Connie Jackaman

IL-2 Intratumoral Immunotherapy Enhances CD8+ T Cells That Mediate Destruction of Tumor Cells and Tumor-Associated Vasculature: A Novel Mechanism for IL-2

Therapeutic use of IL-2 can generate antitumor immunity; however, a variety of different mechanisms have been reported. We injected IL-2 intratumorally (i.t.) at different stages of growth, using our unique murine model of mesothelioma (AE17; and AE17 transfected with secretory OVA (AE17-sOVA)), and systematically analyzed real-time events as they occurred in vivo. The majority of mice with small tumors when treatment commenced displayed complete tumor regression, remained tumor free for >2 mo, and survived rechallenge with AE17 tumor cells. However, mice with large tumors at the start of treatment failed to respond. Timing experiments showed that IL-2-mediated responses were dependent upon tumor size, not on the duration of disease. Although i.t. IL-2 did not alter tumor Ag presentation in draining lymph nodes, it did enhance a previously primed, endogenous, tumor-specific in vivo CTL response that coincided with regressing tumors. Both CD4+ and CD8+ cells were required for IL-2-mediated tumor eradication, because IL-2 therapy failed in CD4+-depleted, CD8+-depleted, and both CD4+- and CD8+-depleted C57BL/6J animals. Tumor-infiltrating CD8+ T cells, but not CD4+ T cells, increased in association with a marked reduction in tumor-associated vascularity. Destruction of blood vessels required CD8+ T cells, because this did not occur in nude mice or in CD8+-depleted C57BL/6J mice. These results show that repeated doses of i.t. (but not systemic) IL-2 mediates tumor regression via an enhanced endogenous tumor-specific CTL response concomitant with reduced vasculature, thereby demonstrating a novel mechanism for IL-2 activity.

Rescue of skeletal muscle alpha-actin-null mice by cardiac (fetal) alpha-actin.

Skeletal muscle α-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac α-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.

Targeting macrophages rescues age-related immune deficiencies in C57BL/6J geriatric mice.

Changes to innate cells, such as macrophages and myeloid‐derived suppressor cells (MDSCs), during aging in healthy or tumor‐bearing hosts are not well understood. We compared macrophage subpopulations and MDSCs from healthy young (6–8 weeks) C57BL/6J mice to those from healthy geriatric (24–28 months) mice. Spleens, lymph nodes, and bone marrow of geriatric hosts contained significantly more M2 macrophages and MDSCs than their younger counterparts. Peritoneal macrophages from geriatric, but not young, mice co‐expressed CD40 and CX3CR1 that are usually mutually exclusively expressed by M1 or M2 macrophages. Nonetheless, macrophages from geriatric mice responded to M1 or M2 stimuli similarly to macrophages from young mice, although they secreted higher levels of TGF‐β in response to IL‐4. We mimicked conditions that may occur within tumors by exposing macrophages from young vs. geriatric mice to mesothelioma or lung carcinoma tumor cell–derived supernatants. While both supernatants skewed macrophages toward the M2‐phenotype regardless of age, only geriatric‐derived macrophages produced IL‐4, suggesting a more immunosuppressive tumor microenvironment will be established in the elderly. Both geriatric‐ and young‐derived macrophages induced allogeneic T‐cell proliferation, regardless of the stimuli used, including tumor supernatant. However, only macrophages from young mice induced T‐cell IFN‐γ production. We examined the potential of an IL‐2/agonist anti‐CD40 antibody immunotherapy that eradicates large tumors in young hosts to activate macrophages from geriatric mice. IL‐2‐/CD40‐activated macrophages rescued T‐cell production of IFN‐γ in geriatric mice. Therefore, targeting macrophages with IL‐2/anti‐CD40 antibody may improve innate and T‐cell immunity in aging hosts.

Actin nemaline myopathy mouse reproduces disease, suggests other actin disease phenotypes and provides cautionary note on muscle transgene expression

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ∼30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations.

Chemotherapy broadens the range of tumor antigens seen by cytotoxic CD8+ T cells in vivo

Cytotoxic chemotherapies may expose the immune system to high levels of tumor antigens and expand the CD8+ T-cell response to include weak or subdominant antigens. Here, we evaluated the in vivo CTL response to tumor antigens using a murine mesothelioma tumor cell line transfected with a neotumor antigen, ovalbumin, that contains a known hierarchy of epitopes for MHC class I molecules. We show that as tumors progress, effector CTLs are generated in vivo that focus on the dominant epitope SIINFEKL, although a weak response was seen to one (KVVRFDKL) subdominant epitope. These CTLs did not prevent tumor growth. Cisplatin treatment slowed tumor growth, slightly improved in vivo SIINFEKL presentation to T cells and reduced SIINFEKL-CTL activity. However, the CTL response to KVVRFDKL was amplified, and a response to another subdominant epitope, NAIVFKGL, was revealed. Similarly, gemcitabine cured most mice, slightly enhanced SIINFEKL presentation, reduced SIINFEKL-CTL activity yet drove a significant CTL response to NAIVFKGL, but not KVVRFDKL. These NAIVFKGL-specific CTLs secreted IFNγ and proliferated in response to in vitro NAIVFKGL stimulation. IL-2 treatment during chemotherapy refocused the response to SIINFEKL and simultaneously degraded the cisplatin-driven subdominant CTL response. These data show that chemotherapy reveals weaker tumor antigens to the immune system, a response that could be rationally targeted. Furthermore, while integrating IL-2 into the chemotherapy regimen interfered with the hierarchy of the response, IL-2 or other strategies that support CTL activity could be considered upon completion of chemotherapy.

CD40-activated B cells contribute to mesothelioma tumor regression

Targeting CD40, a member of the tumor necrosis factor superfamily, using agonist antibodies (Abs) produces dramatic antitumor effects. Indeed, high‐dose intravenous anti‐CD40 Ab ‘licenses’ dendritic cells (DCs) that instruct activated CD8+ cytotoxic T cells to leave lymph nodes (LNs) and penetrate the mesothelioma tumor microenvironment. However, toxic side effects and the potential of an ‘overwhelmed’ immune response warrant an alternative approach. In this study, we show that injecting lower doses of anti‐CD40 Ab directly into the tumor bed avoided toxic side effects and prolonged survival in 60% of mice, with most cured. Unexpectedly, DCs in tumors and LNs ‘disappeared’, CD8+ tumor‐specific T‐cell numbers and function were not enhanced, and T cells did not infiltrate regressing tumors. CD4+ or CD8+ depletion only marginally hindered anti‐CD40 Ab efficacy implying another effector mechanism. B‐cell numbers significantly increased in tumors, draining LNs and spleens during intratumoral anti‐CD40 Ab treatment. CD40 targeting had no effect on splenic B‐1 cells, obliterated marginal zone B cells and promoted follicular (FO) B‐cell activity. Adoptive transfer of tumor antigen‐experienced, CD40‐activated B cells, or their immunoglobulin products, which recognized autoantigens on mesothelioma cells, protected against tumor challenge. Finally, studies using B‐cell knockout mice showed that successful treatment of established tumors required the presence of B cells. Thus, these data suggest that CD40‐activated FO B cells can become an important component of an effective antitumor immune response.

Intratumoral interleukin-2/agonist CD40 antibody drives CD4+-independent resolution of treated-tumors and CD4+-dependent systemic and memory responses

Targeting interleukin-2 (IL-2) and/or agonist anti-CD40 antibody (Ab) into tumors represents an effective vaccination strategy that avoids systemic toxicity and resolves treated-site tumors. Here, we examined IL-2 and/or anti-CD40 Ab-driven local versus systemic T cell function and the installation of T cell memory. Single tumor studies showed that IL-2 induced a potent CD4+ and CD8+ T cell response that was limited to the draining lymph node and treated-site tumor, and lymph node tumor-specific CD8+ T cells did not upregulate CD44. A two-tumor model showed that while IL-2-treated-site tumors resolved, distal tumors continued to grow, implying limited systemic immunity. In contrast, anti-CD40 Ab treatment with or without IL-2 expanded the systemic T cell response to non-draining lymph nodes, and distal tumors resolved. Tumor-specific T cells in lymph nodes of anti-CD40 Ab ± IL-2-treated mice upregulated CD44, demonstrating activation and transition to effector/memory migratory cells. While CD40-activated CD4+ T cells were not required for eradicating treated-site tumors, they, plus CD8+ T cells, were crucial for removing distal tumors. Rechallenge/depletion experiments showed that the effector/memory phase required the presence of previously CD40/IL-2-activated CD4+ and CD8+ T cells to prevent recurrence. These novel findings show that different T cell effector mechanisms can operate for the eradication of local treated-site tumors versus untreated distal tumors and that signaling through CD40 generates a whole of body, effector/memory CD4+ and CD8+ T cell response that is amplified and prolonged via IL-2. Thus, successful immunotherapy needs to generate collaborating CD4+ and CD8+ T cells for a complete long-term protective cure.

Local effector failure in mesothelioma is not mediated by CD4+ CD25+ T-regulator cells

The aim of the present study was to define the point at which mesothelioma T-cell responses fail in order to design better immunotherapies. A murine model of mesothelioma was used which was established with asbestos. Inoculation of tumour cells into syngeneic mice results in progressing tumours with similar histopathology to human mesothelioma. The tumour cells secrete a marker tumour antigen similar to secreted tumour-associated products, such as mesothelin. The mesothelioma microenvironment contains stromal elements including dendritic cells, effector CD8+ and CD4+ T-cells, and CD4+ T-regulatory (Tregs) cells, all of which are activated in situ, implying chronic inflammation. Tumour antigens are rapidly transported to draining lymph nodes wherein tumour-specific T-cell responses are generated. Despite the generation of potent CD8+ cytotoxic lymphocyte in lymphoid organs, those that infiltrate tumours cannot restrain tumour growth suggesting local suppression. Splenic Tregs did not suppress protective responses in adoptive transfer experiments suggesting that systemic Tregs play little role in regulating anti-mesothelioma immune responses. Finally, removal of CD25+ Tregs from the tumour site and lymphoid organs did not alter tumour growth with or without interleukin (IL)-2 or IL-21 immunotherapy. Tregs are not potent regulators of anti-mesothelioma immunity and targeting these cells may not improve results.

Aging and cancer: The role of macrophages and neutrophils

Impaired immune function has been implicated in the declining health and higher incidence of cancer in the elderly. However, age-related changes to immunity are not completely understood. Neutrophils and macrophages represent the first line of defence yet their ability to phagocytose pathogens decrease with aging. Cytotoxic T lymphocytes are critical in eliminating tumors, but T cell function is also compromised with aging. T cell responses can be regulated by macrophages and may depend on the functional phenotype macrophages adopt in response to microenvironmental signals. This can range from pro-inflammatory, anti-tumorigenic M1 to anti-inflammatory, pro-tumorigenic M2 macrophages. Macrophages in healthy elderly adipose and hepatic tissue exhibit a more pro-inflammatory M1 phenotype compared to young hosts whilst immunosuppressive M2 macrophages increase in elderly lymphoid tissues, lung and muscle. These M2-like macrophages demonstrate altered responses to stimuli. Recent studies suggest that neutrophils also regulate T cell function and, like macrophages, neutrophil function is modulated with aging. It is possible that age-modified tissue-specific macrophages and neutrophils contribute to chronic low-grade inflammation that is associated with dysregulated macrophage-mediated immunosuppression, which together are responsible for development of multiple pathologies, including cancer. This review discusses recent advances in macrophage and neutrophil biology in healthy aging and cancer.

Cytokine-armed vaccinia virus infects the mesothelioma tumor microenvironment to overcome immune tolerance and mediate tumor resolution

Intratumoral (i.t.) administration of cytokine genes expressed by viral vectors represents a rational approach that induces cytokine secretion at the site they are needed, and i.t. vaccinia virus (VV) has shown promise in mesothelioma patients. However, we and others have shown that the mesothelioma tumor microenvironment includes macrophages, dendritic cells (DCs), and T cells. Therefore, we investigated which of these cell types are infected after exposure to VV or Fowlpox virus (FPV)-cytokine gene constructs. In vitro studies showed that mesothelioma tumor cells were resistant to FPV infection yet highly permissive to infection by VV vectors resulting in significant cytokine production and impaired proliferation. Macrophages secreted low levels of cytokine suggestive of resistance to overt infection. DCs transiently secreted virally derived cytokines, but did not mature during VV infection. VV inhibition of T cell proliferation was rescued by the interleukin (IL)-2 and IL-12 VV constructs. In vivo studies of murine mesotheliomas showed that i.t. injection of the parent VV could not hinder tumor progression. In contrast, the VV-cytokine constructs induced profound tumor regression. These data suggest that i.t. VV-cytokine gene constructs retard tumor growth by infecting mesothelioma cells and targeting the immune system through tumor-infiltrating DC and T cells.