Distinct developmental pathways generate functionally distinct populations of natural killer cells.

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.

B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

Tumor-derived prostaglandin E2 programs cDC1 dysfunction to impair intratumoral orchestration of anti-cancer T cell responses.

Type 1 conventional dendritic cells (cDC1s) are critical for anti-cancer immunity. Protective anti-cancer immunity is thought to require cDC1s to sustain T cell responses within tumors, but it is poorly understood how this function is regulated and whether its subversion contributes to immune evasion. Here, we show that tumor-derived prostaglandin E2 (PGE2) programmed a dysfunctional state in intratumoral cDC1s, disabling their ability to locally orchestrate anti-cancer CD8+ T cell responses. Mechanistically, cAMP signaling downstream of the PGE2-receptors EP2 and EP4 was responsible for the programming of cDC1 dysfunction, which depended on the loss of the transcription factor IRF8. Blockade of the PGE2-EP2/EP4-cDC1 axis prevented cDC1 dysfunction in tumors, locally reinvigorated anti-cancer CD8+ T cell responses, and achieved cancer immune control. In human cDC1s, PGE2-induced dysfunction is conserved and associated with poor cancer patient prognosis. Our findings reveal a cDC1-dependent intratumoral checkpoint for anti-cancer immunity that is targeted by PGE2 for immune evasion.

Unbiased chemokine receptor screening reveals similar efficacy of lymph node- and tumor-targeted T cell immunotherapy.

Localization is a crucial prerequisite for immune cell function and solid tumors evade immune control by modulating immune cell infiltration into the tumor stroma. Immunosuppressive cells like regulatory T cells are attracted, while cytotoxic CD8+ T cells are excluded. Engineering CD8+ T cells with chemokine receptors is a potent strategy to turn this mechanism of directed immune cell recruitment against the tumor. Here, we utilized fluorescent tagging to track the migratory behavior of tumor-specific T cells engineered with a library of all murine chemokine receptors in vivo. We then asked whether chemokine receptor-mediated redirection of antigen-specific T cells into tumors or tumor-draining lymph nodes showed superior anti-tumoral activity. We found that both targeting approaches showed higher therapeutic efficacy than control T cells. However, multiple receptors conveying the same homing pattern did not augment infiltration. Instead, in the MC38 colon carcinoma model, anti-tumoral efficacy as well as lymph node vs. tumor-homing patterns were mostly driven by CCR4 and CCR6, respectively. Overall, our data, based on fluorescent receptor tagging, identify the tumor-draining lymph node and the tumor itself as viable targets for chemokine receptor-mediated enhancement of adoptive T cell therapy.

Signatures of recent activation identify a circulating T cell compartment containing tumor-specific antigen receptors with high avidity.

T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen-specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma-combined with transgenic reexpression of identified TCRs by CRISPR-Cas9-mediated orthotopic TCR replacement-revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.

Retrogenic Color-Barcoding for Fate Mapping of Single Innate Lymphocytes.

Lymphocyte fate mapping using single-cell transfers has been used to study T and B cell differentiation. Recently, retrogenic color-barcoding has allowed the extension of this approach to single innate lymphocytes such as NK cells. This new and versatile technology is based on the transduction of hematopoietic stem cells (HSCs) with a collection of retroviruses encoding distinct fluorescent proteins. Through combinatorial transduction, fluorescent protein barcodes are generated, which are inherited by the progeny of HSCs after transfer. By sorting individual cells expressing unique color-barcodes from the mature lymphocyte populations derived from these HSCs, it is now possible to track the fate of innate lymphocytes in vivo.

Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy.

Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.

T cells armed with C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours.

The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.

Early emergence of T central memory precursors programs clonal dominance during chronic viral infection.

Chronic cytomegalovirus (CMV) infection leads to long-term maintenance of extraordinarily large CMV-specific T cell populations. The magnitude of this so-called 'memory inflation' is thought to mainly depend on antigenic stimulation during the chronic phase of infection. However, by mapping the long-term development of CD8+ T cell families derived from single naive precursors, we find that fate decisions made during the acute phase of murine CMV infection can alter the level of memory inflation by more than 1,000-fold. Counterintuitively, a T cell family's capacity for memory inflation is not determined by its initial expansion. Instead, those rare T cell families that dominate the chronic phase of infection show an early transcriptomic signature akin to that of established T central memory cells. Accordingly, a T cell family's long-term dominance is best predicted by its early content of T central memory precursors, which later serve as a stem-cell-like source for memory inflation.

Multiplexed whole-animal imaging with reversibly switchable optoacoustic proteins.

We introduce two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria, and tumors in the mouse body and brain. We developed machine learning-based software for commercial imaging systems for temporal unmixed OA imaging, enabling its routine use in life sciences.

Orthotopic replacement of T-cell receptor α- and ß-chains with preservation of near-physiological T-cell function.

Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and ß-chains combined with orthotopic TCR placement leads to accurate αß-pairing and results in TCR regulation similar to that of physiological T cells.

CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes.

Chemokines have crucial roles in organ development and orchestration of leukocyte migration. The chemokine CCL22 is expressed constitutively at high levels in the lymph node, but the functional significance of this expression is so far unknown. Studying a newly established CCL22-deficient mouse, we demonstrate that CCL22 expression by dendritic cells (DCs) promotes the formation of cell-cell contacts and interaction with regulatory T cells (T reg) through their CCR4 receptor. Vaccination of CCL22-deficient mice led to excessive T cell responses that were also observed when wild-type mice were vaccinated using CCL22-deficient DCs. Tumor-bearing mice with CCL22 deficiency showed prolonged survival upon vaccination, and further, CCL22-deficient mice had increased susceptibility to inflammatory disease. In conclusion, we identify the CCL22-CCR4 axis as an immune checkpoint that is crucial for the control of T cell immunity.

PD1-CD28 Fusion Protein Enables CD4+ T Cell Help for Adoptive T Cell Therapy in Models of Pancreatic Cancer and Non-hodgkin Lymphoma.

Background: Interaction of the programmed death receptor 1 (PD-1) and its ligand, PD-L1, suppresses T cell activity and permits tumors to evade T cell-mediated immune surveillance. We have recently demonstrated that antigen-specific CD8+ T cells transduced with a PD1-CD28 fusion protein are protected from PD-1-mediated inhibition. We have now investigated the potential of PD1-CD28 fusion protein-transduced CD4+ T cells alone or in combination with CD8+ T cells for immunotherapy of pancreatic cancer and non-Hodgkin lymphoma. Methods: OVA-specific CD4+ and CD8+ were retrovirally transduced with the PD1-CD28 fusion protein. Cytokine release, proliferation, cytotoxic activity, and phenotype of transduced T cells were assessed in the context of Panc02-OVA (murine pancreatic cancer model) and E.G7-PD-L1 (murine T cell lymphoma model) cells. Results: Stimulation of PD1-CD28 fusion protein-transduced CD4+ T cells with anti-CD3 and recombinant PD-L1 induced specific T cell activation, as measured by IFN-y release and T cell proliferation. Coculture with Panc02-OVA or E.G7-PD-L1 tumor cells also led to specific activation of CD4+ T cells. Cytokine release and T cell proliferation was most effective when tumor cells simultaneously encountered genetically engineered CD4+ and CD8+ T cells. Synergy between both cell populations was also observed for specific tumor cell lysis. T cell cytotoxicity was mediated via granzyme B release and mediated enhanced tumor control in vivo. Transduced CD4+ and CD8+ T cells in co-culture with tumor cells developed a predominant central memory phenotype over time. Different ratios of CD4+ and CD8+ transduced T cells led to a significant increase of IFN-y and IL-2 secretion positively correlating with CD4+ T cell numbers used. Mechanistically, IL-2 and MHC-I were central to the synergistic activity of CD4+ and CD8+ T cells, since neutralization of IL-2 prevented the crosstalk between these cell populations. Conclusion: PD1-CD28 fusion protein-transduced CD4+ T cells significantly improved anti-tumoral effect of fusion protein-transduced CD8+ T cells. Thus, our results indicate that PD1-CD28 fusion protein-transduced CD4+ T cells have the potential to overcome the PD-1-PD-L1 immunosuppressive axis in pancreatic cancer and non-Hodgkin lymphoma.

C-C chemokine receptor type-4 transduction of T cells enhances interaction with dendritic cells, tumor infiltration and therapeutic efficacy of adoptive T cell transfer.

T cell infiltration at the tumor site has been identified as a major predictor for the efficacy of adoptive T cell therapy. The chemokine C-C motif ligand 22 (CCL22) is highly expressed by immune cells in murine and human pancreatic cancer. Expression of its corresponding receptor, C-C chemokine receptor type 4 (CCR4), is restricted to regulatory T cells (Treg). We show that transduction of cytotoxic T cells (CTL) with CCR4 enhances their immigration into a pancreatic cancer model. Further, we show that binding of CCR4 with CCL22 strengthens the binding of T cell LFA-1 to dendritic cell (DC) ICAM-1 and increases CTL activation. In vivo, in a model of subcutaneous pancreatic cancer, treatment of tumor-bearing mice with CCR4-transduced CTL led to the eradication of established tumors in 40% of the mice. In conclusion, CCR4 overexpression in CTL is a promising therapeutic strategy to enhance the efficacy of adoptive T cell transfer (ACT).

Impact of a New Fusion Receptor on PD-1-Mediated Immunosuppression in Adoptive T Cell Therapy.

BACKGROUND: Adoptive T cell transfer (ACT) is currently under investigation for the treatment of metastatic cancer. Recent evidence suggests that the coinhibitory PD-1-PD-L1 axis plays a major role in ACT failure. We hypothesized that a new fusion receptor reverting PD-1-mediated inhibition into CD28 costimulation may break peripheral tolerance. METHODS: Different PD-1-CD28 fusion receptor constructs were created and retrovirally transduced into primary T cell receptor transgenic murine CD8(+) T cells specific for ovalbumin (OT-1). Cytokine release, proliferation, cytotoxicity, and tumor recognition were analyzed in vitro. Antitumor efficacy and mode of action were investigated in mice bearing subcutaneous tumors induced with the pancreatic carcinoma cell line Panc02 expressing the model antigen ovalbumin (Panc-OVA). For antitumoral efficacy, six to eight mice per group were used. All statistical tests are two-sided. RESULTS: Transduction of the PD-1-CD28 receptor constructs mediated enhanced cytokine release, T cell proliferation, and T cell-induced lysis of target tumor cells. The PD-1-CD28 receptor function was dependent on two of the CD28-signaling motifs and IFN-γ release. Treatment of mice with established Panc-OVA tumors with fusion receptor-transduced OT-1 T cells mediated complete tumor regression. Mice rejecting the tumor were protected upon subsequent rechallenge with either ovalbumin-positive or -negative tumors, indicative of a memory response and epitope spreading in nine of 11 mice vs none of the six naïve mice (P < .001). Treatment efficacy was associated with accumulation of IFN-γ-producing T cells and an increased ratio of CD8(+) T cells to immunosuppressive myeloid-derived suppressor cells in the tumors. CONCLUSIONS: Transduction of T cells with this new PD-1-CD28 receptor has the potential of breaking the PD-1-PD-L1-immunosuppressive axis in ACT.

Selective bispecific T cell recruiting antibody and antitumor activity of adoptive T cell transfer.

BACKGROUND: One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. METHODS: SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40(+) and EpCAM(+)). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met(+) human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. RESULTS: The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase(+) melanoma cells by TCR-, as well as of CEA(+) colon cancer cells by chimeric antigen receptor (CAR)-modified T cells. CONCLUSIONS: BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT.