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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 β 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.

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.

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (HCT) for leukemia and myelodysplastic syndrome (MDS). In order to improve the outcome of leukemia patients after allogeneic HCT, it is imperative to identify reliable markers to predict impending relapse. Wilms’ tumor antigen (WT1) is overexpressed in the majority of leukemia and MDS patients and is being considered as a possible universal diagnostic marker for minimal residual disease (MRD), especially since no chromosomal translocation has nearly the frequency of association as WT1 does with leukemia. In this study we prospectively evaluated the prognostic value of MRD monitoring by qRT-PCR for WT1 transcripts. WT1 transcript levels in peripheral blood mononuclear cells (PBMC) were measured utilizing SYBR-Green qRT-PCR on the ABI7300 instrument (Applied Biosystems, Carlsbad, CA) and results were expressed as a ratio of WT1/c-ABL transcript copies normalized by 104 (WT1 ratio: WT1/c-ABLx104). PBMC samples were obtained monthly for 6 months post-HCT, then on alternating monthly schedule until 3 years post-HCT. Patients >18y.o. with confirmed diagnosis of MDS with A total of 83 patients (median age: 54, range: 19-74) with AML (n=39), ALL (n=24), MDS (n=17), or CML (n=3) received allogeneic HCT after fully ablative (n=39) or reduced-intensity (n=44) conditioning. Donor sources were matched related (n=33), unrelated (n=50), or umbilical cord blood (n=2). Fifty-one patients were considered low-risk (AML/ALL in CR1, CML in 1stchronic phase, or MDS-RA/RARS subtypes) while the remaining 32 patients were considered high risk. Sixteen of 83 patients relapsed with a median time of 238 days post-HCT (range: 76-747). The minimum WT1 ratio that gave specificity of 100% in predicting relapse was 50 (95% binomial exact CI: 92.5-100%), as none of the non-relapsed patients crossed this level. Of 16 patients who relapsed, 12 crossed the WT1 ratio of 50, providing a sensitivity of 75% (95% binomial exact CI: 48- 93%). The positive predictive value (PPV) and the negative predictive value (NPV) performance parameters for the WT1 ratio of 50 were 100% and 94.4%, respectively (Table 1). There was an average number of 63 days (SD=29.3) from crossing the WT1 ratio of 50 to hematologic relapse for the 12 relapsed patients. Since PBMC samples from healthy donors consistently demonstrated a WT1 ratio In summary, our data demonstrate that the quantitative measurement of WT1 transcripts is a reliable marker to assess MRD post-HCT for leukemia patients, and its real-time prospective monitoring provides a 2-3 month window of opportunity to introduce medical/immunologic interventions prior to overt hematologic relapse.Table 2WT1 ratio Specificity Sensitivity Positive Predictive Value Negative Predictive Value Days to Relapse (SD)50100%75%100%94.4%63 days (29)4095.5%75%80%94.1%66.9 days (29)3094%75%75%94%71.5 days (30)2085%87.5%58.3%96.6%78 days (28)1056.7%93.8%34.1%97.4%107.5 days (58) Disclosures: No relevant conflicts of interest to declare.

Abstract 953 Wilms' tumor antigen (WT1) is expressed in many solid tumors and hematologic malignancies and serves a physiological role in the maintenance of the tumorigenic phenotype. Immunologic recognition of WT1 has been observed in patients with myeloid and lymphoid leukemia, solid tumors, and even in healthy adults. However, little is known about WT1 expression or WT1-specific immune responses in patients with non-Hodgkin lymphoma (NHL). Current therapeutic modalities for NHL patients are predominantly based on the highly successful application of the anti-CD20 mAb (Rituximab™) or radioactive derivatives (Zevalin™ or Bexxar™) combined with standard chemotherapy. Recent approaches have focused on generating NHL-specific idiotypes as an example of personalized medicine. However, harnessing a T cell response to malignant B or T cells in NHL patients has not succeeded, despite its attractiveness as a universal therapeutic option. We developed a detection system for WT1-specific T cells that is HLA independent by constructing a peptide library of 15mers (11 residues overlapping) corresponding to full length WT1 antigen. We analyzed peripheral blood mononuclear cells (PBMC) after priming with the peptide library for immune responses to the WT1 antigen detectable by flow cytometry based on the CD137 activation marker. Sixty-three NHL patients and 30 healthy donors (HD) were recruited under IRB-approved protocols. The cohort included patients who received allogeneic stem cell transplantation (alloHCT: n=42) or chemotherapy/autologous HCT (n=21). We observed that WT1-specific ex vivo CD8+ T cell responses were significantly greater in our NHL patient cohort (median 0.035%) compared to our randomly chosen HD cohort (0%, p=0.0013)(Figure 1). These WT1-specific CD8+CD137+ T cells co-expressed CD45RA and CD57 surface markers, consistent with them being a TEMRA (Terminal Effector Memory, RA+) population of highly differentiated T cells. Twenty-four of 43 patients (55.8%) with high/intermediate-grade NHL had a positive CD8+ T cell response (CD8+CD137+ ≥0.05%) to the WT1 antigen compared to four of 20 patients (20%) with low-grade NHL (p=0.011). None of the other clinical variables such as patient age, treatment modality (alloHCT vs. autoHCT or chemotherapy), disease status (CR vs. non-CR), prior acute GVHD (only in alloHCT, grade 0-I vs. II–IV) were significantly associated (p≤0.05) with WT1-specific CD8+ T-cell response in NHL patients when we conducted a multivariate analysis. PBMC from one HD and 12 NHL patients were expanded using in vitro stimulation (IVS) with the WT1 peptide library. All PBMC samples expanded in culture after 10–14 days by single IVS (median 7-fold expansion). We observed significantly higher IFN-γ responses in CD8+ T cells when IVS cells were re-stimulated with the WT1 library (median=3.0%, range: 0.08–5.5%) compared with the medium control (median=0.39%, range: 0.05–1.7%, p=0.002). Remarkably, using standard in vitro techniques, the expanded PBMC were cytotoxic to WT1-peptide library-loaded T2 cells and autologous EBV-transformed B-cell lines derived from patients enrolled in the study. These results challenge the notion that central tolerance mechanisms eliminate high affinity WT1-specific T cells that kill tumor cells. In order to distinguish whether WT1-specific T cells are maintained in NHL patients because of an aberration of tolerance mechanisms or the presence of WT1 antigen as a stimulator, we used sensitive real time quantitative PCR (RT-qPCR) methods to detect transcripts in LN from NHL patients and HD. We succeeded in detecting WT1 mRNA transcripts in diseased lymph node (LN) tissues of NHL patients utilizing RT-qPCR technology. There was significantly greater WT1 mRNA in NHL LN (n=6) with an average copy number ratio of 39 (WT1/c-ABL × 104) compared with 0.66 in non-disease LN obtained from individuals without a cancer diagnosis (n=7, p Disclosures: No relevant conflicts of interest to declare.