Trafficking between clonally related peripheral T-helper cells and tissue-resident T-helper cells in chronic GVHD.

Chronic graft-versus-host disease (cGVHD) is an autoimmune-like syndrome. CXCR5-PD-1hi peripheral T-helper (Tph) cells have an important pathogenic role in autoimmune diseases, but the role of Tph cells in cGVHD remains unknown. We show that in patients with cGVHD, expansion of Tph cells among blood CD4+ T cells was associated with cGVHD severity. These cells augmented memory B-cell differentiation and production of immunoglobulin G via interleukin 21 (IL-21). Tph cell expansion was also observed in a murine model of cGVHD. This Tph cell expansion in the blood is associated with the expansion of pathogenic tissue-resident T-helper (Trh) cells that form lymphoid aggregates surrounded by collagen in graft-versus-host disease (GVHD) target tissues. Adoptive transfer experiments showed that Trh cells from GVHD target tissues give rise to Tph cells in the blood, and conversely, Tph cells from the blood give rise to Trh cells in GVHD target tissues. Tph cells in the blood and Trh cells in GVHD target tissues had highly overlapping T-cell receptor α and ß repertoires. Deficiency of IL-21R, B-cell lymphoma 6 (BCL6), or T-bet in donor T cells markedly reduced the proportions of Tph cells in the blood and Trh cells in GVHD target tissues and reduced T-B interaction in the lymphoid aggregates. These results indicate that clonally related pathogenic Tph cells and Trh cells traffic between the blood and cGVHD target tissues, and that IL-21R-BCL6 signaling and T-bet are required for the development and expansion of Tph and Trh cells in the pathogenesis of cGVHD.

Total marrow and lymphoid irradiation as conditioning in haploidentical transplant with posttransplant cyclophosphamide.

Posttransplant cyclophosphamide (PTCy) platform has shown low rates of graft-versus-host disease (GVHD) and nonrelapse mortality (NRM) after haploidentical hematopoietic cell transplantation (HaploHCT). However, because of the limited disease control, relapse rate remains a major cause of treatment failure in high-risk patients. Total marrow and lymphoid irradiation (TMLI) allows for delivery of high radiation to bone marrow and other targeted structures, without increasing off-target radiation exposure and toxicity to end organs. In this phase 1 trial, 31 patients with high-risk and/or active primary refractory leukemias or myelodysplastic syndrome underwent peripheral blood stem cell HaploHCT with TMLI, fludarabine, and cyclophosphamide as the conditioning regimen. Radiation dose was escalated in increments of 200 cGy (1200-2000 cGy). GVHD prophylaxis was PTCy with tacrolimus/mycophenolate mofetil. Grade 2 toxicities by the Bearman scale were mucositis (n = 1), hepatic (n = 3), gastrointestinal (n = 5), and cardiac (n = 2). One patient (1800 cGy) experienced grade 3 pulmonary toxicity (dose-limiting toxicity). At a follow-up duration of 23.9 months for the whole cohort; 2-year NRM was 13%. Cumulative incidence of day 100 grade 2 to 4 and 3 to 4 acute GVHD was 52% and 6%, respectively. Chronic GVHD at 2 years was 35%. For patients treated with 2000 cGy, with a median follow-up duration of 12.3 months, 1-year relapse/progression, progression-free survival, and overall survival rates were 17%, 74%, and 83%, respectively. In conclusion, HaploHCT-TMLI with PTCy was safe and feasible in our high-risk patient population with promising outcomes.

Posttransplant cyclophosphamide as GVHD prophylaxis for peripheral blood stem cell HLA-mismatched unrelated donor transplant.

Efficacy of PTCy after mismatched unrelated donor (MMUD) HCT is unknown. In this pilot clinical trial, we enrolled 38 patients with hematologic malignancies scheduled to undergo MMUD-HCT (≥6/8 HLA-matched donors) onto 1 of 2 conditioning strata: myeloablative using fludarabine and fractionated total body irradiation (n = 19) or reduced intensity with fludarabine/melphalan (n = 19). Graft source was peripheral blood stem cells (PBSCs), and GVHD prophylaxis was PTCy, tacrolimus, and mycophenolate mofetil. Patients' median age was 53 years (range, 21-72 years). Median number of HLA mismatches was 2 (range, 1-4) of 12 loci. Twenty-three patients (61%) were considered racial (n = 12) or ethnic (n = 11) minorities. Median time to neutrophil engraftment was 16 days (range, 13-35 days). With a median follow-up of 18.3 months (range, 4.3-25.0 months) for surviving patients, 1-year overall survival (OS) and GVHD-free/relapse-free survival (GRFS) were 87% (95% confidence interval [CI]: 71-94) and 68% (95% CI: 51-81), respectively. Cumulative incidence of nonrelapse mortality at 100 days and 1 year were 0% and 11% (95% CI: 4-27), respectively, whereas relapse/progression was 11% (95% CI: 4-27). Cumulative incidence of 100-day acute GVHD grades 2-4 and 3-4 and 1-year chronic GVHD were 50% (95% CI: 36-69), 18% (95% CI: 9-36), and 48% (95% CI: 34-68), respectively. The rate of moderate/severe chronic GVHD was 3% in the entire cohort. We showed highly promising OS/GRFS rates with an acceptable risk profile after PBSC-MMUD-HCT with PTCy. This trial was registered at www.clinicaltrials.gov as #NCT03128359.

Salmonella-mediated therapy targeting indoleamine 2, 3-dioxygenase 1 (IDO) activates innate immunity and mitigates colorectal cancer growth.

Patients with colon cancer remain largely refractory to current immunotherapeutic strategies. This is, in part, due to the overexpression of the immune checkpoint protein indoleamine 2,3-dioxygenase 1 (IDO). IDO is an important enzyme contributing to tumor-mediated immunosuppression and also correlates with poor prognosis in colon cancer patients. The aim of this study was to assess the therapeutic efficacy of attenuated Salmonella typhimurium delivering an shRNA plasmid targeting IDO (shIDO-ST) in two mouse models of colorectal cancer. In vitro, the CT26 and MC38 murine colon cancer cell lines were shown to upregulate IDO expression following stimulation with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Transfection of both cell lines with shIDO plasmid reduced IDO protein expression and function. In vivo, shIDO-ST treatment significantly delayed CT26 and MC38 tumor progression compared to mice treated with scrambled shRNA control (shScr-ST) or the clinically tested IDO inhibitor epacadostat. Increased tumor infiltration of neutrophils was found to be the primary immune cell population associated with shIDO-ST treatment, suggesting robust activation of innate immunity. Although increased tumor expression of IDO is associated with resistance to antibody therapy against programed cell death-1 (anti-PD1), co-administration of anti-PD1 with shIDO-ST did not provide additional tumor growth control in either model of colorectal cancer. Altogether, we demonstrate that treatment with shIDO-ST markedly delays tumor growth in two immunocompetent colorectal mouse models and this appears to be a superior therapeutic strategy compared to epacadostat or blocking anti-PD1 antibody therapy in colon cancer.

p53-Reactive T Cells Are Associated with Clinical Benefit in Patients with Platinum-Resistant Epithelial Ovarian Cancer After Treatment with a p53 Vaccine and Gemcitabine Chemotherapy.

Purpose: To conduct a phase I trial of a Modified Vaccinia Ankara vaccine delivering wild-type human p53 (p53MVA) in combination with gemcitabine chemotherapy in patients with platinum-resistant ovarian cancer.Experimental Design: Patients received gemcitabine on days 1 and 8 and p53MVA vaccine on day 15, during the first 3 cycles of chemotherapy. Toxicity was classified using the NCI Common Toxicity Criteria and clinical response assessed by CT scan. Peripheral blood samples were collected for immunophenotyping and monitoring of anti-p53 immune responses.Results: Eleven patients were evaluated for p53MVA/gemcitabine toxicity, clinical outcome, and immunologic response. TOXICITY: there were no DLTs, but 3 of 11 patients came off study early due to gemcitabine-attributed adverse events (AE). Minimal AEs were attributed to p53MVA vaccination. Immunologic and clinical response: enhanced in vitro recognition of p53 peptides was detectable after immunization in both the CD4+ and CD8+ T-cell compartments in 5 of 11 and 6 of 11 patients, respectively. Changes in peripheral T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC) did not correlate significantly with vaccine response or progression-free survival (PFS). Patients with the greatest expansion of p53-reactive T cells had significantly longer PFS than patients with lower p53-reactivity after therapy. Tumor shrinkage or disease stabilization occurred in 4 patients.Conclusions: p53MVA was well tolerated, but gemcitabine without steroid pretreatment was intolerable in some patients. However, elevated p53-reactive CD4+ and CD8+ T-cell responses after therapy correlated with longer PFS. Therefore, if responses to p53MVA can be enhanced with alternative agents, superior clinical responses may be achievable. Clin Cancer Res; 24(6); 1315-25. ©2018 AACR.

MVA vaccine encoding CMV antigens safely induces durable expansion of CMV-specific T cells in healthy adults.

Attenuated poxvirus modified vaccinia Ankara (MVA) is a useful viral-based vaccine for clinical investigation, because of its excellent safety profile and property of inducing potent immune responses against recombinant (r) antigens. We developed Triplex by constructing an rMVA encoding 3 immunodominant cytomegalovirus (CMV) antigens, which stimulates a host antiviral response: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5). We completed the first clinical evaluation of the Triplex vaccine in 24 healthy adults, with or without immunity to CMV and vaccinia virus (previous DryVax smallpox vaccination). Three escalating dose levels (DL) were administered IM in 8 subjects/DL, with an identical booster injection 28 days later and 1-year follow-up. Vaccinations at all DL were safe with no dose-limiting toxicities. No vaccine-related serious adverse events were documented. Local and systemic reactogenicity was transient and self-limiting. Robust, functional, and durable Triplex-driven expansions of CMV-specific T cells were detected by measuring T-cell surface levels of 4-1BB (CD137), binding to CMV-specific HLA multimers, and interferon-γ production. Marked and durable CMV-specific T-cell responses were also detected in Triplex-vaccinated CMV-seronegatives, and in DryVax-vaccinated subjects. Long-lived memory effector phenotype, associated with viral control during CMV primary infection, was predominantly found on the membrane of CMV-specific and functional T cells, whereas off-target vaccine responses activating memory T cells from the related herpesvirus Epstein-Barr virus remained undetectable. Combined safety and immunogenicity results of MVA in allogeneic hematopoietic stem cell transplant (HCT) recipients and Triplex in healthy adults motivated the initiation of a placebo-controlled multicenter trial of Triplex in HCT patients. This trial was registered at www.clinicaltrials.gov as #NCT02506933.

Evaluation of innate and adaptive immunity contributing to the antitumor effects of PD1 blockade in an orthotopic murine model of pancreatic cancer.

Despite the clinical success of anti-PD1 antibody (α-PD1) therapy, the immune mechanisms contributing to the antineoplastic response remain unclear. Here, we describe novel aspects of the immune response involved in α-PD1-induced antitumor effects using an orthotopic Kras (G12D)/p53(R172H)/Pdx1-Cre (KPC) model of pancreatic ductal adenocarcinoma (PDA). We found that positive therapeutic outcome involved both the innate and adaptive arms of the immune system. Adoptive transfer of total splenocytes after short-term (3 d) but not long-term (28 d) PD1 blockade significantly extended survival of non-treated tumor-bearing recipient mice. This protective effect appeared to be mostly mediated by T cells, as adoptive transfer of purified natural killer (NK) cells and/or granulocyte receptor 1 (Gr1)(+) cells or splenocytes depleted of Gr1(+) cells and NK cells did not exhibit transferrable antitumor activity following short-term PD1 blockade. Nevertheless, splenic and tumor-derived CD11b(+)Gr1(+) cells and NK cells showed significant persistence of α-PD1 bound to these cells in the treated primary recipient mice. We observed that short-term inhibition of PD1 signaling modulated the profiles of multifunctional cytokines in the tumor immune-infiltrate, including downregulation of vascular endothelial growth factor A (VEGF-A). Altogether, the data suggest that systemic blockade of PD1 results in rapid modulation of antitumor immunity that differs in the tumor microenvironment (TME) when compared to the spleen. These results demonstrate a key role for early immune-mediated events in controlling tumor progression in response to α-PD1 treatment and warrant further investigation into the mechanisms governing responses to the therapy at the innate-adaptive immune interface.

Ex vivo detection of CD8 T cells specific for H-Y minor histocompatibility antigens in allogeneic hematopoietic stem cell transplant recipients.

Immunologic disparities between minor histocompatibility antigen (mHAg) genes on Y (H-Y) and X (H-X) chromosomes contribute to graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects observed in male recipients of a female donor (FtoM) hematopoietic stem cell transplant (HCT). Using in silico prediction tools, a panel of HLA-A0201 restricted H-Y peptides was synthesized. Expression of CD137 was monitored on CD8(+) T cells after brief stimulation with the H-Y peptides in FtoM HLA-A0201 HCT recipients (N=29), and control groups (HLA-A0201 MtoM [N=18], non-HLA-A0201 FtoM [N=14], and HLA-A0201 female volunteers [N=25]). Specific H-Y responses were significantly greater in HLA-A0201 FtoM than controls. CD8(+) T-cell responses to novel H-Y epitopes were shared among multiple patients, showing marked CD45RA(+)CD27 cytolytic effector profiles. These data represent a proof of concept for our in silico/ex vivo CD8(+) T-cell based approach of prediction and validation of H-Y mHAgs in HCT recipients, which may facilitate prospective studies to identify targets/biomarkers of GVHD/GVL.

Detection and preliminary characterization of CD8+T lymphocytes specific for Wilms' tumor antigen in patients with non-Hodgkin lymphoma.

Wilms' tumor antigen (WT1) is overexpressed in many different solid tumors and hematologic malignancies. However, little is known about WT1 expression or WT1-specific immune responses in patients with non-Hodgkin lymphoma (NHL). In a cross-sectional survey study, we investigated the immune recognition of WT1 by patients with NHL. Utilizing a WT1 overlapping peptide library, we discovered that a large percentage of patients with NHL of all grades maintain WT1-specific T cells. Ex vivo frequencies of these T cells measured from unfractionated samples by the CD137 activation marker assay were high in many patients (some > 1% CD8+). Using standard in vitro techniques we discovered that they were cytotoxic to WT1 peptide library-loaded T2 cells and WT1 antigen-primed autologous Epstein-Barr virus-transformed B cell lines (EBV-LCLs) and expressed interferon gamma (IFN-γ). In addition, we detected WT1 mRNA transcripts in diseased lymph node tissues of patients with NHL utilizing real-time quantitative polymerase chain reaction (RT-qPCR) technology. These results are the first example of strong T cell reactivity against WT1 in patients with NHL which also demonstrate strong cytotoxicity against peptide-loaded tumor cells. The potential for developing WT1 as a target for immunotherapy in NHL deserves further exploration.

Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication.

The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C(pro) to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C(pro) involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C(pro) involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C(pro) is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C(pro)-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

The interaction of cytoplasmic RNA viruses with the nucleus.

Mammalian cells infected with poliovirus, the prototype member of the picornaviridae family, undergo rapid macromolecular and metabolic changes resulting in efficient replication and release of virus from infected cells. Although this virus is predominantly cytoplasmic, it does shut-off transcription of all three cellular transcription systems. Both biochemical and genetic studies have shown that a virally encoded protease, 3C(pro), is responsible for host cell transcription shut-off. The 3C protease cleaves a number of RNA polymerase II transcription factors including the TATA-binding protein (TBP), the cyclic AMP-responsive element binding protein (CREB), the Octamer binding protein (Oct-1), p53, and RNA polymerase III transcription factor IIICalpha, and Polymerase I factor SL-1. Most of these cleavages occur at glutamine-glycine bonds. Additionally, a second viral protease, 2A(pro), also cleaves TBP at a tyrosine-glycine bond. The latter cleavage could be responsible for shut-off of small nuclear RNA transcription. Recent studies indicate that the viral protease-polymerase precursor 3CD can enter nucleus in poliovirus-infected cells. The nuclear localization signal (NLS) present within the 3D sequence appears to play a role in the nuclear entry of 3CD. Thus, 3C may be delivered to the infected cell nucleus in the form the precursor 3CD or other 3C-containing precursors. Auto-proteolytic cleavage of these precursors could then generate 3C. Thus, for a small RNA virus that strictly replicates in the cytoplasm, a portion of its life cycle does include interaction with the host cell nucleus.