CyTOF® for the Masses.

Mass cytometry has revolutionized immunophenotyping, particularly in exploratory settings where simultaneous breadth and depth of characterization of immune populations is needed with limited samples such as in preclinical and clinical tumor immunotherapy. Mass cytometry is also a powerful tool for single-cell immunological assays, especially for complex and simultaneous characterization of diverse intratumoral immune subsets or immunotherapeutic cell populations. Through the elimination of spectral overlap seen in optical flow cytometry by replacement of fluorescent labels with metal isotopes, mass cytometry allows, on average, robust analysis of 60 individual parameters simultaneously. This is, however, associated with significantly increased complexity in the design, execution, and interpretation of mass cytometry experiments. To address the key pitfalls associated with the fragmentation, complexity, and analysis of data in mass cytometry for immunologists who are novices to these techniques, we have developed a comprehensive resource guide. Included in this review are experiment and panel design, antibody conjugations, sample staining, sample acquisition, and data pre-processing and analysis. Where feasible multiple resources for the same process are compared, allowing researchers experienced in flow cytometry but with minimal mass cytometry expertise to develop a data-driven and streamlined project workflow. It is our hope that this manuscript will prove a useful resource for both beginning and advanced users of mass cytometry.

Ex Vivo Expansion of Th2-Polarizing Immunotherapeutic iNKT Cells from Human Peripheral Blood.

iNKT cells, classified as innate lymphocytes with invariant TCRs, have been highlighted as a putative, "off-the-shelf" cellular immunotherapeutic strategy for the treatment of malignant and nonmalignant diseases. However, their paucity in human blood limits their immunotherapeutic applications. Herein we describe a rigorously optimized 21-day ex vivo expansion method to achieve log-fold increases in immunotherapeutic human iNKT cells.

Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity.

BACKGROUND AIMS: Key obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells. METHODS: We delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3+Vα24+Vß11+ iNKT cells. RESULTS: Gene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4+ and CD8+ responder T cells. DISCUSSION: These data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

Innate Immune Determinants of Graft-Versus-Host Disease and Bidirectional Immune Tolerance in Allogeneic Transplantation.

The success of tissue transplantation from a healthy donor to a diseased individual (allo-transplantation) is regulated by the immune systems of both donor and recipient. Developing a state of specific non-reactivity between donor and recipient, while maintaining the salutary effects of immune function in the recipient, is called "immune (transplantation) tolerance". In the classic early post-transplant period, minimizing bidirectional donor ←→ recipient reactivity requires the administration of immunosuppressive drugs, which have deleterious side effects (severe immunodeficiency, opportunistic infections, and neoplasia, in addition to drug-specific reactions and organ toxicities). Inducing immune tolerance directly through donor and recipient immune cells, particularly via subsets of immune regulatory cells, has helped to significantly reduce side effects associated with multiple immunosuppressive drugs after allo-transplantation. The innate and adaptive arms of the immune system are both implicated in inducing immune tolerance. In the present article, we will review innate immune subset manipulations and their potential applications in hematopoietic stem cell transplantation (HSCT) to cure malignant and non-malignant hematological disorders by inducing long-lasting donor ←→ recipient (bidirectional) immune tolerance and reduced graft-versus-host disease (GVHD). These innate immunotherapeutic strategies to promote long-term immune allo-transplant tolerance include myeloid-derived suppressor cells (MDSCs), regulatory macrophages, tolerogenic dendritic cells (tDCs), Natural Killer (NK) cells, invariant Natural Killer T (iNKT) cells, gamma delta T (γδ-T) cells and mesenchymal stromal cells (MSCs).

A sweet alternative: maintaining M2 macrophage polarization.

Glycolytic metabolism functions as a backup mechanism for M2 macrophage polarization when oxidative phosphorylation is disrupted.

Data-Driven Methods for Advancing Precision Oncology.

PURPOSE OF REVIEW: This article discusses the advances, methods, challenges, and future directions of data-driven methods in advancing precision oncology for biomedical research, drug discovery, clinical research, and practice. RECENT FINDINGS: Precision oncology provides individually tailored cancer treatment by considering an individual's genetic makeup, clinical, environmental, social, and lifestyle information. Challenges include voluminous, heterogeneous, and disparate data generated by different technologies with multiple modalities such as Omics, electronic health records, clinical registries and repositories, medical imaging, demographics, wearables, and sensors. Statistical and machine learning methods have been continuously adapting to the ever-increasing size and complexity of data. Precision Oncology supportive analytics have improved turnaround time in biomarker discovery and time-to-application of new and repurposed drugs. Precision oncology additionally seeks to identify target patient populations based on genomic alterations that are sensitive or resistant to conventional or experimental treatments. Predictive models have been developed for cancer progression and survivorship, drug sensitivity and resistance, and identification of the most suitable combination treatments for individual patient scenarios. In the future, clinical decision support systems need to be revamped to better incorporate knowledge from precision oncology, thus enabling clinical practitioners to provide precision cancer care. SUMMARY: Open Omics datasets, machine learning algorithms, and predictive models have enabled the advancement of precision oncology. Clinical decision support systems with integrated electronic health record and Omics data are needed to provide data-driven recommendations to assist clinicians in disease prevention, early identification, and individualized treatment. Additionally, as cancer is a constantly evolving disorder, clinical decision systems will need to be continually updated based on more recent knowledge and datasets.

Bidirectional immune tolerance in nonmyeloablative MHC-mismatched BMT for murine ß-thalassemia.

Nonmyeloablative conditioning using total lymphoid irradiation (TLI) and rabbit antithymocyte serum (ATS) (the murine preclinical equivalent of antithymocyte globulin [ATG]) facilitates immune tolerance after bone marrow transplantation (BMT) across major histocompatibility complex (MHC) disparities and may be a useful strategy for nonmalignant disorders. We previously reported that donor effector T-cell function and graft-versus-host disease (GVHD) are regulated via recipient invariant natural killer T-cell (iNKT) interleukin-4-driven expansion of donor Foxp3+ naturally occurring regulatory T cells (Tregs). This occurs via recipient iNKT- and STAT6-dependent expansion of recipient myeloid dendritic cells (MDCs) that induce contact-dependent expansion of donor Treg through PD-1/PD ligand signaling. After TLI/ATS + BMT, Gr-1lowCD11c+ MDCs and Gr-1highCD11cneg myeloid-derived suppressor cells (MDSCs) were enriched in GVHD target organs. We now report that the recovery of both recipient MDSCs (P < .01) and MDCs (P < .01) is significantly increased when the alkylator cyclophosphamide (CTX) is added to TLI/ATS conditioning. In a BALB/c → B6 lethal GVHD model, adoptive transfer of MDSCs from TLI/ATS/CTX-conditioned recipients is associated with significantly improved GVHD colitis and survival (P < .001), conversion of MDSCs to PD ligand-expressing MDCs, and increased donor naturally occurring Treg recovery (P < .01) compared with control treatment. Using BALB/c donors and ß-thalassemic HW-80 recipients, we found significantly improved rates of engraftment and GVHD following TLI/ATS/CTX compared with TLI/ATS, lethal or sublethal total body irradiation/ATS/CTX, or CTX/ATS conditioning. These data provide preclinical support for trials of TLI/ATG/alkylator regimens for MHC-mismatched BMT for hemoglobinopathies. The data also delineate innate immune mechanisms by which TLI/ATS/CTX conditioning may augment transplantation tolerance.

Recipient myeloid-derived immunomodulatory cells induce PD-1 ligand-dependent donor CD4+Foxp3+ regulatory T cell proliferation and donor-recipient immune tolerance after murine nonmyeloablative bone marrow transplantation.

We showed previously that nonmyeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor-recipient immune tolerance following bone marrow transplantation (BMT) across MHC barriers via recipient invariant NKT (iNKT) cell-derived IL-4-dependent expansion of donor Foxp3(+) naturally occurring regulatory T cells (nTregs). In this study, we report a more specific mechanism. Wild-type (WT) BALB/c (H-2(d)) hosts were administered TLI/ATS and BMT from WT or STAT6(-/-) C57BL/6 (H-2(b)) donors. Following STAT6(-/-) BMT, donor nTregs demonstrated no loss of proliferation in vivo, indicating that an IL-4-responsive population in the recipient, rather than the donor, drives donor nTreg proliferation. In graft-versus-host disease (GVHD) target organs, three recipient CD11b(+) cell subsets (Gr-1(high)CD11c(-), Gr-1(int)CD11c(-), and Gr-1(low)CD11c(+)) were enriched early after TLI/ATS + BMT versus total body irradiation/ATS + BMT. Gr-1(low)CD11c(+) cells induced potent H-2K(b+)CD4(+)Foxp3(+) nTreg proliferation in vitro in 72-h MLRs. Gr-1(low)CD11c(+) cells were reduced significantly in STAT6(-/-) and iNKT cell-deficient Jα18(-/-) BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b(+) cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1(low)CD11c(+) cells to Jα18(-/-) BALB/c recipients of TLI/ATS + BMT restored day-6 donor Foxp3(+) nTreg proliferation and protection from CD8 effector T cell-mediated GVHD. Blockade of programmed death ligand 1 and 2, but not CD40, TGF-ß signaling, arginase 1, or iNOS, inhibited nTreg proliferation in cocultures of recipient-derived Gr-1(low)CD11c(+) cells with donor nTregs. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory dendritic cells are expanded after nonmyeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand-dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.

NKT cells, Treg, and their interactions in bone marrow transplantation.

Bone marrow transplantation (BMT) is a potentially curative treatment for patients with leukemia and lymphoma. Tumor eradication is promoted by the anti-tumor activity of donor T cells contained in the transplant; however, donor T cells also mediate the serious side effect of graft-versus-host disease (GVHD). Separation of GVHD from graft anti-tumor activity is an important goal of research in improving transplant outcome. One approach is to take advantage of the immunomodulatory activity of regulatory NKT cells and CD4(+)CD25(+) Treg of host and/or donor origin. Both host and donor NKT cells and donor Treg are able to prevent GVHD in murine models. In this review, we summarize the mechanisms of NKT cell- and Treg-mediated protection against GVHD in mice while maintaining graft anti-tumor activity. In addition, we also examine the interactions between NKT cells and Treg in the context of BMT, and integrate the data from murine experimental models with the observations made in humans.

Host natural killer T cells induce an interleukin-4-dependent expansion of donor CD4+CD25+Foxp3+ T regulatory cells that protects against graft-versus-host disease.

Although CD4(+)CD25(+) T cells (T regulatory cells [Tregs]) and natural killer T cells (NKT cells) each protect against graft-versus-host disease (GVHD), interactions between these 2 regulatory cell populations after allogeneic bone marrow transplantation (BMT) have not been studied. We show that host NKT cells can induce an in vivo expansion of donor Tregs that prevents lethal GVHD in mice after conditioning with fractionated lymphoid irradiation (TLI) and anti-T-cell antibodies, a regimen that models human GVHD-protective nonmyeloablative protocols using TLI and antithymocyte globulin (ATG), followed by allogeneic hematopoietic cell transplantation (HCT). GVHD protection was lost in NKT-cell-deficient Jalpha18(-/-) hosts and interleukin-4 (IL-4)(-/-) hosts, or when the donor transplant was Treg depleted. Add-back of donor Tregs or wild-type host NKT cells restored GVHD protection. Donor Treg proliferation was lost in IL-4(-/-) hosts or when IL-4(-/-) mice were used as the source of NKT cells for adoptive transfer, indicating that host NKT cell augmentation of donor Treg proliferation after TLI/antithymocyte serum is IL-4 dependent. Our results demonstrate that host NKT cells and donor Tregs can act synergistically after BMT, and provide a mechanism by which strategies designed to preserve host regulatory cells can augment in vivo donor Treg expansion to regulate GVHD after allogeneic HCT.

Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation.

Allogeneic bone marrow transplantation is a curative treatment for leukemia and lymphoma, but graft-vs-host disease (GVHD) remains a major complication. Using a GVHD protective nonmyeloablative conditioning regimen of total lymphoid irradiation and antithymocyte serum (TLI/ATS) in mice that has been recently adapted to clinical studies, we show that regulatory host NKT cells prevent the expansion and tissue inflammation induced by donor T cells, but allow retention of the killing activity of donor T cells against the BCL1 B cell lymphoma. Whereas wild-type hosts given transplants from wild-type donors were protected against progressive tumor growth and lethal GVHD, NKT cell-deficient CD1d-/- and Jalpha-18-/- host mice given wild-type transplants cleared the tumor cells but died of GVHD. In contrast, wild-type hosts given transplants from CD8-/- or perforin-/- donors had progressive tumor growth without GVHD. Injection of host-type NKT cells into Jalpha-18-/- host mice conditioned with TLI/ATS markedly reduced the early expansion and colon injury induced by donor T cells. In conclusion, after TLI/ATS host conditioning and allogeneic bone marrow transplantation, host NKT cells can separate the proinflammatory and tumor cytolytic functions of donor T cells.