Effective immune responses depend on efficient antigen uptake in the periphery, transport of those antigens to, and presentation in draining lymph nodes (LNs). These processes have been studied intensively using stable fluorescent proteins (FPs) as model antigens. To date, ZsGreen is the only FP that can be tracked efficiently towards LNs, hence, it is difficult to compare studies using alternated tracking proteins. Here, we systematically compared six different FPs. We included ZsGreen, ZsYellow, DsRed, AsRed, mCherry, and mRFP based on sequence homology and/or origin species, and generated FP-expressing tumor cell lines. Stability of fluorescent signal was assessed in vitro over time, across different pH environments, and in vivo through FP antigen uptake and transfer to immune cells isolated from tumors and tumor-draining LNs. ZsGreen could be detected in high percentages of all analyzed tumor-infiltrating immune cells, with highest amounts in tumor-associated macrophages (TAMs) and type 2 conventional dendritic cells (cDC2s). ZsYellow, AsRed, and DsRed followed a similar pattern, but percentages of FP-containing immune cells in the tumor were lower than for ZsGreen. Strikingly, mRFP and mCherry demonstrated a 'non-canonical' antigen uptake pattern where percentages of FP-positive tumor-infiltrating immune cells were highest for cDC1s not TAMs and cDC2s despite comparable stabilities and localization of all FPs. Analysis of antigen-containing cells in the LN was hindered by intracellular degradation of FPs. Only ZsGreen could be efficiently tracked to the LN, though some signal was measurable for ZsYellow and DsRed. In summary, we find that detection of antigen uptake and distribution is subject to variabilities related to fluorophore nature. Future experiments need to consider that these processes might be impacted by protein expression, stability, or other unknown factors. Thus, our data sheds light on potential under-appreciated mechanisms regulating antigen transfer and highlights potential uses and necessary caveats to interpretation based on FP use.

Lung metastasis is the lethal determinant in many cancers and a number of lines of evidence point to monocytes and macrophages having key roles in its development. Yet little is known about the immediate fate of incoming tumour cells as they colonize this tissue, and even less known about how they make first contact with the immune system. Primary tumours liberate circulating tumour cells (CTCs) into the blood and we have developed a stable intravital two-photon lung imaging model in mice for direct observation of the arrival of CTCs and subsequent host interaction. Here we show dynamic generation of tumour microparticles in shear flow in the capillaries within minutes of CTC entry. Rather than dispersing under flow, many of these microparticles remain attached to the lung vasculature or independently migrate along the inner walls of vessels. Using fluorescent lineage reporters and flow cytometry, we observed 'waves' of distinct myeloid cell subsets that load differentially and sequentially with this CTC-derived material. Many of these tumour-ingesting myeloid cells collectively accumulated in the lung interstitium along with the successful metastatic cells and, as previously understood, promote the development of successful metastases from surviving tumour cells. Although the numbers of these cells rise globally in the lung with metastatic exposure and ingesting myeloid cells undergo phenotypic changes associated with microparticle ingestion, a consistently sparse population of resident conventional dendritic cells, among the last cells to interact with CTCs, confer anti-metastatic protection. This work reveals that CTC fragmentation generates immune-interacting intermediates, and defines a competitive relationship between phagocyte populations for tumour loading during metastatic cell seeding.

Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to kill cancer. The success of CAR-T cell therapies highlights the promise of programmed immunity and suggests that applying CAR strategies to other immune cell lineages may be beneficial. Here, we engineered a family of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment, which can be modified to direct CAR-P activity towards specific antigens. By screening a panel of engulfment receptor intracellular domains, we found that the cytosolic domains from Megf10 and FcRɣ robustly triggered engulfment independently of their native extracellular domain. We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole human cancer cells. Addition of a tandem PI3K recruitment domain increased cancer cell engulfment. Finally, we show that CAR-P expressing murine macrophages reduce cancer cell number in co-culture by over 40%.

Generation of tumor-infiltrating lymphocytes begins when tumor antigens reach the lymph node (LN) to stimulate T cells, yet we know little of how tumor material is disseminated among the large variety of antigen-presenting dendritic cell (DC) subsets in the LN. Here, we demonstrate that tumor proteins are carried to the LN within discrete vesicles inside DCs and are then transferred among DC subsets. A synapse is formed between interacting DCs and vesicle transfer takes place in the absence of free exosomes. DCs -containing vesicles can uniquely activate T cells, whereas DCs lacking them do not. Understanding this restricted sharing of tumor identity provides substantial room for engineering better anti-tumor immunity.

Intratumoral dendritic cells (DC) bearing CD103 in mice or CD141 in humans drive intratumoral CD8(+) T cell activation. Using multiple strategies, we identified a critical role for these DC in trafficking tumor antigen to lymph nodes (LN), resulting in both direct CD8(+) T cell stimulation and antigen hand-off to resident myeloid cells. These effects all required CCR7. Live imaging demonstrated direct presentation to T cells in LN, and CCR7 loss specifically in these cells resulted in defective LN T cell priming and increased tumor outgrowth. CCR7 expression levels in human tumors correlate with signatures of CD141(+) DC, intratumoral T cells, and better clinical outcomes. This work identifies an ongoing pathway to T cell priming, which should be harnessed for tumor therapies.

The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor (Cre-ERT2) recombinase activity. In this article, we report that 4-hydroxytamoxifen (4OHT) caged with coumarin via a conventional ether linkage led to an unexpected photo-Claisen rearrangement which significantly competed with the release of free 4OHT. The basis for this unwanted reaction appears to be related to the coumarin structure and its radical-based mechanism of uncaging, as it did not occur in ortho-nitrobenzyl (ONB) caged 4OHT that was otherwise linked in the same manner. In an effort to perform design optimization, we introduced a self-immolative linker longer than the ether linkage and identified an optimal linker which allowed rapid 4OHT release by both single-photon and two-photon absorption mechanisms. The ability of this construct to actively control Cre-ERT2 mediated gene modifications was investigated in mouse embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP) reporter dependent gene recombination was controlled by 4OHT release and measured by confocal fluorescence microscopy and flow cytometry. In summary, we report the implications of this photo-Claisen rearrangement in coumarin caged compounds and demonstrate a rational linker strategy for addressing this unwanted side reaction.

Differentiation of proinflammatory CD4⁺ conventional T cells (T_conv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4⁺ T_conv, but then fail to support antitumor CD4⁺ T_conv differentiation. Regulatory T cell (T_reg) depletion enhanced their capacity to elicit strong CD4⁺ T_conv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to T_reg predicts protective ICOS⁺ PD-1^lo CD4⁺ T_conv phenotypes and survival. Further, in melanoma patients with low T_reg abundance, intratumoral cDC2 density alone correlates with abundant CD4⁺ T_conv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4⁺ T_conv abundance and controls tumor growth.

The clinical successes in immunotherapy have been both astounding and at the same time unsatisfactory. Countless patients with varied tumor types have seen pronounced clinical response with immunotherapeutic intervention; however, many more patients have experienced minimal or no clinical benefit when provided the same treatment. As technology has advanced, so has the understanding of the complexity and diversity of the immune context of the tumor microenvironment and its influence on response to therapy. It has been possible to identify different subclasses of immune environment that have an influence on tumor initiation and response and therapy; by parsing the unique classes and subclasses of tumor immune microenvironment (TIME) that exist within a patient's tumor, the ability to predict and guide immunotherapeutic responsiveness will improve, and new therapeutic targets will be revealed.