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Higher γδ T cell counts in patients with malignancies are associated with better survival. However, γδ T cells are rare in the blood and functionally impaired in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and interleukin-2 (IL-2). Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K-562 CD3scFv/CD137L/CD28scFv/IL15RA quadruplet artificial antigen-presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a Good Manufacturing Practice (GMP) compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of coculture with aAPC, which exhibited central (47%) and effector (43%) memory phenotypes. In addition,90% of the expanded γδ T cells expressed NKG2D, although they have low cell surface expression of PD1 and LAG3 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded γδ T cells were effective in killing target cells. Our results demonstrate that large-scale ex vivo expansion of donor-derived γδ T cells in a GMP-like setting can be achieved with the use of quadruplet aAPC and zol/IL-2 for clinical application.

An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1. Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.

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Graft-versus-host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). For decades, GVHD prophylaxis has included calcineurin inhibitors, despite their incomplete efficacy and impairment of graft-versus-leukemia (GVL). Distinct from pharmacologic immune suppression, we have developed what we believe is a novel, human CD83-targeted chimeric antigen receptor (CAR) T cell for GVHD prevention. CD83 is expressed on allo-activated conventional CD4(+) T cells (Tconvs) and proinflammatory dendritic cells (DCs), which are both implicated in GVHD pathogenesis. Human CD83 CAR T cells eradicate pathogenic CD83(+) target cells, substantially increase the ratio of regulatory T cells (Tregs) to allo-activated Tconvs, and provide durable prevention of xenogeneic GVHD. CD83 CAR T cells are also capable of treating xenogeneic GVHD. We show that human acute myeloid leukemia (AML) expresses CD83 and that myeloid leukemia cell lines are readily killed by CD83 CAR T cells. Human CD83 CAR T cells are a promising cell-based approach to preventing 2 critical complications of allo-HCT — GVHD and relapse. Thus, the use of human CD83 CAR T cells for GVHD prevention and treatment, as well as for targeting CD83(+) AML, warrants clinical investigation.

Introduction. Total hip arthroplasty (THA) surgery is one of the most commonly performed and successful orthopedic procedures in the United States. More than 300,000 primary THAs and 40,000 revision THAs performed in the United States every year. While the need for revision surgeries can stem from a variety of causes, there have been, to the author’s knowledge, no studies attempting to correlate the concentrations of certain inflammatory cytokines to metal ion concentrations found in the tissue surrounding the implant, amount of polyethylene wear, or strength of the interface of the modular taper. The purpose of this study was to begin to look at those factors to see if any were indicative of implant survivorship, as well as to see if metal ion content contributes to implant longevity. The testing for this group of well-functioning implants will be useful as a baseline when comparing the same types of testing for failed implants. Methods. A total of nineteen cadaveric total hip implants were obtained from two sources, the Medical Education and Research Institute (Memphis, TN) and RestoreLifeUSA (Elizabethton, TN). The bearings for these implants were either metal on polyethylene or ceramic on polyethylene. Synovial fluid and tissue samples were taken from the joint for testing. Head dissociation was performed, in which an Instron 4505 was used in accordance with ASTM Standard F2009-00 to remove the head from the stem of the implant, recording force. Corrosion scoring was performed on taper surfaces by three scorers. The polyethylene acetabular liner was measured on the superior side with a micrometer to determine how much material loss was evident compared to the inferior side. These three values were then correlated to the testing performed using the synovial fluid and tissue. The synovial fluid was analyzed for inflammatory cytokines IL-6, MCP-1, IL-1β, MIP-3α, M-CSF, IL-8, IL-2, and TNF-α using a premixed Luminex screening assay. These results were given in picograms per milliliter. An anterior and posterior synovial tissue sample was analyzed for the presence of metal ions cobalt, chromium, and titanium using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). All of these results were compiled and analyzed together to search for potential correlations. Results. There were no significant differences in dissociation forces between the groups of implants with head corrosion score 1 and head corrosion score 2. The comparison of MCP-1 to the dissociation force produced a correlation coefficient of 0.64 (p-value 0.05) and the comparison of MIP-3α to the dissociation force produced a correlation coefficient of 0.67 (p-value 0.03). However, when the graphs of these correlations were observed, it seemed likely that this correlation was due to one sample pulling the graph in a positive direction which is demonstrated by the 95% confidence interval (CI) of the correlation coefficient (0.011 to 0.90 for MCP-1, and 0.069 to 0.91 for MIP-3α). When comparing polyethylene wear to the inflammatory cytokine concentrations, no significant correlations were seen. There was a positive correlation between cobalt and chromium levels and dissociation force (r=0.56 for cobalt, r=0.66 for chromium), and a negative correlation between titanium levels and dissociation force (r=-0.30). The positive relationship was opposite of what was expected, as more metal debris should mean the implant surfaces are losing material, which should therefore decrease the strength of the taper connection. The 95% confidence interval for the correlation coefficients included zero for cobalt and titanium, and was fairly wide for chromium (0.11 to 0.90). When observing cytokines and metal ion presence, most relationships were very scattered with low correlation coefficients. However, for cobalt, strong positive relationships were seen for IL-6 (r=0.67, CI: 0.19 to 0.89), MCP-1 (r=0.76, CI: 0.33 to 0.93), and MIP-3α (r=0.60, CI: 0.066 to 0.86). When looking at confidence intervals, there seemed to be a mild correlation between cobalt and IL-6 and a moderate correlation between cobalt and MCP-1. No meaningful relationships were seen for any cytokines with chromium or titanium, so it may be useful to select cytokines known to be responsive to those two metals in particular for future studies. When comparing metal levels between the two corrosion levels seen in the heads, there were no statistically significant differences in any of the metals between implants with a corrosion score of one and those with a corrosion score of two. Discussion. This study was limited by the fact that the sample size for this study was very low. With only nineteen total implants, it was difficult to draw meaningful conclusions. Additional implants are being recruited in order to increase this sample size for future studies. Additionally, it was difficult for meaningful correlations to be seen when comparing any factor to the inflammatory cytokine concentrations, as these values were clustered around the lower limit of detection. However, this was expected with well-functioning implants. While it is difficult to draw meaningful conclusions when used as a correlation, this data will be useful when comparing cytokine concentrations of a group of failed implants. This group is able to serve as a baseline value for each type of testing performed, and will help to make sense of the same testing of failed implants in the future.

BackgroundChimeric antigen receptor (CAR)-T cells for solid tumors have shown modest effectiveness as compared to hematologic malignancies, a consequence of antigen heterogeneity, the immuno-suppressive tumor microenvironment (TME), limited cell persistence, and perhaps most notably, the trafficking of the CAR-T cell to the tumor itself. Early detection of CAR-T cells within a solid tumor has been associated with better outcomes across several clinical trials in diverse tumor settings, suggesting that strategies focused on enhancing CAR-T cell homing to and infiltration into the tumor can yield therapeutic benefit.MethodsHere, we demonstrate that following irradiation or exposure to common chemotherapy drugs, selected tumor cell lines (breast, ovarian, and prostate) specifically upregulate several chemokines, notably the CXCR2 ligand, interleukin (IL)-8, up to 4-fold over baseline control (e.g. 24ng/ml increased to 79ng/ml for SKOV3; 2.9ng/ml increased to 12.5ng/ml for MDA-MB-231). To leverage the upregulation of IL-8 as a mechanism of directing CAR-T cells to the tumor site, we initially engineered primary CAR-T cells to express CXCR2 and demonstrated functional migration, in a dose-dependent manner, to recombinant IL-8 in an in vitro transwell chemotaxis assay; maximal migration of approximately 2-fold over baseline was observed with 10ng/ml of rhIL-8. Similarly, supernatant from pre-conditioned tumor lines also elicited functional enhancements in migration (up to 4-fold specific migration). In addition, ovarian tumors were sub-optimally treated with paclitaxel in vivo, which promoted infiltration of CXCR2+ CAR-T cells and demonstrated enhanced tumor control.ResultsWe then incorporated these findings into our off-the-shelf, iPSC-derived CAR-T cell product platform. Induced pluripotent stem cells (iPSCs) were precisely engineered to co-express CAR and CXCR2 and subsequently differentiated to T cells to generate iPSC-derived CAR-T cells (CAR-iT cells). Like their primary CAR-T cell counterparts, functional chemotaxis of CXCR2+ CAR-iT cells was also observed in response to recombinant IL-8 and preconditioned tumor media. Importantly, CXCR2 expression did not limit CAR-dependent cytolytic function and the specificity of CAR-iT cells, underscoring the compatibility of this approach. Further in vitro and in vivo studies are ongoing and will be presented.ConclusionsCollectively, these data demonstrate that rational engineering of unique chemokine receptors to deliver the ideal chemokine/chemokine receptor match between tumors and effector cells can be leveraged to enhance tumor targeting and trafficking of CAR-iT cells for more effective treatment of solid tumors.Ethics ApprovalThese studies were approved by Fate Therapeutics Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Supplemental Digital Content is available in the text., Adoptive cell therapy with ex vivo expanded tumor infiltrating lymphocytes or gene engineering T cells expressing chimeric antigen receptors (CAR) is a promising treatment for cancer patients. This production utilizes T-cell activation and transduction with activation beads and RetroNectin, respectively. However, the high cost of production is an obstacle for the broad clinical application of novel immunotherapeutic cell products. To facilitate production we refined our approach by using artificial antigen presenting cells (aAPCs) with receptors that ligate CD3, CD28, and the CD137 ligand (CD137L or 41BBL), as well as express the heparin binding domain (HBD), which binds virus for gene-transfer. We have used these aAPC for ex vivo gene engineering and expansion of tumor infiltrating lymphocytes and CAR T cells. We found that aAPCs can support efficacious T-cell expansion and transduction. Moreover, aAPCs expanded T cells exhibit higher production of IFN-γ and lower traits of T-cell exhaustion compared with bead expanded T cells. Our results suggest that aAPC provide a more physiological stimulus for T-cell activation than beads that persistently ligate T cells. The use of a renewable cell line to replace 2 critical reagents (beads and retronectin) for CAR T-cell production can significantly reduce the cost of production and make these therapies more accessible to patients.

Background Although CAR T cells have been shown to be effective and potent in treating several hematologic malignancies, engineered T-cell therapies have had limited success in addressing solid tumors. Unlike liquid tumors where uniformly expressed antigens are accessible and can be effectively targeted, tumor access and antigen heterogeneity are a significant barrier to the successful development of CAR-T cells in solid tumors. Methods Here we demonstrate that the combination of a bi-specific T-cell engager (BiTE) targeting EpCAM with a CAR T cell targeting HER2 enhances the in vitro and in vivo anti-tumor activity against heterogenous solid tumors. Results We observed a dose-dependent enhancement of cytolytic activity when EpCAM-specific BiTEs were titrated alongside 4D5-based HER2-specific CAR T cells against HER2low tumors, enhancing maximal cytolysis by two-fold compared to CAR T cells alone (figure 1). Moreover, the escape of HER2low tumor cells in mixed heterogenous culture systems was circumvented by the combination of HER2-specific CAR T cells and EpCAM-specific BiTEs. The enhancement of efficacy was further demonstrated in an established HER2low MDA-MB-231 xenografts. HER2-specific CAR T cells were unable to contain Her2low tumors, whereas tumor growth was effectively controlled in mice receiving both EpCAM-specific BiTEs and HER2-specific CAR T cells. Conclusions Collectively, these data demonstrate that multi-antigen targeting mediated by BiTEs and CARs extends overall anti-tumor efficacy in preclinical models of heterogenous solid tumors. Fate Therapeutics is currently using its proprietary induced pluripotent stem cell (iPSC) product platform to generate iPSC-derived CAR T cells and iPSC-derived CAR NK cells that secrete BiTEs for the treatment of solid tumors. Ethics Approval These studies were approved by Fate Therapeutics Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1. Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.

Context and Objective CD83 is expressed on activated CD4+ T-cells, dendritic cells, and acute myeloid leukemia (AML), but its expression level on organ-specific normal tissue is not well investigated. A novel human CD83-targeted CAR T-cell has been developed and shown in preclinical work to treat and prevent graft-versus-host disease (GVHD) and confer graft-versus-leukemia (GVL) activity against AML. The purpose of this study is to evaluate CD83 expression in a panel of normal tissue samples to better characterize CD83 expression. Design and Patients An IRB-approved pilot study utilized specimens collected and maintained by Moffitt after informed consent. Thirty samples of representative normal tissues were utilized, with 3 cores taken from each sample, to construct a tissue microarray (TMA). A second TMA was obtained through the Cooperative Human Tissue Network. CD83 expression was assessed by immunohistochemistry (IHC) after antibody validation. IHC expression was scored by hematopathologists, and immunoreactive scores (IRS) were calculated (product of staining intensity and nominal variable denoting expression percentage). IRS scores of 0–1, 2–3, 4–8, and 9–12 correspond to negative, mild, moderate, and strong expression, respectively. Results TMA constructs included 9 normal tissue types with 6–9 cores each and 51 tissue types with 3 cores each in two separate blocks. Strong expression was seen in plasma cells in lymph nodes and tonsils, pancreas, and parathyroid tissue. Moderate expression was seen in lymph nodes, spleen, tonsil, thymus, lung, mesothelium, kidney, liver, thyroid, bladder, cartilage, cerebral cortex, small bowel, stomach, placenta/amniotic membrane, and endocervix. Mild expression was seen in bone marrow, breast, skin, parotid gland, epididymis/seminal vesicle, esophagus, large bowel, anus, uterus, ovary/fallopian tube, synovium, stomach, gallbladder, aorta, and breast. There was no expression in myocardium, adrenal gland, brain white matter, peripheral nerves, smooth muscle, prostate, or ovarian primary oocyte. Conclusions The present study better characterizes CD83 expression on organ-specific normal tissue. Moderate expression was seen in lymphoid tissue. Further work is needed to determine the impact of CD83-targeted therapy on normal tissue expressing CD83. This data will be used in the development of future clinical trials of novel human CD83-targeted therapies with promising preclinical data.

Patients with relapsed or refractory acute myeloid leukemia (AML) are at increased risk of mortality. Higher γδ T cell count in a bone marrow or peripheral blood of patients with leukemia is associated with better survival. However, γδ T cells are rare in the blood and functionally impaired or exhausted in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and IL-2. Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K562 CD3/CD137L/CD28/IL15RA quadruplet artificial antigen presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a GMP-compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of co-culture with aAPC, the majority of which exhibited central (47%) and effector (43%) memory phenotypes. Additionally, >90% of the expanded γδ T cells expressed NKG2D, while they have low cell surface expression of PD1 and LAG2 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded, previously cryopreserved, γδ T cells were effective in killing target cells. Our results demonstrate that large scale ex vivo expansion of donor-derived γδ T cells can be achieved with the use of CD3/CD137L/CD28/IL15RA quadruplet aAPC and zol/IL-2 for clinical application as promising antineoplastic immunotherapy. Figure 1 Disclosures Lancet: Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy. Bejanyan:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees.

Relapse of leukemic cells that do not express the antigen targeted by chimeric antigen receptor (CAR) is still a risk. As is the potential for targeting hematopoietic stem cells (HSCs) that share the same antigen expression, off-tumor on-target toxicity. Further, CAR T cells that bind different epitopes of the same antigen can have different tumor-killing efficacies. Therefore, we screen murine single chain variable fragment (scFv) based for indirect affinity to identify a CAR that targets Acute myeloid leukemia (AML), while minimizing toxicities. Also, recent advances in CAR design have demonstrated that the requirement of two separate tumor antigens to be ligated by CARs can increase the specificity for tumor targets. So designing a CAR that only activates a T cell when it binds two separate AML antigens will allow T cells to enhance safety. Therefore, we set out to develop a affinity based multi-antigen CAR T cell therapy that targets well described antigens for AML, including CD33 and CD123. Mice were immunized with these antigens, spleens collected, and fused with myeloma cell lines. The antibodies of fused hybridomas were screened for binding and activation against antigens by high throughput flow cytometry. After screening, we derived multiple de novo CD33, and CD123 scFvs by sequencing. We incorporated CD33 and CD123 scFvs into standard mono-specific CARs utilizing a 41BB co-stimulatory domain to validate antigen-specificity. Gene transfer assessment of CAR T cells demonstrated about 50-80% transduction efficiency for CD33 and CD123 scFvs. There were no differences in CD4 and CD8 proportions in these CAR T cells. We next examined the CARs for their cytotoxic ability using a Real-Time Cell Analysis (RTCA) system. For the CD33 CARs, 2 (6A11-1 and 27A3-1) out of 5, and for the CD123 CAR, 2 (15A12-11 and 15 A12-12) out of 8, scFv sequences transduced into T cells were highly efficacious at killing target cells and generated significant amounts of cytokines such as IFN-g, TNF- a, and IL-6. CAR T cells with these same scFv sequences were able to proliferate better in response to targeted antigen. To find the best possible combination of CD33 and CD123 scFvs we double transduced T cells with four selected CD33 or CD123 scFvs each with only one co-stimulation domain either CD3z or 4-1BB in "AND" gate fashion. Clear differences in cytotoxic ability and cytokine production were observed. We selected 12 combination of CD33/123 CARs bi-specific CARs to evaluate in vitro efficacy, polyfunctionality, and safety. Finally, to find the best combination of CARs that would be less toxic to HSCs, we performed a Colony Forming Unit (CFU) assay with CD34+ bone marrow stem cells and found 5 bi-specific pairs that were less toxic to HSCs . Based on the CFU assay and PSI index, we were able to select the combination of CD33/123 scFvs that would target AML but minimize the killing of HSCs.

Chimeric antigen receptors (CARs) have an antigen-binding domain fused to transmembrane, costimulatory, and CD3ζ domains. Two CARs with regulatory approval include a CD28 or 4-1BB costimulatory domain. While both CARs achieve similar clinical outcomes, biologic differences have become apparent but not completely understood. Therefore, in this study we aimed to identify mechanistic differences between 4-1BB and CD28 costimulation that contribute to the biologic differences between the 2 CARs and could be exploited to enhance CAR T cell function. Using CD19-targeted CAR T cells with 4-1BB we determined that enhancement of T cell function is driven by NF-κB. Comparison to CAR T cells with CD28 also revealed that 4-1BB is associated with more antiapoptotic proteins and dependence on persistence for B cell killing. While TNF receptor–associated factor 2 (TRAF2) has been presupposed to be required for 4-1BB costimulation in CAR T cells, we determined that TRAF1 and TRAF3 are also critical. We observed that TRAFs impacted CAR T viability and proliferation, as well as cytotoxicity and/or cytokines, in part by regulating NF-κB. Our study demonstrates how 4-1BB costimulation in CAR T cells impacts antitumor eradication and clinical outcomes and has implications for enhanced CAR design.

Respiratory syncytial virus (RSV) is one of the leading causes of bronchiolitis in children, and severe RSV infection early in life has been associated with asthma development. Using a neonatal mouse model, we have shown that down-regulation of IL-4 receptor α (IL-4Rα) with antisense oligonucleotides in the lung during neonatal infection protected from RSV immunopathophysiology. Significant down-regulation of IL-4Rα was observed on pulmonary CD11b+ myeloid dendritic cells (mDCs) suggesting a role for IL-4Rα on mDCs in the immunopathogenesis of neonatal RSV infection. Here, we demonstrated that neonatal CD11b+ mDCs expressed higher levels of IL-4Rα than their adult counterparts. Because CD11b+ mDCs mainly present antigens to CD4+ T cells, we hypothesized that increased expression of IL-4Rα on neonatal CD11b+ mDCs was responsible for Th2 - biased RSV immunopathophysiology. Indeed, when IL-4Rα was selectively deleted from CD11b+ mDCs, the immunopathophysiology typically observed following RSV reinfection was ablated, including Th2 inflammation, airway-mucus hyperproduction, and pulmonary dysfunction. Further, overexpression of IL-4Rα on adult CD11b+ DCs and their adoptive transfer into adult mice was able to recapitulate the Th2-biased RSV immunopathology typically observed only in neonates infected with RSV. IL-4Rα levels on CD11c+ cells were inversely correlated with maturation status of CD11b+ mDCs upon RSV infection. Our data demonstrate that developmentally regulated IL-4Rα expression is critical for the maturity of pulmonary CD11b+ mDCs and the Th2-biased immunopathogenesis of neonatal RSV infection.

Respiratory syncytial virus (RSV) infection is the number one cause of bronchiolitis in infants, yet no vaccines are available because of a lack of knowledge of the infant immune system. Using a neonatal mouse model, we previously revealed that mice initially infected with RSV as neonates develop Th2-biased immunopathophysiologies during reinfection, and we demonstrated a role for enhanced interleukin-4 receptor α (IL-4Rα) expression on T helper cells in these responses. Here we show that RSV infection in neonates induced limited type I interferon (IFN) and plasmacytoid dendritic cell (pDC) responses. IFN alpha (IFN-α) treatment or adoptive transfer of adult pDCs capable of inducing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. A reduced viral load and downregulation of IL-4Rα on Th2 cells were observed in IFN-α-treated neonatal mice, suggesting dual mechanisms of action. IMPORTANCE Respiratory syncytial virus (RSV) is the most significant cause of lower respiratory tract infection in infancy worldwide. Despite the dire need, we have failed to produce efficacious RSV vaccines or therapeutics. Part of the reason for this failure is our lack of understanding of how RSV interacts with the infant immune system to suppress the development of protective immunity. In the study described in the present paper, we used a neonatal mouse model, which more closely mimics human infants, to study the role of the innate immune system, particularly type I interferons (IFNs) and plasmacytoid dendritic cells (pDCs), in the pathogenesis of RSV infection. RSV infection in neonates induced limited type I IFN and pDC responses. IFN-α treatment or adoptive transfer of adult pDCs capable of producing IFN-α prior to neonatal RSV infection decreased Th2-biased immunopathogenesis during reinfection. These data suggest that IFN-α is a promising target for future RSV vaccine design.

An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.

RSV reinfection increases IL-4Rα expression on T helper cells, responsible for the enhanced immunopathologies in mice initially infected as neonates. RSV is the major cause of severe bronchiolitis in infants, and severe bronchiolitis as a result of RSV is associated with subsequent asthma development. A biased Th2 immune response is thought to be responsible for neonatal RSV pathogenesis; however, molecular mechanisms remain elusive. Our data demonstrate, for the first time, that IL-4Rα is up-regulated in vitro on human CD4+ T cells from cord blood following RSV stimulation and in vivo on mouse pulmonary CD4+ T cells upon reinfection of mice, initially infected as neonates. Th cell-specific deletion of Il4ra attenuated Th2 responses and abolished the immunopathophysiology upon reinfection, including airway hyper-reactivity, eosinophilia, and mucus hyperproduction in mice infected initially as neonates. These findings support a pathogenic role for IL-4Rα on Th cells following RSV reinfection of mice initially infected as neonates; more importantly, our data from human cells suggest that the same mechanism occurs in humans.

Background Exposure to elevated levels of particulate matter (PM) is associated with increased risk of morbidity and mortality due to respiratory tract viral infections in infants. Recent identification of environmentally persistent free radicals (EPFRs) in the PM from a variety of combustion sources suggests its role in the enhancement of disease severity of lower respiratory tract infections (LRTI). Our previous studies demonstrated that acute exposure to EPFRs induces pulmonary immunosuppression allowing for enhanced influenza disease severity. Here, we determine the mechanism of EPFR-induced immunosuppression and its impact on the immune response towards influenza infection. Methods Neonatal mice (3 days old) were acutely exposed to DCB (combustion derived PM with chemisorbed EPFR) for seven consecutive days. Four days post-exposure (dpe), mice were infected with influenza virus. Pulmonary T cell phenotypes including regulatory T cells (Tregs) were analyzed by flow cytometry. The role of IL10 in EPFR-induced exacerbation of influenza disease severity was determined by administering recombinant IL10 (rIL10) to wild type mice or by using IL10 deficient (IL10−/−) neonatal mice. Mice were assessed for morbidity by measuring percent weight change and pulmonary viral load. Results Neonatal mice exposed to EPFRs had a significant increase in pulmonary Tregs and the immunosuppressive cytokine IL10 following influenza infection, which coincided with decreased protective T cell responses to influenza infection at 6 dpi. Depletion of Tregs in EPFR-exposed neonatal mice resulted in increased protective, adaptive T cell responses, whereas adoptive transfer of Tregs from EPFR-exposed neonates to air-exposed neonatal mice suppressed adaptive T cell responses towards influenza infection. Further, treatment with rIL10 could recapitulate EPFR-induced exacerbation of morbidity and pulmonary viral load compared to air exposed and influenza infected mice, whereas, EPFR-exposed IL10−/− neonates exhibited significant reductions in morbidity, pulmonary viral load and adaptive T cell responses following influenza infection. Conclusions Neonatal exposure to EPFRs induced Tregs and IL10 resulting in suppressed adaptive T cell responses and enhanced influenza disease severity in neonatal mice. Depletion of Tregs increased adaptive T cell responses and deficiency of IL10 reduced morbidity and conferred enhanced protection against influenza virus. Electronic supplementary material The online version of this article (doi:10.1186/s12931-016-0487-4) contains supplementary material, which is available to authorized users.

Pneumonia due to methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of morbidity and mortality in infants particularly following lower respiratory tract viral infections such as Respiratory Syncytial Virus (RSV). However, the mechanisms by which co-infection of infants by MRSA and RSV cause increased lung pathology are unknown. Because the infant immune system is qualitatively and quantitatively different from adults we developed a model of infant MRSA pneumonia which will allow us to investigate the effects of RSV co-infection on disease severity. We infected neonatal and adult mice with increasing doses of MRSA and demonstrate that neonatal mice have delayed kinetics in clearing the bacteria in comparison to adult mice. There were differences in recruitment of immune cells into the lung following infection. Adult mice exhibited an increase in neutrophil recruitment that coincided with reduced bacterial titers followed by an increase in macrophages. Neonatal mice, however, exhibited an early increase in neutrophils that did not persist despite continued presence of the bacteria. Unlike the adult mice, neonatal mice failed to exhibit an increase in macrophages. Neonates exhibited a decrease in phagocytosis of MRSA suggesting that the decrease in clearance was partially due to deficient phagocytosis of the bacteria. Both neonates and adults responded with an increase in pro-inflammatory cytokines following infection. However, in contrast to the adult mice, neonates did not express constitutive levels of the anti-microbial peptide Reg3γ in the lung. Infection of neonates did not stimulate expression of the co-stimulatory molecule CD86 by dendritic cells and neonates exhibited a diminished T cell response compared to adult mice. Overall, we have developed a neonatal model of MRSA pneumonia that displays a similar delay in bacterial clearance as is observed in the neonatal intensive care unit and will be useful for performing co-infection studies.

The therapeutic promise of chimeric antigen receptor (CAR) T cells was realized when complete remission rates of 90% were reported after treating B cell acute lymphoblastic leukemia (B-ALL) with CD19-targeted CAR T cells. However, a major obstacle with continued clinical development of CAR T cells is the limited understanding of CAR T cell biology and its mechanisms of immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation causing acute toxicities, but also lead to T cell exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. We hypothesized that by incorporating null mutations of the CD28 subdomains (YMNM, PRRP, or PYAP) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ (Fig1A), IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytoxicity was enhanced with the PYAP only CAR T cells compared to non-mutated CARs (Fig1B). When we examined the PYAP (mut06) only mutant in an immune competent mouse model we found similar B cell aplasia and CAR T cell persistence compared to non-mutated CD28 CAR T cells. Additionally, PYAP only CAR T cells injected into mice had decreased (82% to 62%) expression of PD1 in the BM. Using a pre-clinical immunocompetent mouse tumor model we found the PYAP only CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells, with 100% survival of mice given PAYP only CAR T cells compared to 50% survival of mice given non-mutated CAR T cells (Fig1C). We next sought to determine what role CAR T cell exhaustion was playing using a Rag knockout mouse system. CAR T cells were given to Rag-/- mice and 1 week later mice were challenged with tumor. Studies in Rag-/- mice also showed PYAP only CAR T cells were increased 35% in the BM and 92% in the spleen compared to non-mutated CD28 CAR T cells. We also found PYAP only CAR T cells had significantly less expression of PD1 compared to non-mutated CAR T cells (Fig1D). We then co-cultured CAR T cells with target cells expressing CD19 and PDL1 and found PYAP only CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This shows that PYAP only CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. Using Nur77, pAkt, and pmTOR to measure CAR signaling we found PYAP only CAR T cells had significantly reduced levels of Nur77 while still having higher expression then first generation CAR T cells. We then examined what affect the PYAP only CAR had on transcription factors. We found similar AP1 and NF-kB expression between PYAP only and non-mutated CD28 CAR T cells but a significant reduction of NFAT in the PYAP only mutants compared to non-mutated CD28 CAR T cells. This suggests reduced NFAT expression contributes to the PYAP only CAR's resistance to exhaustion. Finally, we made human CAR constructs of the PYAP only mutant. We found PYAP only human CAR T cells had increased cytoxicity and decreased exhaustion in vitro compared to non-mutated human CD28 CAR T cells. NFAT levels in human PYAP only CAR T cells were significantly reduced compared to non-mutated CAR T cells supporting our findings in mice. Our results demonstrate that CAR T cells with only a PYAP CD28 subdomain have better cytoxicity and decreased exhaustion compared to non-mutated CD28 CAR T cells. Our results suggest this is the result of decreased CAR and NFAT signaling. Additionally, we were able to validate these findings using human CAR constructs. This work allows for development of an enhanced 2nd and 3rd generation CAR T cell therapies for B cell malignancies by optimizing CAR T cell activation and persistence which may reduce relapse rates and severe toxicities. Figure 1 Figure 1. Disclosures Davila: Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The therapeutic promise of chimeric antigen receptor (CAR) T cells was realized when complete remission rates of 90% were reported after treating B cell acute lymphoblastic leukemia (B-ALL) with CD19-targeted CAR T cells. However, with increasing numbers of patients treated, challenges have become evident, especially regarding poor CAR T cell responses, which might be caused by exhaustion. Detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. We hypothesized that by mutating the CD28 subdomains YMNM and PRRP and leaving only the PYAP CD28 subdomain active we would optimize CAR T cell signaling and reduce exhaustion. We found mutated CAR T cells with only a functional PYAP subdomain secrete 50% less IFNγ, IL6, and TNFα after 24hr stimulation as well as having significantly less TCR signaling as measured by Nur77 compared to non-mutated CD28 CAR T cells. Additionally, mutated CAR T cells injected into mice had decreased expression of PD1 (82% to 62%) in the BM after 2 months. Studies in Rag−/− mice also showed mutated CAR T cells were increased 35% in the BM and 92% in the spleen compared to non-mutated CD28 CAR T cells. After challenge with tumor, exhausted mutant CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that CAR T cells with only the PYAP subdomain are able to persist and be effective longer because they have optimized CD28 signaling. This work allows for development of an enhanced 2nd generation CAR T cell therapy for B cell malignancies by optimizing CAR T cell activation and persistence which may reduce relapse rates and severe toxicities.

The prototypical second-generation CD19-targeted CAR (chimeric antigen receptor) features a costimulatory domain, either CD28 or 41BB, with both designs achieving comparable clinical outcomes against B cell malignancies. However, clinical application of CAR T cells has outpaced the understanding of their functions in vivo. We evaluated fully murine CD19-targeted CAR T cells containing CD28 or 41BB endodomain in an immune competent B-ALL (B cell acute lymphoblastic leukemia) mouse model. Mouse 41BB CAR T cells imparted inferior survival to mouse CD28 CAR T cells at a stress-test dose. Replacing mouse 41BB endodomain with human 41BB sequence significantly increased NF-κB signaling in 41BB CAR T cells and enhanced survival and persistence to match the mouse CD28 CAR T cells. TRAF binding and its subsequent NF-κB signaling are more important to 41BB than CD28 CAR T functions as evidenced by TRAF1 deficient 41BB but not CD28 CAR T cells showing inferior in vivo B cell killing and CAR T persistence to wild type CAR T cells. Also, 41BB CAR T cells with impaired TRAF binding showed remarkably decreased NF-κB signaling, viability and proliferation in vitro. Over-expressing TRAF2 or TRAF3 but not TRAF1 significantly enhanced CD19 targeted 41BB CAR T functions, such as cytotoxicity, viability and proliferation. Our study demonstrates how co-stimulatory domains impact CAR T functions and selective TRAF over-expression could be a new strategy to improve current CAR T therapy.

The infant immune response to respiratory syncytial virus (RSV) remains incompletely understood. Here we review the use of a neonatal mouse model of RSV infection to mimic severe infection in human infants. We describe numerous age-specific responses, organized by cell type, observed in RSV-infected neonatal mice and draw comparisons (when possible) to human infants.

Respiratory syncytial virus (RSV) is the number one cause of lower respiratory tract infection in infants; and there are no vaccines or specific antiviral drugs against RSV. The pathogenesis of RSV remains elusive. This is partly due to the fact that the mouse is semi-permissive for human RSV. The present study sought to determine if a better neonatal mouse model of RSV infection could be obtained using a recombinant virus in which the F protein of standard laboratory strain A2 was replaced with that from the line 19 isolate (rA2-19F). Five-day-old pups were infected with A2 or rA2-19F. rA2-19F caused significantly more lung inflammation and mucus than A2. More type II T helper (Th2) cells and less type I (Th1) cells and effector CD8+ T cells (Tc1) were observed in the rA2-19F infected lungs compared to A2. A cohort of infected neonates were allowed to mature to adulthood and reinfected with the same strain at 4 wks post primary infection. Similarly, secondary infection with rA2-19F caused exaggerated Th2...

Background Exposures to elevated levels of particulate matter (PM) enhance severity of influenza virus infection in infants. The biological mechanism responsible for this phenomenon is unknown. The recent identification of environmentally persistent free radicals (EPFRs) associated with PM from a variety of combustion sources suggests its role in the enhancement of influenza disease severity. Methods Neonatal mice (< seven days of age) were exposed to DCB230 (combustion derived PM with a chemisorbed EPFR), DCB50 (non-EPFR PM sample), or air for 30 minutes/day for seven consecutive days. Four days post-exposure, neonates were infected with influenza intranasally at 1.25 TCID50/neonate. Neonates were assessed for morbidity (% weight gain, peak pulmonary viral load, and viral clearance) and percent survival. Lungs were isolated and assessed for oxidative stress (8-isoprostanes and glutathione levels), adaptive immune response to influenza, and regulatory T cells (Tregs). The role of the EPFR was also assessed by use of transgenic mice expressing human superoxide dismutase 2. Results Neonates exposed to EPFRs had significantly enhanced morbidity and decreased survival following influenza infection. Increased oxidative stress was also observed in EPFR exposed neonates. This correlated with increased pulmonary Tregs and dampened protective T cell responses to influenza infection. Reduction of EPFR-induced oxidative stress attenuated these effects. Conclusions Neonatal exposure to EPFR containing PM resulted in pulmonary oxidative stress and enhanced influenza disease severity. EPFR-induced oxidative stress resulted in increased presence of Tregs in the lungs and subsequent suppression of adaptive immune response to influenza. Electronic supplementary material The online version of this article (doi:10.1186/s12989-014-0057-1) contains supplementary material, which is available to authorized users.

Elevated levels of combustion-derived particulate matter (CDPM) are a risk factor for the development of lung diseases such as asthma. Studies have shown that CDPM exacerbates asthma, inducing acute lung dysfunction and inflammation; however, the impact of CDPM exposure on early immunological responses to allergens remains unclear. To determine the effects of early-life CDPM exposure on allergic asthma development in infants, we exposed infant mice to CDPM and then induced a mouse model of asthma using house dust mite (HDM) allergen. Mice exposed to CDPM+HDM failed to develop a typical asthma phenotype including airway hyper-responsiveness, T-helper type 2 (Th2) inflammation, Muc5ac expression, eosinophilia, and HDM-specific immunoglobulin (Ig) compared with HDM-exposed mice. Although HDM-specific IgE was attenuated, total IgE was twofold higher in CDPM+HDM mice compared with HDM mice. We further demonstrate that CDPM exposure during early life induced an immunosuppressive environment in the lung, concurrent with increases in tolerogenic dendritic cells and regulatory T cells, resulting in the suppression of Th2 responses. Despite having early immunosuppression, these mice develop severe allergic inflammation when challenged with allergen as adults. These findings demonstrate a mechanism whereby CDPM exposure modulates adaptive immunity, inducing specific antigen tolerance while amplifying total IgE, and leading to a predisposition to develop asthma upon rechallenge later in life.

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma., Author Summary IL-33 is responsible for the immunopathophysiological response observed following neonatal RSV infection in mice. Its presence in nasal aspirates of human infants with severe RSV and suggests its role in disease severity and asthma.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and severe disease is associated with asthma development in later life. We have shown that neonatal mice respond to primary RSV infection with T helper type 2 (Th2) biased immune responses which are enhanced following reinfection. Our lab has demonstrated elevated levels of IL-4Rα on neonatal mDCs compared to adults. Therefore, we investigated the role of IL-4Rα on mDCs in the RSV induced Th2 biased response in neonates. CD4+ T cells from adult mice were co-cultured with RSV infected CD11c+ cells (DCs) from neonatal wild type or IL-4Rα-/- mice and resulting CD4+ T cell sub phenotypes were analyzed. DCs from IL-4Rα-/- mice induced more Th1 and less Th2 cells compared to DCs from wild type mice. Thus, we hypothesized that IL-4Rα on neonatal mDCs is responsible for the pathogenicity in RSV infection. To test this hypothesis, we used transgenic mice in which IL-4Rα is specifically depleted on CD11c+ cells. These mice were infected with RSV as neonates and reinfected four weeks later. We then analyzed the pulmonary T cell profile, cytokines, and pathophysiological responses. Mice in which IL-4Rα was deleted on mDCs showed significant reductions in their pathological Th2 responses. Together, these data demonstrate that the asthmatic Th2 biased immune phenotype induced during neonatal RSV infection is mediated in part by IL-4Rα expression and signaling on mDCs.

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and represents an enormous health burden worldwide. Severe RSV infections are a significant risk factor for childhood asthma, but the pathogenic mechanisms responsible for RSV induced airway disease remain elusive. We and others have shown that age at time of initial infection is a crucial determinant of RSV immunopathogenesis (i.e., Th2 inflammation, eosinophilia, airway hyperresponsiveness, mucus). Using an age-appropriate mouse model of RSV that phenotypically recapitulates severe Th2-biased RSV seen in human infants, we show that this age-related disparity in immunopathogenesis is related to enhanced IL-33 production and increased group 2 innate lymphoid cells (ILC2s) in lungs of neonatal mice compared to adult mice. In nasal aspirates from RSV-infected human infants, IL-33 was elevated in the youngest of patients which further indicates age-variable regulation in response to RSV infection. These data provide a previously unknown mechanism involving ILC2s and IL-33 in RSV mediated human asthma, and represent the first evidence of differential ILC2/IL-33 regulation with respect to very early age.

Respiratory syncytial virus (RSV) remains a major cause of bronchiolitis in infants. Infants developing severe RSV disease have increased risk of developing asthma; the causes of which are unknown. We and others have previously demonstrated that RSV infection results in a skewed Th2 response, which may be responsible for this phenomenon. Here, we show that neonatal mice (< 7 d) expressed limited amounts of type I IFNs in the lung, which was associated with low numbers of pulmonary plasmacytoid dendritic cells (pDCs; a major source of type I IFNs during viral infections) following RSV infection. When neonatal mice were pre-treated with IFNa, the typical Th2 skewed response was decreased compared to untreated control mice. Neonatal mice treated with IFNa demonstrated decreased pulmonary Th2 (IFNg+CD3+CD4+) and Tc2 (IFNg+CD3+CD8+) cells, decreased eosinophils and mucus hyperproduction in the airways, and decreased inflammation in the lung parenchyma upon reinfection. More importantly, pulmonary function of the IFNa treated mice was significantly improved compared to the control mice. We conclude that the limited amount of type I IFNs in the neonates, possibly due to an inchoate pulmonary pDC response, may be partially responsible for the Th2 skewed immune response resulting from neonatal RSV infection. Our study pinpoints IFNa as a possible therapeutic target during infant RSV infection.

Effect of Treg depletion on IL10 levels in the lungs of EPFR exposed and influenza infected neonatal mice. IL10 levels in the lungs of Treg depleted and EPFR exposed neonatal mice at 6 dpi after infection with influenza virus (DCB/PC61/Flu) in comparison to Air/Flu, DCB/Flu, and EPFR exposed neonatal mice treated with rat IgG isotype control and infected with influenza virus (DCB/Isotype/flu). Data are plotted as means ± SEM, *p

Background Respiratory syncytial virus (RSV) is the number one cause of lower respiratory tract infection in infants; and severe RSV infection in infants is associated with asthma development. Today, there are still no vaccines or specific antiviral therapies against RSV. The mechanisms of RSV pathogenesis in infants remain elusive. This is partly due to the fact that the largely-used mouse model is semi-permissive for RSV. The present study sought to determine if a better neonatal mouse model of RSV infection could be obtained using a chimeric virus in which the F protein of A2 strain was replaced with the F protein from the line 19 clinical isolate (rA2-19F). Methods Five-day-old pups were infected with the standard laboratory strain A2 or rA2-19F and various immunological and pathophysiological parameters were measured at different time points post infection, including lung histology, bronchoalveolar lavage fluid (BALF) cellularity and cytokines, pulmonary T cell profile, and lung viral load. A cohort of infected neonates were allowed to mature to adulthood and reinfected. Pulmonary function, BALF cellularity and cytokines, and T cell profiles were measured at 6 days post reinfection. Results The rA2-19F strain in neonatal mice caused substantial lung pathology including interstitial inflammation and airway mucus production, while A2 caused minimal inflammation and mucus production. Pulmonary inflammation was characterized by enhanced Th2 and reduced Th1 and effector CD8+ T cells compared to A2. As with primary infection, reinfection with rA2-19F induced similar but exaggerated Th2 and reduced Th1 and effector CD8+ T cell responses. These immune responses were associated with increased airway hyperreactivity, mucus hyperproduction and eosinophilia that was greater than that observed with A2 reinfection. Pulmonary viral load during primary infection was higher with rA2-19F than A2. Conclusions Therefore, rA2-19F caused enhanced lung pathology and Th2 and reduced effector CD8+ T cell responses compared to A2 during initial infection in neonatal mice and these responses were exacerbated upon reinfection. The exact mechanism is unknown but appears to be associated with increased pulmonary viral load in rA2-19F vs. A2 infected neonatal lungs. The rA2-19F strain represents a better neonatal mouse model of RSV infection. Electronic supplementary material The online version of this article (doi:10.1186/s12931-015-0244-0) contains supplementary material, which is available to authorized users.

Effect of Treg depletion on IL10 levels in the lungs of EPFR exposed and influenza infected neonatal mice. IL10 levels in the lungs of Treg depleted and EPFR exposed neonatal mice at 6 dpi after infection with influenza virus (DCB/PC61/Flu) in comparison to Air/Flu, DCB/Flu, and EPFR exposed neonatal mice treated with rat IgG isotype control and infected with influenza virus (DCB/Isotype/flu). Data are plotted as means ± SEM, *p