been explored. To directly compare several common gamma cytokines bulk T cells from C57BL/6 mice were cultured with transient CD3/CD28 stimulation in the presence of wild-type IL2, OMCP-IL2 or combination IL15/IL7 at 100IU/ml. While both IL2 and IL15/IL7 resulted in substantial T cell expansion by day 12 of culture (5.8±0.7 and 3.4±0.16-fold expansion respectively) OMCP-IL2 treated cultures resulted in 10±0.8 fold expansion by the same time point. Furthermore, IL2 and IL15/IL7-treated cultures did not expand further after two weeks of cytokine treatment while OMCP-IL2 treated cultures expanded for 40 days (reaching a 27±0.7-fold expansion). Flow cytometric analysis revealed that the higher CD8+ T cell numbers in OMCP-IL2 treated cultures were due to both increased proliferation (%KI67 positivity in 49±9%, 70±2% and 86±3% of CD8+ T cells for IL2, IL15/IL7 and OMCP-IL2 treated cultures respectively) and improved viability (27±2%, 20±2% and 79±1.8% viable cells for IL2, IL15/IL7 and OMCP-IL2-treated cultures respectively). While wild-type IL2 expanded both CD8+ and CD4+ T cells OMCP-IL2 preferentially expanded CD8+ T cells. Phenotypic analysis revealed that the majority of CD8+ T cells expanded in OMCP-IL2 were CD44hiCD62Lhi central memory T cells while those expanded in IL2 were primarily CD44hiCD62Llow effector cells. Consistent with this a starting population of 1 million T cells resulted in 60,756±21,813; 368,756±30,217; and 7,605,870±872,870 CD8+ central memory T cells after three weeks of culture in IL2, IL15/7 and OMCP-IL2 respectively. Furthermore, both IL2 and IL15/7 expanded cells expressed high levels of surface PD-1 (67.8% and 40.9% respectively) while minimum PD-1 was expressed on OMCP-IL2 expanded cells (18.4%) In summary, we now demonstrate that the use of an NKG2D-targeted form of IL2 provides a significant quantitative and qualitative advantage for CD8+ T cell expansion in vitro. Such an advantage might be the result of precise delivery of IL2 to cytotoxic T cells or novel signaling pathways induced by engagement of both the NKG2D and IL2 receptor. Since the ability to obtain a significant number of CAR T cells limits the clinical application of this technology the possibility of quickly and efficiently expanding T cells will be key to improving clinical therapy. Citation Format: Kang Li, Lei Shi, Qing Wang, Oscar Onyema, Yizhan Guo, Alexander Sasha Krupnick. Superior expansion of central memory CD8+ T cells using NKG2D-targeted delivery of IL-2: Implications for adoptive T cell immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A47.

Multivariable regression models are widely used in medical literature for the purpose of diagnosis or prediction. Conventionally, the adequacy of these models is assessed using metrics of diagnostic performances such as sensitivity and specificity, which fail to account for clinical utility of a specific model. Decision curve analysis (DCA) is a widely used method to measure this utility. In this framework, a clinical judgment of the relative value of benefits (treating a true positive case) and harms (treating a false positive case) associated with prediction models is made. As such, the preferences of patients or policy-makers are accounted for by using a metric called threshold probability. A decision analytic measure called net benefit is then calculated for each possible threshold probability, which puts benefits and harms on the same scale. The article is a technical note on how to perform DCA in R environment. The decision curve is depicted with the ggplot2 system. Correction for overfitting is done via either bootstrap or cross-validation. Confidence interval and P values for the comparison of two models are calculated using bootstrap method. Furthermore, we describe a method for computing area under net benefit for the comparison of two models. The average deviation about the probability threshold (ADAPT), which is a more recently developed index to measure the utility of a prediction model, is also introduced in this article.

Introduction Based on the accumulation of eosinophils (eos) in rejected lung grafts, it was widely held that eos perpetuate lung graft rejection. However, we showed recently in a mechanistic study that eos mediate lung graft tolerance. As eos are also present in rejected lungs, we therefore explore the possibility of a different role of eos in lung graft rejection. Method Using orthotopic left lung transplantation of MHC-1 mismatched Balb/c (H2Kd) donors to C57BL/6J (H2Kb) recipients, with or without co-stimulatory blockade immunosuppression, we quantified the number of eos in lung grafts at day 7 post-transplant (D7). Th1, Th2 and Th17 cytokine profiles of D4 lung grafts were analyzed by quantitative PCR. D7 eos were also analyzed for their expression of Th1, Th2 and Th17 signature genes. The role of eos was examined in iPHIL mice that experience selective ablation of eos after Diphtheria toxin (DT) administration. Balb/c left lungs transplanted into DT or vehicle treated iPHIL mice without immunosuppression were analyzed histologically and flow cytometrically at D4. Results We observed similar numbers of eos, higher Th1 polarization, lower IL33, a Th2 cytokine and less Th17 polarization of the lung microenvironment, and higher expression of the Th1 signature genes among eos, in rejecting compared to accepting lung grafts. Eos deficiency was associated with more rapid lung graft rejection, evidenced in higher CD8+ T cell proliferation, lower CD4/CD8 ratio, and enhanced T cell differentiation to effector memory phenotype. Conclusion Our results indicate that eos directly ameliorate lung graft rejection even in the absence of immunosuppression. This calls for a revalidation of the importance of eos in lung allograft pathology.

Given the maternal physiological changes, the morbidity rate of cerebral venous thrombosis (CVT) is higher during pregnancy and puerperium. Intracranial cryptococcal infection is a severe central nervous system (CNS) infection caused by Cryptococcus which is mostly found in immunocompromised patients. Nevertheless, there was no report on human immunodeficiency virus (HIV)-negative pregnant woman presenting with both CVT and intracranial cryptococcal infection simultaneously. Here we present a case of CVT and intracranial cryptococcal infection on a HIV-negative pregnant woman who manifested recurrent headache.

Individuals with robust natural killer (NK) cell function incur lower rates of malignancies. To expand our understanding of genetic factors contributing to this phenomenon, we analyzed NK cells from cancer resistant and susceptible strains of mice. We identified a correlation between NK levels of the X-chromosome-located adaptor protein SLy1 and immunologic susceptibility to cancer. Unlike the case for T or B lymphocytes, where SLy1 shuttles between the cytoplasm and nucleus to facilitate signal transduction, in NK cells SLy1 functions as a ribosomal protein and is located solely in the cytoplasm. In its absence, ribosomal instability results in p53-mediated NK cell senescence and decreased clearance of malignancies. NK defects are reversible under inflammatory conditions and viral clearance is not impacted by SLy1 deficiency. Our work defines a previously unappreciated X-linked ribosomopathy that results in a specific and subtle NK cell dysfunction leading to immunologic susceptibility to cancer.