Kai Sun

IFN-γ mediates CD4 + T-cell loss and impairs secondary antitumor responses after successful initial immunotherapy

Protective cell-mediated immune responses in cancer are critically dependent on T-helper type 1 (TH1) cytokines such as interferon-γ (IFN-γ). We have previously shown that the combination of CD40 stimulation and interleukin-2 (IL-2) leads to synergistic antitumor responses in several models of advanced metastatic disease. We now report that after this treatment and other immunotherapy regimens, the CD4+ T-cell population, in contrast to CD8+ T cells, did not significantly increase but rather exhibited a substantial level of apoptosis that was dependent on IFN-γ. Mice immunized with tumor cells and treated with an immunotherapy regimen that was initially protective were later unable to mount effective memory responses compared with immunized mice not receiving immunotherapy. Immunotherapy given to tumor-bearing Ifngr−/− mice resulted in restoration of secondary responses. Thus, although immunotherapeutic regimens inducing strong IFN-γ responses can lead to successful early antitumor efficacy, they may also impair the development of durable antitumor responses.

Differential effects of donor T-cell cytokines on outcome with continuous bortezomib administration after allogeneic bone marrow transplantation

Dissociating graft-versus-tumor (GVT) effect from acute graft-versus-host disease (GVHD) still remains a great challenge in allogeneic bone marrow transplantation (allo-BMT). Bortezomib, a proteasome inhibitor, has shown impressive efficacy as a single agent in patients with hematologic malignancies but can result in toxicity when administered late after allogeneic transplantation in murine models of GVHD. In the current study, the effects of T-cell subsets and their associated cytokines on the efficacy of bortezomib in murine allogeneic BMT were investigated. Increased levels of serum tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) were observed after allo-BMT and continuous bortezomib administration. Bortezomib-induced GVHD-dependent mortality was preventable by depletion of CD4(+) but not CD8(+) T cells from the donor graft. The improved survival correlated with markedly reduced serum TNFalpha but not IFNgamma levels. Transfer of Tnf(-/-) T cells also protected recipients from bortezomib-induced GVHD-dependent toxicity. Importantly, prolonged administration of bortezomib after transplantation of purified CD8(+) T cells resulted in enhanced GVT response, which was dependent on donor CD8(+) T cell-derived IFNgamma. These results indicate that decreased toxicity and increased efficacy of bortezomib in murine allo-BMT can be achieved by removal of CD4(+) T cells from the graft or by inhibiting TNFalpha.

Immunomodulation and pharmacological strategies in the treatment of graft-versus-host disease

Background: Allogeneic hematopoietic stem cell transplantation offers great promise for the treatment of a variety of diseases including malignancies and other diseases of hematopoietic origin. However, morbidity and mortality due to graft-versus-host disease (GVHD) remain a major barrier to its application. Objective: This review will provide an overview of the pathophysiology of GVHD and discuss the recent advances in GVHD management in both preclinical and clinical studies. Methods: An extensive literature search on PubMed from 1995 to 2008 was performed. Results/conclusion: There has been much progress in our understanding of GVHD and finding new means to control acute GVHD. While these approaches hold promise, as yet none has been able to replace the standard methods we may use routinely to decrease the incidence of the condition.

Ex vivo Inhibition of NF-κB Signaling in Alloreactive T-cells Prevents Graft-versus-host Disease

The ex vivo induction of alloantigen‐specific hyporesponsiveness by costimulatory pathway blockade or exposure to immunoregulatory cytokines has been shown to inhibit proliferation, IL‐2 production, and the graft‐versus‐host disease (GVHD) capacity of adoptively transferred T‐cells. We hypothesized that inhibition of the intracellular NF‐κB pathway in alloreactive T‐cells, which is critical for T‐cell activation events including IL‐2 transcription, could lead to alloantigen hyporesponsiveness and loss of GVHD capacity. We demonstrate that treatment of mixed lymphocyte reaction (MLR) cultures with PS1145, a potent inhibitor of NF‐κB activation, can induce T‐cell hyporesponsiveness to alloantigen in primary and secondary responses while preserving in vitro responses to potent mitogenic stimulation. GVHD lethality in recipients of ex vivo PS1145‐treated cells was profoundly inhibited. Parking of control or PS1145‐treated MLR cells in syngeneic Rag−/− recipients resulted in intact contact hypersensitivity (CHS) responses. However, GVHD lethality capacity also was restored, suggesting that lymphopenic expansion uncoupled alloantigen hyporesponsiveness. These results indicate that the NF‐κB pathway is a critical regulator of alloresponses and provide a novel small molecule inhibitor based approach that is effective in preventing early posttransplant GVHD lethality but that also permits donor T‐cell responses to recover after a period of lymphopenic expansion.

In Vivo Models for the Study of Graft-vs-Host Disease and Graft-vs-Tumor Effects

While nonprimate studies of graft-vs-host disease (GVHD) most commonly use rodent models, the field of allogeneic transplantation and treatment of GVHD also owes much to its study in dogs. Donnall Thomas, in collaboration with other investigators at Fred Hutchinson Cancer Center, pioneered the field of allogeneic bone marrow transplantation with his work in beagles. The use of outbred animals requires detailed knowledge of the major histocompatibility complex (MHC) complex for these species and the availability of reagents for tissue typing. More recently, the miniature swine model has been added to the repertoire of models available to investigators. While this model has not gained prominence with investigators of GVHD, it is used to study mechanisms of tolerance in allogeneic and xenogeneic hematopoietic cell and/or organ transplantation. The swine model has advantages for translational work because its body size and composition is comparable to humans Likewise, the sheep model has been used to study issues of tolerance in xenogeneic hematopoietic cell transplantation.

Donor HSCs with a preexisting ASXL1-mutation led to the development of FLT3-ITD positive AML in the donor and FLT3-ITD negative AML in the recipient after unrelated transplant

Recently, it has been shown that hematopoietic stem cells (HSCs) acquire somatic mutations over time, most commonly involving DNMT3A, ASXL1, and TET2 genes [1]. These mutation-bearing stem cells contribute to normal hematopoiesis harbor a selective growth advantage compared to their non-mutated normal stem/progenitor counterparts, leading to the development of clonal hematopoiesis, and can subsequently gain additional mutations that confer full malignant potential [1–3]. Although normal individuals with clonal hematopoiesis are at increased risk of developing hematologic malignancy, there are fewer studies to directly demonstrate pre-leukemic clone carrying a single epigenetic founder mutation can promote evolution to AML in vivo. In some patients, donor HSCs may contribute to the development of acute leukemia in the recipient. This is called donor cell leukemia (DCL) and almost always the leukemia is AML [4, 5]. Hahn et al. [6] reported that a pre-leukemic clone carrying a single epigenetic founder mutation (DNMT3AR882H) was transplanted from an apparently healthy marrow donor to his brother. After transplant, the sibling donor first developed a normal karyotype AML with mutations in DNMT3A, NPM1, and FLT3, and 4 months later, the recipient developed a normal karyotype AML with mutations in DNMT3A and NPM1, but wild-type FLT3 [6]. This report presents direct evidence that HSCs carrying a single pre-leukemic mutation can evolve to AML [6]. Here, we report an unusual case. A female patient underwent successful, unrelated allogeneic hematopoietic stem cell transplantation (allo-HSCT) for B-ALL (Supplementary Fig. I). Twenty months after transplant of donor HSCs with a preexisting ASXL1-mutation, the recipient was diagnosed with FLT3-ITD-negative AML (Supplementary Fig. I). In addition, 84 months after transplant (64 months after the recipient was diagnosed with AML), the donor developed FLT3-ITD-positive AML carrying the same ASXL1-mutation (Supplementary Fig. I). Before allo-HSCT, the recipient and the donor gave their written informed consent for “potential usage of specimens for medical research” in accordance with the Declaration of Helsinki. The 28-year-old female was diagnosed with B-ALL by classical morphology and immunophenotype (Supplementary Fig. I). Cytogenetic evaluation showed a normal 46, XX. The patient achieved CR with induction therapy. After consolidation therapy, an allo-HSCT from an HLAidentical unrelated donor (33-year-old male, donorrecipient matched by high resolution HLA typing at HLAA, -B, -C, DRB1, and DQB1, 10/10 matches) was performed (Supplementary Table 1). The recipient received conditioning with busulfan, 4 mg/kg/day orally for 3 days; cyclophosphamide, 50 mg/kg/day for 2 days; cytarabine, 2 g/m/day for 1 day; semustine, 250 mg/m/day orally for 1 * Kai Sun sunkai@cellscience.org

Low Total Body Irradiation (TBI) Augments the Synergistic Anti-Tumor Effects of Anti-CD40 and IL2 Immunotherapy

Anti-Tumor Effects of Anti-CD40 and IL2 Immunotherapy Kai Sun, Lisbeth A Welniak, Robert Wiltrout, Bruce R Blazar, William J Murphy. Department of Microbiology and Immunology, University of Nevada, Reno, NV; Division of Basic Research, Nevada Cancer Institute, Las Vegas, NV; Laboratory of Experimental Immunology, NCI at Frederick, Frederick, MD; Division of Bone Marrow Transplantation, University of Minnesota Cancer Center, Minneapolis, MN. We have previously demonstrated that agonist antibodies to CD40, when given with IL2, results in synergistic anti-tumor effects in mice bearing advanced metastatic renal cell carcinomas. This combination was shown to result in significant increases in dendritic cells and tumor-specific CD8+ T cell responses. However, this synergy necessitated the use of relatively large amounts of IL2, but unless this was administered, no anti-tumor effects were observed in a model of significant Renca tumor burden at the time of treatment. It has previously been demonstrated that prior TBI of tumor-bearing recipients can augment the anti-tumor effects of CD40 stimulation. In this study, we assessed the anti-tumor effects of CD40 stimulation with IL2 in tumor-bearing mice given low dose TBI with the hypothesis that activation of Toll-like receptors and induction of inflammatory cytokines will enhance the ability of anti-CD40 to promote dendritic cell development and function. Mice were given Renca intravenously to localize the tumor to the lung. After four days the mice were treated with 100 ug anti-CD40 (FGK115) and 1 million units IL2 with or without low dose TBI (200 cGy). This TBI dose is much lower than other studies in which TBI was given with CD40 stimulus. AntiCD40 and IL2 administration was repeated on day 7 post-TBI followed by anti-CD40 and 500,000 units IL-2 on days 11–13 post-TBI. The results demonstrated that using this tumor burden and regimen, modest but significant anti-tumor effects were observed when anti-CD40 and IL2 were used. Administration of TBI with anti-CD40 or IL2 given singly gave no protective effects when compared to anti-CD40 and IL2 without TBI. The addition of low dose TBI with anti-CD40 and IL2 resulted in significantly enhanced protection. Importantly, the addition of low dose TBI also allowed for much lower amounts of IL2 to be administered and still result in significant protection. No toxicities were observed after this regimen. Thus, the combination of low dose TBI can markedly enhance the synergy observed when CD40 stimulation is administered with IL2 and allow for lower amounts of IL2 to be administered. This results in less toxicity, which can be observed when high amounts of IL2 are given, as well as provide even greater anti-tumor effects presumably through greater activation of the dendritic cell compartments. Supported in part by RO1CA95572 and NCI/NIH Contract NO1-CO-12400.