The aged tumor microenvironment limits T cell control of cancer.

The etiology and effect of age-related immune dysfunction in cancer is not completely understood. Here we show that limited priming of CD8+ T cells in the aged tumor microenvironment (TME) outweighs cell-intrinsic defects in limiting tumor control. Increased tumor growth in aging is associated with reduced CD8+ T cell infiltration and function. Transfer of T cells from young mice does not restore tumor control in aged mice owing to rapid induction of T cell dysfunction. Cell-extrinsic signals in the aged TME drive a tumor-infiltrating age-associated dysfunctional (TTAD) cell state that is functionally, transcriptionally and epigenetically distinct from canonical T cell exhaustion. Altered natural killer cell-dendritic cell-CD8+ T cell cross-talk in aged tumors impairs T cell priming by conventional type 1 dendritic cells and promotes TTAD cell formation. Aged mice are thereby unable to benefit from therapeutic tumor vaccination. Critically, myeloid-targeted therapy to reinvigorate conventional type 1 dendritic cells can improve tumor control and restore CD8+ T cell immunity in aging.

Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop.

Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.

CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

Dendritic cells as shepherds of T cell immunity in cancer.

In cancer patients, dendritic cells (DCs) in tumor-draining lymph nodes can present antigens to naive T cells in ways that break immunological tolerance. The clonally expanded progeny of primed T cells are further regulated by DCs at tumor sites. Intratumoral DCs can both provide survival signals to and drive effector differentiation of incoming T cells, thereby locally enhancing antitumor immunity; however, the paucity of intratumoral DCs or their expression of immunoregulatory molecules often limits antitumor T cell responses. Here, we review the current understanding of DC-T cell interactions at both priming and effector sites of immune responses. We place emerging insights into DC functions in tumor immunity in the context of DC development, ontogeny, and functions in other settings and propose that DCs control at least two T cell-associated checkpoints of the cancer immunity cycle. Our understanding of both checkpoints has implications for the development of new approaches to cancer immunotherapy.

CXCL10 chemokine regulates heterogeneity of the CD8+ T cell response and viral set point during chronic infection.

CD8+ T cells responding to chronic infection adapt an altered differentiation program that provides some restraint on pathogen replication yet limits immunopathology. This adaptation is imprinted in stem-like cells and propagated to their progeny. Understanding the molecular control of CD8+ T cell differentiation in chronic infection has important therapeutic implications. Here, we find that the chemokine receptor CXCR3 is highly expressed on viral-specific stem-like CD8+ T cells and that one of its ligands, CXCL10, regulates the persistence and heterogeneity of responding CD8+ T cells in spleens of mice chronically infected with lymphocytic choriomeningitis virus. CXCL10 is produced by inflammatory monocytes and fibroblasts of the splenic red pulp, where it grants stem-like cells access to signals promoting differentiation and limits their exposure to pro-survival niches in the white pulp. Consequently, functional CD8+ T cell responses are greater in Cxcl10-/- mice and are associated with a lower viral set point.

Cancer cells relax and resist cytotoxic attack.

When it comes to cancer evading the immune response, antigen presentation usually gets all the attention. In this issue of Immunity, Tello-Lafoz et al. reveal that cancer cells have another card up their sleeve: by regulating gene expression to "soften" their actin cytoskeleton, cancer cells limit susceptibility to lymphocyte-mediated cytotoxic attack.

Behavioural immune landscapes of inflammation.

Transcriptional and proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues1,2. These approaches, however, do not describe dynamic scenarios in which cells continuously change their biochemical properties and downstream 'behavioural' outputs3-5. Here we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamics of individual leukocytes at sites of active inflammation. By analysing more than 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioural descriptors of individual cells and used these high-dimensional datasets to build behavioural landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and uncovered a continuum of neutrophil states inside blood vessels, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioural screening in 24 mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and interference with Fgr protected mice from inflammatory injury. Thus, behavioural landscapes report distinct properties of dynamic environments at high cellular resolution.

Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy.

The transcription factor NFAT promotes exhaustion of activated CD8⁺ T cells.

During persistent antigen stimulation, CD8(+) T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8(+) T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8(+) T cells and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8(+) T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1.

Guidance factors orchestrating regulatory T cell positioning in tissues during development, homeostasis, and response.

Over their lifetime, regulatory T cells (Treg) recalibrate their expression of trafficking receptors multiple times as they progress through development, respond to immune challenges, or adapt to the requirements of functioning in various non-lymphoid tissue environments. These trafficking receptors, which include chemokine receptors and other G-protein coupled receptors, integrins, as well as selectins and their ligands, enable Treg not only to enter appropriate tissues from the bloodstream via post-capillary venules, but also to navigate these tissues to locally execute their immune-regulatory functions, and finally to seek out the right antigen-presenting cells and interact with these, in part in order to receive the signals that sustain their survival, proliferation, and functional activity, in part in order to execute their immuno-regulatory function by altering antigen presenting cell function. Here, we will review our current knowledge of when and in what ways Treg alter their trafficking properties. We will focus on the chemokine system and try to identify specialized, non-redundant roles of individual receptors as well as similarities and differences to the conventional T cell compartment.

The transcription factor NFAT exhibits signal memory during serial T cell interactions with antigen-presenting cells.

Interactions with antigen-presenting cells (APCs) interrupt T cell migration through tissues and trigger signaling pathways that converge on the activation of transcriptional regulators, including nuclear factor of activated T cells (NFAT), which control T cell function and differentiation. Both stable and unstable modes of cognate T cell-APC interactions have been observed in vivo, but the functional significance of unstable, serial contacts has remained unclear. Here we used multiphoton intravital microscopy in lymph nodes and tumors to show that while NFAT nuclear import was fast (t(1/2 max)∼1 min), nuclear export was slow (t(1/2)∼20 min) in T cells. During delayed export, nuclear NFAT constituted a short-term imprint of transient TCR signals and remained transcriptionally active for the T cell tolerance gene Egr2, but not for the effector gene Ifng, which required continuous TCR triggering for expression. This provides a potential mechanistic basis for the observation that a predominance of unstable APC interactions correlates with the induction of T cell tolerance.

Antigen availability determines CD8⁺ T cell-dendritic cell interaction kinetics and memory fate decisions.

T cells are activated by antigen (Ag)-bearing dendritic cells (DCs) in lymph nodes in three phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely correlated with Ag dose. The second phase, characterized by stable DC-T cell contacts, is believed to be necessary for full-fledged T cell activation. Here we have shown that this is not the case. CD8⁺ T cells interacting with DCs presenting low-dose, short-lived Ag did not transition to phase 2, whereas higher Ag dose yielded phase 2 transition. Both antigenic constellations promoted T cell proliferation and effector differentiation but yielded different transcriptome signatures at 12 hr and 24 hr. T cells that experienced phase 2 developed long-lived memory, whereas conditions without stable contacts yielded immunological amnesia. Thus, T cells make fate decisions within hours after Ag exposure, resulting in long-term memory or abortive effector responses, correlating with T cell-DCs interaction kinetics.

CXCR3 chemokine receptor-ligand interactions in the lymph node optimize CD4+ T helper 1 cell differentiation.

Differentiation of naive CD4(+) T cells into T helper (Th) cells is a defining event in adaptive immunity. The cytokines and transcription factors that control Th cell differentiation are understood, but it is not known how this process is orchestrated within lymph nodes (LNs). Here we have shown that the CXCR3 chemokine receptor was required for optimal generation of interferon-γ (IFN-γ)-secreting Th1 cells in vivo. By using a CXCR3 ligand reporter mouse, we found that stromal cells predominately expressed the chemokine ligand CXCL9 whereas hematopoietic cells expressed CXCL10 in LNs. Dendritic cell (DC)-derived CXCL10 facilitated T cell-DC interactions in LNs during T cell priming while both chemokines guided intranodal positioning of CD4(+) T cells to interfollicular and medullary zones. Thus, different chemokines acting on the same receptor can function locally to facilitate DC-T cell interactions and globally to influence intranodal positioning, and both functions contribute to Th1 cell differentiation.

Bone degradation machinery of osteoclasts: An HIV-1 target that contributes to bone loss.

Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.

Orchestrating the orchestrators: chemokines in control of T cell traffic.

The understanding of how chemokines orchestrate the trafficking and activity of immune cells has increased considerably. So far, over 50 chemokines and 20 chemokine receptors have been identified. Detailed analyses have demonstrated the function of chemokine receptors on T cell subsets, the temporal and spatial expression patterns of chemokines in vivo and the phenotypes of animals genetically deficient in one component or several components of the chemokine-chemokine receptor system. New microscopy modalities for studying the influence of chemokines on the migratory activity of T cells in the lymph node have also brought new insights. Here we review such advances with particular emphasis on control of the migration of T cell subsets in lymph nodes and in peripheral tissues in homeostasis and inflammation.

T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation.

After homing to lymph nodes, CD8+ T cells are primed by dendritic cells (DCs) in three phases. During phase one, T cells undergo brief serial contacts with DCs for several hours, whereas phase two is characterized by stable T cell-DC interactions. We show here that the duration of phase one and T cell activation kinetics correlated inversely with the number of complexes of cognate peptide and major histocompatibility complex (pMHC) per DC and with the density of antigen-presenting DCs per lymph node. Very few pMHC complexes were necessary for the induction of full-fledged T cell activation and effector differentiation. However, neither T cell activation nor transition to phase two occurred below a threshold antigen dose determined in part by pMHC stability. Thus, phase one permits T cells to make integrated 'measurements' of antigen dose that determine subsequent T cell participation in immune responses.

Tumor Tolerance-Promoting Function of Regulatory T Cells Is Optimized by CD28, but Strictly Dependent on Calcineurin.

Regulatory T cells (Treg) restrain immune responses against malignant tumors, but their global depletion in cancer patients will likely be limited by systemic autoimmune toxicity. Instead, approaches to "tune" their activities may allow for preferential targeting of tumor-reactive Treg. Although Ag recognition regulates Treg function, the roles of individual TCR-dependent signaling pathways in enabling Treg to promote tumor tolerance are not well characterized. In this study, we examined in mouse tumor models the role of calcineurin, a key mediator of TCR signaling, and the role of the costimulatory receptor CD28 in the differentiation of resting central Treg into effector Treg endowed with tumor tropism. We find that calcineurin, although largely dispensable for suppressive activity in vitro, is essential for upregulation of ICOS and CTLA-4 in Treg, as well as for expression of chemokine receptors driving their accumulation in tumors. In contrast, CD28 is not critical, but optimizes the formation of tumor-homing Treg and their fitness in tumor tissue. Accordingly, although deletion of either CnB or CD28 strongly impairs Treg-mediated tumor tolerance, lack of CnB has an even more pronounced impact than lack of CD28. Hence, our studies reveal distinct roles for what has classically been defined as signal 1 and signal 2 of conventional T cell activation in the context of Treg-mediated tumor tolerance.

B Cells Drive Autoimmunity in Mice with CD28-Deficient Regulatory T Cells.

Follicular regulatory T (TFR) cells are a newly defined regulatory T cell (Treg) subset that suppresses follicular helper T cell-mediated B cell responses in the germinal center reaction. The precise costimulatory signal requirements for proper TFR cell differentiation and function are still not known. Using conditional knockout strategies of CD28, we previously demonstrated that loss of CD28 signaling in Tregs caused autoimmunity in mice (termed CD28-ΔTreg mice), characterized by lymphadenopathy, accumulation of activated T cells, and cell-mediated inflammation of the skin and lung. In this study, we show that CD28 signaling is required for TFR cell differentiation. Treg-specific deletion of CD28 caused a reduction in TFR cell numbers and function, which resulted in increased germinal center B cells and Ab production. Moreover, residual CD28-deficient TFR cells showed a diminished suppressive capacity as assessed by their ability to inhibit Ab responses in vitro. Surprisingly, genetic deletion of B cells in CD28-ΔTreg mice prevented the development of lymphadenopathy and CD4+ T cell activation, and autoimmunity that mainly targeted skin and lung tissues. Thus, autoimmunity occurring in mice with CD28-deficient Tregs appears to be driven primarily by loss of TFR cell differentiation and function with resulting B cell-driven inflammation.

T cell-intrinsic S1PR1 regulates endogenous effector T-cell egress dynamics from lymph nodes during infection.

Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1(-/-) effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1(-/-) T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1(-/-) effector T cells failed to enter the sinuses. We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN.

Conduits mediate transport of low-molecular-weight antigen to lymph node follicles.

To track drainage of lymph-borne small and large antigens (Ags) into the peripheral lymph nodes and subsequent encounter by B cells and follicular dendritic cells, we used the approach of multiphoton intravital microscopy. We find a system of conduits that extend into the follicles and mediate delivery of small antigens to cognate B cells and follicular dendritic cells. The follicular conduits provide an efficient and rapid mechanism for delivery of small antigens and chemokines such as CXCL13 to B cells that directly contact the conduits. By contrast, large antigens were bound by subcapsular sinus macrophages and subsequently transferred to follicular B cells as previously reported. In summary, the findings identify a unique pathway for the channeling of small lymph-borne antigens and chemoattractants from the subcapsular sinus directly to the B cell follicles. This pathway could be used for enhancing delivery of vaccines or small molecules for improvement of humoral immunity.