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In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20+CD27+IgD− isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine–cytokine receptor interactions, cytotoxic T-cell activation, and T-cell–dependent B-cell activation. TIL-B–upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR–immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B–rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses.Significance:Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor–driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers.

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Supplementary materials and methods and Supplementary figure legends revised

Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognizing cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B-cell activation factor (BAFF), and the toll-like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation, and antibody production. Combined IL-17+BAFF+CpG prolonged B-cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19+CD20+CD27+IgD- B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha [FOLR]) led to secreted antibodies recognizing the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B-cell precursors (CD27-CD38-IgD-), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B-cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B-cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses.

The unmet clinical need for effective treatments in ovarian cancer has yet to be addressed using monoclonal antibodies (mAbs), which have largely failed to overcome tumour-associated immunosuppression, restrict cancer growth, and significantly improve survival. In recent years, experimental mAb design has moved away from solely targeting ovarian tumours and instead sought to modulate the wider tumour microenvironment (TME). Tumour-associated macrophages (TAMs) may represent an attractive therapeutic target for mAbs in ovarian cancer due to their high abundance and close proximity to tumour cells and their active involvement in facilitating several pro-tumoural processes. Moreover, the expression of several antibody crystallisable fragment (Fc) receptors and broad phenotypic plasticity of TAMs provide opportunities to modulate TAM polarisation using mAbs to promote anti-tumoural phenotypes. In this review, we discuss the role of TAMs in ovarian cancer TME and the emerging strategies to target the contributions of these cells in tumour progression through the rationale design of mAbs.

The application of monoclonal antibodies (mAbs) for the treatment of melanoma has significantly improved the clinical management of this malignancy over the last decade. Currently approved mAbs for melanoma enhance T cell effector immune responses by blocking immune checkpoint molecules PD-L1/PD-1 and CTLA-4. However, more than half of patients do not benefit from treatment. Targeting the prominent myeloid compartment within the tumor microenvironment, and in particular the ever-abundant tumor-associated macrophages (TAMs), may be a promising strategy to complement existing therapies and enhance treatment success. TAMs are a highly diverse and plastic subset of cells whose pro-tumor properties can support melanoma growth, angiogenesis and invasion. Understanding of their diversity, plasticity and multifaceted roles in cancer forms the basis for new promising TAM-centered treatment strategies. There are multiple mechanisms by which macrophages can be targeted with antibodies in a therapeutic setting, including by depletion, inhibition of specific pro-tumor properties, differential polarization to pro-inflammatory states and enhancement of antitumor immune functions. Here, we discuss TAMs in melanoma, their interactions with checkpoint inhibitor antibodies and emerging mAbs targeting different aspects of TAM biology and their potential to be translated to the clinic.

B cells are emerging as key players of anti-tumor adaptive immune responses. We investigated regulatory and pro-inflammatory cytokine-expressing B cells in patients with melanoma by flow cytometric intracellular cytokine, CyTOF, transcriptomic, immunofluorescence, single-cell RNA-seq, and B:T cell co-culture analyses. We found enhanced circulating regulatory (TGF-β

Triple-negative breast cancers (TNBC) expressing PD-L1 qualify for checkpoint inhibitor immunotherapy. Cyclin E/CDK2 is a potential target axis in TNBC; however, small-molecule drugs at efficacious doses may be associated with toxicity, and treatment alongside immunotherapy requires investigation. We evaluated CDK inhibition at suboptimal levels and its anti-tumor and immunomodulatory effects. Transcriptomic analyses of primary breast cancers confirmed higher cyclin E/CDK2 expression in TNBC compared with non-TNBC. Out of the three CDK2-targeting inhibitors tested, the CDK 2, 7 and 9 inhibitor SNS-032 was the most potent in reducing TNBC cell viability and exerted cytotoxicity against all eight TNBC cell lines evaluated in vitro. Suboptimal SNS-032 dosing elevated cell surface PD-L1 expression in surviving TNBC cells. In mice engrafted with human immune cells and challenged with human MDA-MB-231 TNBC xenografts in mammary fat pads, suboptimal SNS-032 dosing partially restricted tumor growth, enhanced the tumor infiltration of human CD45+ immune cells and elevated cell surface PD-L1 expression in surviving cancer cells. In tumor-bearing mice engrafted with human immune cells, the anti-PD-L1 antibody avelumab, given sequentially following suboptimal SNS-032 dosing, reduced tumor growth compared with SNS-032 alone or with avelumab without prior SNS-032 priming. CDK inhibition at suboptimal doses promotes immune cell recruitment to tumors, PD-L1 expression by surviving TNBC cells and may complement immunotherapy.

Introduction: The treatment of cutaneous melanoma has been revolutionized by the development of small-molecule inhibitors targeting the MAPK pathway, including inhibitors of BRAF (BRAFi) and MEK (MEKi), and immune checkpoint blockade antibodies, occurring in tandem. Despite these advances, the 5-year survival rate for patients with advanced melanoma remains only around 50%. Although not designed to alter immune responses within the tumor microenvironment (TME), MAPK pathway inhibitors (MAPKi) exert a range of effects on the host immune compartment that may offer opportunities for therapeutic interventions. Areas covered: We review the effects of MAPKi, especially BRAFi, on the TME, focusing on alterations in inflammatory cytokine secretion, recruitment of immune cells and their functions, both during response to BRAFi treatment and as resistance develops. We outline potential combinations of MAPKi with established and experimental treatments. Expert opinion: MAPKi in combination or in sequence with established treatments such as checkpoint inhibitors, anti-angiogenic agents, or new therapies such as adoptive cell therapies, may augment their immunological effects, reverse tumor-associated immune suppression, and offer the prospect of longer-lived clinical responses. Refining therapeutic tools at our disposal and embracing ‘old friends’ in the melanoma treatment arsenal, alongside new target identification, may improve the chances of therapeutic success.

Simple Summary Despite significant advances in immunotherapy seen in the last decade, melanoma accounts for 2500 deaths a year in the UK. There remains an unmet clinical need to improve melanoma treatment. Melanoma is known as the “archetypal immunogenic tumour”, with a dense immune infiltrate. Chemokines are chemoattractant cytokines, essential for the positioning of all immune cells. This review outlines how the interplay of chemokine networks can enable melanoma tumours to survive, grow, metastasise, and evade anticancer immune responses. By better understanding how melanomas can exploit chemokine pathways, new targets to therapy may be revealed. Abstract The incidence of cutaneous malignant melanoma is rising globally and is projected to continue to rise. Advances in immunotherapy over the last decade have demonstrated that manipulation of the immune cell compartment of tumours is a valuable weapon in the arsenal against cancer; however, limitations to treatment still exist. Cutaneous melanoma lesions feature a dense cell infiltrate, coordinated by chemokines, which control the positioning of all immune cells. Melanomas are able to use chemokine pathways to preferentially recruit cells, which aid their growth, survival, invasion and metastasis, and which enhance their ability to evade anticancer immune responses. Aside from this, chemokine signalling can directly influence angiogenesis, invasion, lymph node, and distal metastases, including epithelial to mesenchymal transition-like processes and transendothelial migration. Understanding the interplay of chemokines, cancer cells, and immune cells may uncover future avenues for melanoma therapy, namely: identifying biomarkers for patient stratification, augmenting the effect of current and emerging therapies, and designing specific treatments to target chemokine pathways, with the aim to reduce melanoma pathogenicity, metastatic potential, and enhance immune cell-mediated cancer killing. The chemokine network may provide selective and specific targets that, if included in current therapeutic regimens, harbour potential to improve outcomes for patients.

BackgroundCancer immunotherapy with monoclonal antibodies and chimeric antigen receptor (CAR) T cell therapies can benefit from selection of new targets with high levels of tumor specificity and from early assessments of efficacy and safety to derisk potential therapies.MethodsEmploying mass spectrometry, bioinformatics, immuno-mass spectrometry and CRISPR/Cas9 we identified the target of the tumor-specific SF-25 antibody. We engineered IgE and CAR T cell immunotherapies derived from the SF-25 clone and evaluated potential for cancer therapy.ResultsWe identified the target of the SF-25 clone as the tumor-associated antigen SLC3A2, a cell surface protein with key roles in cancer metabolism. We generated IgE monoclonal antibody, and CAR T cell immunotherapies each recognizing SLC3A2. In concordance with preclinical and, more recently, clinical findings with the first-in-class IgE antibody MOv18 (recognizing the tumor-associated antigen Folate Receptor alpha), SF-25 IgE potentiated Fc-mediated effector functions against cancer cells in vitro and restricted human tumor xenograft growth in mice engrafted with human effector cells. The antibody did not trigger basophil activation in cancer patient blood ex vivo, suggesting failure to induce type I hypersensitivity, and supporting safe therapeutic administration. SLC3A2-specific CAR T cells demonstrated cytotoxicity against tumor cells, stimulated interferon-γ and interleukin-2 production in vitro. In vivo SLC3A2-specific CAR T cells significantly increased overall survival and reduced growth of subcutaneous PC3-LN3-luciferase xenografts. No weight loss, manifestations of cytokine release syndrome or graft-versus-host disease, were detected.ConclusionsThese findings identify efficacious and potentially safe tumor-targeting of SLC3A2 with novel immune-activating antibody and genetically modified cell therapies.

Monoclonal antibodies are at the forefront of novel therapeutic strategies for cancer and have made a positive impact on the clinical management of several malignancies. Success in the clinic was originally demonstrated in hematological cancers with the regulatory approval of the anti-CD20 monoclonal antibody rituximab for the treatment of non-Hodgkin’s lymphoma in 1997 and subsequently with the anti-HER2 trastuzumab for the treatment of breast cancer in 1998. Early research and development efforts concentrated on the design of antibodies that selectively target cancer cells or tumor-associated vasculature. In the last decade, two further antibody classes have emerged: checkpoint inhibitors capable of targeting checkpoint molecules on immune cells to activate immune responses against tumors and antibody-drug conjugates, comprising monoclonal antibodies conjugated to toxic payloads to deliver these specifically to tumor cells. A clearer understanding of an antibody’s functional profile may pave the way for identification of the most highly efficacious agents for specific patient groups.

The incidence of pre-malignant skin disease has been rising in recent decades, with varying rates of progression to invasive disease. This article describes the most common forms of pre-malignant lesions: actinic keratoses, Bowen's disease, dysplastic naevi and melanoma-in-situ. We discuss their clinical significance and management. We also discuss the importance of early detection of malignant melanoma by long-term follow-up in high-risk patients.

In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20+CD27+IgD− isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine–cytokine receptor interactions, cytotoxic T-cell activation, and T-cell–dependent B-cell activation. TIL-B–upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR–immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B–rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. Significance: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor–driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers.

IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory (CD40, CD80, CD86), and reduced expression of regulatory (CD163, CD206, MerTK) monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators (TNF&alpha, MCP-1, IL-10, CXCL-10, IL-1&beta, IL-6, IL-23). High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric Fc&epsilon, RI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric Fc&epsilon, RI signalling. These included recently-identified Fc&epsilon, RI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer.

Cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and the Programmed Death Receptor 1 (PD-1) are immune checkpoint molecules that are well-established targets of antibody immunotherapies for the management of malignant melanoma. The monoclonal antibodies, Ipilimumab, Pembrolizumab, and Nivolumab, designed to interfere with T cell inhibitory signals to activate immune responses against tumors, were originally approved as monotherapy. Treatment with a combination of immune checkpoint inhibitors may improve outcomes compared to monotherapy in certain patient groups and these clinical benefits may be derived from unique immune mechanisms of action. However, treatment with checkpoint inhibitor combinations also present significant clinical challenges and increased rates of immune-related adverse events. In this review, we discuss the potential mechanisms attributed to single and combined checkpoint inhibitor immunotherapies and clinical experience with their use.

Summary The in-depth understanding of skin resident memory CD8+ T lymphocytes (TRM) may help to uncover strategies for their manipulation during disease. We investigated isolated TRM from healthy human skin, which expressed the residence marker CD69, and compared them to circulating CD8+ T cell populations from the same donors. There were significantly increased proportions of CD8+CD45RA−CD27− T cells in the skin that expressed low levels of killer cell lectin-like receptor G1 (KLRG1), CD57, perforin and granzyme B. The CD8+ TRM in skin were therefore phenotypically distinct from circulating CD8+CD45RA−CD27− T cells that expressed high levels of all these molecules. Nevertheless, the activation of CD8+ TRM with T cell receptor (TCR)/CD28 or interleukin (IL)-2 or IL-15 in vitro induced the expression of granzyme B. Blocking signalling through the inhibitory receptor programmed cell death 1 (PD)-1 further boosted granzyme B expression. A unique feature of some CD8+ TRM cells was their ability to secrete high levels of tumour necrosis factor (TNF)-α and IL-2, a cytokine combination that was not seen frequently in circulating CD8+ T cells. The cutaneous CD8+ TRM are therefore diverse, and appear to be phenotypically and functionally distinct from circulating cells. Indeed, the surface receptors used to distinguish differentiation stages of blood T cells cannot be applied to T cells in the skin. Furthermore, the function of cutaneous TRM appears to be stringently controlled by environmental signals in situ.

The immune system employs several checkpoint pathways to regulate responses, maintain homeostasis and prevent self-reactivity and autoimmunity. Tumor cells can hijack these protective mechanisms to enable immune escape, cancer survival and proliferation. Blocking antibodies, designed to interfere with checkpoint molecules CTLA-4 and PD-1/PD-L1 and counteract these immune suppressive mechanisms, have shown significant success in promoting immune responses against cancer and can result in tumor regression in many patients. While inhibitors to CTLA-4 and the PD-1/PD-L1 axis are well-established for the clinical management of melanoma, many patients do not respond or develop resistance to these interventions. Concerted efforts have focused on combinations of approved therapies aiming to further augment positive outcomes and survival. While CTLA-4 and PD-1 are the most-extensively researched targets, results from pre-clinical studies and clinical trials indicate that novel agents, specific for checkpoints such as A2AR, LAG-3, IDO and others, may further contribute to the improvement of patient outcomes, most likely in combinations with anti-CTLA-4 or anti-PD-1 blockade. This review discusses the rationale for, and results to date of, the development of inhibitory immune checkpoint blockade combination therapies in melanoma. The clinical potential of new pipeline therapeutics, and possible future therapy design and directions that hold promise to significantly improve clinical prognosis compared with monotherapy, are discussed.

Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo.

Malignant melanoma is associated with poor clinical prognosis; however, novel molecular and immune therapies are now improving patient outcomes. Almost 50% of melanomas harbor targetable activating mutations of BRAF that promote RAS–RAF–MEK–ERK pathway activation and melanoma proliferation. Recent evidence also indicates that melanomas bearing mutant BRAF may also have altered immune responses, suggesting additional avenues for treatment of this patient group. The small molecule inhibitors selective for mutant BRAF induce significant but short-lived clinical responses in a proportion of patients, but also lead to immune stimulatory bystander events, which then subside with the emergence of resistance to inhibition. Simultaneous BRAF and MEK inhibition, and especially combination of BRAF inhibitors with new immunotherapies such as checkpoint blockade antibodies, may further enhance immune activation, or counteract immunosuppressive signals. Preclinical evaluation and ongoing clinical trials should provide novel insights into the role of immunity in the therapy of BRAF-mutant melanoma. Mol Cancer Ther; 13(12); 2769–83. ©2014 AACR.

Identification of mutations in the gene encoding the serine/threonine-protein kinase, BRAF, and constitutive activation of the mitogen-activated protein kinase (MAPK) pathway in around 50% of malignant melanomas have led to the development and regulatory approval of targeted pathway inhibitor drugs. A proportion of patients are intrinsically resistant to BRAF inhibitors, and most patients who initially respond, acquire resistance within months. In this review, we discuss pathway inhibitors and their mechanisms of resistance, and we focus on numerous efforts to improve clinical benefits through combining agents with disparate modes of action, including combinations with checkpoint inhibitor antibodies. We discuss the merits of combination strategies based on enhancing immune responses or overcoming tumor-associated immune escape mechanisms. Emerging insights into mechanisms of action, resistance pathways and their impact on host-tumor relationships will inform the design of optimal combinations therapies to improve outcomes for patients who currently do not benefit from recent treatment breakthroughs.

Evidence of tumor-resident mature B cell and antibody compartments and reports of associations with favorable prognosis in malignant melanoma suggest that humoral immunity could participate in antitumor defense. Likely striving to confer immunological protection while being subjected to tumor-promoting immune tolerance, B cells may engender multiple functions, including antigen processing and presentation, cytokine-mediated signaling, antibody class switching, expression and secretion. We review key evidence in support of multifaceted immunological mechanisms by which B cells may counter or contribute to malignant melanoma, and we discuss their potential translational implications. Dissecting the contributions of tumor-associated humoral responses can inform future treatment avenues.

Monoclonal antibodies are an established modality for the clinical management of many cancers. To date, all antibodies approved for cancer treatment are of the IgG class, and many are targeted towards non-solid tumors. We hypothesized that antibodies of the IgE class may be able to recruit and activate immune cells against tissue resident tumors based on very high affinity of IgE for cognate Fcε Receptors on potent effector cells such as masT-cells, monocytes and macrophages. A novel IgE-based antibody against folate receptor alpha (FRα) for ovarian carcinoma was developed and demonstrated to engender superior anti-tumor effector functions compared with those triggered by its IgG counterpart in different in vivo models of cancer. We have now translated this concept to a first-in-man clinical trial. We have previously reported that monocytes and macrophages are important IgE effector cells against tumors. However, the underlying mechanisms of how IgE exerts antitumor immunity by engaging these effector cells are insufficiently characterized. Here, we sought to investigate the immune profile signatures triggered by the engagement of tumor antigen-specific IgE with Fc-receptors on the surface of monocytes, a key immune effector cell recruited by IgE antibodies. In order to investigate the above aim, the following methods were utilized: Antibody dependenT-cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP) were measured by flow cytometry. Cell stimulation studies with cytokines or by cross-linking of IgE monoclonal antibodies on the surface of monocytes were conducted. Expression and secretion of immune mediators were assessed using quantitative-PCR and ELISA. Toxicity of cytokines towards immune and tumor cells were investigated by cell viability (MTS) assays. As a result, tumor antigen-specific IgE-mediated tumor cell cytotoxicity mediated by human monocytes correlated with increased levels of TNFα, MCP-1 and IL-10. Cross-linking of IgE, but not IgG, on the monocyte surface, triggered up-regulation of the proinflammatory mediator TNFα. TNFα stimulation of monocytes and of tumor cells triggered stimulation of MCP-1 by both cells. Neither cross-linking of IgE or IgG antibodies on tumor cells or monocytes, nor stimulation of these cells with TNFα or MCP-1, was sufficient to upregulate IL-10. However, IL-10 expression was induced by monocytic cells when stimulated by TNFα and MCP-1 in combination, and by stimulation with IL-10 in an autocrine manner. None of these stimulation conditions was sufficient to upregulate IL-10 in cancer cell lines of different origins. None of the three cytokines had direct cytotoxic effects towards immune or tumor cells. However, blockade of TNFα receptor on monocytes reduced ADCC and increased levels of the chemoattractant MCP-1 recruited monocytes in chemotaxis assays in vitro and macrophages in tumor lesions in a rat model of cancer treated with anti-tumor IgE. These findings suggest that IgE-dependent monocyte-mediated tumor cell cytotoxicity triggers TNFα, MCP-1 and IL-10, an axis normally associated with IgE-mediated anti-parasite immune functions. This cascade appears to be responsible for a prominent recruitment of monocytes and macrophages towards tumor cells but may not be solely responsible for IgE-mediated antitumor cytotoxic functions. Identifying the immune profiles involved in IgE-mediated monocyte antitumor mechanisms may help identify markers of effector cell activation and support the development of IgE class as a novel immuno-oncology strategy. Citation Format: Mano Nakamura, Heather J. Bax, James F. Spicer, Katie E. Lacy, Sophia Tsoka, Debra H. Josephs, Sophia Karagiannis. Discovering the immune profiles of a novel antifolate receptor alpha IgE antibody associated with monocyte-mediated antitumor functions [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A095.

Skin cancer is the most common type of cancer in the world and its incidence is increasing rapidly. Skin cancers are broadly divided into melanoma skin cancer and non-melanoma skin cancer, the majority of which are derived from cells within the epidermis. Melanoma originates from melanocytes whereas non-melanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma, originate from cells of keratinocyte lineage. Exposure to ultraviolet light is the most important aetiological factor in the development of skin cancers. This article describes the most common forms of skin cancer and their management.

Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10-driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4(+)-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell-mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches.

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.

Immunity declines during aging, however the mechanisms involved in this decline are not known. In this study, we show that cutaneous delayed type hypersensitivity (DTH) responses to recall antigens are significantly decreased in older individuals. However, this is not related to CC chemokine receptor 4, cutaneous lymphocyte-associated antigen, or CD11a expression by CD4+ T cells or their physical capacity for migration. Instead, there is defective activation of dermal blood vessels in older subject that results from decreased TNF-α secretion by macrophages. This prevents memory T cell entry into the skin after antigen challenge. However, isolated cutaneous macrophages from these subjects can be induced to secrete TNF-α after stimulation with Toll-like receptor (TLR) 1/2 or TLR 4 ligands in vitro, indicating that the defect is reversible. The decreased conditioning of tissue microenvironments by macrophage-derived cytokines may therefore lead to defective immunosurveillance by memory T cells. This may be a predisposing factor for the development of malignancy and infection in the skin during aging.

Whereas our understanding of the skin immune system has increased exponentially in recent years, the role of B cells in cutaneous immunity remains poorly defined. Recent studies have revealed the presence of B cells within lymphocytic infiltrates in chronic inflammatory skin diseases and cutaneous malignancies including melanoma, and have examined their functional significance in these settings. We review these findings and discuss them in the context of the current understanding of the role of B cells in normal skin physiology, as well as in both animal and human models of skin pathology. We integrate these findings into a model of cutaneous immunity wherein crosstalk between B cells and other skin-resident immune cells plays a central role in skin immune homeostasis.

The Mantoux Test (MT) is a classical delayed-type hypersensitivity (DTH) response to the intradermal injection of tuberculin purified protein derivative (PPD). It represents a cutaneous T cell mediated memory recall immune response. The test is typically used to determine immunity to tuberculosis in humans and positive reactions develop in individuals previously exposed to Mycobacterium tuberculosis, and those immunised with the Bacillus of Calmette and Guérin (BCG) vaccine. In view of its relative accessibility human skin represents a convenient tissue for the investigation of human immune responses. Using the MT, we have been able to determine that significant cellular proliferation and clonal expansion occur at the site of antigen deposition in the skin. Furthermore, cells undergoing proliferation in the skin also undergo accelerated differentiation. Taken together with other studies, in humans and in mice, these observations shed new light on the importance of the microenvironment at the site of the immune response for the proliferation and differentiation of memory T cells.

Reactivation of the varicella zoster virus (VZV) increases during aging. Although the effects of VZV reactivation are observed in the skin (shingles), the number and functional capacity of cutaneous VZV-specific T cells have not been investigated. The numbers of circulating IFN-γ-secreting VZV-specific CD4(+) T cells are significantly decreased in old subjects. However, other measures of VZV-specific CD4(+) T cells, including proliferative capacity to VZV antigen stimulation and identification of VZV-specific CD4(+) T cells with an major histocompatibility complex class II tetramer (epitope of IE-63 protein), were similar in both age groups. The majority of T cells in the skin of both age groups expressed CD69, a characteristic of skin-resident T cells. VZV-specific CD4(+) T cells were significantly increased in the skin compared with the blood in young and old subjects, and their function was similar in both age groups. In contrast, the number of Foxp3(+) regulatory T cells and expression of the inhibitory receptor programmed cell death -1 PD-1 on CD4(+) T cells were significantly increased in the skin of older humans. Therefore, VZV-specific CD4(+) T cells in the skin of older individuals are functionally competent. However, their activity may be restricted by multiple inhibitory influences in situ.

The interface between malignant melanoma and patient immunity has long been recognised and efforts to treat this most lethal form of skin cancer by activating immune responses with cytokine, vaccine and also antibody immunotherapies have demonstrated promise in limited subsets of patients. In the present study, we discuss different antibody immunotherapy approaches evaluated in the context of melanoma, each designed to act on distinct targets and to employ different mechanisms to restrict tumour growth and spread. Monoclonal antibodies recognising melanoma-associated antigens such as CSPG4/MCSP and targeting elements of tumour-associated vasculature (VEGF) have constituted long-standing translational approaches aimed at reducing melanoma growth and metastasis. Recent insights into mechanisms of immune regulation and tumour-immune cell interactions have helped to identify checkpoint molecules on immune (CTLA4, PD-1) and tumour (PD-L1) cells as promising therapeutic targets. Checkpoint blockade with antibodies to activate immune responses and perhaps to counteract melanoma-associated immunomodulatory mechanisms led to the first clinical breakthrough in the form of an anti-CTLA4 monoclonal antibody. Novel modalities to target key mechanisms of immune suppression and to redirect potent effector cell subsets against tumours are expected to improve clinical outcomes and to provide previously unexplored avenues for therapeutic interventions.

The title of Torjesen’s article could be misleading.1 Sentinel node biopsy (SNB) is a staging tool used to provide patients with the best available information,2 and is a well established prognostic test. Our research shows that patients want information on prognosis.3 Lymph node status is the most meaningful prognostic indicator and SNB the most sensitive …

We investigated the relationship between varicella zoster virus (VZV)–specific memory CD4+ T cells and CD4+Foxp3+ regulatory T cells (Tregs) that accumulate after intradermal challenge with a VZV skin test Ag. VZV-specific CD4+ T cells were identified with a MHC class II tetramer or by intracellular staining for either IFN-γ or IL-2 after Ag rechallenge in vitro. VZV-specific T cells, mainly of a central memory (CD45RA−CD27+) phenotype, accumulate at the site of skin challenge compared with the blood of the same individuals. This resulted in part from local proliferation because >50% of tetramer defined Ag-specific CD4+ T cells in the skin expressed the cell cycle marker Ki67. CD4+Foxp3+ T cells had the characteristic phenotype of Tregs, namely CD25hiCD127loCD39hi in both unchallenged and VZV challenged skin and did not secrete IFN-γ or IL-2 after antigenic restimulation. The CD4+Foxp3+ T cells from unchallenged skin had suppressive activity, because their removal led to an increase in cytokine secretion after activation. After VZV Ag injection, Foxp3+CD25hiCD127loCD39hi T cells were also found within the VZV tetramer population. Their suppressive activity could not be directly assessed by CD25 depletion because activated T cells in the skin were also CD25+. Nevertheless, there was an inverse correlation between decreased VZV skin responses and proportion of CD4+Foxp3+ T cells present, indicating indirectly their inhibitory activity in vivo. These results suggest a linkage between the expansion of Ag-specific CD4+ T cells and CD4+ Tregs that may provide controlled responsiveness during Ag-specific stimulation in tissues.

Antibodies have been detected against known melanoma-associated antigens in patients with malignant disease. While much is known about cancer-specific T cell responses, the nature of cancer-specific B cells and their antibody repertoires are less well understood. We investigated circulating skin-homing B cells (CD19+CD22+CLA+) in healthy volunteers (n = 24) and melanoma patients (n = 49) and examined the presence of CD22+ B cells in melanoma lesions (n = 173) and normal skins tissue (n = 16). We detected mature IgG mRNA in 13 of 27 normal skin and 9 of 21 cutaneous melanoma samples and we detected mRNA for the enzyme Activation-induced cytidine deaminase (AID). IgG variable heavy chain sequences from melanomas (n = 51) and normal skins (n = 29) featured lower IgG1/IgGtotal subclass representation compared with circulating B cell antibody sequences (n = 36). The presence of affinity-matured cutaneous antibody repertoires in malignant skin was supported by evidence of somatic hypermutation, class-switching and B cell clonal family trees in melanoma lesions and a subset of melanoma-associated clones featuring shorter CDR3 region lengths relative to circulating B cell sequences. Homology modelling also indicated differential putative binding site patterns in melanoma compared to normal skin-resident antibodies, suggesting distinct melanoma-associated repertoires and potentially, antigen recognition profiles. These findings support the presence of a mature tumor-resident B cell compartment with characteristics distinct to that of B cells in normal skin and the circulation. Citation Format: Louise Saul, Kristina M. Ilieva, Heather J. Bax, Panagiotis Karagiannis, Isabel Correa, Irene Rodriguez-Hernandez, Debra H. Josephs, Isabella Tosi, Isioma U. Egbuniwe, Sara Lombardi, Silvia Crescioli, Carl Hobbs, Federica Villanova, Anthony Cheung, Jenny LC Geh, Ciaran Healy, Mark Harries, Victoria Sanz-Moreno, David Fear, James F. Spicer, Katie E. Lacy, Frank O. Nestle, Sophia N. Karagiannis. IgG antibody switching and clonal expansion in melanoma and normal skin microenvironments [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A116.