Deleterious Changes To The T Cell Compartment Following Immunotherapy

Abstract: The combination of anti-CD40 and interleukin-2 is a potent immunotherapy regimen that results in synergistic anti-tumor responses. This has been demonstrated in multiple murine tumor models of metastatic disease with various tumor types. The primary anti-tumor responses elicited by this combination are capable of inducing tumor regression and prolonged survival. However, the generation of secondary T cell responses after irradiated tumor vaccine is abrogated after anti-CD40 and IL-2. This abrogation also occurs after other immunotherapeutic approaches that prompt the production of large amounts of interferon-gamma (IFNγ). These observations correlated with a significant skewing of the T cell compartment. First, we observed a selective decreased of conventional CD4+ T cells following immunotherapy. Second, we observed a more than five fold expansion of memory phenotype cells which were incapable of generating responses to new antigens. The data presented here suggest that despite initial tumor regression, potent systemic immunotherapy may impair responses to new immunological challenges.Selective CD4+ T cell death after immunotherapy results in an alteration in the ratio of CD4+ T cells to CD8+ T cells and impairs the generation of a secondary immune response. Our data suggest that this phenomenon after immunotherapy is the result of the selective upregulation of programmed death-1 (PD-1) and its IFNγ responsive ligand, B7-H1. We show that the expression of PD-1 is restricted to the surface of Foxp3neg CD4+ T cells and that CD8+ T cells and CD4+ Foxp3+ regulatory T cells remain PD-1 low after immunotherapy. Furthermore, the expression of PD-1 correlates with CD4+ T cell death after immunotherapy. In the absence of IFNγ either by the use of mice lacking IFNγ (IFNγ-/-) or the receptor for IFNγ (IFNγR-/-), B7-H1 remains low after immunotherapy. Subsequently, CD4+ T cells expand in response to immunotherapy in the absence of IFNγ responsive B7-H1. We observed a significant expansion of memory phenotype T cells after cytokine based immunotherapy which correlated with impairment of proliferative responses to new antigens. Memory T cells are more sensitive to cytokine stimulation than naïve T cells. Therefore, we used a young thymectomized mouse model to determine if pre-existing memory T cells were preferentially expanded by immunotherapy. The thymectomized mouse model allowed us to evaluate long term T cell responses to immunotherapy in the absence of de novo T cell generation. Using this model, we observed expansion of memory T cells, within both the CD4+ and CD8+ T cell compartments without a major sacrifice of the size of the naïve T cell compartment. Compared to memory T cell expansion, there was relatively small change in the naïve T cell compartment. Naïve CD8+ T cell numbers were unchanged by immunotherapy and naïve CD4+ T cells were decreased by less than half. Memory T cells were still significantly expanded after 30 days of rest. Furthermore, the persistent expansion of memory T cells correlated with a maintained decrease in proliferative function to new antigens. Taken together, these data demonstrate a long term consequence of immunotherapy to the phenotypic makeup and, importantly, the function of the T cell compartment

Clinical Cancer Therapy by NK Cells via Antibody-Dependent Cell-Mediated Cytotoxicity

Natural killer (NK) cells are powerful effector cells that can be directed to eliminate tumor cells through tumor-targeted monoclonal antibodies (mAbs). Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malignancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs) designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumor-targeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination immunotherapy regimens to pursue for distinct clinical settings

Increasing the Clinical Efficacy of NK and Antibody-Mediated Cancer Immunotherapy: Potential Predictors of Successful Clinical Outcome Based on Observations in High-Risk Neuroblastoma

Disease recurrence is frequent in high-risk neuroblastoma (NBL) patients even after multi-modality aggressive treatment [a combination of chemotherapy, surgical resection, local radiation therapy, autologous stem cell transplantation, and cis-retinoic acid (CRA)]. Recent clinical studies have explored the use of monoclonal antibodies (mAbs) that bind to disialoganglioside (GD2), highly expressed in NBL, as a means to enable immune effector cells to destroy NBL cells via antibody-dependent cell-mediated cytotoxicity (ADCC). Preclinical data indicate that ADCC can be more effective when appropriate effector cells are activated by cytokines. Clinical studies have pursued this by administering anti-GD2 mAb in combination with ADCC-enhancing cytokines (IL2 and GM-CSF), a regimen that has demonstrated improved cancer-free survival. More recently, early clinical studies have used a fusion protein that consists of the anti-GD2 mAb directly linked to IL2, and anti-tumor responses were seen in the Phase II setting. Analyses of genes that code for receptors that influence ADCC activity and natural killer (NK) cell function [Fc receptor (FcR), killer immunoglublin-like receptor (KIR), and KIR-ligand (KIR-L)] suggest patients with anti-tumor activity are more likely to have certain genotype profiles. Further analyses will need to be conducted to determine whether these genotypes can be used as predictive markers for favorable therapeutic outcome. In this review, we discuss factors that affect response to mAb-based tumor therapies such as hu14.18-IL2. Many of our observations have been made in the context of NBL; however, we will also include some observations made with mAbs targeting other tumor types that are consistent with results in NBL. Therefore, we hypothesize that the NBL observations discussed here may also be relevant to mAb therapy for other cancers, in which ADCC is known to play a role

Out-of-Sequence Signal 3 Paralyzes Primary CD4+ T-Cell-Dependent Immunity

SummaryPrimary T cell activation involves the integration of three distinct signals delivered in sequence: (1) antigen recognition, (2) costimulation, and (3) cytokine-mediated differentiation and expansion. Strong immunostimulatory events such as immunotherapy or infection induce profound cytokine release causing “bystander” T cell activation, thereby increasing the potential for autoreactivity and need for control. We show that during strong stimulation, a profound suppression of primary CD4+ T-cell-mediated immune responses ensued and was observed across preclinical models and patients undergoing high-dose interleukin-2 (IL-2) therapy. This suppression targeted naive CD4+ but not CD8+ T cells and was mediated through transient suppressor of cytokine signaling-3 (SOCS3) inhibition of the STAT5b transcription factor signaling pathway. These events resulted in complete paralysis of primary CD4+ T cell activation, affecting memory generation and induction of autoimmunity as well as impaired viral clearance. These data highlight the critical regulation of naive CD4+ T cells during inflammatory conditions

Aging predisposes to acute inflammatory induced pathology after tumor immunotherapy.

Cancer commonly occurs in the elderly and immunotherapy (IT) is being increasingly applied to this population. However, the majority of preclinical mouse tumor models assessing potential efficacy and toxicities of therapeutics use young mice. We assessed the impact of age on responses to systemic immune stimulation. In contrast to young mice, systemic cancer IT regimens or LPS given to aged mice resulted in rapid and lethal toxicities affecting multiple organs correlating with heightened proinflammatory cytokines systemically and within the parenchymal tissues. This inflammatory response and increased morbidity with age was independent of T cells or NK cells. However, prior in vivo depletion of macrophages in aged mice resulted in lesser cytokine levels, increased survival, and decreased liver histopathology. Furthermore, macrophages from aged mice and normal human elderly volunteers displayed heightened TNF and IL-6 production upon in vitro stimulation. Treatment of both TNF knockout mice and in vivo TNF blockade in aged mice resulted in significant increases in survival and lessened pathology. Importantly, TNF blockade in tumor-bearing, aged mice receiving IT displayed significant anti-tumor effects. These data demonstrate the critical role of macrophages in the age-associated hyper-inflammatory cytokine responses to systemic immunostimulation and underscore the importance of performing preclinical assessments in aged mice

Aging predisposes to acute inflammatory induced pathology after tumor immunotherapy.

Cancer commonly occurs in the elderly and immunotherapy (IT) is being increasingly applied to this population. However, the majority of preclinical mouse tumor models assessing potential efficacy and toxicities of therapeutics use young mice. We assessed the impact of age on responses to systemic immune stimulation. In contrast to young mice, systemic cancer IT regimens or LPS given to aged mice resulted in rapid and lethal toxicities affecting multiple organs correlating with heightened proinflammatory cytokines systemically and within the parenchymal tissues. This inflammatory response and increased morbidity with age was independent of T cells or NK cells. However, prior in vivo depletion of macrophages in aged mice resulted in lesser cytokine levels, increased survival, and decreased liver histopathology. Furthermore, macrophages from aged mice and normal human elderly volunteers displayed heightened TNF and IL-6 production upon in vitro stimulation. Treatment of both TNF knockout mice and in vivo TNF blockade in aged mice resulted in significant increases in survival and lessened pathology. Importantly, TNF blockade in tumor-bearing, aged mice receiving IT displayed significant anti-tumor effects. These data demonstrate the critical role of macrophages in the age-associated hyper-inflammatory cytokine responses to systemic immunostimulation and underscore the importance of performing preclinical assessments in aged mice