CAR+ and CAR- T cells share a differentiation trajectory into an NK-like subset after CD19 CAR T cell infusion in patients with B cell malignancies.

Chimeric antigen receptor (CAR) T cell therapy is effective in treating B cell malignancies, but factors influencing the persistence of functional CAR+ T cells, such as product composition, patients' lymphodepletion, and immune reconstitution, are not well understood. To shed light on this issue, here we conduct a single-cell multi-omics analysis of transcriptional, clonal, and phenotypic profiles from pre- to 1-month post-infusion of CAR+ and CAR- T cells from patients from a CARTELL study (ACTRN12617001579381) who received a donor-derived 4-1BB CAR product targeting CD19. Following infusion, CAR+ T cells and CAR- T cells shows similar differentiation profiles with clonally expanded populations across heterogeneous phenotypes, demonstrating clonal lineages and phenotypic plasticity. We validate these findings in 31 patients with large B cell lymphoma treated with CD19 CAR T therapy. For these patients, we identify using longitudinal mass-cytometry data an association between NK-like subsets and clinical outcomes at 6 months with both CAR+ and CAR- T cells. These results suggest that non-CAR-derived signals can provide information about patients' immune recovery and be used as correlate of clinically relevant parameters.

High-dimensional intravital microscopy reveals major changes in splenic immune system during postnatal development.

Spleen is a key organ for immunologic surveillance, acting as a firewall for antigens and parasites that spread through the blood. However, how spleen leukocytes evolve across the developmental phase, and how they spatially organize and interact in vivo is still poorly understood. Using a novel combination of selected antibodies and fluorophores to image in vivo the spleen immune environment, we described for the first time the dynamics of immune development across postnatal period. We found that spleens from adults and infants had similar numbers and arrangement of lymphoid cells. In contrast, splenic immune environment in newborns is sharply different from adults in almost all parameters analysed. Using this in vivo approach, B cells were the most frequent subtype throughout the development. Also, we revealed how infections - using a model of malaria - can change the spleen immune profile in adults and infants, which could become the key to understanding different severity grades of infection. Our new imaging solutions can be extremely useful for different groups in all areas of biological investigation, paving a way for new intravital approaches and advances.

Progenitor Hierarchy of Chronic Myelomonocytic Leukemia Identifies Inflammatory Monocytic-Biased Trajectory Linked to Worse Outcomes.

Myeloblast expansion is a hallmark of disease progression and comprises CD34+ hematopoietic stem and progenitor cells (HSPC). How this compartment evolves during disease progression in chronic myeloid neoplasms is unknown. Using single-cell RNA sequencing and high-parameter flow cytometry, we show that chronic myelomonocytic leukemia (CMML) CD34+ HSPC can be classified into three differentiation trajectories: monocytic, megakaryocyte-erythroid progenitor (MEP), and normal-like. Hallmarks of monocytic-biased trajectory were enrichment of CD120b+ inflammatory granulocyte-macrophage progenitor (GMP)-like cells, activated cytokine receptor signaling, phenotypic hematopoietic stem cell (HSC) depletion, and adverse outcomes. Cytokine receptor diversity was generally an adverse feature and elevated in CD120b+ GMPs. Hypomethylating agents decreased monocytic-biased cells in CMML patients. Given the enrichment of RAS pathway mutations in monocytic-biased cells, NRAS-competitive transplants and LPS-treated xenograft models recapitulated monocytic-biased CMML, suggesting that hematopoietic stress precipitates the monocytic-biased state. Deconvolution of HSPC compartments in other myeloid neoplasms and identifying therapeutic strategies to mitigate the monocytic-biased differentiation trajectory should be explored. SIGNIFICANCE: Our findings establish that multiple differentiation states underlie CMML disease progression. These states are negatively augmented by inflammation and positively affected by hypomethylating agents. Furthermore, we identify HSC depletion and expansion of GMP-like cells with increased cytokine receptor diversity as a feature of myeloblast expansion in inflammatory chronic myeloid neoplasms. This article is highlighted in the In This Issue feature, p. 476.

High-throughput and high-dimensional single-cell analysis of antigen-specific CD8+ T cells.

Although critical to T cell function, antigen specificity is often omitted in high-throughput multiomics-based T cell profiling due to technical challenges. We describe a high-dimensional, tetramer-associated T cell antigen receptor (TCR) sequencing (TetTCR-SeqHD) method to simultaneously profile cognate antigen specificities, TCR sequences, targeted gene expression and surface-protein expression from tens of thousands of single cells. Using human polyclonal CD8+ T cells with known antigen specificity and TCR sequences, we demonstrate over 98% precision for detecting the correct antigen specificity. We also evaluate gene expression and phenotypic differences among antigen-specific CD8+ T cells and characterize phenotype signatures of influenza- and Epstein-Barr virus-specific CD8+ T cells that are unique to their pathogen targets. Moreover, with the high-throughput capacity of profiling hundreds of antigens simultaneously, we apply TetTCR-SeqHD to identify antigens that preferentially enrich cognate CD8+ T cells in patients with type 1 diabetes compared to healthy controls and discover a TCR that cross-reacts with diabetes-related and microbiome antigens. TetTCR-SeqHD is a powerful approach for profiling T cell responses in humans and mice.

Single cell RNA sequencing of AML initiating cells reveals RNA-based evolution during disease progression.

The prognosis of most patients with AML is poor due to frequent disease relapse. The cause of relapse is thought to be from the persistence of leukemia initiating cells (LIC's) following treatment. Here we assessed RNA based changes in LICs from matched patient diagnosis and relapse samples using single-cell RNA sequencing. Previous studies on AML progression have focused on genetic changes at the DNA mutation level mostly in bulk AML cells and demonstrated the existence of DNA clonal evolution. Here we identified in LICs that the phenomenon of RNA clonal evolution occurs during AML progression. Despite the presence of vast transcriptional heterogeneity at the single cell level, pathway analysis identified common signaling networks involving metabolism, apoptosis and chemokine signaling that evolved during AML progression and become a signature of relapse samples. A subset of this gene signature was validated at the protein level in LICs by flow cytometry from an independent AML cohort and functional studies were performed to demonstrate co-targeting BCL2 and CXCR4 signaling may help overcome therapeutic challenges with AML heterogeneity. It is hoped this work will facilitate a greater understanding of AML relapse leading to improved prognostic biomarkers and therapeutic strategies to target LIC's.

Identification and characterization of a SARS-CoV-2 specific CD8+ T cell response with immunodominant features.

The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8+ T cells. Here, we analyze samples from 31 patients with COVID-19 for CD8+ T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8+ T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8+ T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8+ T cell responses are detectable up to 5 months after recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8+ T cells during convalescence.

Zebularine elevates STING expression and enhances cGAMP cancer immunotherapy in mice.

DNA methylation abnormality is closely related to tumor occurrence and development. Chemical inhibitors targeting DNA methyltransferase (DNMTis) have been used in treating cancer. However, the impact of DNMTis on antitumor immunity has not been well elucidated. In this study, we show that zebularine (a demethylating agent) treatment of cancer cells led to increased levels of interferon response in a cyclic guanosine monophosphate-AMP (cGAMP) synthase (cGAS)- and stimulator of interferon genes (STING)-dependent manner. This treatment also specifically sensitized the cGAS-STING pathway in response to DNA stimulation. Incorporation of zebularine into genomic DNA caused demethylation and elevated expression of a group of genes, including STING. Without causing DNA damage, zebularine led to accumulation of DNA species in the cytoplasm of treated cells. In syngeneic tumor models, administration of zebularine alone reduced tumor burden and extended mice survival. This effect synergized with cGAMP and immune checkpoint blockade therapy. The efficacy of zebularine was abolished in nude mice and in cGAS-/- or STING-/- mice, indicating its dependency on host immunity. Analysis of tumor cells indicates upregulation of interferon-stimulated genes (ISGs) following zebularine administration. Zebularine promoted infiltration of CD8 T cells and natural killer (NK) cells into tumor and therefore suppressed tumor growth. This study unveils the role of zebularine in sensitizing the cGAS-STING pathway to promote anti-tumor immunity and provides the foundation for further therapeutic development.

OMIP 071: A 31-Parameter Flow Cytometry Panel for In-Depth Immunophenotyping of Human T-Cell Subsets Using Surface Markers.

Dissecting the functional diversity of T cells is critical in elucidating mechanisms and in developing therapies for various diseases. Here, we designed a 31-parameter (29-color) panel to enable the characterization of T-cell subsets and immunophenotyping of the human peripheral blood and lymph nodes using cell surface staining. In addition to adaptive T-cell markers, TCR Vα24-Jα18, TCR γδ, TCR Vɑ7.2, and CD161 were included to identify iNKT, γδ T, and MAIT cells, respectively, which are innate-like T cells. C-X-C chemokine receptors (CXCR3, CXCR4, CXCR5, CXCR6) and C-C motif chemokine receptors (CCR4, CCR6, CCR7) were included to enable the identification of Th cell subsets (Th1, Th2, Th17), Tfh cell subsets (Tfh1, Tfh2, Tfh17), and Th cells with specific homing capacities. Furthermore, in this panel, we also used markers for assessing cell differentiation (CD45RO, CD7), activation (CD57, CD95, HLA-DR) and the expression of some cosignaling molecules (PD-1, NKG2D, CD28). Particularly, CD69 and CD103 were included for the further analysis of tissue resident memory T (Trm) cells. This panel would enable the in-depth immunophenotyping of human T-cell subsets, and may be applied in the monitoring, prognosis, and mechanistic studies of various immune-related diseases.

Tissue Infiltrating LTi-Like Group 3 Innate Lymphoid Cells and T Follicular Helper Cells in Graves' and Hashimoto's Thyroiditis.

Background: Hashimoto's thyroiditis (HT) and Graves' disease (GD) are autoimmune thyroid disorders (AITDs). These conditions have been associated to abnormalities in circulating regulatory T cells (Tregs). We postulated that immune perturbations could be more pronounced at the thyroid tissue level. Methods: The phenotype of PBMCs and immune cells infiltrating thyroid tissue from 19 patients with HT, 21 patients with GD, and 30 controls has been analyzed by flow cytometry. Results: We report that blood and thyroid Treg cell subsets are similarly represented in all AITDs patients and controls. Increased Lymphoid tissue inducer (LTi)-like ILC3 and CXCR5+ PD-1hi CD4+ T follicular helper cells (Tfh) tissue-infiltrating cells, together with the prevalence of tertiary lymphoid structures (TLS) and germinal centers (GCs) represented a typical immune signature in all HT and 60% of GD patients. In the remaining group of GD patients, the absence of the aforementioned abnormalities was associated with a higher prevalence of ophthalmopathy. Conclusion: Tissue infiltrating Lymphoid Tissue inducer-like group 3 Innate Lymphoid cells and T follicular helper cells are increased in most thyroid autoimmune disease.

Cycling CD4+ T cells in HIV-infected immune nonresponders have mitochondrial dysfunction.

Immune nonresponder (INR) HIV-1-infected subjects are characterized by their inability to reconstitute the CD4+ T cell pool after antiretroviral therapy. This is linked to poor clinical outcome. Mechanisms underlying immune reconstitution failure are poorly understood, although, counterintuitively, INRs often have increased frequencies of circulating CD4+ T cells in the cell cycle. While cycling CD4+ T cells from healthy controls and HIV+ patients with restored CD4+ T cell numbers complete cell division in vitro, cycling CD4+ T cells from INRs do not. Here, we show that cells with the phenotype and transcriptional profile of Tregs were enriched among cycling cells in health and in HIV infection. Yet there were diminished frequencies and numbers of Tregs among cycling CD4+ T cells in INRs, and cycling CD4+ T cells from INR subjects displayed transcriptional profiles associated with the impaired development and maintenance of functional Tregs. Flow cytometric assessment of TGF-ß activity confirmed the dysfunction of Tregs in INR subjects. Transcriptional profiling and flow cytometry revealed diminished mitochondrial fitness in Tregs among INRs, and cycling Tregs from INRs had low expression of the mitochondrial biogenesis regulators peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α) and transcription factor A for mitochondria (TFAM). In vitro exposure to IL-15 allowed cells to complete division, restored the expression of PGC1α and TFAM, and regenerated mitochondrial fitness in the cycling Tregs of INRs. Our data suggest that rescuing mitochondrial function could correct the immune dysfunction characteristic of Tregs in HIV-1-infected subjects who fail to restore CD4+ T cells during antiretroviral therapy.

CTLA-4+PD-1- Memory CD4+ T Cells Critically Contribute to Viral Persistence in Antiretroviral Therapy-Suppressed, SIV-Infected Rhesus Macaques.

Antiretroviral therapy (ART) suppresses viral replication in HIV-infected individuals but does not eliminate the reservoir of latently infected cells. Recent work identified PD-1+ follicular helper T (Tfh) cells as an important cellular compartment for viral persistence. Here, using ART-treated, SIV-infected rhesus macaques, we show that CTLA-4+PD-1- memory CD4+ T cells, which share phenotypic markers with regulatory T cells, were enriched in SIV DNA in blood, lymph nodes (LN), spleen, and gut, and contained replication-competent and infectious virus. In contrast to PD-1+ Tfh cells, SIV-enriched CTLA-4+PD-1- CD4+ T cells were found outside the B cell follicle of the LN, predicted the size of the persistent viral reservoir during ART, and significantly increased their contribution to the SIV reservoir with prolonged ART-mediated viral suppression. We have shown that CTLA-4+PD-1- memory CD4+ T cells are a previously unrecognized component of the SIV and HIV reservoir that should be therapeutically targeted for a functional HIV-1 cure.

CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology.

CD (cluster of differentiation) Ags are cell surface molecules expressed on leukocytes and other cells relevant for the immune system. CD nomenclature has been universally adopted by the scientific community and is officially approved by the International Union of Immunological Societies and sanctioned by the World Health Organization. It provides a unified designation system for mAbs, as well as for the cell surface molecules that they recognize. This nomenclature was established by the Human Leukocyte Differentiation Antigens Workshops. In addition to defining the CD nomenclature, these workshops have been instrumental in identifying and determining the expression and function of cell surface molecules. Over the past 30 y, the data generated by the 10 Human Leukocyte Differentiation Antigens Workshops have led to the characterization and formal designation of more than 400 molecules. CD molecules are commonly used as cell markers, allowing the identification and isolation of leukocyte populations, subsets, and differentiation stages. mAbs against these molecules have proven to be essential for biomedical research and diagnosis, as well as in biotechnology. More recently, they have been recognized as invaluable tools for the treatment of several malignancies and autoimmune diseases. In this article, we describe how the CD nomenclature was established, present the official updated list of CD molecules, and provide a rationale for their usefulness in the 21st century.

Sialyl Lewis x (CD15s) identifies highly differentiated and most suppressive FOXP3high regulatory T cells in humans.

CD4(+) regulatory T (Treg) cells expressing CD25 and the transcription factor forkhead box P3 (FOXP3) are indispensable for immunological self-tolerance and homeostasis. FOXP3(+)CD25(+)CD4(+) T cells in humans, however, are heterogeneous in function and differentiation status, including suppressive or nonsuppressive cells as well as resting or activated Treg cells. We have searched for cell surface markers specific for suppression-competent Treg cells by using a panel of currently available monoclonal antibodies reactive with human T cells. We found that CD15s (sialyl Lewis x) was highly specific for activated, terminally differentiated, and most suppressive FOXP3(high) effector Treg (eTreg) cells and able to differentiate them in various clinical settings from nonsuppressive FOXP3(+) T cells secreting inflammatory cytokines. For example, CD15s(+)FOXP3(+) eTreg cells were increased in sarcoidosis, whereas it was nonsuppressive CD15s(-)FOXP3(+) T cells that were expanded in lupus flares. FOXP3(+) cells induced from conventional CD4(+) T cells by T-cell receptor stimulation hardly expressed CD15s. CD15s(+)CD4(+) T-cell depletion was sufficient to evoke and enhance in vitro immune responses against tumor or viral antigens. Collectively, we have identified CD15s as a biomarker instrumental in both phenotypic and functional analysis of FOXP3(+)CD4(+) T-cell subpopulations in health and disease. It allows specific targeting of eTreg cells, rather than whole FOXP3(+)CD4(+) T cells, in controlling immune responses.

Multidimensional single-cell analysis of BCR signaling reveals proximal activation defect as a hallmark of chronic lymphocytic leukemia B cells.

PURPOSE: Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. We aimed to model differential signaling behavior between B cells from CLL and healthy individuals to pinpoint modes of dysregulation. EXPERIMENTAL DESIGN: We developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we modeled BCR signaling in 67 CLL patients using Partial Least Squares Regression (PLSR). Results from multidimensional modeling were validated using an independent test cohort of 38 patients. RESULTS: We identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLCγ2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLCγ2, as maximally predictive and sufficient to distinguish CLL from healthy samples, pointing to this juncture in the signaling pathway as a hallmark of CLL B cells. Specific BCR pathway signaling signatures that correlate with the disease and its degree of aggressiveness were identified. Heterogeneity in the PLSR response variable within the B cell population is both a characteristic mark of healthy samples and predictive of disease aggressiveness. CONCLUSION: Single-cell multidimensional analysis of BCR signaling permitted focused analysis of the variability and heterogeneity of signaling behavior from patient-to-patient, and from cell-to-cell. Disruption of the pSYK/pPLCγ2 relationship is uncovered as a robust hallmark of CLL B cell signaling behavior. Together, these observations implicate novel elements of the BCR signal transduction as potential therapeutic targets.

Human plasma cells express granzyme B.

While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116). Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

Macrophages induce differentiation of plasma cells through CXCL10/IP-10.

In tonsils, CD138(+) plasma cells (PCs) are surrounded by CD163(+) resident macrophages (Ms). We show here that human Ms (isolated from tonsils or generated from monocytes in vitro) drive activated B cells to differentiate into CD138(+)CD38(++) PCs through secreted CXCL10/IP-10 and VCAM-1 contact. IP-10 production by Ms is induced by B cell-derived IL-6 and depends on STAT3 phosphorylation. Furthermore, IP-10 amplifies the production of IL-6 by B cells, which sustains the STAT3 signals that lead to PC differentiation. IP-10-deficient mice challenged with NP-Ficoll show a decreased frequency of NP-specific PCs and lower titers of antibodies. Thus, our results reveal a novel dialog between Ms and B cells, in which IP-10 acts as a PC differentiation factor.

CD160 and PD-1 co-expression on HIV-specific CD8 T cells defines a subset with advanced dysfunction.

Chronic viral infections lead to persistent CD8 T cell activation and functional exhaustion. Expression of programmed cell death-1 (PD-1) has been associated to CD8 T cell dysfunction in HIV infection. Herein we report that another negative regulator of T cell activation, CD160, was also upregulated on HIV-specific CD8 T lymphocytes mostly during the chronic phase of infection. CD8 T cells that expressed CD160 or PD-1 were still functional whereas co-expression of CD160 and PD-1 on CD8 T cells defined a novel subset with all the characteristics of functionally exhausted T cells. Blocking the interaction of CD160 with HVEM, its natural ligand, increased HIV-specific CD8 T cell proliferation and cytokine production. Transcriptional profiling showed that CD160(-)PD-1(+)CD8 T cells encompassed a subset of CD8(+) T cells with activated transcriptional programs, while CD160(+)PD-1(+) T cells encompassed primarily CD8(+) T cells with an exhausted phenotype. The transcriptional profile of CD160(+)PD-1(+) T cells showed the downregulation of the NFκB transcriptional node and the upregulation of several inhibitors of T cell survival and function. Overall, we show that CD160 and PD-1 expressing subsets allow differentiating between activated and exhausted CD8 T cells further reinforcing the notion that restoration of function will require multipronged approaches that target several negative regulators.

Loss of memory B cells during chronic HIV infection is driven by Foxo3a- and TRAIL-mediated apoptosis.

Loss of memory B cells occurs from the onset of HIV-1 infection and persists into the chronic stages of infection. Lack of survival of these cells, even in subjects being treated, could primarily be the consequence of an altered local microenvironment induced by HIV infection. In this study we showed that memory B cell survival was significantly decreased in aviremic successfully treated (ST) subjects compared with subjects who control viral load as a result of natural immunity (elite controller [EC]) or with uninfected control (HIV-) subjects. The lower survival levels observed in memory B cells from ST subjects were the result of disrupted IL-2 signaling that led to increased transcriptional activity of Foxo3a and increased expression of its proapoptotic target TRAIL. Notably, memory B cell survival in ST subjects was significantly enhanced by the addition of exogenous IL-2 in a Foxo3a-dependent manner. We further showed that Foxo3a silencing by siRNA resulted in decreased expression of TRAIL and apoptosis levels in memory B cells from ST subjects. Our results thus establish a direct role for Foxo3a/TRAIL signaling in the persistence of memory B cells and provide a mechanism for the reduced survival of memory B cells during HIV infection. This knowledge could be exploited for the development of therapeutic and preventative HIV vaccines.