Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity.

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.

Author Correction: Tissue-resident memory CD8+ T cells promote melanoma-immune equilibrium in skin.

Panel j was inadvertently labelled as panel k in the caption to Fig. 4. Similarly, 'Fig. 4k' should have been 'Fig. 4j' in the sentence beginning 'TNF-α-deficient gBT-I cells were…'. In addition, the surname of author Umaimainthan Palendira was misspelled 'Palendria'. These errors have been corrected online.

Tissue-resident memory CD8+ T cells promote melanoma-immune equilibrium in skin.

The immune system can suppress tumour development both by eliminating malignant cells and by preventing the outgrowth and spread of cancer cells that resist eradication1. Clinical and experimental data suggest that the latter mode of control-termed cancer-immune equilibrium1-can be maintained for prolonged periods of time, possibly up to several decades2-4. Although cancers most frequently originate in epithelial layers, the nature and spatiotemporal dynamics of immune responses that maintain cancer-immune equilibrium in these tissue compartments remain unclear. Here, using a mouse model of transplantable cutaneous melanoma5, we show that tissue-resident memory CD8+ T cells (TRM cells) promote a durable melanoma-immune equilibrium that is confined to the epidermal layer of the skin. A proportion of mice (~40%) transplanted with melanoma cells remained free of macroscopic skin lesions long after epicutaneous inoculation, and generation of tumour-specific epidermal CD69+ CD103+ TRM cells correlated with this spontaneous disease control. By contrast, mice deficient in TRM formation were more susceptible to tumour development. Despite being tumour-free at the macroscopic level, mice frequently harboured melanoma cells in the epidermal layer of the skin long after inoculation, and intravital imaging revealed that these cells were dynamically surveyed by TRM cells. Consistent with their role in melanoma surveillance, tumour-specific TRM cells that were generated before melanoma inoculation conferred profound protection from tumour development independently of recirculating T cells. Finally, depletion of TRM cells triggered tumour outgrowth in a proportion (~20%) of mice with occult melanomas, demonstrating that TRM cells can actively suppress cancer progression. Our results show that TRM cells have a fundamental role in the surveillance of subclinical melanomas in the skin by maintaining cancer-immune equilibrium. As such, they provide strong impetus for exploring these cells as targets of future anticancer immunotherapies.

PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours.

Although adoptive T-cell therapy has shown remarkable clinical efficacy in haematological malignancies, its success in combating solid tumours has been limited. Here, we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T-cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8+ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling, thereby enhancing both CAR T-cell activation and homing to CXCL9/10-expressing tumours to eradicate HER-2+ mammary tumours in vivo. Our findings define PTPN2 as a target for bolstering T-cell-mediated anti-tumour immunity and CAR T-cell therapy against solid tumours.

NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8⁺ T cells.

Memory T cells exert antigen-independent effector functions, but how these responses are regulated is unclear. We discovered an in vivo link between flagellin-induced NLRC4 inflammasome activation in splenic dendritic cells (DCs) and host protective interferon-γ (IFN-γ) secretion by noncognate memory CD8(+) T cells, which could be activated by Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that CD8α(+) DCs were particularly efficient at sensing bacterial flagellin through NLRC4 inflammasomes. Although this activation released interleukin 18 (IL-18) and IL-1ß, only IL-18 was required for IFN-γ production by memory CD8(+) T cells. Conversely, only the release of IL-1ß, but not IL-18, depended on priming signals mediated by Toll-like receptors. These findings provide a comprehensive mechanistic framework for the regulation of noncognate memory T cell responses during bacterial immunity.

Tumour draining lymph node-generated CD8 T cells play a role in controlling lung metastases after a primary tumour is removed but not when adjuvant immunotherapy is used.

Surgical resection of cancer remains the frontline therapy for millions of patients annually, but post-operative recurrence is common, with a relapse rate of around 45% for non-small cell lung cancer. The tumour draining lymph nodes (dLN) are resected at the time of surgery for staging purposes, and this cannot be a null event for patient survival and future response to immune checkpoint blockade treatment. This project investigates cancer surgery, lymphadenectomy, onset of metastatic disease, and response to immunotherapy in a novel model that closely reflects the clinical setting. In a murine metastatic lung cancer model, primary subcutaneous tumours were resected with associated dLNs remaining intact, completely resected or partially resected. Median survival after surgery was significantly shorter with complete dLN resection at the time of surgery (49 days (95%CI)) compared to when lymph nodes remained intact (> 88 days; p < 0.05). Survival was partially restored with incomplete lymph node resection and CD8 T cell dependent. Treatment with aCTLA4 whilst effective against the primary tumour was ineffective for metastatic lung disease. Conversely, aPD-1/aCD40 treatment was effective in both the primary and metastatic disease settings and restored the detrimental effects of complete dLN resection on survival. In this pre-clinical lung metastatic disease model that closely reflects the clinical setting, we observe decreased frequency of survival after complete lymphadenectomy, which was ameliorated with partial lymph node removal or with early administration of aPD-1/aCD40 therapy. These findings have direct relevance to surgical lymph node resection and adjuvant immunotherapy in lung cancer, and perhaps other cancer, patients.

Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells.

Skin-derived dendritic cells (DCs) include Langerhans cells, classical dermal DCs and a langerin-positive CD103(+) dermal subset. We examined their involvement in the presentation of skin-associated viral and self antigens. Only the CD103(+) subset efficiently presented antigens of herpes simplex virus type 1 to naive CD8(+) T cells, although all subsets presented these antigens to CD4(+) T cells. This showed that CD103(+) DCs were the migratory subset most efficient at processing viral antigens into the major histocompatibility complex class I pathway, potentially through cross-presentation. This was supported by data showing only CD103(+) DCs efficiently cross-presented skin-derived self antigens. This indicates CD103(+) DCs are the main migratory subtype able to cross-present viral and self antigens, which identifies another level of specialization for skin DCs.

IFNß inhibits the development of allergen tolerance and is conducive to the development of asthma on subsequent allergen exposure.

Asthma is a chronic disease affecting up to 10% of the Australian population for which medical treatment is solely aimed at relief of symptoms rather than prevention of disease. Evidence from animal and human studies demonstrates a strong link between viral respiratory infections, atopy and the development of asthma. Type I IFNs include IFNα and IFNß, with subtype expression tailored toward the specific viral infection. We hypothesized that exposure to type I IFNs and allergen may interfere with the healthy response to innocuous airway antigen exposure. In this study, we use an ovalbumin (OVA)-induced BALB/c model of experimental allergic airways disease, where pre-exposure of the airways to OVA is protective against allergen sensitization, leading to allergen tolerance. We investigated airways pre-exposure with OVA and type I IFNs on development of allergic airways disease. We demonstrate restoration of allergic airways disease on pre-exposure with allergen and IFNß, and not IFNα. Dysfunction in tolerance led to changes in dendritic cell antigen capture/traffic, T-cell and B-cell responses. Furthermore, exposure to IFNß with ongoing allergen exposure led to the development of hallmark asthma features, including OVA-specific IgE and airways eosinophilia. Data indicate a role for IFNß in linking viral infection and allergy.

Skin tumor immunity: Site does matter for antigen presentation by DCs.

The immune system has the ability to specifically identify and eliminate tumors, but the underlying mechanisms responsible for this phenomenon are not fully understood. A study published in this issue of the European Journal of Immunology now provides new insights into this important problem. Joncker et al. [Eur. J. Immunol. 2016. 46: 609-618] show that the timely mobilization of tumor antigen-bearing dendritic cells (DCs) from the periphery to the lymph nodes is critical for effective antitumor T-cell immunity, and that DCs present tumor antigens much more efficiently when encountered in the skin rather than in the subcutaneous tissues.

T cells recognizing a 11mer influenza peptide complexed to H-2D(b) show promiscuity for peptide length.

T-cell repertoire is selected according to self peptide-MHC (major histocompatibility complex) complexes in the thymus. Although most peripheral T cells recognize specific pathogen-derived peptides complexed to self-MHC exclusively, some possess cross-reactivity to other self or foreign peptides presented by self-MHC molecules; a phenomenon often termed T-cell receptor (TCR) promiscuity or degeneracy. TCR promiscuity has been attributed to various autoimmune conditions. On the other hand, it is considered a mechanism for a relatively limited TCR repertoire to deal with a potentially much larger antigenic peptide repertoire. Such property has also been utilized to bypass self-tolerance for cancer vaccine development. Although many studies explored such degeneracy for peptide of the same length, few studies reported such properties for peptides of different length. In this study, we finely characterized the CD8(+) T-cell response specific for a 11mer peptide derived from influenza A viral polymerase basic protein 2. The short-term T-cell line, despite possessing highly biased TCR, was able to react with multiple peptides of different length sharing the same core sequence. Out data clearly showed the importance of detailed and quantitative assessments for such T-cell specificity. Our data also emphasize the importance of biochemical demonstration of the naturally presented minimal peptide.

Mixed proteasomes function to increase viral peptide diversity and broaden antiviral CD8+ T cell responses.

The three proteasome subunits with proteolytic activity are encoded by standard or immunoproteasome genes. Many proteasomes expressed by normal cells and cells exposed to cytokines are "mixed", that is, contain both standard and immunoproteasome subunits. Using a panel of 38 defined influenza A virus-derived epitopes recognized by C57BL/6 mouse CD8(+) T cells, we used mice with targeted disruption of ß1i, ß2i, or ß5i/ß2i genes to examine the contribution of mixed proteasomes to the immunodominance hierarchy of antiviral CD8(+) T cells. We show that each immunoproteasome subunit has large effects on the primary and recall immunodominance hierarchies due to modulating both the available T cell repertoire and generation of individual epitopes as determined both biochemically and kinetically in Ag presentation assays. These findings indicate that mixed proteasomes function to enhance the diversity of peptides and support a broad CD8(+) T cell response.

Altered immunity and dendritic cell activity in the periphery of mice after long-term engraftment with bone marrow from ultraviolet-irradiated mice.

Alterations to dendritic cell (DC) progenitors in the bone marrow (BM) may contribute to long-lasting systemic immunosuppression (>28 d) following exposure of the skin of mice to erythemal UV radiation (UVR). DCs differentiated in vitro from the BM of mice 3 d after UVR (8 kJ/m(2)) have a reduced capacity to initiate immunity (both skin and airways) when adoptively transferred into naive mice. Studies in IL-10(-/-) mice suggested that UV-induced IL-10 was not significantly involved. To investigate the immune capabilities of peripheral tissue DCs generated in vivo from the BM of UV-irradiated mice, chimeric mice were established. Sixteen weeks after reconstitution, contact hypersensitivity responses were significantly reduced in mice reconstituted with BM from UV-irradiated mice (UV-chimeric). When the dorsal skin of UV-chimeric mice was challenged with innate inflammatory agents, the hypertrophy induced in the draining lymph nodes was minimal and significantly less than that measured in control-chimeric mice challenged with the same inflammatory agent. When DCs were differentiated from the BM of UV-chimeric mice using FLT3 ligand or GM-CSF + IL-4, the cells maintained a reduced priming ability. The diminished responses in UV-chimeric mice were not due to different numerical or proportional reconstitution of BM or the hematopoietic cells in blood, lymph nodes, and skin. Erythemal UVR may imprint a long-lasting epigenetic effect on DC progenitors in the BM and alter the function of their terminally differentiated progeny.

CD4+ T cell immunity against cutaneous melanoma encompasses multifaceted MHC II-dependent responses.

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor-α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II-dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

Optimal conditions required for influenza A infection-enhanced cross-priming of CD8⁺ T cells specific to cell-associated antigens.

Dendritic cells can take up exogenous tumor antigens and present their antigenic epitopes to CD8⁺ T cells (T(CD8⁺)), a process called cross-presentation. Cross-presentation is especially important in antitumor immunity because tumor cells, although carrying tumor antigens, do not activate naive T cells efficiently because of a lack of co-stimulatory molecules. Our group has recently shown that influenza A virus (IAV) infection of allogeneic cells lead to enhanced cross-priming of T(CD8⁺) specific to cellular antigens. To develop this into a potential vaccine strategy, in this study, we have systematically investigated the numbers of allogeneic cells infected by IAV, IAV doses and their infectious activity, the length of in vitro infection and other associated factors. We have defined the optimal immune-enhancing conditions and we have also shown in vivo that such enhanced cross-priming did lead to enhanced tumor protection. The knowledge should be useful for developing more robust cancer vaccine.

Single cell transcriptomics reveals cell type specific features of developmentally regulated responses to lipopolysaccharide between birth and 5 years.

Background: Human perinatal life is characterized by a period of extraordinary change during which newborns encounter abundant environmental stimuli and exposure to potential pathogens. To meet such challenges, the neonatal immune system is equipped with unique functional characteristics that adapt to changing conditions as development progresses across the early years of life, but the molecular characteristics of such adaptations remain poorly understood. The application of single cell genomics to birth cohorts provides an opportunity to investigate changes in gene expression programs elicited downstream of innate immune activation across early life at unprecedented resolution. Methods: In this study, we performed single cell RNA-sequencing of mononuclear cells collected from matched birth cord blood and 5-year peripheral blood samples following stimulation (18hrs) with two well-characterized innate stimuli; lipopolysaccharide (LPS) and Polyinosinic:polycytidylic acid (Poly(I:C)). Results: We found that the transcriptional response to LPS was constrained at birth and predominantly partitioned into classical proinflammatory gene upregulation primarily by monocytes and Interferon (IFN)-signaling gene upregulation by lymphocytes. Moreover, these responses featured substantial cell-to-cell communication which appeared markedly strengthened between birth and 5 years. In contrast, stimulation with Poly(I:C) induced a robust IFN-signalling response across all cell types identified at birth and 5 years. Analysis of gene regulatory networks revealed IRF1 and STAT1 were key drivers of the LPS-induced IFN-signaling response in lymphocytes with a potential developmental role for IRF7 regulation. Conclusion: Additionally, we observed distinct activation trajectory endpoints for monocytes derived from LPS-treated cord and 5-year blood, which was not apparent among Poly(I:C)-induced monocytes. Taken together, our findings provide new insight into the gene regulatory landscape of immune cell function between birth and 5 years and point to regulatory mechanisms relevant to future investigation of infection susceptibility in early life.

Influenza A infection enhances cross-priming of CD8+ T cells to cell-associated antigens in a TLR7- and type I IFN-dependent fashion.

The initiation of antitumor immunity relies on dendritic cells (DCs) to cross-present cell-associated tumor Ag to CD8(+) T cells (T(CD8+)) due to a lack of costimulatory molecules on tumor cells. Innate danger signals have been demonstrated to enhance cross-priming of T(CD8+) to soluble as well as virally encoded Ags; however, their effect on enhancing T(CD8+) cross-priming to cell genome-encoded Ags remains unknown. Furthermore, influenza A virus (IAV) has not been shown to enhance antitumor immunity. Using influenza-infected allogeneic cell lines, we show in this study that T(CD8+) responses to cell-associated Ags can be dramatically enhanced due to enhanced T(CD8+) expansion. This enhanced cross-priming in part involves TLR7- but not TLR3-mediated sensing of IAV and is entirely dependent on MyD88 and IFN signaling pathways. We also showed that the inflammasome-induced IL-1 and IFN-γ did not play a role in enhancing cross-priming in our system. We further demonstrated in our ex vivo system that CD8(+) DCs are the only APCs able to prime TCR-transgenic T(CD8+). Importantly, plasmacytoid DCs and CD8(-) DCs were both able to enhance such priming when provided in coculture. These observations suggest that IAV infection of tumor cells may facilitate improved cross-presentation of tumor Ags and may be used to augment clinical vaccine efficacy.

Tissue destruction caused by cytotoxic T lymphocytes induces deletional tolerance.

Autoimmune diseases tend to be chronic and progressive, but how these responses are sustained is not clear. One cell type that might contribute to autoimmunity is the cytotoxic T lymphocyte (CTL), which, as a consequence of causing tissue destruction and production of cytokines, could provide a sustained supply of antigen and inflammatory signals for dendritic cells to maintain immune stimulation. Here we examined whether such CTL-mediated tissue damage alone could provide antigen in the right context to recruit immune effectors and sustain autoimmunity. We show that while CTL-mediated tissue damage caused the release of self-antigens that stimulated the proliferation of naive autoreactive CD8(+) T cells, such responses failed to precipitate disease and, instead, led to deletional tolerance. These findings indicate that despite the capacity of CTLs to produce inflammatory cytokines and to cause tissue damage, their responses are not sustaining, but instead favor induction of self-tolerance.

Tissue-resident memory T cells in the era of (Neo) adjuvant melanoma management.

Tissue-resident memory T (TRM) cells have emerged as key players in the immune control of melanoma. These specialized cells are identified by expression of tissue retention markers such as CD69, CD103 and CD49a with downregulation of egress molecules such as Sphingosine-1-Phosphate Receptor-1 (S1PR1) and the lymphoid homing receptor, CD62L. TRM have been shown to be integral in controlling infections such as herpes simplex virus (HSV), lymphocytic choriomeningitis virus (LCMV) and influenza. More recently, robust pre-clinical models have also demonstrated TRM are able to maintain melanoma in a dormant state without progression to macroscopic disease reminiscent of their ability to control viral infections. The discovery of the role these cells play in anti-melanoma immunity has coincided with the advent of immune checkpoint inhibitor (ICI) therapy which has revolutionized the treatment of cancers. ICIs that target programmed death protein-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) have led to substantial improvements in outcomes for patients with metastatic melanoma and have been rapidly employed to reduce recurrences in the resected stage III setting. While ICIs mediate anti-tumor activity via CD8+ T cells, the specific subsets that facilitate this response is unclear. TRM invariably exhibit high expression of immune checkpoints such as PD-1, CTLA-4 and lymphocyte activating gene-3 (LAG-3) which strongly implicates this CD8+ T cell subset as a crucial mediator of ICI activity. In this review, we present pre-clinical and translational studies that highlight the critical role of TRM in both immune control of primary melanoma and as a key CD8+ T cell subset that mediates anti-tumor activity of ICIs for the treatment of melanoma.

Type I interferon subtypes differentially activate the anti-leukaemic function of natural killer cells.

Natural killer (NK) cells have an intrinsic ability to detect and eliminate leukaemic cells. Cellular therapies using cytokine-activated NK cells have emerged as promising treatments for patients with advanced leukaemia. However, not all patients respond to current NK cell therapies, and thus improvements in efficacy are required. Type I interferons (IFN-I) are a family of potent immunomodulatory cytokines with a known ability to modulate NK cell responses against cancer. Although the human IFN-I family comprises 16 distinct subtypes, only IFNα2 has been widely explored as an anti-cancer agent. Here, we investigated the individual immunomodulatory effects each IFNα subtype and IFNß had on NK cell functionality to determine whether a particular subtype confers enhanced effector activity against leukaemia. Importantly, IFNα14 and IFNß were identified as superior activators of NK cell effector function in vitro. To test the ability of these subtypes to enhance NK cell activity in vivo, IFN-I stimulation was overlaid onto a standard ex vivo expansion protocol to generate NK cells for adoptive cell therapy. Interestingly, infusion of NK cells pre-activated with IFNα14, but not IFNß, significantly prolonged survival in a preclinical model of leukaemia compared to NK cells expanded without IFN-I. Collectively, these results highlight the diverse immunomodulatory potencies of individual IFN-I subtypes and support further investigation into the use of IFNα14 to favourably modulate NK cells against leukaemia.