Faecal microbial transfer and complex carbohydrates mediate protection against COPD.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DESIGN: Using an in vivo mouse model of cigarette smoke (CS)-induced COPD and faecal microbial transfer (FMT), we characterised the faecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in 16 patients with COPD using a randomised, double-blind, placebo-controlled pilot study of inulin supplementation. RESULTS: FMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation, and transfer of CS-associated microbiota after antibiotic-induced microbiome depletion was sufficient to increase lung inflammation while suppressing colonic immunity in the absence of CS exposure. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in CS-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. CONCLUSION: The gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically.

MHCII restriction demonstrates B cells have very limited capacity to activate tumour-specific CD4+ T cells in vivo.

There has been growing interest in the role of B cells in antitumour immunity and potential use in adoptive cellular therapies. To date, the success of such therapies is limited. The intrinsic capacity of B cells to specifically activate tumour-specific CD4+ T cells in vivo via TCR-dependent interactions remains poorly defined. We have developed an in vivo tumour model that utilizes MHCII I-E restriction which limits antigen presentation to tumour-specific CD4 T cells to either tumour-specific B cells or host myeloid antigen presenting cells (APCs) in lymphopenic RAG-/-mice. We have previously shown that these naive tumour-specific CD4+ T cells can successfully eradicate established tumours in this model when activated by host APCs. When naïve tumour-specific B cells are the only source of I-E+ APC, very limited proliferation of naïve CD4+ T cells is observed, whereas host I-E+ APCs are potent T cell activators. B cells pre-activated with an anti-CD40 agonistic antibody in vivo support increased T cell proliferation, although far less than host APCs. CD4+ T cells that have already differentiated to an effector/central memory phenotype proliferate more readily in response to naïve B cells, although still 100-fold less than in response to host APCs. This study demonstrates that even in a significantly lymphopenic environment, myeloid APCs are the dominant primary activators of tumour-specific T cells, in contrast to the very limited capacity of tumour-specific B cells. This suggests that future anti-tumour therapies that incorporate activated B cells should also include mechanisms that activate host APCs.

A High-Throughput Colorimetric Microplate Assay for Determination of Plasma Arginase Activity.

Arginase, an enzyme involved in the urea cycle, is gaining attention as a critical player in numerous chronic pathologies. Additionally, increased activity of this enzyme has been shown to correlate with poor prognosis in a range of cancers. Colorimetric assays that measure the conversion of arginine to ornithine have long been used to determine the activity of arginase. However, this analysis is hindered by a lack of standardization across protocols. Here, we describe in detail a novel revision of the Chinard's colorimetric assay used to determine arginase activity. Dilution series of patient plasma are plotted to form a logistic function, from which activity can be interpolated by comparison to an ornithine standard curve. Inclusion of patient dilution series rather than a single point increases the robustness of the assay. This high-throughput microplate assay analyzes 10 samples per plate to produce highly reproducible results.

Immunoprofiling reveals cell subsets associated with the trajectory of cytomegalovirus reactivation post stem cell transplantation.

Human cytomegalovirus reactivation is a major opportunistic infection after allogeneic haematopoietic stem cell transplantation and has a complex relationship with post-transplant immune reconstitution. Here, we use mass cytometry to define patterns of innate and adaptive immune cell reconstitution at key phases of human cytomegalovirus reactivation in the first 100 days post haematopoietic stem cell transplantation. Human cytomegalovirus reactivation is associated with the development of activated, memory T-cell profiles, with faster effector-memory CD4+ T-cell recovery in patients with low-level versus high-level human cytomegalovirus DNAemia. Mucosal-associated invariant T cell levels at the initial detection of human cytomegalovirus DNAemia are significantly lower in patients who subsequently develop high-level versus low-level human cytomegalovirus reactivation. Our data describe distinct immune signatures that emerged with human cytomegalovirus reactivation after haematopoietic stem cell transplantation, and highlight Mucosal-associated invariant T cell levels at the first detection of reactivation as a marker that may be useful to anticipate the magnitude of human cytomegalovirus DNAemia.

Mass cytometry reveals immune signatures associated with cytomegalovirus (CMV) control in recipients of allogeneic haemopoietic stem cell transplant and CMV-specific T cells.

OBJECTIVES: Cytomegalovirus (CMV) is known to have a significant impact on immune recovery post-allogeneic haemopoietic stem cell transplant (HSCT). Adoptive therapy with donor-derived or third-party virus-specific T cells (VST) can restore CMV immunity leading to clinical benefit in prevention and treatment of post-HSCT infection. We developed a mass cytometry approach to study natural immune recovery post-HSCT and assess the mechanisms underlying the clinical benefits observed in recipients of VST. METHODS: A mass cytometry panel of 38 antibodies was utilised for global immune assessment (72 canonical innate and adaptive immune subsets) in HSCT recipients undergoing natural post-HSCT recovery (n = 13) and HSCT recipients who received third-party donor-derived CMV-VST as salvage for unresponsive CMV reactivation (n = 8). RESULTS: Mass cytometry identified distinct immune signatures associated with CMV characterised by a predominance of innate cells (monocytes and NK) seen early and an adaptive signature with activated CD8+ T cells seen later. All CMV-VST recipients had failed standard antiviral pharmacotherapy as a criterion for trial involvement; 5/8 had failed to develop the adaptive immune signature by study enrolment despite significant CMV antigen exposure. Of these, VST administration resulted in development of the adaptive signature in association with CMV control in three patients. Failure to respond to CMV-VST in one patient was associated with persistent absence of the adaptive immune signature. CONCLUSION: The clinical benefit of CMV-VST may be mediated by the recovery of an adaptive immune signature characterised by activated CD8+ T cells.

Rapidly expanded partially HLA DRB1-matched fungus-specific T cells mediate in vitro and in vivo antifungal activity.

Invasive fungal infections are a major cause of disease and death in immunocompromised hosts, including patients undergoing allogeneic hematopoietic stem cell transplant (HSCT). Recovery of adaptive immunity after HSCT correlates strongly with recovery from fungal infection. Using initial selection of lymphocytes expressing the activation marker CD137 after fungal stimulation, we rapidly expanded a population of mainly CD4+ T cells with potent antifungal characteristics, including production of tumor necrosis factor α, interferon γ, interleukin-17, and granulocyte-macrophage colony stimulating factor. Cells were manufactured using a fully good manufacturing practice-compliant process. In vitro, the T cells responded to fungal antigens presented on fully and partially HLA-DRB1 antigen-matched presenting cells, including when the single common DRB1 antigen was allelically mismatched. Administration of antifungal T cells lead to reduction in the severity of pulmonary and cerebral infection in an experimental mouse model of Aspergillus. These data support the establishment of a bank of cryopreserved fungus-specific T cells using normal donors with common HLA DRB1 molecules and testing of partially HLA-matched third-party donor fungus-specific T cells as a potential therapeutic in patients with invasive fungal infection after HSCT.

Brick plots: an intuitive platform for visualizing multiparametric immunophenotyped cell clusters.

BACKGROUND: The advent of mass cytometry has dramatically increased the parameter limit for immunological analysis. New approaches to analysing high parameter cytometry data have been developed to ease analysis of these complex datasets. Many of these methods assign cells into population clusters based on protein expression similarity. RESULTS: Here we introduce an additional method, termed Brick plots, to visualize these cluster phenotypes in a simplified and intuitive manner. The Brick plot method generates a two-dimensional barcode that displays the phenotype of each cluster in relation to the entire dataset. We show that Brick plots can be used to visualize complex mass cytometry data, both from fundamental research and clinical trials, as well as flow cytometry data. CONCLUSION: Brick plots represent a new approach to visualize complex immunological data in an intuitive manner.

Pretreatment Innate Cell Populations and CD4 T Cells in Blood Are Associated With Response to Immune Checkpoint Blockade in Melanoma Patients.

The development of changes in T cells, referred to as T cell exhaustion, has been suggested as a cause of primary or acquired resistance to immunotherapy by immune checkpoint blockade (ICB). A limited number of studies, largely performed on tumor infiltrating lymphocytes (TILs), has provided evidence in support of this hypothesis, but whether similar changes occur in circulating blood lymphocytes has received little attention. In the present study, a comprehensive analysis of peripheral blood leukocytes from 42 patients taken over the course of treatment with anti-PD-1 was undertaken. The patients included those grouped as responders (who did not progress), primary non-responders (primary resistance) and those with acquired resistance (who initially responded then subsequently progressed). Analysis included surface markers of exhaustion, production of cytokines following in vitro stimulation, and assessment of transcription factor levels associated with T cell exhaustion. There were differences in innate cell populations between responders and non-responders at baseline and maintained throughout therapy. Frequencies of total and classical CD14+CD16- monocytes were higher and the major subset of NK cells (CD16hiCD56+) was significantly smaller in the primary resistance group compared with responders. However, differences in peripheral blood expression of exhaustion markers were not evident between the treatment groups. T cell exhaustion markers were expressed in practically all patients and the major observation was an increase in CD39 on CD4 T cells during treatment. The results confirm the association of Eomes transcription factor with T cell exhaustion but levels of expression and the ratio with T-bet over Eomes did not differ between the patient groups. Thus, peripheral blood expression of T cell exhaustion markers does not distinguish between responders and non-responders to anti-PD-1 therapy. CD4 T cell expression of IFNγ also differed in pre-treatment samples, indicating that predictors of response unrelated to exhaustion may be present in peripheral blood. The association of response with innate cell populations and CD4 T cell responses requires further study.

Pembrolizumab for anaplastic thyroid cancer: a case study.

Blockade of the PD-1/PD-L1 pathway with targeted monoclonal antibodies has demonstrated encouraging anti-tumour activity in multiple cancer types. We present the case of a patient with BRAF-negative stage IVC anaplastic thyroid cancer (ATC) treated with the anti-PD-1 monoclonal antibody, pembrolizumab, following radiographic progression on chemoradiation. Blood samples were collected prior to and at four time points during treatment with pembrolizumab. Mass cytometry was used to determine expression of relevant biomarkers by peripheral blood mononuclear cells. Faecal samples were collected at baseline and 4 weeks following treatment initiation; taxonomic profiling using 16S ribosomal RNA (rRNA) gene sequencing was performed. Following treatment, a marked expansion in CD20+ B cell, CD16+ CD56lo NK cell and CD45RO+ CCR7+ central memory CD4+ T-cell populations was observed in the peripheral blood. Proportions of cells expressing the co-receptors TIGIT, OX40 and CD86 also increased during treatment. A high abundance of bacteria of the order Bacteroidales, specifically from the Bacteroidaceae and Rikenellaceae families, was identified in the faecal microbiota. Moreover, the patient's microbiome was enriched in Clostridiales order members Ruminococcaceae, Veillonellaceae and Lachnospiraceae. Alpha diversity of the gut microbiome was significantly higher following initiation of checkpoint therapy as assessed by the Shannon and Simpson index. Our results suggest that treatment with pembrolizumab promotes expansion of T-, B- and NK cell populations in the peripheral blood at the time of tumour regression and have the potential to be implemented as predictive biomarkers in the context of checkpoint blockade therapy. Larger studies to confirm these findings are warranted.

Mass Cytometry Discovers Two Discrete Subsets of CD39-Treg Which Discriminate MGUS From Multiple Myeloma.

Multiple Myeloma (MM) is preceded by the clinically stable condition monoclonal gammopathy of undetermined significance (MGUS). Critical immune events that discriminate MGUS from newly diagnosed MM (ND)MM patients remain unknown, but may involve changes in the regulatory T cell (Treg) compartment that favor myeloma growth. To address this possibility, we used mass cytometry and the unsupervised clustering algorithm Flow self-organizing map (FlowSOM) to interrogate the distribution of multiple subsets within CD25+CD127low/negTreg in matched bone marrow (BM) and peripheral blood (PB) of MGUS and NDMM patients. Both mass cytometry and flow cytometry confirmed a trend toward prevalence of CD39-Treg within the Treg compartment in BM and PB of NDMM patients compared to CD39-Treg in MGUS patients. FlowSOM clustering displayed a phenotypic organization of Treg into 25 metaclusters that confirmed Treg heterogeneity. It identified two subsets which emerged within CD39-Treg of NDMM patients that were negligible or absent in CD39-Treg of MGUS patients. One subset was found in both BM and PB which phenotypically resembled activated Treg based on CD45RO, CD49d, and CD62L expression; another subset resembled BM-resident Treg based on its tissue-resident CD69+CD62L-CD49d- phenotype and restricted location within the BM. Both subsets co-expressed PD-1 and TIGIT, but PD-1 was expressed at higher levels on BM-resident Treg than on activated Treg. Within BM, both subsets had limited Perforin and Granzyme B production, whilst activated Treg in PB acquired high Perforin and Granzyme B production. In conclusion, the use of mass cytometry and FlowSOM clustering discovered two discrete subsets of CD39-Treg which are discordant in MGUS and NDMM patients and may be permissive of myeloma growth which warrants further study. Understanding the regulatory properties of these subsets may also advance MGUS and MM diagnosis, prognosis, and therapeutic implications for MM patients.

High-Dimensional Mass Cytometric Analysis Reveals an Increase in Effector Regulatory T Cells as a Distinguishing Feature of Colorectal Tumors.

T cell infiltration of tumors plays an important role in determining colorectal cancer disease progression and has been incorporated into the Immunoscore prognostic tool. In this study, mass cytometry was used to demonstrate a significant increase in the frequency of both conventional CD25+FOXP3+CD127lo regulatory T cells (Tregs) as well as BLIMP-1+ Tregs in the tumor compared with nontumor bowel (NTB) of the same patients. Network cluster analyses using SCAFFoLD, VorteX, and CITRUS revealed that an increase in BLIMP-1+ Tregs was a single distinguishing feature of the tumor tissue compared with NTB. BLIMP-1+ Tregs represented the most significantly enriched T cell population in the tumor compared with NTB. The enrichment of ICOS, CD45RO, PD-1, PDL-1, LAG-3, CTLA-4, and TIM-3 on BLIMP-1+ Tregs suggests that BLIMP-1+ Tregs have a more activated phenotype than conventional Tregs and may play a role in antitumor immune responses.

Widespread alterations in the peripheral blood innate immune cell profile in cystic fibrosis reflect lung pathology.

Cystic fibrosis (CF) is caused by mutations to the CF transmembrane conductance regulator (CFTR) gene. CFTR is known to be expressed on multiple immune cell subtypes, dendritic cells, monocytes/macrophages, neutrophils and lymphocytes. We hypothesized that the lack of CFTR expression on peripheral blood innate immune cells would result in an altered cell profile in the periphery and that this profile would reflect lung pathology. We performed a flow cytometric phenotypic investigation of innate immune cell proportions in peripheral blood collected from 17 CF patients and 15 age-matched healthy controls. We observed significant differences between CF patients and controls in the relative proportions of natural killer (NK) cells, monocytes and their subsets, with significant correlations observed between proportions of NK and monocyte cell subsets and lung function (forced expiratory volume in 1 sec, % predicted; FEV1% predicted) in CF patients. This study demonstrates the widespread nature of immune dysregulation in CF and provides a basis for identification of potential therapeutic targets. Modulation of the distinct CF-related immune cell phenotype identified could also be an important biomarker for evaluating CFTR-targeted drug efficacy.

Mass Cytometry for the Assessment of Immune Reconstitution After Hematopoietic Stem Cell Transplantation.

Mass cytometry, or Cytometry by Time-Of-Flight, is a powerful new platform for high-dimensional single-cell analysis of the immune system. It enables the simultaneous measurement of over 40 markers on individual cells through the use of monoclonal antibodies conjugated to rare-earth heavy-metal isotopes. In contrast to the fluorochromes used in conventional flow cytometry, metal isotopes display minimal signal overlap when resolved by single-cell mass spectrometry. This review focuses on the potential of mass cytometry as a novel technology for studying immune reconstitution in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Reconstitution of a healthy donor-derived immune system after HSCT involves the coordinated regeneration of innate and adaptive immune cell subsets in the recipient. Mass cytometry presents an opportunity to investigate immune reconstitution post-HSCT from a systems-level perspective, by allowing the phenotypic and functional features of multiple cell populations to be assessed simultaneously. This review explores the current knowledge of immune reconstitution in HSCT recipients and highlights recent mass cytometry studies contributing to the field.

Anti-PD-1-induced high-grade hepatitis associated with corticosteroid-resistant T cells: a case report.

Effective treatment or prevention of immune side effects associated with checkpoint inhibitor therapy of cancer is an important goal in this new era of immunotherapy. Hepatitis due to immunotherapy with antibodies against PD-1 is uncommon and generally of low severity. We present an unusually severe case arising in a melanoma patient after more than 6 months uncomplicated treatment with anti-PD-1 in an adjuvant setting. The hepatitis rapidly developed resistance to high-dose steroids, requiring anti-thymocyte globulin (ATG) to achieve control. Mass cytometry allowed comprehensive phenotyping of circulating lymphocytes and revealed that CD4+ T cells were profoundly depleted by ATG, while CD8+ T cells, B cells, NK cells and monocytes were relatively spared. Multiple abnormalities in CD4+ T cell phenotype were stably present in the patient before disease onset. These included a population of CCR4-CCR6- effector/memory CD4+ T cells expressing intermediate levels of the Th1-related chemokine receptor CXCR3 and abnormally high multi-drug resistance type 1 transporter (MDR1) activity as assessed by a rhodamine 123 excretion assay. Expression of MDR1 has been implicated in steroid resistance and may have contributed to the severity and lack of a sustained steroid response in this patient. The number of CD4+ rhodamine 123-excreting cells was reduced > 3.5-fold after steroid and ATG treatment. This case illustrates the need to consider this form of steroid resistance in patients failing treatment with corticosteroids. It also highlights the need for both better identification of patients at risk and the development of treatments that involve more specific immune suppression.

Immunotherapy-induced sarcoidosis in patients with melanoma treated with PD-1 checkpoint inhibitors: Case series and immunophenotypic analysis.

AIM: Sarcoidosis is a multisystem granulomatous disease. This condition has a documented association with the diagnosis of melanoma and can be induced in melanoma patients receiving anti-neoplastic therapy. We evaluated a case series of melanoma patients who developed immunotherapy-induced sarcoidosis. METHODS: Three patients with melanoma (n = 1 resected Stage III, n = 2 metastatic) treated with anti-programmed cell death (PD)-1 antibody therapy at two institutions developed biopsy-proven sarcoidosis. We used mass cytometry to determine expression of the relevant chemokine receptors (CR) by peripheral blood mononuclear cells for two of the three patients who developed sarcoidosis and 13 melanoma patients who did not. Blood samples were collected before receiving PD-1 checkpoint inhibitor therapy. RESULTS: Immunophenotypic analysis demonstrated abnormally high numbers of circulating Th17.1 (CCR6+ CCR4- CXCR3+ CCR10- ) cells prior to commencing PD-1 checkpoint inhibitor therapy in five of 15 melanoma patients, including both the patients who developed sarcoidosis during the course of therapy. CONCLUSION: Our findings support prior literature implicating Th17.1 cells in the pathogenesis of sarcoidosis. However, we demonstrate these findings in patients with melanoma prior to administration of checkpoint therapy and before the onset of clinically symptomatic sarcoidosis. The identification of elevated Th17.1 cells in melanoma patients who have not developed sarcoidosis may reflect the established association between melanoma and sarcoidosis. With some patients receiving these agents over a prolonged period, the clinical course of immunotherapy-induced sarcoidosis is uncertain.

The Analysis of CD83 Expression on Human Immune Cells Identifies a Unique CD83+-Activated T Cell Population.

CD83 is a member of the Ig gene superfamily, first identified in activated lymphocytes. Since then, CD83 has become an important marker for defining activated human dendritic cells (DC). Several potential CD83 mRNA isoforms have been described, including a soluble form detected in human serum, which may have an immunosuppressive function. To further understand the biology of CD83, we examined its expression in different human immune cell types before and after activation using a panel of mouse and human anti-human CD83 mAb. The mouse anti-human CD83 mAbs, HB15a and HB15e, and the human anti-human CD83 mAb, 3C12C, were selected to examine cytoplasmic and surface CD83 expression, based on their different binding characteristics. Glycosylation of CD83, the CD83 mRNA isoforms, and soluble CD83 released differed among blood DC, monocytes, and monocyte-derived DC, and other immune cell types. A small T cell population expressing surface CD83 was identified upon T cell stimulation and during allogeneic MLR. This subpopulation appeared specifically during viral Ag challenge. We did not observe human CD83 on unstimulated human natural regulatory T cells (Treg), in contrast to reports describing expression of CD83 on mouse Treg. CD83 expression was increased on CD4+, CD8+ T, and Treg cells in association with clinical acute graft-versus-host disease in allogeneic hematopoietic cell transplant recipients. The differential expression and function of CD83 on human immune cells reveal potential new roles for this molecule as a target of therapeutic manipulation in transplantation, inflammation, and autoimmune diseases.

Pro- and anti-tumour effects of B cells and antibodies in cancer: a comparison of clinical studies and preclinical models.

The primary immune role of B cells is to produce antibodies, but they can also influence T cell function via antigen presentation and, in some contexts, immune regulation. Whether their roles in tumour immunity are similar to those in other chronic immune responses such as autoimmunity and chronic infection, where both pro- and anti-inflammatory roles have been described, remains controversial. Many studies have aimed to define the role of B cells in antitumor immune responses, but despite this considerable body of work, it is not yet possible to predict how they will affect immunity to any given tumour. In many human cancers, the presence of tumour-infiltrating B cells and tumour-reactive antibodies correlates with extended patient survival, and this clinical observation is supported by data from some animal models. On the other hand, T cell responses can be adversely affected by B cell production of immunoregulatory cytokines, a phenomenon that has been demonstrated in humans and in animal models. The isotype and concentration of tumour-reactive antibodies may also influence tumour progression. Recruitment of B cells into tumours may directly reflect the subtype and strength of the anti-tumour T cell response. As the response becomes chronic, B cells may attenuate T cell responses in an attempt to decrease host damage, similar to their described role in chronic infection and autoimmunity. Understanding how B cell responses in cancer are related to the effectiveness of the overall anti-tumour response is likely to aid in the development of new therapeutic interventions against cancer.

EZH2 as a mediator of treatment resistance in melanoma.

Direct treatments of cancer such as chemotherapy, radiotherapy and targeted therapy have been shown to depend on recruitment of the immune system for their effectiveness. Recent studies have shown that development of resistance to direct therapies such as BRAF inhibitors in melanoma is associated with suppression of immune responses. We point to emerging data that implicate activation of the polycomb repressive complex 2 (PRC2) and its catalytic component-enhancer of zeste homolog 2 (EZH2)-in progression of melanoma and suppression of immune responses. EZH2 appears to have an important role in differentiation of CD4 T cells and particularly in the function of T regulatory cells, which suppress immune responses to melanoma. We review mechanisms of EZH2 activation at the genomic level and from activation of the MAP kinase, E2F or NF-kB2 pathways. These studies are consistent with activation of EZH2 as a common mechanism for induction of immune suppression in patients failing direct therapies and suggest EZH2 inhibitors may have a role in combination with immunotherapy and targeted therapies to prevent development of immunosuppression.

Collaboration between tumor-specific CD4+ T cells and B cells in anti-cancer immunity.

The role of B cells and antibodies in anti-tumor immunity is controversial, with both positive and negative effects reported in animal models and clinical studies. We developed a murine B16.F10 melanoma model to study the effects of collaboration between tumor-specific CD4+ T cells and B cells on tumor control. By incorporating T cell receptor transgenic T cells and B cell receptor isotype switching B cells, we were able to track the responses of tumor-reactive T and B cells and the development of anti-tumor antibodies in vivo. In the presence of tumor-specific B cells, the number of tumor-reactive CD4+ T cells was reduced in lymphoid tissues and the tumor itself, and this correlated with poor tumor control. B cells had little effect on the Th1 bias of the CD4+ T cell response, and the number of induced FoxP3+ regulatory cells (iTregs) generated from within the original naive CD4+ T cell inoculum was unrelated to the degree of B cell expansion. In response to CD4+ T cell help, B cells produced a range of isotype-switched anti-tumor antibodies, principally IgG1, IgG2a/c and IgG2b. In the absence of CD4+ T cells, B cells responded to agonistic anti-CD40 administration by switching to production of IgG2a/c and, to a lesser extent, IgG1, IgG3, IgA and IgE, which reduced the number of lung metastases after i.v. tumor inoculation but had no effect on the growth of subcutaneous tumors.

Tumour-specific CD4 T cells eradicate melanoma via indirect recognition of tumour-derived antigen.

The importance of CD4 T cells in tumour immunity has been increasingly recognised, with recent reports describing robust CD4 T cell-dependent tumour control in mice whose immune-regulatory mechanisms have been disturbed by irradiation, chemotherapy, immunomodulatory therapy and/or constitutive immunodeficiency. Tumour control in such models has been attributed in large part to direct Major Histocompatibility Complex (MHC) class II-dependent CD4 T cell killing of tumour cells. To test whether CD4 T cells can eradicate tumours without directly killing tumour cells, we developed an animal model in which tumour-derived antigen could be presented to T-cell receptor (TCR)-transgenic CD4 T cells by host but not tumour MHC class II molecules. In I-E(+) mice bearing I-E(null) tumours, naive I-E-restricted CD4 T cells proliferated locally in tumour-draining lymph nodes after recognising tumour-derived antigen on migratory dendritic cells. In lymphopaenic but not immunosufficient hosts, CD4 T cells differentiated into polarised T helper type 1 (Th1) cells expressing interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα) and interleukin (IL)-2 but little IL-17, and cleared established tumours. Tumour clearance was enhanced by higher TCR affinity for tumour antigen-MHC class II and was critically dependent on IFNγ, as demonstrated by early tumour escape in animals treated with an IFNγ blocking antibody. Thus, CD4 T cells and IFNγ can control tumour growth without direct T-cell killing of tumour cells, and without requiring additional adaptive immune cells such as CD8 T cells and B cells. Our results support a role for effective CD4 T cell-dependent tumour immunity against MHC class II-negative tumours.