Robert N. Reger

Adoptively transferred effector cells derived from naïve rather than central memory CD8+ T cells mediate superior antitumor immunity

Effector cells derived from central memory CD8+ T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve, rather than central memory T cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1− phenotype, naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8+ T cells may allow superior efficacy upon adoptive transfer.

Th17 Cells Are Long Lived and Retain a Stem Cell-like Molecular Signature

Th17 cells have been described as short lived, but this view is at odds with their capacity to trigger protracted damage to normal and transformed tissues. We report that Th17 cells, despite displaying low expression of CD27 and other phenotypic markers of terminal differentiation, efficiently eradicated tumors and caused autoimmunity, were long lived, and maintained a core molecular signature resembling early memory CD8(+) cells with stem cell-like properties. In addition, we found that Th17 cells had high expression of Tcf7, a direct target of the Wnt and β-catenin signaling axis, and accumulated β-catenin, a feature observed in stem cells. In vivo, Th17 cells gave rise to Th1-like effector cell progeny and also self-renewed and persisted as IL-17A-secreting cells. Multipotency was required for Th17 cell-mediated tumor eradication because effector cells deficient in IFN-γ or IL-17A had impaired activity. Thus, Th17 cells are not always short lived and are a less-differentiated subset capable of superior persistence and functionality.

IL-12 triggers a programmatic change in dysfunctional myeloid-derived cells within mouse tumors.

Solid tumors are complex masses with a local microenvironment, or stroma, that supports tumor growth and progression. Among the diverse tumor-supporting stromal cells is a heterogeneous population of myeloid-derived cells. These cells are alternatively activated and contribute to the immunosuppressive environment of the tumor; overcoming their immunosuppressive effects may improve the efficacy of cancer immunotherapies. We recently found that engineering tumor-specific CD8(+) T cells to secrete the inflammatory cytokine IL-12 improved their therapeutic efficacy in the B16 mouse model of established melanoma. Here, we report the mechanism underlying this finding. Surprisingly, direct binding of IL-12 to receptors on lymphocytes or NK cells was not required. Instead, IL-12 sensitized bone marrow-derived tumor stromal cells, including CD11b(+)F4/80(hi) macrophages, CD11b(+)MHCII(hi)CD11c(hi) dendritic cells, and CD11b(+)Gr-1(hi) myeloid-derived suppressor cells, causing them to enhance the effects of adoptively transferred CD8(+) T cells. This reprogramming of myeloid-derived cells occurred partly through IFN-γ. Surprisingly, direct presentation of antigen to the transferred CD8(+) T cells by tumor was not necessary; however, MHCI expression on host cells was essential for IL-12-mediated antitumor enhancements. These results are consistent with a model in which IL-12 enhances the ability of CD8(+) T cells to collapse large vascularized tumors by triggering programmatic changes in otherwise suppressive antigen-presenting cells within tumors and support the use of IL-12 as part of immunotherapy for the treatment of solid tumors.

Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

T-cell-based immunotherapies can be effective in the treatment of large vascularized tumors, but they rely on adoptive transfer of substantial numbers ( approximately 20 million) of tumor-specific T cells administered together with vaccination and high-dose interleukin (IL)-2. In this study, we report that approximately 10,000 T cells gene-engineered to express a single-chain IL-12 molecule can be therapeutically effective against established tumors in the absence of exogenous IL-2 and vaccine. Although IL-12-engineered cells did not perist long-term in hosts, they exhibited enhanced functionality and were detected in higher numbers intratumorally along with increased numbers of endogenous natural killer and CD8(+) T cells just before regression. Importantly, transferred T cells isolated from tumors stably overproduced supraphysiologic amounts of IL-12, and the therapeutic effect of IL-12 produced within the tumor microenvironment could not be mimicked with high doses of exogenously provided IL-12. Furthermore, antitumor effects could be recapitulated by engineering wild-type open-repertoire splenocytes to express both the single-chain IL-12 and a recombinant tumor-specific T-cell receptor (TCR), but only when individual cells expressed both the TCR and IL-12, indicating that arrested migration of T cells at the tumor site was required for their activities. Successful tumor eradication was dependent on a lymphodepleting preconditioning regimen that reduced the number of intratumoral CD4(+) Foxp3(+) T regulatory cells. Our findings reveal an approach to genetically modify T cells to reduce the cell number needed, eliminate the need for vaccines or systemic IL-2, and improve immunotherapy efficacy based on adoptive transfer of gene-engineered T cells.

Repression of the DNA-binding inhibitor Id3 by Blimp-1 limits the formation of memory CD8+ T cells

The transcriptional repressor Blimp-1 promotes the differentiation of CD8+ T cells into short-lived effector cells (SLECs) that express the lectin-like receptor KLRG-1, but how it operates remains poorly defined. Here we show that Blimp-1 bound to and repressed the promoter of the gene encoding the DNA-binding inhibitor Id3 in SLECs. Repression of Id3 by Blimp-1 was dispensable for SLEC development but limited the ability of SLECs to persist as memory cells. Enforced expression of Id3 was sufficient to restore SLEC survival and enhanced recall responses. Id3 function was mediated in part through inhibition of the transcriptional activity of E2A and induction of genes regulating genome stability. Our findings identify the Blimp-1–Id3–E2A axis as a key molecular switch that determines whether effector CD8+ T cells are programmed to die or enter the memory pool.

Checkpoint Inhibition of KIR2D with the Monoclonal Antibody IPH2101 Induces Contraction and Hyporesponsiveness of NK-cells in Patients with Myeloma

Purpose: Immune checkpoint inhibitors have recently revolutionized cancer immunotherapy. On the basis of data showing KIR-ligand mismatched natural killer (NK) cells reduce the risk of leukemia and multiple myeloma relapse following allogeneic hematopoietic stem cell transplantation, investigators have developed a checkpoint inhibition antibody that blocks KIR on NK cells. Although in vitro studies suggest the KIR2D-specific antibody IPH2101 induces KIR-ligand mismatched tumor killing by NK cells, our single-arm phase II clinical trial in patients with smoldering multiple myeloma was prematurely terminated due to lack of clinical efficacy. This study aimed at unveiling the underlying mechanisms behind the lack of clinical efficacy. Experimental Design: Treatment-naïve patients received an intravenous infusion of 1 mg/kg IPH2101 every other month for up to a year. Peripheral blood was collected at baseline and 24 hours after first infusion, followed by weekly samples for the first month and monthly samples thereafter. NK cell phenotype and function was analyzed using high-resolution flow cytometry. Results: Unexpectedly, infusion of IPH2101 resulted in rapid reduction in both NK cell responsiveness and KIR2D expression on the NK cell surface. In vitro assays revealed KIR2D molecules are removed from the surface of IPH2101-treated NK cells by trogocytosis, with reductions in NK cell function directly correlating with loss of free KIR2D surface molecules. Although IPH2101 marginally augmented the antimyeloma cytotoxicity of remaining KIR2Ddull patient NK cells, the overall response was diminished by significant contraction and reduced function of KIR2D-expressing NK cells. Conclusions: These data raise concerns that the unexpected biological events reported in this study could compromise antibody-based strategies designed at augmenting NK cell tumor killing via checkpoint inhibition. Clin Cancer Res; 22(21); 5211–22. ©2016 AACR. See related commentary by Felices and Miller, p. 5161

Effect of high-dose plerixafor on CD34+ cell mobilization in healthy stem cell donors: results of a randomized crossover trial

Hematopoietic stem cells can be mobilized from healthy donors using single-agent plerixafor without granulocyte colony-stimulating factor and, following allogeneic transplantation, can result in sustained donor-derived hematopoiesis. However, when a single dose of plerixafor is administered at a conventional 240 μg/kg dose, approximately one-third of donors will fail to mobilize the minimally acceptable dose of CD34+ cells needed for allogeneic transplantation. We conducted an open-label, randomized trial to assess the safety and activity of high-dose (480 μg/kg) plerixafor in CD34+ cell mobilization in healthy donors. Subjects were randomly assigned to receive either a high dose or a conventional dose (240 μg/kg) of plerixafor, given as a single subcutaneous injection, in a two-sequence, two-period, crossover design. Each treatment period was separated by a 2-week minimum washout period. The primary endpoint was the peak CD34+ count in the blood, with secondary endpoints of CD34+ cell area under the curve (AUC), CD34+ count at 24 hours, and time to peak CD34+ following the administration of plerixafor. We randomized 23 subjects to the two treatment sequences and 20 subjects received both doses of plerixafor. Peak CD34+ count in the blood was significantly increased (mean 32.2 versus 27.8 cells/μL, P=0.0009) and CD34+ cell AUC over 24 hours was significantly increased (mean 553 versus 446 h cells/μL, P<0.0001) following the administration of the 480 μg/kg dose of plerixafor compared with the 240 μg/kg dose. Remarkably, of seven subjects who mobilized poorly (peak CD34+ ≤20 cells/μL) after the 240 μg/kg dose of plerixafor, six achieved higher peak CD34+ cell numbers and all achieved higher CD34+ AUC over 24 hours after the 480 μg/kg dose. No grade 3 or worse drug-related adverse events were observed. This study establishes that high-dose plerixafor can be safely administered in healthy donors and mobilizes greater numbers of CD34+ cells than conventional-dose plerixafor, which may improve CD34+ graft yields and reduce the number of apheresis procedures needed to collect sufficient stem cells for allogeneic transplantation. (ClinicalTrials.gov, identifier: NCT00322127)

Efficient mRNA-Based Genetic Engineering of Human NK Cells with High-Affinity CD16 and CCR7 Augments Rituximab-Induced ADCC against Lymphoma and Targets NK Cell Migration toward the Lymph Node-Associated Chemokine CCL19

For more than a decade, investigators have pursued methods to genetically engineer natural killer (NK) cells for use in clinical therapy against cancer. Despite considerable advances in viral transduction of hematopoietic stem cells and T cells, transduction efficiencies for NK cells have remained disappointingly low. Here, we show that NK cells can be genetically reprogramed efficiently using a cGMP-compliant mRNA electroporation method that induces rapid and reproducible transgene expression in nearly all transfected cells, without negatively influencing their viability, phenotype, and cytotoxic function. To study its potential therapeutic application, we used this approach to improve key aspects involved in efficient lymphoma targeting by adoptively infused ex vivo-expanded NK cells. Electroporation of NK cells with mRNA coding for the chemokine receptor CCR7 significantly promoted migration toward the lymph node-associated chemokine CCL19. Further, introduction of mRNA coding for the high-affinity antibody-binding receptor CD16 (CD16-158V) substantially augmented NK cell cytotoxicity against rituximab-coated lymphoma cells. Based on these data, we conclude that this approach can be utilized to genetically modify multiple modalities of NK cells in a highly efficient manner with the potential to improve multiple facets of their in vivo tumor targeting, thus, opening a new arena for the development of more efficacious adoptive NK cell-based cancer immunotherapies.

Documenting stress in caregivers of transplantation patients: initial evidence of HPA dysregulation.

Abstract There is growing evidence linking caregiver stress with an increased risk for morbidity and mortality. While the emotional and practical burden experienced by caregivers is well established, the physiological changes that may affect the caregiver’s health are less understood. This study sought to compare self-reported stress, anxiety and depression along with neuroendocrine and immune markers of stress among adult caregivers of allogeneic hematopoietic stem cell transplantation patients during the acute transplant recovery period to matched non-caregivers controls. Biomarkers and self-reported data were collected at three points during the patient’s HSCT: (1) before transplant, (2) after initial transplantation discharge (±7 days) and (3) 6 weeks after initial transplantation discharge. Mixed linear modeling was used to examine differences by group and time. Twenty-one caregivers and 20 controls completed all study procedures. The majority of caregivers were female (57% or 57.1%) and married (95.2%), with a mean age of 52 ± 11.4 years. Caregiver perceived stress, anxiety and depression scores were significantly higher than controls (p < 0.001) with effect sizes (ES) ranging from 1.37 to 1.80 and they did not change over time (p > 0.05) for either group. Caregivers had significantly lower serum cortisol levels than controls at both discharge (p = 0.013; ES = 0.81) and 6 weeks after discharge (p = 0.028; ES = 0.72) but exhibited no significant relationship between self-reported stress and serum cortisol. In addition, caregivers showed a significant inverse relationship between stress and epinephrine levels (rs=−0.654, p = 0.021). These findings support the evidence of the caregiving experience being stressful. The counter-intuitive relationship between cortisol and epinephrine might suggest dysregulation of the HPA axis and central nervous system but additional research on the physiological impact of caregiving is warranted.

Allogeneic transplantation using CD34+selected peripheral blood progenitor cells combined with non-mobilized donor T cells for refractory severe aplastic anaemia

Allogeneic haematopoietic stem cell transplantation is curative for severe aplastic anaemia (SAA) unresponsive to immunosuppressive therapy. To reduce chronic graft‐versus‐host disease (GVHD), which occurs more frequently after peripheral blood stem cell (PBSC) transplantation compared to bone‐marrow transplantation (BMT), and to prevent graft rejection, we developed a novel partial T‐cell depleted transplant that infuses high numbers of granulocyte colony‐stimulating factor‐mobilized CD34+ selected PBSCs combined with a BMT‐equivalent dose of non‐mobilized donor T‐cells. Fifteen patients with refractory SAA received cyclophosphamide, anti‐thymocyte globulin and fludarabine conditioning, and were transplanted with a median 8 × 106 CD34+ cells/kg and 2 × 107 non‐mobilized CD3+ T‐cells/kg from human leucocyte antigen‐matched sibling donors. All achieved sustained engraftment with only two developing acute and two developing chronic GVHD. With a 3·5‐year median follow‐up, 86% of patients survived and were transfusion‐independent. When compared to a retrospective cohort of 56 bone‐marrow failure patients that received the identical transplant preparative regimen and GVHD prophylaxis with the exception that the allograft contained unmanipulated PBSCs, partial T‐cell depleted transplant recipients had delayed donor T‐cell chimerism and relative reduction of 75% in the incidence of acute grade II‐IV GVHD (13% vs. 52%; P = 0·010) and of 82% in chronic GVHD (13% vs. 72%; P = 0·0004). In multivariate analysis, partial T‐cell depleted transplants remained significantly associated with a reduced risk of GVHD. In conclusion, for patients with refractory SAA, this novel transplant strategy achieves excellent engraftment and survival when compared to unmanipulated PBSC transplants and dramatically reduces the incidence of both acute and chronic GVHD.