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Supplementary Figure 1 shows [18F]F-AraG Structure and Schematic of AraG Uptake into T Cells Supplementary Figure 2 shows [18F]F-AraG and [3H]F-AraC Uptake and Retention Across Various Immune Cell Lines Supplementary Figure 3 shows AraG Competitive Inhibition of [18F]F-AraG Uptake Across Various Immune Cell Lines Supplementary Figure 4 shows Comparison of [18F]F-AraG and [3H]F-AraC Uptake and Retention Across Various Solid Tumor Cell Lines Supplementary Figure 5 shows Western Blot Analysis of dCK Expression in Wild-type and Mutant CCRF-CEM cells Supplementary Figure 6 shows dGK overexpression in CHO-K1 cells leads to increased [18F]F-AraG uptake and retention Supplementary Figure 7 shows [3H]F-AraC Accumulates in Cells via dCK Activity Supplementary Figure 8 shows Uptake of [18F]F-AraG in Activated Versus Resting Murine T Cells Supplementary Figure 9 shows Isolation and Activation of Primary Human T Cells Supplementary Figure 10 shows Uptake and Retention of [3H]AraG and [3H]F-AraC in Resting and Activated Human Primary T Cells Supplementary Figure 11 shows Evaluation of GFP+ Donor T cells in Spleens and Cervical Lymph Nodes (CLNs) at Day 3 and Day 10 of Acute GVHD Supplementary Figure 12 shows Evaluation of Proliferation of Donor T Cells in Cervical Lymph Nodes

A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2′-deoxy-2′-[18F]fluoro-9-β-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893–902. ©2017 AACR.

Day4_Control

Day7_Control

Purpose:To develop a prognostic model and cytogenetic risk classification for previously treated patients with chronic lymphocytic leukemia (CLL) undergoing reduced intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT).Experimental Design:We performed a retrospective analysis of outcomes of 606 patients with CLL who underwent RIC allogeneic HCT between 2008 and 2014 reported to the Center for International Blood and Marrow Transplant Research.Results:On the basis of multivariable models, disease status, comorbidity index, lymphocyte count, and white blood cell count at HCT were selected for the development of prognostic model. Using the prognostic score, we stratified patients into low-, intermediate-, high-, and very-high-risk [4-year progression-free survival (PFS) 58%, 42%, 33%, and 25%, respectively, P < 0.0001; 4-year overall survival (OS) 70%, 57%, 54%, and 38%, respectively, P < 0.0001]. We also evaluated karyotypic abnormalities together with del(17p) and found that del(17p) or ≥5 abnormalities showed inferior PFS. Using a multivariable model, we classified cytogenetic risk into low, intermediate, and high (P < 0.0001). When the prognostic score and cytogenetic risk were combined, patients with low prognostic score and low cytogenetic risk had prolonged PFS (61% at 4 years) and OS (75% at 4 years).Conclusions:In this large cohort of patients with previously treated CLL who underwent RIC HCT, we developed a robust prognostic scoring system of HCT outcomes and a novel cytogenetic-based risk stratification system. These prognostic models can be used for counseling patients, comparing data across studies, and providing a benchmark for future interventions. For future study, we will further validate these models for patients receiving targeted therapies prior to HCT.

Day4_GVHD

Supplementary Material

CHO Cell Transfection Tracer Uptake and Efflux Assays for Adherent Cells Western Blot Immunofluorescence Staining of Lymphoid Organs Ex Vivo Donor T Cell Proliferation Analysis Automated preparation of [18F]FAraG for human imaging using the Neptis perform PET synthesizer Imaging Studies in Humans Statistics

Supplemental Figures & Legends

Allogeneic hematopoietic cell transplantation (HCT) remains an important treatment for adults with acute lymphoblastic leukemia (ALL). We hypothesized that advances in ALL and transplantation have resulted in improved HCT outcomes in recent years. In this study, we evaluated the characteristics and outcomes of adult ALL patients undergoing allogeneic HCT over the last decade. Patients with ALL aged 18 years and older who underwent allogeneic HCT at Stanford University between 2008 and 2019 were included in this study. Patients were divided into 2 eras based on year of HCT: 2008 to 2013 (earlier era) and 2014 to 2019 (later era). A total of 285 patients were included: 119 patients underwent HCT in the earlier era and 166 in the later era. Patients who underwent transplantation in the later era were more likely to be Hispanic (38% versus 21%) and to have an HCT-comorbidity index ≥3 (31% versus 18%). Donor source for HCT also differed with an increase in the use of HLA-mismatched donor sources (38% versus 24%), notably umbilical cord blood in the later era (16% versus 0%). Patients in the later era were less likely to undergo transplantation with active disease (4% versus 16%); pre-HCT rates of measurable residual disease were similar across the eras (38% versus 40%). In unadjusted analyses, overall survival (OS) improved across eras, with 2-year estimates for the later and earlier eras of 73% (95% confidence interval [CI], 66%-80%) versus 55% (95% CI, 46%-64%), respectively. Multivariable analysis confirmed the association between later era and OS (hazard ratio = 0.52, 95% CI, 0.34-0.78). Finally, among patients relapsing after HCT (25% in later era and 33% in earlier era), the use of novel immunotherapies increased in the later era (44% versus 3%), as did the median OS after post-HCT relapse (16 months versus 8 months, P.001). OS after HCT for adult ALL has improved in recent years. This is due, in part, to a significant improvement in the ability to effectively salvage adults with ALL relapsing after HCT.

Failure of hematopoietic stem cell transplantation (HSCT) has been associated with the immunodeficiency that follows shortly after therapy, which renders patients susceptible to opportunistic infections and cancer relapse. Thus, strategies that induce more robust immune recovery are needed which may improve outcomes. Natural killer (NK) cells have been utilized to improve HSCT given their importance in eliminating infected and transformed cells. We explored whether the infusion of recently described NK cell-committed progenitor (NKP:CD3-CD19-Ly6D-NK1.1-CD27+CD244+cKit+CD127+CD135-CD122+/-) subsets along with unfractioned bone marrow cells (uBMC) accelerated NK cell reconstitution following allogeneic HSCT (alloHSCT). Administration of donor-type NKP (H2b) along with uBMC in alloHSCT settings resulted in increased number and percentages of donor-type precursor NK cells (CD3-CD122+NKG2D+NK1.1-DX5-) compared to recipients that received uBMC alone or in combination with common lymphoid progenitors (CLP:CD135+CD122-) at day 14 post-HSCT. By day 21, NK cell numbers were significantly higher and displayed a more mature phenotype (CD3-CD122+NKG2D+NK1.1+DX5+) with co-expression of CD27 and CD11b. These data demonstrate faster NK cell development when NKP are given after alloHSCT. More importantly, infusion of NKP during alloHSCT to accelerate NK cell reconstitution could result in stronger anti-tumor responses while reducing graft vs. host disease making alloHSCT more effective.

Natural Killer (NK) cells have been used as a cancer therapy against multiple cancers. Despite initial promises, NK-based immunotherapy has resulted in limited success and therefore, new approaches are needed. Our group described the induction of NK cell exhaustion (NCE) upon tumor exposure that resulted in reduced cytotoxic function associated with lower expression of several molecules such as NKG2D and the transcription factors Eomesodermin and T-bet. Here we show that NCE induced by tumor cells is MHC-I independent and is not mediated by the expression of NKG2D ligands (NKG2DL) on tumor cells. However, when NKG2D-deficient NK cells were exposed to tumor cells limited proliferation and higher IFNg production was observed suggesting a role for NKG2D in NCE. Moreover, NCE patters were observed at later stages of in vitro IL2-activation that were reduced in NKG2D-deficient NK cells. Interestingly, concomitant downregulation of NKG2D and upregulation of NKG2DL (MULT1, Rae-1) was also detected. Upregulation of NKGDL expression was particularly seen after the cells were acid-stripped which was shown to disrupt MHC-I-Ly49s cis-interaction complexes. NCE was also observed after prolonged in vivo IL2 stimulation that impaired NK cell maturation. In conclusion, we demonstrated a role for NKG2D in the induction of NCE that could represent a self-regulatory mechanism important in controlling NK cell activation utilized by tumor cells to prevent NK cell anti-tumor responses.

In recipients of allogeneic hematopoietic-cell transplants, peripheral-blood cells mobilized with the use of filgrastim (recombinant granulocyte colony-stimulating factor) engraft more rapidly than bone marrow. However, the relative effects of these techniques on the rates of acute and chronic graft-versus-host disease, overall survival, and disease-free survival have not been determined in randomized studies.Between March 1996 and July 1999, 172 patients (12 to 55 years of age) with hematologic cancer were randomly assigned to receive either bone marrow or filgrastim-mobilized peripheral-blood cells from HLA-identical relatives for hematopoietic rescue after the treatment of hematologic cancer with high doses of chemotherapy, with or without radiation.The recovery of both neutrophils and platelets was faster with peripheral-blood cells than with marrow (P0.001 for both comparisons). The cumulative incidence of grade II, III, or IV acute graft-versus-host disease at 100 days was 64 percent with peripheral-blood cells and 57 percent with marrow (hazard ratio, 1.21; 95 percent confidence interval, 0.81 to 1.81; P=0.35). The cumulative incidence of chronic graft-versus-host disease was 46 percent with peripheral-blood cells and 35 percent with marrow (hazard ratio, 1.16; 95 percent confidence interval, 0.71 to 1.90; P=0.54). The estimated overall probability of survival at two years was 66 percent with peripheral-blood cells and 54 percent with marrow (hazard ratio for death, 0.62; 95 percent confidence interval, 0.38 to 1.02; P=0.06). The rate of disease-free survival at two years was 65 percent with peripheral-blood cells and 45 percent with marrow (hazard ratio for relapse or death, 0.60; 95 percent confidence interval, 0.38 to 0.95; P=0.03).In patients given high-dose chemotherapy, with or without radiation, for the treatment of hematologic cancer, allogeneic peripheral-blood cells used for hematopoietic rescue restore blood counts faster than allogeneic bone marrow, without increasing the risk of graft-versus-host disease.

Adoptive transfer of CD4+CD25+FoxP3+ regulatory T cells (Tregs) prevents graft versus host disease (GvHD) in several animal models and following allogeneic hematopoietic cell transplantation (HCT) in clinical trials. In these models donor derived Tregs have been mainly used as they share the same major histocompatibility complex (MHC) with conventional CD4+ and CD8+ T cells (Tcons) that are primarily responsible for GvHD onset and persistence. Third-party derived Tregs are a promising alternative tool for cellular therapy as they can be prepared in advance, screened for pathogens and activity and banked. In this study we explored MHC disparities between Tregs and Tcons in HCT to evaluate the impact of these different cell populations in GvHD prevention and survival after transplant. Methods and Results We evaluated the ability of highly purified Treg to suppress proliferation of C57BL/6 (H-2b) Tcons following exposure to irradiated splenocytes from BALB/C (H-2d) mice in vitro in a mixed lymphocyte reaction (MLR). Either donor derived C57BL/6 (H-2b) or third party FVB (H-2q) Tregs suppressed Tcon proliferation at the Treg/Tcon ratios of 1:2 and 1:4. The same Treg population effectively suppressed different MHC derived Tcons where BALB/C (H-2d) or FVB (H-2q, third-party) Tcons were incubated with irradiated splenocytes from C57BL/6 (H-2b) mice and were effectively suppressed with BALB/C (H-2d) Tregs. In the MLR, third-party Tregs present the same activation molecule expression patterns as MHC matched Tregs: CTLA4 and LAG3 expression is enhanced after stimulation with interleukin-2 (IL-2) and anti-CD3/CD28 beads, while MHC class II molecule expression is increased after 3-4 days of culture with Tcons and irradiated splenocytes. Furthermore third-party and MHC matched Tregs express the same levels of interleukin-10 (IL-10). We translated these results to in vivo studies in animal models. In these studies T cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) mice was injected into lethally irradiated (total body irradiation, 8 Gy) BALB/C (H-2d) recipient mice. 2 days later GvHD was induced by injecting luc+ donor derived Tcons (1x106/mouse). Using this model GvHD was evaluated following the adoptive transfer of freshly isolated CD4+CD25+FoxP3+ Tregs derived from BALB/C (H-2d, host type), C57BL/6 (H-2b, donor type), FVB (H-2q, third-party) or BALB/B (H-2b, minor mismatched with the donor, major mismatched with the host) mice at the different Treg/Tcon ratios of 1:1, 1:2 and 1:4. As expected, donor Tregs exerted the strongest dose dependent GvHD protection (p = 0.028), while host Tregs did not improve mouse survival (p = 0.58). Third-party and minor mismatched with the donor Tregs improved mouse survival (third-party and minor mismatched with the donor respectively, p = 0.028 and p = 0.17) but mice had worse GvHD score profiles (both p< 0.001) and could not recover their weight as well as mice treated with donor Tregs (both p< 0.001). In vivoTcon bioluminescent imaging confirmed these results showing a reduced Tcon proliferation in mice treated with donor, third-party and minor mismatched with the donor Tregs, the first exerting the strongest effect (after 6 weeks of observation, p< 0.001). Conclusions Our studies indicate that MHC disparities between Tregs and Tcons do not represent an insurmountable barrier for Treg function. In vitro and in vivo data strongly suggest that Tregs can suppress Tcon proliferation without requiring MHC matching. In vivo GvHD prevention efficiency was affected by MHC disparities with donor derived Treg being the most effective, however, third party Treg also resulted in GvHD attenuation. These studies indicate that both donor and third party Treg could be effective in clinical application raising the possibility of screening and banking Treg for use. Further, these studies highlight the need for activation of the Treg on host tissues to effectively suppress conventional T cell proliferation and GvHD induction. Disclosures: No relevant conflicts of interest to declare.

Regulatory T cells with the phenotype of CD4+CD25+FoxP3+ (Treg) decrease acute graft-versus-host disease (GVHD) while preserving graft-versus-leukemia effects. However, their low frequency limits clinical translation. One approach has been to expand Treg ex vivo using cytokines (IL-2/TGFb) and antibodies (anti-CD3/anti-CD28). Another approach may be to develop strategies to expand Treg in vivo. Death Receptor 3 (DR3) is a member of the TNF superfamily and its agonistic antibody (4C12) was reported to preferentially activate Treg. We investigated the role of DR3 stimulation in expanding Treg and decreasing acute GVHD in a murine model. Two approaches were investigated. The first approach was to utilize 4C12 to treat B6 donor mice (CD45.1/luc+) and isolate conventional T cells (Tcon) four days later from lymph nodes and spleen using anti-CD4 and anti-CD8 beads. Tcon from 4C12-treated donors were consistently enriched with an increased percentage and absolute numbers of Foxp3+CD4+ cells compared to Tcon from isotype-treated donors (25.6% vs. 12.6%, p The second approach was to treat lethally irradiated recipient Balb/c mice with 4C12 or isotype antibody intraperitoneally one hour after the injection of 5x10^6 T cell-depleted BM cells from wild-type B6 mice. Tcon (1x10^6) from CD45.1 luc+ B6 mice were injected on Day 2. Recipient mice treated with 4C12 had significantly lower GVHD-related score and better survival than isotype-treated recipients (n=5 for each group, p In summary, stimulation of Treg through DR3 resulted in enhanced number and function of Treg which was associated with less GVHD by either treating the donor mice or recipients at the time of transplant. This approach of Treg stimulation could serve as an alternative approach to ex vivo Treg expansion to enhance Treg function resulting in less morbidity and better survival with reduced GVHD risk. Together our data suggest that agonistic anti-DR3 antibody stimulation can effectively activate and expand Treg resulting in decreased acute GVHD in a murine GVHD model. Disclosures: No relevant conflicts of interest to declare.

CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-versus-host disease (GVHD). However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit monoclonal antibody against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25–30% of CD4+CD25+ Treg and a small population of conventional memory T cells. CD101+ Treg displayed greater in vitro suppression of alloantigen driven T cell proliferation as compared to CD101 negative (CD101-) Treg. In a model of GVHD induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101+ Treg, compared to CD101− Treg. Moreover, treatment with CD101+ Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.

CD4+CD25+ regulatory T cells (Treg) have been demonstrated to reduce the severity of acute graft-versus-host disease (aGvHD) in murine models of bone marrow transplantation. However, the surface molecules that are critical for suppression are unclear. The TNF-R superfamily member CD30 has been shown to be expressed on regulatory T cells that down-modulate nickel specific immune responses and to be relevant for Treg mediated protection from allograft rejection. Deficiency of the CD30 molecule (CD30−/−) is associated with impaired thymic negative selection and augmented T cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg function in an aGvHD model. Treg derived from CD30−/ − animals were significantly less effective in preventing aGvHD lethality (wt vs. CD30−/ − p=0.002). Signal intensity derived from expanding luciferase expressing alloreactive conventional T cells (Tconv) was significantly higher if CD30−/ − Treg as compared to wt Treg (p=0.007) were transferred as assessed by bioluminescencent based imaging. Blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb during the early (days −2 to +4) but not late (days +4 to +10) phase of adoptive Treg transfer reduced Treg mediated protection from proinflammatory cytokine accumulation and apoptosis of donor-type CD4 and CD8 T cells. In vivo bioluminescence imaging demonstrated intact Treg homing, but reduced expansion when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg increased with alloantigen exposure and CD153 expression on recipient-type dendritic cells increased in the presence of an irradiation induced proinflammatory environment but not when T cell depleted bone marrow and Tconv were transferred into non-irradiated Rag 2−/ −γc−/ − recipients. These data are the first to demonstrate that early CD30 signaling is relevant for Treg mediated aGvHD protection after major MHC mismatch bone marrow transplantation.