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Previous work from our group and others has shown that patients with breast cancer can generate a T cell response against specific human epidermal growth factor 2 (HER2) epitopes. In addition, preclinical work has shown that this T cell response can be augmented by Ag-directed mAb therapy. This study evaluated the activity and safety of a combination of dendritic cell (DC) vaccination given with mAb and cytotoxic therapy. We performed a phase I/II study using autologous DCs pulsed with two different HER2 peptides given with trastuzumab and vinorelbine to a study cohort of patients with HER2-overexpressing and a second with HER2 nonoverexpressing metastatic breast cancer. Seventeen patients with HER2-overexpressing and seven with nonoverexpressing disease were treated. Treatment was well tolerated, with one patient removed from therapy because of toxicity and no deaths. Forty-six percent of patients had stable disease after therapy, with 4% achieving a partial response and no complete responses. Immune responses were generated in the majority of patients but did not correlate with clinical response. However, in one patient, who has survived >14 y since treatment in the trial, a robust immune response was demonstrated, with 25% of her T cells specific to one of the peptides in the vaccine at the peak of her response. These data suggest that autologous DC vaccination when given with anti-HER2–directed mAb therapy and vinorelbine is safe and can induce immune responses, including significant T cell clonal expansion, in a subset of patients.

Cytokines and Chemokines

Supplemental Methods

Immune suppression, exhaustion, and senescence are frequently seen throughout disease progression in acute myeloid leukemia (AML). We conducted a phase II study of high-dose cytarabine followed by pembrolizumab 200 mg i.v. on day 14 to examine whether PD-1 inhibition improves clinical responses in relapsed/refractory (R/R) AML. Overall responders could receive pembrolizumab maintenance up to 2 years. Among 37 patients enrolled, the overall response rate, composite complete remission (CRc) rate (primary endpoint), and median overall survival (OS) were 46%, 38%, and 11.1 months, respectively. Patients with refractory/early relapse and those receiving treatment as first salvage had encouraging outcomes (median OS, 13.2 and 11.3 months, respectively). Grade ≥3 immune-related adverse events were rare (14%) and self-limiting. Patients who achieved CRc had a higher frequency of progenitor exhausted CD8+ T cells expressing TCF-1 in the bone marrow prior to treatment. A multifaceted correlative approach of genomic, transcriptomic, and immunophenotypic profiling offers insights on molecular correlates of response and resistance to pembrolizumab.Significance:Immune-checkpoint blockade with pembrolizumab was tolerable and feasible after high-dose cytarabine in R/R AML, with encouraging clinical activity, particularly in refractory AML and those receiving treatment as first salvage regimen. Further study of pembrolizumab and other immune-checkpoint blockade strategies after cytotoxic chemotherapy is warranted in AML.See related commentary by Wei et al., p. 551.This article is highlighted in the In This Issue feature, p. 549

Testing of T cell–based cancer therapeutics often involves measuring cancer antigen–specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8+ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer+ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor β (TCRβ) sequencing, we identified recurrent UNC-CDK4-1 tetramer–associated TCRβ clonotypes in a patient with a UNC-CDK4-1 tetramer+ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRβ repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer–associated TCRβ clonotypes represented >17% of the entire TCRβ repertoire—far in excess of the UNC-CDK4-1 tetramer+ frequency—indicating that the recurrent TCRβ clonotypes identified from UNC-CDK-4-1 tetramer+ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1–driven clonal T-cell expansion. Mapping recurrent TCRβ clonotype sequences onto TCRβ repertoires can help confirm or refute antigen-specific T-cell expansion in vivo. Cancer Immunol Res; 3(3); 228–35. ©2015 AACR.

The document contains 4 supplemental tables: 1. RT-PCR Primers for TCRbeta sequencing 2. HPLC-MS identified peptide sequences 3. The amino acid sequences of recurrent TCRbeta clonotypes 4. The distribution of the CDR3 amino acid length in the analyzed patient. There are 2 supplementary figures: 1. Tetramer flow cytometry showing the generation of an oligoclonal T-cell population with a UNC-CDK4-1 high affinity T-cell population. 2. More detailed information regarding the tetramer gating strategy employed.

Purpose:Although cisplatin plus radiotherapy is a standard treatment of locally advanced head and neck squamous cell carcinoma (LA-HNSCC), cisplatin contraindication is common. Radiation elicits and promotes tumor-directed immune stimulation, which may potentiate anti–PD-1 therapy. We provide the first efficacy report of combined pembrolizumab and definitive radiotherapy in LA-HNSCC.Patients and Methods:This single-arm, multi-institution, phase II study (NCT02609503) enrolled 29 cisplatin-ineligible patients. Patients received radiotherapy concurrently with three cycles of pembrolizumab 200 mg every 3 weeks followed by three adjuvant cycles. The primary endpoint was a progression-free survival (PFS) of ≥16 months. Correlative studies included peripheral blood flow cytometry and Luminex cytokine profiling.Results:Reasons for cisplatin ineligibility included otopathy (69.0%), nephropathy (20.7%), and neuropathy (6.9%). With median follow-up of 21 months, estimated 24-month PFS and overall survival rates were 71% (95% confidence interval, 49%–84%) and 75% (51%–88%). The primary PFS endpoint has exceeded the hypothesis and its median has not been reached. Toxicities were typical of radiotherapy; however, high rates of grade 3/4 lymphopenia (58.6%) were observed. Flow cytometry revealed a relative decline in CD4 T cells and B cells, but not CD8 T cells. Upon treatment, frequencies of transitional B cells and tissue-like memory B cells increased, while resting memory B cells decreased. Patients with progression had greater percentages of baseline naïve B cells and fewer marginal zone B cells.Conclusions:Pembrolizumab and radiotherapy is efficacious in LA-HNSCC and should be evaluated in a randomized trial. The observed changes in B-cell markers deserve further study both as potential biomarkers and as therapeutic targets.

T cell gating

PFS and OS by AJCC 8

B cell gating

Immune suppression, exhaustion, and senescence are frequently seen throughout disease progression in acute myeloid leukemia (AML). We conducted a phase II study of high-dose cytarabine followed by pembrolizumab 200 mg i.v. on day 14 to examine whether PD-1 inhibition improves clinical responses in relapsed/refractory (R/R) AML. Overall responders could receive pembrolizumab maintenance up to 2 years. Among 37 patients enrolled, the overall response rate, composite complete remission (CRc) rate (primary endpoint), and median overall survival (OS) were 46%, 38%, and 11.1 months, respectively. Patients with refractory/early relapse and those receiving treatment as first salvage had encouraging outcomes (median OS, 13.2 and 11.3 months, respectively). Grade ≥3 immune-related adverse events were rare (14%) and self-limiting. Patients who achieved CRc had a higher frequency of progenitor exhausted CD8+ T cells expressing TCF-1 in the bone marrow prior to treatment. A multifaceted correlative approach of genomic, transcriptomic, and immunophenotypic profiling offers insights on molecular correlates of response and resistance to pembrolizumab. Significance: Immune-checkpoint blockade with pembrolizumab was tolerable and feasible after high-dose cytarabine in R/R AML, with encouraging clinical activity, particularly in refractory AML and those receiving treatment as first salvage regimen. Further study of pembrolizumab and other immune-checkpoint blockade strategies after cytotoxic chemotherapy is warranted in AML. See related commentary by Wei et al., p. 551. This article is highlighted in the In This Issue feature, p. 549

Background IL-2 inducible kinase (ITK) is highly expressed in metastatic melanomas and its inhibition suppresses melanoma cell proliferation. We hypothesize that ibrutinib has a direct antitumor effect in melanoma cell lines and that treatment of metastatic melanomas with ibrutinib induces antitumor responses. Methods We assessed the ibrutinib effect on melanoma cell proliferation, apoptosis, and motility. Patients with metastatic melanoma refractory to PD-1 and MAPK inhibitors (if BRAFV600-mutant) were treated with ibrutinib, 840 mg PO QD, as part of a phase II clinical trial (clinicaltrials.gov NCT02581930). Results Melanoma cell lines frequently express ITK, YES1, and EGFR. Ibrutinib suppressed cell motility and proliferation in most cell lines. Eighteen patients (13 male; median age 63.5 years, range 37-82; 12 with ipilimumab resistance) were enrolled. The most frequent side effects were fatigue (61%), anorexia (50%), hyponatremia (28%), nausea, and vomiting (22% each). No antitumor responses were seen. At a median follow-up of 6 months (0.3-35.8 months), the median progression-free survival was 1.3 months (range 0.2-5.5 months). Fifteen patients were discontinued from the study due to progression, and 14 patients had died from metastatic melanoma. All archived tumors expressed ITK, 41% had no expression of p16 and PTEN, and 61% had absent tumor-infiltrating lymphocytes (TILs). Ibrutinib significantly suppressed proliferating (Ki67+) CD19+ peripheral blood mononuclear cells and had no significant effect on other lymphocyte subsets. Conclusion Ibrutinib did not induce any meaningful clinical benefit. ITK expression may not be clinically relevant. Treatment-refractory metastatic melanomas have other fundamental defects (i.e. absent PTEN and p16 expression, absent TILs) that may contribute to an adverse prognosis.

Purpose: Although cisplatin plus radiotherapy is a standard treatment of locally advanced head and neck squamous cell carcinoma (LA-HNSCC), cisplatin contraindication is common. Radiation elicits and promotes tumor-directed immune stimulation, which may potentiate anti–PD-1 therapy. We provide the first efficacy report of combined pembrolizumab and definitive radiotherapy in LA-HNSCC. Patients and Methods: This single-arm, multi-institution, phase II study (NCT02609503) enrolled 29 cisplatin-ineligible patients. Patients received radiotherapy concurrently with three cycles of pembrolizumab 200 mg every 3 weeks followed by three adjuvant cycles. The primary endpoint was a progression-free survival (PFS) of ≥16 months. Correlative studies included peripheral blood flow cytometry and Luminex cytokine profiling. Results: Reasons for cisplatin ineligibility included otopathy (69.0%), nephropathy (20.7%), and neuropathy (6.9%). With median follow-up of 21 months, estimated 24-month PFS and overall survival rates were 71% (95% confidence interval, 49%–84%) and 75% (51%–88%). The primary PFS endpoint has exceeded the hypothesis and its median has not been reached. Toxicities were typical of radiotherapy; however, high rates of grade 3/4 lymphopenia (58.6%) were observed. Flow cytometry revealed a relative decline in CD4 T cells and B cells, but not CD8 T cells. Upon treatment, frequencies of transitional B cells and tissue-like memory B cells increased, while resting memory B cells decreased. Patients with progression had greater percentages of baseline naïve B cells and fewer marginal zone B cells. Conclusions: Pembrolizumab and radiotherapy is efficacious in LA-HNSCC and should be evaluated in a randomized trial. The observed changes in B-cell markers deserve further study both as potential biomarkers and as therapeutic targets.

Background: While immunotherapy holds promise in the treatment of mTNBC, response rates (RR) in unselected patients (pts) are approximately 20%. Strategies to augment response to immunotherapy include depletion of regulatory T cells (Tregs). A single dose of cyclophosphamide (Cy) given prior to checkpoint inhibition achieved this goal in preclinical models of TNBC (Taylor et al., JCI, 2017). Thus, we designed a phase II study to evaluate this strategy in the clinical setting of mTNBC. Patients/Methods: In cycle 1 (C1), eligible pts with mTNBC received a single dose of Cy 300mg/m2 IV on day 1 (C1D1) followed by pembrolizumab 200mg IV on day 2 (C1D2), then every 3 weeks thereafter. The co-primary objectives were (1) progression free survival (PFS, null 1.9 mos vs. 2.9 mos, 80% power, alpha 0.05) and (2) reduction in Tregs in peripheral blood measured by flow cytometry. Secondary endpoints were response rate (RR), survival (OS), and RNA-based correlative endpoints. Results: 40 patients were evaluable for efficacy: mean age 54.5 yrs (33 – 82 yrs), 75% white, 22% black, 3% American Indian. All patients had received 1 prior line of chemotherapy in the metastatic setting; 29% received 5 or more prior lines. The most common grade 3 adverse events (AE's), all 5%, were neutropenia, anemia, elevated AST, and fatigue. Immune-related grade 3 AE's, all 3%, included colitis, dry mouth, pneumonitis. Overall RR was 21% (0 CR, 8 PR), 3 pts had stable disease. Median PFS was 1.8 months (mos) (95% CI 1.4–2.5) and OS was 6.3 mos (95% CI 2.8–8.4). There was a non-significant decrease in Tregs from C1D1 to C1D2 (-3.3%, p=0.19); but from C1D2 to C2D1, Tregs increased 21.7% (p=0.005). There was no association between changes in Tregs or number of prior lines of therapy with RR (p>0.09), while immune-related AE's were associated with response (p=0.02). Correlatives studies illustrate B cell immune gene signature expression and B cell receptor repertoire diversity were enriched in responders, while genes/pathways associated with neutrophils, anti-apoptosis, PI3K/AKT and down-regulation of MHC class 1 were associated with non-response. Conclusions: While pembroliuzumab plus Cy was well-tolerated among pts with mTNBC, efficacy was similar to historical control, likely due to minimal effect of Cy on Tregs. Correlative analyses illustrate that study of adaptive immune features, including B cell biology, is a promising strategy for understanding response to PD-1 inhibition in breast cancer. Further strategies to deplete Tregs in a more sustained manner are worthy of future exploration (NCT02768701). Citation Format: Anders CK, Moore D, Sambade M, Cuaboy L, Garrett A, Woodcock M, McKinnon K, Cowens K, Bortone D, Calhoun B, Carey L, Dees C, Jolly T, Muss H, Reeder-Hayes K, Kaltman R, Jankowitz R, Gudena V, Olajide O, Perou C, Vincent B, Serody J. LCCC 1525: A phase II study of a priming dose of cyclophosphamide prior to pembrolizumab to treat metastatic triple negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-05.

Background In metastatic triple negative breast cancer (mTNBC), median progression-free survival (PFS) with chemotherapy alone is approximately 2–4 months1 and improvements with single agent checkpoint inhibitors (CI) are limited by modest response rates. Murine breast cancer models have demonstrated a role for intratumoral regulatory T cells (Tregs) in modulating response to CIs.2 A phase II clinical trial was conducted to test the hypothesis that a single, priming dose of cyclophosphamide prior to pembrolizumab would improve PFS in mTNBC. Here we present the correlative genomic and immunologic analyses from this study. Methods This trial (https://clinicaltrials.gov/ct2/show/NCT02768701) recruited 40 patients with largely pretreated mTNBC. Response was defined as >30% decrease in imaging-assessed disease burden. Clinical benefit was defined as treatment response or stable disease. Tumor specimens were collected prior to enrollment, and peripheral blood mononuclear cell (PBMC) samples taken prior to cyclophosphamide and before each cycle of pembrolizumab. RNA sequencing was performed on tumor samples for gene expression and immune repertoire reconstruction. Targeted sequencing of the T-cell beta chain, IG kappa, lambda and heavy chain (TRB, IGK, IGL, and IGH, respectively) on PBMCs captured the peripheral immune repertoire. Whole exome sequencing was performed on tumor samples with PBMCs serving as a matched normal. Results Of 40 patients enrolled, 31 patients had tumor RNA-seq and at least 15 had matched PBMC-derived immune chains capturing both pre and post treatment. When preliminary RNA-seq samples (n=22) revealed upregulation in B-cell receptor pathways and related gene signatures (figure 1), we updated our planned analysis to exclude tumor specimens collected from lymph nodes. In our final analysis, response to therapy (4 of 25, 16%) was associated in tumor RNA-Seq with gene pathways involving programmed cell death and MAPK activation, while non-responding tumors were enriched in G-protein signaling and inhibition of insulin secretion (figure 2a,b, table 1). Immune gene signatures related to NK cells and B-cell activation, signaling and interaction with T follicular helper cells,3–7 were associated with response (figure 2g). Pre-treatment immune repertoire measures demonstrated a significant association between increased peripheral IGH abundance and richness, and both future clinical benefit and response to therapy (figure 3a-d). Conclusions Response to CI therapy was associated with immunogenomic features of programmed cell death and B-cell activation. Pre-treatment circulating immunoglobulin diversity measures (high IGH abundance and IGH richness) also correlated with future response to therapy. Taken together, these data suggest that B-cell activity contributes significantly to response to CI therapy in mTNBC. Acknowledgements UNC Office of Clinical and Translational Research (OCTR), High Throughput Sequencing Facility (HTSF), and UNC Bioinformatics Core. We also thank the patients in this study and their families, without whom this study would not have been possible. Trial Registration Clinical Trials. gov: NCT02768701. Ethics Approval All patients provided written informed consent, and the study was approved by each institution’s institutional review board (No. NCT02768701). References Tutt A, Tovey H, Cheang MCU, et al. Carboplatin in BRCA1/2-mutated and triple-negative breast cancer BRCAness subgroups: the TNT Trial. Nat Med 2018;24(5):628–637. doi:10.1038/s41591-018-0009-7 Taylor NA, Vick SC, Iglesia MD, et al. Treg depletion potentiates checkpoint inhibition in claudin-low breast cancer. J Clin Invest 2017;127(9):3472–3483. doi:10.1172/JCI90499 Hollern TFH B cell: Hollern DP, Xu N, Thennavan A, et al. B Cells and T Follicular Helper cells mediate response to checkpoint inhibitors in high mutation burden mouse models of breast cancer. Cell 2019;179(5):1191–1206.e21. doi:10.1016/j.cell.2019.10.028 Iglesia B cell: Iglesia MD, Vincent BG, Parker JS, et al. Prognostic B-cell signatures using mRNA-seq in patients with subtype-specific breast and ovarian cancer. Clin Cancer Res 2014;20(14):3818–3829. doi:10.1158/1078-0432.CCR-13-3368 Fan IGG: Fan C, Prat A, Parker JS, et al. Building prognostic models for breast cancer patients using clinical variables and hundreds of gene expression signatures. BMC Med Genomics2011;4:3. Published 2011 Jan 9. doi:10.1186/1755-8794-4-3 Bindea: Bindea G, Mlecnik B, Tosolini M, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity 2013;39(4):782–795. doi:10.1016/j.immuni.2013.10.003 Prat Claudin: Prat A, Parker JS, Karginova O, et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 2010;12(5):R68. doi:10.1186/bcr2635

Chronic graft-versus-host disease (cGVHD) causes significant morbidity and mortality in patients after allogeneic bone marrow (BM) or stem cell transplantation (allo-SCT). Recent work has indicated that both T and B lymphocytes play an important role in the pathophysiology of cGVHD. Previously, our group showed a critical role for the germinal center response in the function of B cells using a bronchiolitis obliterans (BO) model of cGVHD. Here, we demonstrated for the first time that cGVHD is associated with severe defects in the generation of BM B lymphoid and uncommitted common lymphoid progenitor cells. We found an increase in the number of donor CD4+ T cells in the BM of mice with cGVHD that was negatively correlated with B-cell development and the frequency of osteoblasts and Prrx-1–expressing perivascular stromal cells, which are present in the B-cell niche. Use of anti-DR3 monoclonal antibodies to enhance the number of donor regulatory T cells (Tregs) in the donor T-cell inoculum ameliorated the pathology associated with BO in this model. This correlated with an increased number of endosteal osteoblastic cells and significantly improved the generation of B-cell precursors in the BM after allo-SCT. Our work indicates that donor Tregs play a critical role in preserving the generation of B-cell precursors in the BM after allo-SCT. Approaches to enhance the number and/or function of donor Tregs that do not enhance conventional T-cell activity may be important to decrease the incidence and severity of cGVHD in part through normal B-cell lymphopoiesis.

Neisseria gonorrhoeae is an exclusive human pathogen that evades the host immune system through multiple mechanisms. We have shown that N. gonorrhoeae suppresses the capacity of antigen-presenting cells to induce CD4(+) T cell proliferation. In this study, we sought to determine the gonococcal factors involved in this adaptive immune suppression. We show that suppression of the capacity of antigen-pulsed dendritic cells to induce T cell proliferation is recapitulated by administration of a high-molecular-weight fraction of conditioned medium from N. gonorrhoeae cultures, which includes outer membrane vesicles that are shed during growth of the bacteria. N. gonorrhoeae PorB is the most abundant protein in N. gonorrhoeae–derived vesicles, and treatment of dendritic cells with purified recombinant PorB inhibited the capacity of the cells to stimulate T cell proliferation. This immunosuppressive feature of purified PorB depended on proper folding of the protein. PorB from N. gonorrhoeae, as well as other Neisseria species and other Gram-negative bacterial species, are known to activate host Toll-like receptor 2 (TLR2) signaling. Published studies have demonstrated that purified Neisseria PorB forms proteinacious nanoparticles, termed proteosomes, when detergent micelles are removed. Unlike folded, detergent-solubilized PorB, PorB proteosomes stimulate immune responses. We now demonstrate that the formation of PorB proteosomes from structurally intact PorB eliminates the immunosuppressive property of the protein while enhancing TLR2 stimulation. These findings suggest that gonococcal PorB present in shed outer membrane vesicles plays a role in suppression of adaptive immune responses to this immune-evasive pathogen.

Immunotherapy holds tremendous promise for improving cancer treatment. To administer radiotherapy with immunotherapy has been shown to improve immune responses and can elicit the 'abscopal effect'. Unfortunately, response rates for this strategy remain low. Herein we report an improved cancer immunotherapy approach that utilizes antigen-capturing nanoparticles (AC-NPs). We engineered several AC-NP formulations and demonstrated that the set of protein antigens captured by each AC-NP formulation is dependent on the NP surface properties. We showed that AC-NPs deliver tumour-specific proteins to antigen-presenting cells (APCs) and significantly improve the efficacy of αPD-1 (anti-programmed cell death 1) treatment using the B16F10 melanoma model, generating up to a 20% cure rate compared with 0% without AC-NPs. Mechanistic studies revealed that AC-NPs induced an expansion of CD8

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that affects the function and development of immune cells. Here, we show that recipient mice receiving AhR-/- T cells have improved survival and decreased acute graft-versus-host disease (aGVHD) in 2 different murine allogeneic bone marrow transplant (BMT) models. We also show that CD4+ T cells lacking AhR demonstrate reduced accumulation in secondary lymphoid tissue because of low levels of proliferation 4 days after BMT. Additionally, we found a significant increase in the quantity of peripherally induced regulatory donor T (pTreg) cells in the colon of recipients transplanted with AhR-/- T cells 14 days after transplant. Blockade of AhR using a clinically available AhR antagonist greatly enhanced the in vitro generation of inducible Treg (iTreg) cells from naive CD4+ human T cells. We have identified AhR as a novel target on donor T cells that is critical to the pathogenesis of aGVHD.

Immunotherapy holds tremendous promise for improving cancer treatment. To administer radiotherapy with immunotherapy has been shown to improve immune responses and can elicit the 'abscopal effect'. Unfortunately, response rates for this strategy remain low. Herein we report an improved cancer immunotherapy approach that utilizes antigen-capturing nanoparticles (AC-NPs). We engineered several AC-NP formulations and demonstrated that the set of protein antigens captured by each AC-NP formulation is dependent on the NP surface properties. We showed that AC-NPs deliver tumour-specific proteins to antigen-presenting cells (APCs) and significantly improve the efficacy of αPD-1 (anti-programmed cell death 1) treatment using the B16F10 melanoma model, generating up to a 20% cure rate compared with 0% without AC-NPs. Mechanistic studies revealed that AC-NPs induced an expansion of CD8+ cytotoxic T cells and increased both CD4+T/Treg and CD8+T/Treg ratios (Treg, regulatory T cells). Our work presents a novel strategy to improve cancer immunotherapy with nanotechnology.

Triple-negative breast cancer (TNBC) has few therapeutic options, and alternative approaches are urgently needed. Stimulator of IFN genes (STING) is becoming an exciting target for therapeutic adjuvants. However, STING resides inside the cell, and the intracellular delivery of CDNs, such as cGAMP, is required for the optimal activation of STING. We show that liposomal nanoparticle-delivered cGAMP (cGAMP-NP) activates STING more effectively than soluble cGAMP. These particles induce innate and adaptive host immune responses to preexisting tumors in both orthotopic and genetically engineered models of basal-like TNBC. cGAMP-NPs also reduce melanoma tumor load, with limited responsivity to anti-PD-L1. Within the tumor microenvironment, cGAMP-NPs direct both mouse and human macrophages (M), reprograming from protumorigenic M2-like phenotype toward M1-like phenotype; enhance MHC and costimulatory molecule expression; reduce M2 biomarkers; increase IFN-γ-producing T cells; augment tumor apoptosis; and increase CD4+ and CD8+ T cell infiltration. Activated T cells are required for tumor suppression, as their depletion reduces antitumor activity. Importantly, cGAMP-NPs prevent the formation of secondary tumors, and a single dose is sufficient to inhibit TNBC. These data suggest that a minimal system comprised of cGAMP-NP alone is sufficient to modulate the tumor microenvironment to effectively control PD-L1-insensitive TNBC.

Claudin-low breast cancer is an aggressive subtype that confers poor prognosis and is found largely within the clinical triple-negative group of breast cancer patients. Here, we have shown that intrinsic and immune cell gene signatures distinguish the claudin-low subtype clinically as well as in mouse models of other breast cancer subtypes. Despite adaptive immune cell infiltration in claudin-low tumors, treatment with immune checkpoint inhibitory antibodies against cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death receptor 1 (PD-1) were ineffective in controlling tumor growth. CD4+FoxP3+ Tregs represented a large proportion of the tumor-infiltrating lymphocytes (TILs) in claudin-low tumors, and Tregs isolated from tumor-bearing mice were able to suppress effector T cell responses. Tregs in the tumor microenvironment highly expressed PD-1 and were recruited partly through tumor generation of the chemokine CXCL12. Antitumor efficacy required stringent Treg depletion combined with checkpoint inhibition; delays in tumor growth were not observed using therapies that modestly diminished the number of Tregs in the tumor microenvironment. This study provides evidence that the recruitment of Tregs to the tumor microenvironment inhibits an effective antitumor immune response and highlights early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint blockade antibodies in specific subtypes of cancer that are heavily infiltrated with adaptive immune cells.

Acute graft-versus-host disease (aGVHD) is the most common complication for patients undergoing allogeneic stem cell transplantation. Despite extremely aggressive therapy targeting donor T cells, patients with grade III or greater aGVHD of the lower GI tract, who do not respond to therapy with corticosteroids, have a dismal prognosis. Thus, efforts to improve understanding of the function of local immune and non-immune cells in regulating the inflammatory process in the GI tract during aGVHD are needed. Here, we demonstrate, using murine models of allogeneic BMT, that type 2 innate lymphoid cells (ILC2s) in the lower GI tract are sensitive to conditioning therapy and show very limited ability to repopulate from donor bone marrow. Infusion of donor ILC2s was effective in reducing the lethality of aGVHD and in treating lower GI tract disease. ILC2 infusion was associated with reduced donor proinflammatory Th1 and Th17 cells, accumulation of donor myeloid-derived suppressor cells (MDSCs) mediated by ILC2 production of IL-13, improved GI tract barrier function, and a preserved graft-versus-leukemia (GVL) response. Collectively, these findings suggest that infusion of donor ILC2s to restore gastrointestinal tract homeostasis may improve treatment of severe lower GI tract aGVHD.

Background: Outcomes remain dismal for patients (pts) with relapsed/refractory (R/R) AML. Programmed Death-1 (PD-1), an inhibitory receptor on T and B cells, suppresses immune activation. We hypothesized that administration of pembrolizumab, a monoclonal antibody targeting PD-1, after high dose cytarabine (HiDAC) salvage chemotherapy would stimulate a T-cell mediated anti-leukemic immune response leading to improved efficacy in R/R AML. Methods: We are conducting a multicenter phase II study of HiDAC ( Results: To date, 26 pts are evaluable for safety and response (Table 1). Grade ≥3 immune-related adverse events have been rare and self-limiting. The overall CR rate is 35%. Of the 9 CR pts, 5 (56%) had no evidence of minimal residual disease (MRD) by standard monitoring. Notably, 2/3 primary refractory pts with inv(3) cytogenetics achieved CR and underwent SCT. Additionally, 1 pt with relapsed AML with most recent treatment refractory to HiDAC salvage achieved CR with no evidence of MRD. Five pts received maintenance pembrolizumab: 3 relapsed (median duration of CR = 2.8 months; range: 2-5.7 months), 1 proceeded to SCT after 2 cycles, and 1 initially achieved a partial remission (PR) and had stable disease for 12 cycles of maintenance pembrolizumab before progressing. Four pts received a SCT in CR (n=3) and morphologic leukemia free state (MLFS) (n=1). Grade II acute graft-versus host disease (GVHD) and moderate chronic GVHD was seen in 2/4 (50%) pts, respectively. With a median follow up of 10.8 months to date, median overall survival is 10.5 months (range: 1.8-20.8 months). We performed B cell receptor amplicon sequencing, T cell receptor (TCR) amplicon sequencing from CD8+ T cells, and RNA-seq from enriched blasts and non-blast fractions in bone marrow (BM) from 3 CR and 3 non-responders (NR) prior to HiDAC. From the BM blast enriched fraction, expression of innate immune genes such as NLRP12, C3, S100A9, S100A12 and CD14 correlated with response. Gene Set Enrichment Analysis (GSEA) demonstrated that CR correlated with expression of genes in the Toll Pathway, Lysosome pathway, and adaptive immune system while NRs were associated with expression of the PAR1 and GATA3 pathways. From the non-blast BM population, CR correlated with expression of cell-cycle genes such as CCNE1, CCNB2, E2F2, KIF18B and CDKN3. Not surprisingly, GSEA revealed that expression of cell cycle pathways correlated with response. CR correlated with increased expression of B cell metagenes and the inverse IPRES signature in the non-blast BM fraction. As previously reported, there was a significant increase in peripheral blood (PB) TCR diversity in CR pts. CR was also significantly associated with the abundance and richness of the non-blast BM fraction of the B cell heavy and both light chains suggesting that a broader immune response at baseline may be critical for response to HiDAC and pembrolizumab. Finally, CR correlated with increased expression of CD300E, CCR7, CCR4, CCR8 and CCL7 in PB CD8+ T cells suggesting that migration of monocytes, T and B cells is associated with response. Conclusions: Our findings demonstrate that pembrolizumab is well tolerated after HiDAC in an ongoing study in R/R AML. An encouraging response rate has been seen in a high-risk patient population without apparent additive toxicity post-SCT. Our RNA-seq and amplicon sequencing data indicate that biomarkers of response are present prior to therapy. Increased expression of innate immune genes expressed by leukemic blasts and cell cycle genes by the non-blast fraction correlated with response to therapy. Additionally, CR was associated with increased measures of B and T cell diversity and immune cell migration. Further immunogenomic biomarker correlates are ongoing to determine predictors of response to pembrolizumab after HiDAC in R/R AML. Disclosures Zeidner: Tolero: Honoraria, Other: Travel Fees, Research Funding; Asystbio Laboratories: Consultancy; Merck: Research Funding; Takeda: Other: Travel fees, Research Funding; Rafael Pharmaceuticals: Other: Travel Fees; Celgene: Honoraria. Vincent:Merck: Research Funding. Foster:Celgene: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding; Shire: Honoraria. Coombs:Incyte: Other: Travel fees; AROG: Other: Travel fees; DAVA Oncology: Honoraria; Abbvie: Consultancy; H3 Biomedicine: Honoraria. Luznik:WIndMIL Therapeutics: Equity Ownership, Patents & Royalties. Gojo:Merck inc: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Serody:Merck: Research Funding.

The infusion of donor regulatory T cells (Tregs) has been used to prevent acute graft-versus-host disease (GVHD) in mice and has shown promise in phase 1 clinical trials. Previous work suggested that early Treg migration into lymphoid tissue was important for GVHD prevention. However, it is unclear how and where Tregs function longitudinally to affect GVHD. To better understand their mechanism of action, we studied 2 Treg-associated chemokine receptors in murine stem cell transplant models. CC chemokine receptor (CCR) 4 was dispensable for donor Treg function in the transplant setting. Donor Tregs lacking CCR8 (CCR8(-/-)), however, were severely impaired in their ability to prevent lethal GVHD because of increased cell death. By itself, CCR8 stimulation was unable to rescue Tregs from apoptosis. Instead, CCR8 potentiated Treg survival by promoting critical interactions with dendritic cells. In vivo, donor bone marrow-derived CD11c(+) antigen-presenting cells (APCs) were important for promoting donor Treg maintenance after transplant. In contrast, host CD11c(+) APCs appeared to be dispensable for early activation and expansion of donor Tregs. Collectively, our data indicate that a sustained donor Treg presence is critical for their beneficial properties, and that their survival depends on CCR8 and donor but not host CD11c(+) APCs.

Donor T cells lacking AhR demonstrate decreased aGVHD because of reduced donor T-cell proliferation early after transplant.Absence of AhR on donor cells increased pTreg cells in the colon; in vitro blockade increased the number of human iTreg from CD4+ T cells.

Current immune checkpoint inhibitors (eg. PD-L1) shows limited efficacy for varies cancer types, especially the difficult-to-treat triple-negative breast cancer. This study uses liposomal nanoparticles (NPs) to deliver the STING agonist, cGAMP, as an alternative approach to amplify innate immune activation and anti-tumor treatment. We studied the impact of cGAMP-NPs on macrophage reprograming and activation by determining M1/M2 biomarkers on polarized M2(+) cells and cytokines from secreted supernatants. The critical role of STING/IFNAR signaling in cGAMP-NP-mediated response was also examined. The efficacy was further evaluated in an orthotopically-transplanted model (C3(1)Tag model) for basal-like triple-negative breast cancer (TNBC) and a spontaneous genetic engineered mouse (GEM) model of basal-like TNBC (C3(1)Tag GEM). Both of these preclinical models that are resistant to PD-L1 checkpoint blockade and most other therapies, which parallels TNBC in humans. cGAMP-NPs were shown to activate STING and induce both innate and adaptive host immune responses. Also, cGAMP-NPs accumulated within macrophages at the tumor site and reversed a pro-tumorigenic microenvironment. Effective tumor suppression was achieved by intravenous delivery in orthotopic and GEM of TNBC with no responsivity to anti-PD-L1 and in B16F10 melanoma with limited responsivity to anti-PD-L1. The anti-tumor immunity was achieved in a STING-dependent fashion that relies on T cells and macrophages but does not require prior knowledge of the tumor antigen. cGAMP-NPs were shown to induce M2-like macrophages to skew towards a M1-like phenotype, cytokine production, MHC and co-stimulatory molecule expression, enhanced CD4+ and CD8+ T cells infiltration, and tumor apoptosis. Moreover, cGAMP-NPs alone induced durable anti-tumor T cell responses and prevented the formation of secondary tumors. We demonstrated the amplified efficacy of liposome formulation relative to soluble cGAMP as an anti-tumor therapeutic that obviates the need of intratumoral injection. Our data suggested that cGAMP-NPs are a potent treatment regimen to modulate the microenvironment of tumors with limited or no responsivity to anti-PD-L1. Citation Format: Ning Cheng, Rebecca Watkins-Schulz, Robert Junkins, Clément David, Brandon M. Johnson, Stephanie A. Montgomery, Kevin J. Peine, David B. Darr, Hong Yuan, Karen P. McKinnon, Qi Liu, Lei Miao, Leaf Huang, Eric M. Bachelder, Kristy M. Ainslie, Jenny P-y Ting. Nanoparticle-incorporated STING activator as an immunotherapeutic for PD-L1 resistant triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-126.

Approximately 500,000 people are hospitalized with severe dengue illness annually. Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is believed to contribute to the pathogenic cytokine storm described in severe dengue patients, but the precise signaling pathways contributing to elevated cytokine production are not elucidated. IL-1β is a potent inflammatory cytokine that is frequently elevated during severe dengue, and the unique dual regulation of IL-1β provides an informative model to study ADE-induced cytokines. This work utilizes patient-derived anti-DENV mAbs and primary human monocytes to study ADE-induced IL-1β and other cytokines. ADE of DENV serotype 2 (DENV-2) elevates mature IL-1β secretion by monocytes independent of DENV replication by 4 h postinoculation (hpi). Prior to this, DENV immune complexes activate spleen tyrosine kinase (Syk) within 1 hpi. Syk induces elevated IL1B, TNF, and IL6 mRNA by 2 hpi. Syk mediates elevated IL-1β secretion by activating ERK1/2, and both Syk and ERK1/2 inhibitors ablated ADE-induced IL-1β secretion. Maturation of pro-IL-1β during ADE requires caspase-1 and NLRP3, but caspase-1 is suboptimally increased by ADE and can be significantly enhanced by a typical inflammasome agonist, ATP. Importantly, this inflammatory Syk-ERK signaling axis requires DENV immune complexes, because DENV-2 in the presence of serotype-matched anti-DENV-2 mAb, but not anti-DENV-1 mAb, activates Syk, ERK, and IL-1β secretion. This study provides evidence that DENV-2 immune complexes activate Syk to mediate elevated expression of inflammatory cytokines. Syk and ERK may serve as new therapeutic targets for interfering with ADE-induced cytokine expression during severe dengue.

Testing of T cell–based cancer therapeutics often involves measuring cancer antigen–specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8+ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer+ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor β (TCRβ) sequencing, we identified recurrent UNC-CDK4-1 tetramer–associated TCRβ clonotypes in a patient with a UNC-CDK4-1 tetramer+ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRβ repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer–associated TCRβ clonotypes represented >17% of the entire TCRβ repertoire—far in excess of the UNC-CDK4-1 tetramer+ frequency—indicating that the recurrent TCRβ clonotypes identified from UNC-CDK-4-1 tetramer+ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1–driven clonal T-cell expansion. Mapping recurrent TCRβ clonotype sequences onto TCRβ repertoires can help confirm or refute antigen-specific T-cell expansion in vivo. Cancer Immunol Res; 3(3); 228–35. ©2015 AACR.

The possibility of engineering the immune system in a targeted fashion using biomaterials such as nanoparticles has made considerable headway in recent years. However, little is known as to how modulating the spatial presentation of a ligand augments downstream immune responses. In this report we show that geometric manipulation of phosphatidylserine (PS) through fabrication on rod-shaped PLGA nanoparticles robustly dampens inflammatory responses from innate immune cells while promoting T regulatory cell abundance by impeding effector T cell expansion. This response depends on the geometry of PS presentation as both PS liposomes and 1 micron cylindrical PS-PLGA particles are less potent signal inducers than 80 × 320 nm rod-shaped PS-PLGA particles for an equivalent dose of PS. We show that this immune tolerizing effect can be co-opted for therapeutic benefit in a mouse model of multiple sclerosis and an assay of organ rejection using a mixed lymphocyte reaction with primary human immune cells. These data provide evidence that geometric manipulation of a ligand via biomaterials may enable more efficient and tunable programming of cellular signaling networks for therapeutic benefit in a variety of disease states, including autoimmunity and organ rejection, and thus should be an active area of further research.

Ideal nanoparticle (NP)-based drug and vaccine delivery vectors should be free of inherent cytotoxic or immunostimulatory properties. Therefore, determining baseline immune responses to nanomaterials is of utmost importance when designing human therapeutics. We characterized the response of human immune cells to hydrogel NPs fabricated using Particle Replication in Non-wetting Templates (PRINT) technology. We found preferential NP uptake by primary CD14(+) monocytes, which was significantly reduced upon PEGylation of the NP surface. Multiplex cytokine analysis of NP treated primary human peripheral blood mononuclear cells suggests that PRINT based hydrogel NPs do not evoke significant inflammatory responses nor induce cytotoxicity or complement activation. We furthered these studies using an in vivo humanized mouse model and similarly found preferential NP uptake by human CD14(+) monocytes without systemic inflammatory cytokine responses. These studies suggest that PRINT hydrogel particles form a desirable platform for vaccine and drug delivery as they neither induce inflammation nor toxicity. From the clinical editor: The authors here fabricated hydrogel nanorods using the PRINT (Particle Replication In Nonwetting Templates) fabrication process. They tested the interaction of human immune cells with these particles and found no immunoreactivity. This finding would suggest that monodisperse PRINT particles of identical shape and size could serve a variety of clinical applications.

CD4+CD25+ regulatory T cells (Tregs) have been shown to inhibit graft-versus-host disease (GVHD) in murine models, and this suppression was mediated by Tregs expressing the lymphoid homing molecule l-selectin. Here, we demonstrate that Tregs lacking expression of the chemokine receptor CCR5 were far less effective in preventing lethality from GVHD. Survival of irradiated recipient animals given transplants supplemented with CCR5-/- Tregs was significantly decreased, and GVHD scores were enhanced compared with animals receiving wild-type (WT) Tregs. CCR5-/- Tregs were functional in suppressing T-cell proliferation in vitro and ex vivo. However, although the accumulation of Tregs within lymphoid tissues during the first week after transplantation was not dependent on CCR5, the lack of function of CCR5-/- Tregs correlated with impaired accumulation of these cells in the liver, lung, spleen, and mesenteric lymph node, more than one week after transplantation. These data are the first to definitively demonstrate a requirement for CCR5 in Treg function, and indicate that in addition to their previously defined role in inhibiting effector T-cell expansion in lymphoid tissues during GVHD, later recruitment of Tregs to both lymphoid tissues and GVHD target organs is important in their ability to prolong survival after allogeneic bone marrow transplantation.

Introduction: Breast cancer can be separated into five intrinsic subtypes based on differences in the transcriptome of the tumor. We propose that the intrinsic differences of specific tumor subytpes lead to extrinsic differences in the tumor microenvironment. Methods: We utilized human clinical and genetically engineered mouse model (GEMM) samples of the intrinsic subtypes luminal A, basal-like, and claudin-low breast cancers to evaluate the immune landscape in the tumor microenvironment by histology and microarray analysis. Our claudin-low GEMM was derived from BRCA-/-/p53-/- mice. The HER-2 overexpressing, basal-like, and luminal A models have been previously descried. We utilized the FoxP3-DTR transgenic mouse model as a method of regulatory T cell (Treg) depletion to evaluate their function in these GEMMs. Results: The claudin-low human tumors were heavily infiltrated with immune cells, with CD4+ T cells being the most prominent, when compared to the luminal A subtype (P = 0.01). There were also increased focal areas of Tregs in human claudin-low tumors. To evaluate the mechanism for these findings, we utilized a GEMM of claudin-low tumors in addition HER-2 overexpressing, basal-like, and luminal A models. Mice with claudin-low tumors recruited elevated numbers of immune cells to the tumor microenvironment when compared to other breast cancer subtypes (P=0.01). Additionally, there was increased expression of multiple chemokine ligands in the tumor microenvironment among claudin-low tumors, with CXCL12 being the most highly overexpressed. Because the claudin-low tumors were heavily immune infiltrated, we hypothesized that blockade of the inhibitory checkpoint receptors programmed death-1 (PD-1) and cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) could delay tumor growth and improve anti-tumor immune response. Surprisingly, we saw no delay in tumor growth in the claudin-low model using checkpoint inhibition. To investigate if presence of Tregs limited the function of checkpoint inhibitors, mice with claudin-low tumors were treated with AMD3100, a CXCR4 inhibitor. This decreased Treg infiltration into the tumor but did not alter tumor growth. We then utilized the FoxP3-DTR transgenic mouse model, where depletion of Tregs alone resulted in a very modest decrease in tumor growth, while depletion of Tregs plus checkpoint inhibition significantly improved survival (P = 0.03) and increased cytokine production by CD8+ T cells. Conclusion: We found that an effective anti-tumor immune response in claudin-low tumors is inhibited by the recruitment of Tregs to the tumor microenvironment. These data highlight early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint inhibition therapy for claudin-low breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Sarah C. Vick, Nicholas A. Taylor, Michael D. Iglesia, W June Brickey, Lisa A. Carey, Bentley R. Midkiff, Karen P. McKinnon, Shannon Reisdorf, Joel S. Parker, Charles M. Perou, Benjamin G. Vincent, Jonathan S. Serody. Regulatory T cell recruitment limits the effectiveness of checkpoint inhibition for claudin-low breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 568. doi:10.1158/1538-7445.AM2017-568

Most FDA-approved adjuvants for infectious agents boost humoral but not cellular immunity, and have poorly-understood mechanisms. Stimulator of interferon genes (STING, also known as MITA, MPYS, or ERIS) is an exciting adjuvant target due to its role in cyclic dinucleotide (CDN)-driven anti-viral immunity; however, a major hindrance is STING's cytosolic localization which requires intracellular delivery of its agonists. As a result, STING agonists administered in a soluble form have elicited suboptimal immune responses. Delivery of STING agonists via particle platforms has proven a more successful strategy, but the opportunity for improved formulations and bioactivity remains. In this study we evaluated the adjuvant activity of the potent STING agonist, CDN 3′3′-cGAMP (cGAMP), encapsulated in acid-sensitive acetalated dextran (Ace-DEX) polymeric microparticles (MPs) which passively target antigen-presenting cells for intracellular release. This formulation was superior to all particle delivery systems evaluated and maintained its bioactivity following a sterilizing dose of gamma irradiation. Compared to soluble cGAMP, the Ace-DEX cGAMP MPs enhanced type-I interferon responses nearly 1000-fold in vitro and 50-fold in vivo , caused up to a 10 4 -fold boost in antibody titers, increased Th1-associated responses, and expanded germinal center B cells and memory T cells. Furthermore, the encapsulated cGAMP elicited no observable toxicity in animals and achieved protective immunity against a lethal influenza challenge seven months post-immunization when using CDN adjuvant doses up to 100-fold lower than previous reports. For these reasons, Ace-DEX MP-encapsulated cGAMP represents a potent vaccine adjuvant of humoral and cellular immunity.

The reconstitution of specific immune niches after bone marrow transplant is mediated by donor immune cells repopulating specific microenvironments. Here, we demonstrate that type 2 innate lymphoid cells (ILC2) in the GI tract are rapidly eliminated by irradiation and/or chemotherapy and are not repopulated in the first month post-transplant by donor bone marrow cells. In contrast, ILC2 cells in the lung are not sensitive to conditioning therapy and quickly recover quantitatively post-transplant. Donor ILC2 cell infusion with donor T cells and TCD bone marrow has been shown by us to markedly diminish lethality and clinical score associated with acute GvHD. However, the mechanism for that finding was unclear. Here, we demonstrate that donor ILC2 infusion enhances the persistence of GI tract, but not splenic or pulmonary MDSCs (Fig 1a). This persistence required the expression of IL-13 by the ILC2 cells (Fig 1b). Absence of IL-13 production by ILC2 cells greatly diminished the activity of those cells to prevent GvHD (Fig 1c). ILC2 cell infusion decreased the number of T cells generating IFN-γ and IL-17A in the GI tract. However, we found no difference in the number of IFN-γ or IL-17A generating T cells in the GI tract after the infusion of IL-13-/- ILC2 cells. To evaluate the function of ILC2 cells in MDSC biology we performed in vitro quantitation of MDSCs in co-culture with ILC2 cells. MDSCs cultured with WT ILC2s, survived significantly better than MDSCs cultured alone, MDSCs cultured with IL-13 and MDSC cultured in the presence of IL-7 and IL-33. In addition, MDSCs co-cultured with WT ILC2s survived significantly better than those cultured with IL-13-/- ILC2s. Surprisingly, enhancement of MDSC survival requires cell-to-cell contact with ILC2s, as seen when either WT or IL-13-/- ILC2s and MDSCs are cultured in plates across semi-permeable membranes. ILC2s have been shown to have a role in intestinal barrier repair via amphiregulin (Areg). When compared to untreated recipients, ILC2 treated recipients showed significantly enhance barrier function which can be observed as early as 12 days post-transplant and as late as day 20. Interestingly, recipients of Areg deficient ILC2s show improved survival compared to control treatment but diminished survival compared to recipients of WT ILC2 cells. Several cell types have been shown to suppress aGvHD, including MDSCs and regulatory T cells (Treg). To evaluate the function of ILC2 cells as treatment, we compared them to MDSC and Treg infusion. When used to treat aGvHD on day 7 post-transplant, IL-13 derived MDSCs have no effect on recipient survival. In contrast ILC2 infusion results in a significant prolongation of survival compared to MDSC infusion with approximately 35% of recipient mice surviving past day 50. When compared to Treg infusion (1:1 ratio with Tcons) both ILC2 and Treg infusions block aGvHD. However, unlike ILC2 infusion in which no mice develop P815 tumors post-transplant, 80% of mice given Tregs developed tumors. At a lower Treg dose (1:4) the GvL response was restored but without the beneficial effect on GvHD. In summary, ILC2s represent a novel cell population that are required for recovery of GI tract homeostasis following allo-SCT. ILC2s play a pivotal role in the development of donor MDSCs and intestinal barrier repair. Additional investigation should be focused on the recovery of specific immunosuppressive cell populations from donor bone marrow cells after allo-SCT. IL-13 dependent ILC2 induced expansion of donor MDSCs. (a) Frequencies CD11b+/GR-1+/Ly-6C+/Ly-6G+ MDSCs in the LP of the colon of allo-SCT recipients 12 days post-transplant with or without WT ILC2s. These represent 2 independent experiments, bar graphs are average ± SEM, student's t test using GraphPad Prism, * p Disclosures No relevant conflicts of interest to declare.

Umbilical cord blood (UCB) stem cells from related and unrelated allogeneic donors have emerged as novel treatment for patients with hematologic malignancies. The incidence and severity of acute graft-versus-host disease (GVHD) after UCB transplantation compares favorably with that observed in recipients of matched unrelated donor allogeneic grafts, but remains a major cause of morbidity and mortality. It has been shown that stimulated lymphocytes from UCB have reduced production of cytokines including interferon-γ (IFN-γ) and tumor necrosis factor- (TNF-), which play a role in GVHD pathophysiology. We investigated the molecular mechanisms underlying this reduced cytokine production by analyzing expression of nuclear factor of activated T cells-1 (NFAT1) in UCB T cells. We detected no constitutive expression of NFAT1 protein in unstimulated UCB T cells compared with adult T cells. Moreover, although NFAT1 expression in UCB T cells was upregulated after prolonged (40 hours) T-cell stimulation, it was only partially upregulated when compared with adult controls. Our observation of minimal NFAT1 expression after stimulation correlated with reduced cytoplasmic IFN-γ and TNF- production in UCB T cells studied simultaneously. Reduced NFAT1 expression may blunt amplification of donor UCB T-cell alloresponsiveness against recipient antigens, thereby potentially limiting GVHD incidence and severity after allogeneic UCB transplantation.

Background: The use of immunosuppressive therapies after solid organ transplantation has been shown to increase a patient's risk for Epstein-Barr virus (EBV)–associated lymphoma. A potential therapy for this disorder is the adoptive transfer of EBV-specific cytotoxic T lymphocytes (CTLs). We proposed that dendritic cells (DCs) could be loaded with EBV antigens and be used to improve the in vitro generation of EBV-specific CTLs. Methods: Autologous EBV-transformed B-lymphoblastoid cell lines (BLCLs) were generated from normal donors, and CTLs were initiated by culturing peripheral blood mononuclear cells with DCs alone, disrupted BLCLs alone, intact, irradiated BLCLs alone, and DCs loaded with disrupted BLCLs. Lytic activities were determined with a 4-hour chromium-release assay against autologous BLCLs, and statistical calculations were performed by a Student t test assuming equal variance. Results: The lytic activity of CTLs generated with DCs loaded with disrupted BLCLs reached 78% and was statistically significant (P < .01) at all effector/target ratios compared with CTLs generated with DCs alone, disrupted BLCLs alone, or intact BLCLs alone. Total numbers of CTLs were also greater than those of control groups for DCs loaded with disrupted BLCLs. Conclusions: DCs improved the in vitro generation of EBV-specific CTLs as evidenced by this group's significantly increased lytic activity over that of the control group. The improved lytic activity of DC-generated EBV-CTLs suggests that adoptive transfer of these cells could lead to a more effective immunotherapeutic response against posttransplantation EBV-associated lymphoma. (Surgery 1998;124:171-6.)

Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. We therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of our data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genome-wide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM.

Dengue virus is a major global health threat and can lead to life-threatening hemorrhagic complications due to immune activation and cytokine production. Cross-reactive antibodies to an earlier dengue virus infection are a recognized risk factor for severe disease. These antibodies bind heterologous dengue serotypes and enhance infection into Fc-receptor-bearing cells, a process known as antibody-dependent enhancement of infection. One crucial cytokine seen elevated in severe dengue patients is IL-1β, a potent inflammatory cytokine matured by the inflammasome. We used a highly-physiologic system by studying antibody-dependent enhancement of IL-1β in primary human monocytes with anti-dengue human monoclonal antibodies isolated from patients. Antibody-enhancement increased viral replication in primary human monocytes inoculated with supernatant harvested from Vero cells infected with dengue virus serotype 2 (DENV-2) 16681. Surprisingly, IL-1β secretion induced by infectious supernatant harvested from two independent Vero cell lines was not enhanced by antibody. Secretion of multiple other inflammatory cytokines was also independent of antibody signaling. However, IL-1β secretion did require NLRP3 and caspase-1 activity. Immunodepletion of dengue virions from the infectious supernatant confirmed that virus was not the main IL-1β-inducing agent, suggesting that a supernatant component(s) not associated with the virion induced IL-1β production. We excluded RNA, DNA, contaminating LPS, viral NS1 protein, complement, and cytokines. In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction. Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion. This study indicates that Vero cells infected with DENV-2 16681 may produce inflammatory components during dengue virus propagation that mask the virus-specific immune response. Thus, the choice of host cell and viral purity should be carefully considered, while insect-derived virus represents a system that elicits antibody-dependent cytokine responses to dengue virus with fewer confounding issues.

The SecA protein is present in all bacteria, and it is a central component of the general Sec-dependent protein export pathway. An unusual property ofMycobacterium tuberculosisis the presence of two SecA proteins: SecA1, the essential “housekeeping” SecA, and SecA2, the accessory secretion factor. Here, we report that a ΔsecA2mutant ofM. tuberculosiswas defective for growth in the early stages of low-dose aerosol infection of C57BL/6 mice, a time during which the bacillus is primarily replicating in macrophages. Consistent with this in vivo phenotype, we found that the ΔsecA2mutant was defective for growth in macrophages from C57BL/6 mice. The ΔsecA2mutant was also attenuated for growth in macrophages from phox−/−mice and from NOS2−/−mice. These mice are defective in the reactive oxygen intermediate (ROI)-generating phagocyte oxidase and the reactive nitrogen intermediate (RNI)-generating inducible nitric oxide synthase, respectively. This indicated a role for SecA2 in the intracellular growth ofM. tuberculosisthat is independent of protecting against these ROIs or RNIs. Macrophages infected with the ΔsecA2mutant produced higher levels of tumor necrosis factor alpha, interleukin-6, RNI, and gamma interferon-induced major histocompatibility complex class II. This demonstrated a function forM. tuberculosisSecA2 in suppressing macrophage immune responses, which could explain the role of SecA2 in intracellular growth. Our results provide another example of a relationship betweenM. tuberculosisvirulence and inhibition of the host immune response.

We recently identified expression of the semaphorin receptor, plexin-A1, in dendritic cells (DCs); however, its function in these cells remains to be elucidated. To investigate function and maximize physiological relevance, we devised a retroviral approach to ablate plexin-A1 gene expression using small hairpin RNA (shRNA) in primary bone marrow-derived DCs. We show that plexin-A1 localizes within the cytoplasm of immature DCs, becomes membrane-associated, and is enriched at the immune synapse in mature DCs. Reducing plexin-A1 expression with shRNA greatly reduced actin polarization as well as Rho activation without affecting Rac or Cdc42 activation. A Rho inhibitor, C3, also reduced actin polarization. These changes were accompanied by the near-ablation of T cell activation. We propose a mechanism of adaptive immune regulation in which plexin-A1 controls Rho activation and actin cytoskeletal rearrangements in DCs that is associated with enhanced DC-T cell interactions.

Dendritic cell (DC)-based vaccines are increasingly used for the treatment of patients with malignancies. Although these vaccines are typically safe, consistent and lasting generation of tumor-specific immunity has been rarely demonstrated. Improved methods for delivering tumor Ags to DCs and approaches for overcoming tolerance or immune suppression to self-Ags are critical for improving immunotherapy. Viral vectors may address both of these issues, as they can be used to deliver intact tumor Ags to DCs, and have been shown to inhibit the suppression mediated by CD4+CD25+ regulatory T cells. We have evaluated the potential use of Venezuelan equine encephalitis virus replicon particles (VRPs) for in vitro Ag delivery to human monocyte-derived DCs. VRPs efficiently transduced immature human DCs in vitro, with ∼50% of immature DCs expressing a vector-driven Ag at 12 h postinfection. VRP infection of immature DCs was superior to TNF-α treatment at inducing phenotypic maturation of DCs, and was comparable to LPS stimulation. Additionally, VRP-infected DC cultures secreted substantial amounts of the proinflammatory cytokines IL-6, TNF-α, and IFN-α. Finally, DCs transduced with a VRP encoding the influenza matrix protein (FMP) stimulated 50% greater expansion of FMP-specific CD8+ CTL when compared with TNF-α-matured DCs pulsed with an HLA-A*0201-restricted FMP peptide. Thus, VRPs can be used to deliver Ags to DCs resulting in potent stimulation of Ag-specific CTL. These findings provide the rationale for future studies evaluating the efficacy of VRP-transduced DCs for tumor immunotherapy.

The coordinated expression of chemokines and receptors may be important in the directed migration of alloreactive T cells during graft-vs-host disease (GVHD). Recent work demonstrated in a murine model that transfer of CCR5-deficient (CCR5−/−) donor cells to nonconditioned haploidentical recipients resulted in reduced donor cell infiltration in liver and lymphoid tissues compared with transfer of CCR5+/+ cells. To investigate the function of CCR5 during GVHD in conditioned transplant recipients, we transferred CCR5−/− or wild-type C57BL/6 (B6) T cells to lethally irradiated B6D2 recipients. Unexpectedly, we found an earlier time to onset and a worsening of GVHD using CCR5−/− T cells, which was associated with significant increases in the accumulation of alloreactive CD4+ and CD8+ T cells in liver and lung. Conversely, the transfer of CCR5−/− donor cells to nonirradiated recipients led to reduced infiltration of target organs, confirming previous studies and demonstrating that the role of CCR5 on donor T cells is dependent on conditioning of recipients. Expression of proinflammatory chemokines in target tissues was dependent on conditioning of recipients, such that CXCL10 and CXCL11 were most highly expressed in tissues of irradiated recipients during the first week post-transplant. CCR5−/− T cells were shown to have enhanced migration to CXCL10, and blocking this ligand in vivo improved survival in irradiated recipients receiving CCR5−/− T cells. Our data indicate that the effects of inhibiting CCR5/ligand interaction on donor T cells during GVHD differ depending on conditioning of recipients, a finding with potentially important clinical significance.