Adoptive immunotherapy with Tregs is a promising approach for prevention or treatment of type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B-chain 10-23 peptide presented in the context of the IAg7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR re-directed NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Co-transfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In wild type NOD mice, InsB-g7 CAR Tregs prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising new therapeutic approach for the prevention of autoimmune diabetes.
Justin A. Spanier, Vivian Fung, Christine M. Wardell, Mohannad H. Alkhatib, Yixin Chen, Linnea A. Swanson, Alexander J. Dwyer, Matthew E. Weno, Nubia Silva, Jason S. Mitchell, Paul C. Orban, Majid Mojibian, C. Bruce Verchere, Brian T. Fife, Megan K. Levings
BACKGROUND. Recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) is often an incurable disease, with patients experiencing median survival of under ten months and significant morbidity. While immune checkpoint blockade (ICB) drugs are effective in ~20% of patients, the remaining experience limited clinical benefit and are exposed to potential adverse effects and financial costs. Clinically approved biomarkers, such as tumor mutational burden (TMB), have modest predictive value in HNSCC. METHODS. We analyze clinical and genomic features, generated using whole-exome sequencing, in 133 ICB-treated R/M HNSCC patients, of whom 69 had virus-associated and 64 had non-virus-associated tumors. RESULTS. Hierarchical clustering of genomic data revealed six molecular subtypes characterized by a wide range of objective response rates and survival after ICB therapy. The prognostic importance of these 6 subtypes was validated in an external cohort. A random forest-based predictive model, using several clinical and genomic features, predicted progression-free survival (PFS), overall survival (OS), and response with greater accuracy than a model based on TMB alone. Recursive partitioning analysis identified three features (systemic inflammatory response index, TMB, and smoking signature) that classified patients into risk groups with accurate discrimination of PFS and OS. CONCLUSION. These findings shed light on the immunogenomic characteristics of HNSCC tumors that drive differential response to ICB and identify a clinical-genomic classifier that outperforms the current clinically approved biomarker of TMB. This validated predictive tool may help with clinical risk stratification in patients with R/M HNSCC for whom ICB is being considered.
Cristina Valero, Mahdi Golkaram, Joris L. Vos, Bin Xu, Conall Fitzgerald, Mark Lee, Shannon K. Kaplan, Catherine Y Han, Xin Pei, Reith Sarkar, Lillian A. Boe, Abhinav Pandey, Elizabeth S. Koh, Charlotte L. Zuur, David B. Solit, Traci Pawlowski, Li Liu, Alan L. Ho, Diego Chowell, Nadeem Riaz, Timothy A. Chan, Luc G.T. Morris
BACKGROUND. Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function CXCR4 mutations. Myelokathexis is neutropenia from neutrophil retention in bone marrow and is associated with lymphopenia and monocytopenia. The CXCR4 antagonist plerixafor mobilizes leukocytes to the blood; however, safety and efficacy in WHIM syndrome are undefined. METHODS. In this investigator-initiated, single-center, randomized, quadruple-masked phase 3 crossover trial, we compared the total infection severity score (TISS) as primary endpoint in an intent-to-treat manner in 19 WHIM patients for 12-months on plerixafor versus 12-months on G-CSF, the standard-of-care for severe congenital neutropenia. RESULTS. Plerixafor was non-superior to G-CSF for TISS (p=0.65). In exploratory endpoints, plerixafor was non-inferior to G-CSF for maintaining neutrophil counts >500 cells/microliter (p=0.023) and was superior to G-CSF for maintaining lymphocyte counts >1000 cells/microliter (p<0.0001). Complete regression of a subset of large wart areas occurred on plerixafor in 5 of 7 patients with major wart burdens at baseline. Transient rash occurred on plerixafor, and bone pain was more common on G-CSF. There were no significant differences in drug preference or quality of life, or the incidence of drug failure or serious adverse events. CONCLUSIONS. Plerixafor was not superior in WHIM patients to G-CSF for TISS, the primary endpoint. Together with wart regression and hematologic improvement, the infection severity results support continued study of plerixafor as a potential treatment for WHIM syndrome.(Funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases; clinicaltrials.gov registration number, NCT02231879)
David H. McDermott, Daniel Velez, Elena Cho, Edward W. Cowen, John J. DiGiovanna, Diana V. Pastrana, Christopher B. Buck, Katherine R. Calvo, Pamela J. Gardner, Sergio D. Rosenzweig, Pamela Stratton, Melissa A. Merideth, H. Jeffrey Kim, Carmen Brewer, James D. Katz, Douglas B. Kuhns, Harry L. Malech, Dean Follmann, Michael P. Fay, Philip M. Murphy
Entry of antigen-specific T cells into human tumors is critical for immunotherapy, but the underlying mechanisms are poorly understood. Here, we combined high-dimensional spatial analyses with in vitro and in vivo modeling to study the mechanisms underlying immune infiltration in human multiple myeloma (MM) and its precursor monoclonal gammopathy of undetermined significance (MGUS). Clustered tumor growth was a feature of MM but not MGUS biopsies, and this growth pattern was reproduced in humanized mouse models. MM biopsies exhibited intralesional as well as spatial heterogeneity, with coexistence of T cell–rich and T cell–sparse regions and the presence of areas of T cell exclusion. In vitro studies demonstrated that T cell entry into MM clusters was regulated by agonistic signals and CD2-CD58 interactions. Upon adoptive transfer, antigen-specific T cells localized to the tumor site but required in situ DC–mediated antigen presentation for tumor entry. C-type lectin domain family 9 member A–positive (CLEC9A+) DCs appeared to mark portals of entry for gradients of T cell infiltration in MM biopsies, and their proximity to T cell factor 1–positive (TCF1+) T cells correlated with disease state and risk status. These data illustrate a role for tumor-associated DCs and in situ activation in promoting the infiltration of antigen-specific T cells in MM and provide insights into spatial alterations in tumor/immune cells with malignant evolution.
M. Hope Robinson, Nancy Y. Villa, David L. Jaye, Ajay K. Nooka, Alyssa Duffy, Samuel S. McCachren, Julia Manalo, Jeffrey M. Switchenko, Sierra Barnes, Sayalee Potdar, Maryam I. Azeem, Ava A. Horvat, Vaunita C. Parihar, Jingjing Gong, Yan Liang, Geoffrey H. Smith, Vikas A. Gupta, Lawrence H. Boise, Jonathan L. Kaufman, Craig C. Hofmeister, Nisha S. Joseph, Sagar Lonial, Kavita M. Dhodapkar, Madhav V. Dhodapkar
Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11β-HSD1 (encoded by Hsd11b1). Here we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth but reduced in vivo tumor progression, which corresponded with increased frequencies of tumor-infiltrating CD8+ T cells (TIL) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of Tconv activation in tumor-infiltrating Treg. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11β-HSD1 reduced tumor growth to the same degree as gene knockout, and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11β-HSD1 is well-tolerated in clinical studies, these data suggest that targeting 11β-HSD1 may be a beneficial adjunct in cancer therapy.
Matthew D. Taves, Shizuka Otsuka, Michaela A. Taylor, Kaitlynn M. Donahue, Thomas J. Meyer, Margaret C. Cam, Jonathan D. Ashwell
Extracellular cold-inducible RNA-binding protein (eCIRP) is a key mediator of severity and mortality in sepsis. We found that stimulation of mouse bone marrow–derived neutrophils (BMDNs) with eCIRP generated a distinct neutrophil subpopulation, characterized by cell surface markers of both antigen-presenting cells and aged neutrophils as well as expression of IL-12, which we named antigen-presenting aged neutrophils (APANs). The frequency of APANs was significantly increased in the blood, spleen, and lungs of WT mice subjected to cecal ligation and puncture–induced sepsis but not in CIRP–/– mice. Patients with sepsis had a significant increase in circulating APAN counts compared with healthy individuals. Compared with non–APAN-transfered mice, APAN-transferred septic mice had increased serum levels of injury and inflammatory markers, exacerbated acute lung injury (ALI), and worsened survival. APANs and CD4+ T cells colocalized in the spleen, suggesting an immune interaction between these cells. APANs cocultured with CD4+ T cells significantly induced the release of IFN-γ via IL-12. BMDNs stimulated with eCIRP and IFN-γ underwent hyper-NETosis. Stimulating human peripheral blood neutrophils with eCIRP also induced APANs, and stimulating human neutrophils with eCIRP and IFN-γ caused hyper-NETosis. Thus, eCIRP released during sepsis induced APANs to aggravate ALI and worsen the survival of septic animals via CD4+ T cell activation, Th1 polarization, and IFN-γ–mediated hyper-NETosis.
Hui Jin, Monowar Aziz, Atsushi Murao, Molly Kobritz, Andrew J. Shih, Robert P. Adelson, Max Brenner, Ping Wang
Recognition of pathogen-associated molecular patterns can trigger the IRE1α arm of the endoplasmic reticulum (ER) stress response in innate immune cells. This process maintains ER homeostasis and also coordinates diverse immunomodulatory programs during bacterial and viral infections. However, the role of innate IRE1α signaling in response to fungal pathogens remains elusive. Here, we report that systemic infection with the human opportunistic fungal pathogen Candida albicans induces proinflammatory IRE1α hyperactivation in myeloid cells that leads to fatal kidney immunopathology. Mechanistically, simultaneous activation of the TLR/IL-1R adaptor protein MyD88 and the C-type lectin receptor Dectin-1 by C. albicans induced NADPH oxidase-driven generation of reactive oxygen species that caused ER stress and IRE1α-dependent overexpression of key inflammatory mediators such as IL-1β, IL-6, CCL5, PGE2 and TNFα. Selective ablation of IRE1α in leukocytes, or treatment with an IRE1α pharmacological inhibitor, mitigated kidney inflammation and prolonged the survival of mice with systemic C. albicans infection. Therefore, controlling IRE1α hyperactivation may be useful for impeding the immunopathogenic progression of disseminated candidiasis.
Deepika Awasthi, Sahil Chopra, Byuri A. Cho, Alexander Emmanuelli, Tito A. Sandoval, Sung-Min Hwang, Chang-Suk Chae, Camilla Salvagno, Chen Tan, Liliana Vasquez-Urbina, Jose J. Fernandez Rodriguez, Sara F. Santagostino, Takao Iwawaki, E. Alfonso Romero-Sandoval, Mariano Sanchez Crespo, Diana K. Morales, Iliyan D. Iliev, Tobias M. Hohl, Juan R. Cubillos-Ruiz
RATIONALE. Food allergy (FA) is a growing health problem requiring physiologic confirmation via the oral food challenge (OFC). Many OFCs result in clinical anaphylaxis, causing discomfort and risk while limiting OFC utility. Transepidermal water loss (TEWL) measurement provides a potential solution to detect food anaphylaxis in real time prior to clinical symptoms. We evaluated whether TEWL changes during an OFC could predict anaphylaxis onset. METHODS. Physicians and nurses blind to TEWL results conducted and adjudicated the results of all 209 OFCs in this study. A study coordinator measured TEWL throughout the OFC and had no input on OFC conduct. TEWL was measured two ways in two separate groups. First, TEWL was measured using static, discrete measurements. Second, TEWL was measured using continuous monitoring. Participants who consented gave blood before and after OFCs for biomarker analyses. RESULTS. TEWL rose significantly (2.93 g/m2/h) during reactions and did not rise during non-reacting OFCs (-1.00 g/m2/h). Systemic increases in tryptase and interleukin-3 were also detected during reactions, providing supporting biochemical evidence of anaphylaxis. The TEWL rise occurred 48 minutes earlier than clinically evident anaphylaxis. Continuous monitoring detected a significant rise in TEWL that presaged positive OFCs, but no rise was seen in OFCs with no reaction, providing high predictive specificity (96%) for anaphylaxis against non-reactions 38 minutes prior to anaphylaxis. CONCLUSIONS. During OFCs, a TEWL rise anticipates a positive clinical challenge. TEWL presents a novel monitoring modality that may predict food anaphylaxis and facilitate improvements in OFC safety and tolerability.
Charles F. Schuler, Kelly M. O'Shea, Jonathan P. Troost, Bridgette Kaul, Christopher M. Launius, Jayme Cannon, David M. Manthei, George E. Freigeh, Georgiana Sanders, Simon P. Hogan, Nicholas W. Lukacs, James R. Baker Jr
BACKGROUND. IgE-mediated anaphylaxis is a potentially fatal systemic allergic reaction for which there are no currently FDA-approved preventative therapies. Bruton’s tyrosine kinase (BTK) is an essential enzyme for IgE-mediated signaling pathways, and is an ideal pharmacologic target to prevent allergic reactions. In this open-label trial, we evaluated the safety and efficacy of acalabrutinib, a BTK inhibitor that is FDA-approved to treat some B cell malignancies, in preventing clinical reactivity to peanut in adults with peanut allergy. METHODS. After undergoing graded oral peanut challenge to establish their baseline level of clinical reactivity, 10 patients had a 6-week rest period, then received four standard doses of 100 mg acalabrutinib twice daily and underwent repeat food challenge. The primary endpoint was the change in patients’ threshold dose of peanut protein to elicit an objective clinical reaction. RESULTS. At baseline, patients tolerated a median of 29 mg of peanut protein before objective clinical reaction. During subsequent food challenge on acalabrutinib, patients’ median tolerated dose significantly increased to 4,044 mg (range, 444 – 4,044 mg). Seven patients tolerated the maximum protocol amount (4,044 mg) of peanut protein with no clinical reaction, and the other 3 patients’ peanut tolerance increased between 32- and 217-fold. Three patients experienced a total of 4 adverse events that were considered to be possibly related to acalabrutinib; all events were transient and nonserious. CONCLUSION. Acalabrutinib pretreatment achieved clinically-relevant increases in patients’ tolerance to their food allergen, thereby supporting the need for larger, placebo-controlled trials. TRIAL REGISTRATION. ClinicalTrials.gov NCT05038904 FUNDING. AstraZeneca Pharmaceuticals, the Johns Hopkins Institute for Clinical and Translational Research, the Ludwig Family Foundation, and NIH grants AI143965 and AI106043.
Ragha V. Suresh, Collin Dunnam, Dhananjay Vaidya, Robert A. Wood, Bruce S. Bochner, Donald W. MacGlashan, Jr., Melanie C. Dispenza
There is no vaccine to protect from cryptosporidiosis, a leading cause of diarrhea in infants in low and middle income countries. Here we comprehensively identified parasite antigens associated with protection from reinfection. A Cryptosporidium protein microarray was constructed by in vitro transcription and translation of 1761 C. parvum, C. hominis or C. meleagridis antigens, including proteins with a signal peptide and/or a transmembrane domain. Plasma IgG and/or IgA from Bangladeshi children longitudinally followed for cryptosporidiosis from birth to three years of age, identified 233 seroreactive proteins. Seven of these were associated with protection from reinfection. These included Cp23 and Cp17, Gp900 and four additional antigens (CpSMP1, CpMuc8, CpCorA and CpCCDC1). Infection in the first year of life however often resulted in no detectable antigen-specific antibody response, and antibody responses, when detected, were (i) specific to the infecting parasite genotype, and (ii) decayed in the months post-infection. In conclusion humoral immune responses against specific parasite antigens were associated with acquired immunity. While antibody decay over time and parasite genotype-specificity may limit natural immunity, this work serves as a foundation for antigen selection for vaccine design.
Carol A. Gilchrist, Joseph J. Campo, Jozelyn V. Pablo, Jennie Z. Ma, Andy Teng, Amit Oberai, Adam D. Shandling, Masud Alam, Mamun Kabir, Abu S.G. Faruque, Rashidul Haque, William A. Petri Jr.