Justin Kline

Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8α+ dendritic cells

The generation of antitumor CD8+ T cell responses requires type I interferon responsiveness in host antigen-presenting cells

PD-1/PD-L1 interactions inhibit antitumor immune responses in a murine acute myeloid leukemia model

Negative regulatory mechanisms within the solid tumor microenvironment inhibit antitumor T-cell function, leading to evasion from immune attack. One inhibitory mechanism is up-regulation of programmed death-ligand 1 (PD-L1) expressed on tumor or stromal cells which binds to programmed death-1 (PD-1) on activated T cells. PD-1/PD-L1 engagement results in diminished antitumor T-cell responses and correlates with poor outcome in murine and human solid cancers. In contrast to available data in solid tumors, little is known regarding involvement of the PD-1/PD-L1 pathway in immune escape by hematopoietic cancers, such as acute myeloid leukemia (AML). To investigate this hypothesis, we used the murine leukemia, C1498. When transferred intravenously, C1498 cells grew progressively and apparently evaded immune destruction. Low levels of PD-L1 expression were found on C1498 cells grown in vitro. However, PD-L1 expression was up-regulated on C1498 cells when grown in vivo. PD-1(-/-) mice challenged with C1498 cells generated augmented antitumor T-cell responses, showed decreased AML burden in the blood and other organs, and survived significantly longer than did wild-type mice. Similar results were obtained with a PD-L1 blocking antibody. These data suggest the importance of the PD-1/PD-L1 pathway in immune evasion by a hematologic malignancy, providing a rationale for clinical trials targeting this pathway in leukemia patients.

CD47 Blockade Triggers T cell-mediated Destruction of Immunogenic Tumors

Macrophage phagocytosis of tumor cells mediated by CD47-specific blocking antibodies has been proposed to be the major effector mechanism in xenograft models. Here, using syngeneic immunocompetent mouse tumor models, we reveal that the therapeutic effects of CD47 blockade depend on dendritic cell but not macrophage cross-priming of T cell responses. The therapeutic effects of anti-CD47 antibody therapy were abrogated in T cell–deficient mice. In addition, the antitumor effects of CD47 blockade required expression of the cytosolic DNA sensor STING, but neither MyD88 nor TRIF, in CD11c+ cells, suggesting that cytosolic sensing of DNA from tumor cells is enhanced by anti-CD47 treatment, further bridging the innate and adaptive responses. Notably, the timing of administration of standard chemotherapy markedly impacted the induction of antitumor T cell responses by CD47 blockade. Together, our findings indicate that CD47 blockade drives T cell–mediated elimination of immunogenic tumors.

Geriatric assessment to predict survival in older allogeneic hematopoietic cell transplantation recipients

Allogeneic hematopoietic cell transplantation is increasingly utilized in older adults. This study prospectively evaluated the prognostic utility of geriatric assessment domains prior to allogeneic transplantation in recipients aged 50 years and over. Geriatric assessment was performed prior to transplant, and included validated measures across domains of function and disability, comorbidity, frailty, mental health, nutritional status, and systemic inflammation. A total of 203 patients completed geriatric assessment and underwent transplant. Median age was 58 years (range 50–73). After adjusting for established prognostic factors, limitations in instrumental activities of daily living (HR 2.38, 95%CI: 1.59–3.56; P<0.001), slow walk speed (HR 1.80, 95%CI: 1.14–2.83; P=0.01), high comorbidity by hematopoietic cell transplantation-specific comorbidity index (HR 1.56, 95%CI: 1.07–2.28; P=0.02), low mental health by short-form-36 mental component summary (HR 1.67, 95%CI: 1.13–2.48; P=0.01), and elevated serum C-reactive protein (HR 2.51, 95%CI: 1.54–4.09; P<0.001) were significantly associated with inferior overall survival. These associations were more pronounced in the cohort 60 years and over. Geriatric assessment measures confer independent prognostic utility in older allogeneic transplant recipients. Implementation of geriatric assessment prior to allogeneic transplantation may aid appropriate selection of older adults.

Impact of disease burden at time of allogeneic stem cell transplantation in adults with acute myeloid leukemia and myelodysplastic syndromes

Summary:The impact of disease burden on the outcome of patients with acute myeloid leukemia (AML) undergoing allogeneic stem cell transplantation (SCT) has not been well defined. Data from several retrospective series suggest that overt leukemia at the time of transplant increases the risk of relapse. We reviewed the outcomes of 68 consecutive adults with AML (n=60) or myelodysplastic syndromes (MDS) (n=8) who received an allogeneic SCT at the University of Chicago between May 1986 and October 2002 to confirm the importance of currently recognized risk factors for overall survival (OS) and progression-free survival (PFS). In addition, we wanted to determine whether quantification of residual disease by blast percentage or cytogenetic abnormalities at the time of SCT was correlated with outcome. AML subtypes based on the FAB classification were as follows: M0=9, M1=9, M2=16, M3=2, M4=16, M5=3, M6=5. Cytogenetic analysis was available from 52 patients. Using standard morphologic criteria, 34 patients were in complete remission (CR) and 34 had visible leukemia present. The majority of donors were HLA-identical siblings (n=55). In all, 56 patients received myeloablative conditioning regimens and 12 received a reduced-intensity, fludarabine-based conditioning regimen. OS and PFS times were 7.1 months (95% CI, 4.8–10.4) and 5.1 months (95% CI, 3.2–7.8), respectively. Median follow-up from SCT was 4.6 years (range, 0.6–17.0) for survivors. In multivariate analysis, the following factors were found to be associated with worse survival: (1) increased percentage of blasts in the bone marrow at the time of SCT, (2) presence of acute graft-versus-host disease, (3) mismatched donor, (4) Zubrod performance score of ⩾2, and (5) age ⩾45 years. We also found a trend towards improved outcome among patients in cytogenetic remission as compared to those who had residual cytogenetic abnormalities and those in overt relapse. These data support an association between pre-transplant disease burden and poor outcome after SCT.

Leukemia cell–targeted STAT3 silencing and TLR9 triggering generate systemic antitumor immunity

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and immune checkpoint commonly activated in cancer cells and in tumor-associated immune cells. We previously developed an immunostimulatory strategy based on targeted Stat3 silencing in Toll-like receptor 9 (TLR9)-positive hematopoietic cells using CpG-small interfering RNA (siRNA) conjugates. Here, we assessed the therapeutic effect of systemic STAT3 blocking/TLR9 triggering in disseminated acute myeloid leukemia (AML). We used mouse Cbfb-MYH11/Mpl-induced leukemia model, which mimics human inv(16) AML. Our results demonstrate that intravenously delivered CpG-Stat3 siRNA, but not control oligonucleotides, can eradicate established AML and impair leukemia-initiating potential. These antitumor effects require hosts effector T cells but not TLR9-positive antigen-presenting cells. Instead, CpG-Stat3 siRNA has direct immunogenic effect on AML cells in vivo upregulating major histocompatibility complex class-II, costimulatory and proinflammatory mediators, such as interleukin-12, while downregulating coinhibitory PD-L1 molecule. Systemic injections of CpG-Stat3 siRNA generate potent tumor antigen-specific immune responses, increase the ratio of tumor-infiltrating CD8(+) T cells to regulatory T cells in various organs, and result in CD8(+) T-cell-dependent regression of leukemia. Our findings underscore the potential of using targeted STAT3 inhibition/TLR9 triggering to break tumor tolerance and induce immunity against AML and potentially other TLR9-positive blood cancers.

Autologous graft-versus-host disease: harnessing anti-tumor immunity through impaired self-tolerance.

The absence of a graft-versus-malignancy (GVM) effect may be responsible for the higher relapse rate seen after autologous hematopoietic cell transplantation (auto-HCT) compared with allogeneic hematopoietic cell transplantation (allo-HCT). Acute GVHD developing after allo-HCT, however, is associated with significant morbidity and mortality. An autoimmune syndrome similar to acute GVHD has been reported to occur after auto-HCT and has been termed the ‘auto-aggression’ syndrome or autologous GVHD (auto-GVHD). Auto-GVHD tends to be milder than classical GVHD, most commonly involves the skin (rarely the gastrointestinal tract, liver or both) and often is self-limited. Auto-GVHD has been reported to occur both spontaneously and in subjects receiving post transplant immune modulation with CsA, IFN-γ or the combination. The development of auto-GVHD depends upon the derangement of self tolerance either through administration of post transplant CsA, depletion of regulatory T cells following the preparative chemoradiotherapy or both. Self-reactive CD8+ T cells paradoxically are able to recognize a self peptide antigen presented by MHC class II molecules and appear to mediate the syndrome. Many clinical trials have been performed using CsA with or without IFN-γ in an attempt to induce auto-GVHD. While many patients do indeed develop the syndrome, any associated anti-tumor effect remains questionable to date. New strategies to exploit auto-GVHD and enhance a GVM effect such as through the depletion of regulatory T cells or through use of newer immunomodulatory agents may improve the efficacy of auto-HCT.

Pre-transplant ganciclovir and post transplant high-dose valacyclovir reduce CMV infections after alemtuzumab-based conditioning

Alemtuzumab (Campath-1H)-based conditioning regimens are effective in preventing GVHD, but are associated with very high rates of cytomegalovirus (CMV) infection, a major limitation to their use. We evaluated 85 patients receiving conditioning with fludarabine 30 mg/m2/day (day −7 to day −3), alemtuzumab 20 mg/day (day −7 to day −3), and melphalan 140 mg/m2 on day −2. The initial patients received post transplant CMV prophylaxis with high-dose acyclovir. A very high incidence of CMV viremia was observed as has been commonly reported after alemtuzumab-based conditioning. Sixty-seven subsequent patients received pre-transplant ganciclovir and high-dose valacyclovir after engraftment. The cumulative incidence of CMV infection in the valacyclovir cohort was 29%. This compared favorably to the cumulative incidence of 53% in patients receiving only acyclovir (P=0.004) and to literature data. CMV prophylaxis with pre-transplant ganciclovir and high-dose valacyclovir after engraftment appears effective in preventing the excessive incidence of CMV infection after alemtuzumab-based conditioning regimens.

PD-1 regulates extrathymic regulatory T-cell differentiation

Treg cells and the programed death‐1/programed death ligand‐1 (PD‐1/PD‐L1) pathway are both critical for maintaining peripheral tolerance to self‐Ags. A significant subset of Treg cells constitutively expresses PD‐1, which prompted an investigation into the role of PD‐1/PD‐L1 interactions in Treg‐cell development, function, and induction in vivo. The phenotype and abundance of Treg cells was not significantly altered in PD‐1‐deficient mice. The thymic development of polyclonal and monospecific Treg cells was not negatively impacted by PD‐1 deficiency. The suppressive function of PD‐1−/− Treg cells was similar to their PD‐1+/+ counterparts both in vitro and in vivo. However, in three different in vivo experimental settings, PD‐1−/− conventional CD4+ T cells demonstrated a strikingly diminished tendency toward differentiation into peripherally induced Treg (pTreg) cells. Our results demonstrate that PD‐1 is dispensable for thymic Treg‐cell development and suppressive function, but is critical for the extrathymic differentiation of pTreg cells in vivo. These data suggest that Ab blockade of the PD‐1/PD‐L1 pathway may augment T‐cell responses by acting directly on conventional T cells, and also by suppressing the differentiation of pTreg cells.

Therapeutic Activity of High-Dose Intratumoral IFN-β Requires Direct Effect on the Tumor Vasculature

Endogenous type I IFN production after innate immune recognition of tumor cells is critical for generating natural adaptive immune responses against tumors in vivo. We recently have reported that targeting low doses of IFN-β to the tumor microenvironment using tumor-specific mAbs can facilitate antitumor immunity, which could be augmented further with PD-L1/PD-1 blockade. However, sustained high doses of type I IFNs in the tumor microenvironment, which are potently therapeutic alone, may function through distinct mechanisms. In the current report, we demonstrate that high-dose intratumoral type I IFNs indeed exerted a profound therapeutic effect in the murine B16 model, which unexpectedly did not increase T cell responses. Moreover, bone marrow chimeras revealed a role for type I IFN signaling on nonhematopoietic cells, and most of the therapeutic effect was retained in mice deficient in T, B, and NK cells. Rather, the tumor vasculature was ablated with high-dose intratumoral IFN-β, and conditional deletion of IFN-α/βR in Tie2-positive vascular endothelial cells eliminated most of the antitumor activity. Therefore, the major component of the antitumor activity of sustained high doses of type I IFNs occurs through a direct antiangiogenic effect. Our data help resolve conditions under which distinct antitumor mechanisms of type I IFNs are operational in vivo.