William L. Redmond

Distinct Requirements for Deletion versus Anergy during CD8 T Cell Peripheral Tolerance In Vivo

Activation of naive T cells by quiescent APCs results in tolerance through deletion and anergy. The underlying basis for these distinct fates is unclear. Using clone 4 TCR transgenic animals as a source of naive CD8 T cells, we examined the requirements for peripheral deletion in vivo. Our results demonstrate that independent of the amount of Ag used for stimulation, a single dose was insufficient to achieve complete clonal deletion. Instead, further antigenic exposure was required to completely eliminate all of the activated T cells. Additionally, consecutive stimulations with low doses of Ag were highly effective in promoting deletion. In contrast, although stimulation with high doses of Ag initially led to the apoptosis of many of the activated T cells, it induced hyporesponsiveness in a portion of the responding cells, thereby sparing them from further activation and deletion. These data explain why some conditions promote tolerance through clonal deletion whereas others promote anergy. Furthermore, these data provide a framework to devise protocols for effective deletion of potentially autoreactive T cells.

Cutting Edge: T Cell-Mediated Pathology in Murine Lyme Borreliosis

Even in the absence of an appropriate model or direct evidence, T cells have been hypothesized to exacerbate the manifestations of Lyme disease. To define definitely the role of T cells in Lyme disease, the course of disease in immunocompetent and B cell-deficient mice was compared. By 8 wk postinoculation, immunocompetent mice resolved both carditis and arthritis, whereas foci of myocarditis and severe destructive arthritis characterized disease of B cell-deficient mice. Cell transfer experiments using infected B6-Rag1 knock out mice demonstrated that: 1) innate immunity mediated the initial sequelae of infection, 2) transferring both naive T cells and B cells induced resolution of carditis and arthritis, 3) infected mice reconstituted with T cells developed myocarditis and severe destructive arthritis, and 4) CD4+ T cells were responsible for the observed immune-mediated pathology. These data demonstrate directly the deleterious effect of T cells in Lyme disease.

CD8+ T Cell Tolerance in Nonobese Diabetic Mice Is Restored by Insulin-Dependent Diabetes Resistance Alleles

Although candidate genes controlling autoimmune disease can now be identified, a major challenge that remains is defining the resulting cellular events mediated by each locus. In the current study we have used NOD-InsHA transgenic mice that express the influenza hemagglutinin (HA) as an islet Ag to compare the fate of HA-specific CD8+ T cells in diabetes susceptible NOD-InsHA mice with that observed in diabetes-resistant congenic mice having protective alleles at insulin-dependent diabetes (Idd) 3, Idd5.1, and Idd5.2 (Idd3/5 strain) or at Idd9.1, Idd9.2, and Idd9.3 (Idd9 strain). We demonstrate that protection from diabetes in each case is correlated with functional tolerance of endogenous islet-specific CD8+ T cells. However, by following the fate of naive, CFSE-labeled, islet Ag-specific CD8+ (HA-specific clone-4) or CD4+ (BDC2.5) T cells, we observed that tolerance is achieved differently in each protected strain. In Idd3/5 mice, tolerance occurs during the initial activation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes where CD25+ regulatory T cells (Tregs) effectively prevent their accumulation. In contrast, resistance alleles in Idd9 mice do not prevent the accumulation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes, indicating that tolerance occurs at a later checkpoint. These results underscore the variety of ways that autoimmunity can be prevented and identify the elimination of islet-specific CD8+ T cells as a common indicator of high-level protection.

Deletion of Naive CD8 T Cells Requires Persistent Antigen and Is Not Programmed by an Initial Signal from the Tolerogenic APC

Activation of naive CD8 T cells in vivo requires the recognition of cognate peptide-MHC complexes on APCs. Depending upon the activation status of the APC, such recognition will promote either a vigorous immune response or T cell tolerance and deletion. Recent studies suggest that the initial signals provided by APCs are sufficient to program the proliferation of naive CD8 T cells and their differentiation into effector cells. In this study, we sought to determine whether an initial encounter with tolerogenic APCs was sufficient to program deletion of naive CD8 T cells. Surprisingly, we find that regardless of whether naive CD8 T cells were stimulated by activated or quiescent APCs, transfer of the activated T cells into an Ag-free host was sufficient to ensure survival. Thus, although the extent of clonal expansion and development of effector function is determined by the activation status of the stimulatory APC, peripheral clonal deletion requires persistent Ag and is not determined by the initial stimulatory event.

B7-2 (CD86) Controls the Priming of Autoreactive CD4 T Cell Response against Pancreatic Islets

The B7-1/2-CD28 system provides the critical signal for the generation of an efficient T cell response. We investigated the role played by B7-2 in influencing pathogenic autoimmunity from islet-reactive CD4 T cells in B7-2 knockout (KO) NOD mice which are protected from type 1 diabetes. B7-2 deficiency caused a profound diminishment in the generation of spontaneously activated CD4 T cells and islet-specific CD4 T cell expansion. B7-2 does not impact the effector phase of the autoimmune response as adoptive transfer of islet Ag-specific BDC2.5 splenocytes stimulated in vitro could easily induce disease in B7-2KO mice. CD4 T cells showed some hallmarks of hyporesponsiveness because TCR/CD28-mediated stimulation led to defective activation and failure to induce disease in NODscid recipients. Furthermore, CD4 T cells exhibited enhanced death in the absence of B7-2. Interestingly, we found that B7-2 is required to achieve normal levels of CD4+CD25+CD62L+ T regulatory cells because a significant reduction of these T regulatory cells was observed in the thymus but not in the peripheral compartments of B7-2KO mice. In addition, our adoptive transfer experiments did not reveal either pathogenic or regulatory potential associated with the B7-2KO splenocytes. Finally, we found that the lack of B7-2 did not induce a compensatory increase in the B7-1 signal on APC in the PLN compartment. Taken together these results clearly indicate that B7-2 plays a critical role in priming islet-reactive CD4 T cells, suggesting a simplified, two-cell model for the impact of this costimulatory molecule in autoimmunity against islets.

The Apoptotic Pathway Contributing to the Deletion of Naive CD8 T Cells during the Induction of Peripheral Tolerance to a Cross-Presented Self-Antigen

The maintenance of T cell tolerance in the periphery proceeds through several mechanisms, including anergy, immuno-regulation, and deletion via apoptosis. We examined the mechanism underlying the induction of CD8 T cell peripheral tolerance to a self-Ag expressed on pancreatic islet β-cells. Following adoptive transfer, Ag-specific clone 4 T cells underwent deletion independently of extrinsic death receptors, including Fas, TNFR1, or TNFR2. Additional experiments revealed that the induction of clone 4 T cell apoptosis during peripheral tolerance occurred via an intrinsic death pathway that could be inhibited by overexpression of Bcl-2 or targeted deletion of the proapoptotic molecule, Bim, thereby resulting in accumulation of activated clone 4 T cells. Over-expression of Bcl-2 in clone 4 T cells promoted the development of effector function and insulitis whereas Bim−/− clone 4 cells were not autoaggressive. Examination of the upstream molecular mechanisms contributing to clone 4 T cell apoptosis revealed that it proceeded in a p53, E2F1, and E2F2-independent manner. Taken together, these data reveal that initiation of clone 4 T cell apoptosis during the induction of peripheral tolerance to a cross-presented self-Ag occurs through a Bcl-2-sensitive and at least partially Bim-dependent mechanism.

Preliminary analysis of immune responses in patients enrolled in a Phase II trial of Cyclophosphamide with Allogenic DRibble Vaccine Alone (DPV-001) or with GM-CSF or Imiquimod for adjuvant treatment of Stage IIIA or IIIB NSCLC

Preliminary analysis of immune responses in patients enrolled in a Phase II trial of cyclophosphamide with allogenic dribble vaccine alone (DPV-001) or with GM-CSF or imiquimod for adjuvant treatment of stage IIIa or IIIb NSCLC Rachel Sanborn, Brian Boulmay, Rui Li, Bradley Spieler, Kyle Happel, Christopher Paustian, Tarsem Mougdil, Zipei Feng, Christopher Dubay, Brenda Fisher, Yoshinobu Koguchi, Sandra Aung, Eileen Mederos, Carlo Bifulco, Michael McNamara, Keith S Bahjat, William Redmond, Augusto C Ochoa, Hong Ming Hu, Bernard Fox, Walter Urba, Traci Hilton

Galectin-3 inhibition using novel inhibitor GR-MD-02 improves survival and immune function while reducing tumor vasculature.

Immunosuppression and reduced cytolytic function of tumor-infiltrating lymphocytes are major obstacles to creating effective therapies for patients. It is known that Galectin-3 (Gal3), a lectin family member, is expressed and secreted by numerous cancers and immune cell subsets. Serum Gal3 expression is higher in patients with metastatic versus non-metastatic disease, and is associated with reduced survival in metastatic melanoma. Furthermore, Gal3 has been implicated in disease progression via the promotion of angiogenesis and metastasis. Interestingly, extracellular Gal3 inhibits the function of tumor-infiltrating lymphocytes (TIL) by associating with and restricting movement of the TCR. Gal3 has also been shown to promote M2 polarization in macrophages and mobilize myeloid cells from the bone marrow to promote a metastatic niche within the tumor. We hypothesized that Gal3 inhibition would improve TIL function while inhibiting the growth and metastasis of tumors. Through collaboration with Galectin Therapeutics, we tested Gal3 inhibition in cancer using the novel Gal3 inhibitor, GR-MD-02, with agonist anti-OX40 therapy. We observed that Gal3 inhibition with GR-MD-02 promoted antigen specific T cell expansion in vivo. Moreover, Gal3 inhibition/OX40 agonism improved survival in the MCA-205 sarcoma and 4T-1 mammary carcinoma models, and prolonged survival in the TRAMP-C1 prostate cancer model. Importantly, Gal3 inhibition/OX40 agonism reduced lung metastases in the 4T-1 model. Within the tumor, we observed an increase in the number of proliferating and IFNg-producing TIL. Gal3 inhibition/OX40 agonism was also associated with a decrease in functional tumor vasculature, as determined by CD31 staining by IHC within the tumors. These studies, along with clinical data from a liver fibrosis trial, supported testing GR-MD-02 in combination with ipilimumab in a Phase I trial for patients with metastatic melanoma.

A phase I/II study of pembrolizumab (P) with gemcitabine (G) in patients (Pts) with previously-treated advanced non-small cell lung cancer (NSCLC): Phase I safety results.

e20605Background: P, an anti-PD1 antibody active in advanced NSCLC, has optimal benefit in tumors with high PD-L1 expression. G may increase antigen cross-presentation and prime CD8 T cell responses via increased tumor cell apoptosis. The combination of antigen cross-presentation and T cell activation may have a synergistic effect with concurrent anti-PD1 immunotherapy. This study is evaluating P + G in pts with previously-treated advanced NSCLC with goal to establish feasible dosing prior to phase II evaluation. Methods: The phase I portion enrolled sequential cohorts of 2 pts each with advanced NSCLC (1-3 prior lines of therapy), regardless of PD-L1 status. Treatment consisted of G, 1250 mg/m2, Days (D) 1 & 8 + P 200 mg D 1; 21-day cycles. Pts received G to max 6 cycles, P to max 2 years. Each cohort closed until pts completed 6 weeks therapy (2 cycles). Dose limiting toxicity (DLT) was defined as Gr 4 hematologic or Gr 3/4 non-hematologic toxicity at least possibly treatment-related within the first 6 ...

Abstract 1604: NKTR-214 synergizes with radiotherapy to drive tumor regression

The purpose of this study was to investigate therapeutic and mechanistic synergies between single-dose radiotherapy and systemic administration of NKTR-214. NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol. NKTR-214 is designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Regulatory CD4 T cells. Preclinical models demonstrated NKTR-214 preferentially expands effector CD8+ T cells and NK cells within the tumor resulting in marked tumor growth suppression as a single-agent and in combination with checkpoint inhibitors. A phase I/II trial is in progress to evaluate NKTR-214 safety and efficacy in an outpatient setting. Radiation therapy can induce antigen-release and epitope spreading, while NKTR-214 can active and expand antigen-specific effector populations. We hypothesized that the combination of systemic NKTR-214 and local radiotherapy would generate better therapeutic responses than either treatment alone. In this study, we evaluated the combination of systemic NKTR-214 treatment with single fraction high-dose radiotherapy (20 Gy) in multiple murine models. We used flow cytometry, multi-spectral histology, and whole tumor mRNA profiling to investigate local, systemic and potential abscopal immune responses. We used Nur77-GFP reporter mice to enable detection of T cell receptor ligation in vivo and to evaluate the effects of NKTR-214+RT on tumor-reactive T cells. The results from these studies indicate that the combination of NKTR-214 and radiotherapy is synergistic, providing significantly better anti-tumor responses than either monotherapy. Consistent with previous observations, NKTR-214 alone induces expression of a wide range of activation markers expressed by CD4 and CD8 T cells as well as NK cells in the blood, lymph nodes and tumor. The combination of radiotherapy and NKTR-214 was found to have several unique effects including a significant increase (>75%) in the absolute numbers of lymphocytes in the peripheral blood, increased expression of activation markers (CD25, PD-1) by CD8 T cells in the blood and tumor, and increased density of tumor-infiltrating NK cells. Evaluation of tumor infiltrating lymphocytes (TIL) in Nur77-GFP reporter mice revealed that the combination of NKTR-214+RT resulted in a higher frequency of recently activated (Nur77-GFP+) CD8 T cells in treated (irradiated) and abscopal (non-irradiated) tumors. Whole tumor mRNA profiling and multi-spectral histology of these tumors is being assessed to identify key differences in the tumor-microenvironment that may help to define the underlying mechanism of action. Taken together, these data provide evidence of synergy between localized radiotherapy and systemic NKTR-214 treatment via an expansion of activated, tumor-specific CD8 T cells. Citation Format: Michael J. Mcnamara, Melissa Kasiewicz, Ian Hilgart-Martiszus, Ute Hoch, Deborah H. Charych, William L. Redmond. NKTR-214 synergizes with radiotherapy to drive tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1604. doi:10.1158/1538-7445.AM2017-1604