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Background: The Network for Pancreatic Organ donors with Diabetes-Kidney (nPOD-K) project was initiated to assess the feasibility of using kidneys from organ donors to enhance understanding of diabetic kidney disease (DKD) progression., Methods: Traditional and digital pathology approaches were employed to characterize the nPOD-K cohort. Periodic acid-Schiff- and Hematoxylin and Eosin-stained sections were used to manually examine and score each nPOD-K case. Brightfield and fluorescently labelled whole slide images of nPOD-K sections were used to train, validate, and test deep learning compartment segmentation and machine learning image analysis tools within Visiopharm software. These digital pathology tools were subsequently employed to evaluate kidney cell-specific markers and pathological indicators., Results: Digital quantitation of mesangial expansion, tubular atrophy, kidney injury molecule (KIM)-1 expression, cellular infiltration, and fibrosis index aligned with histological DKD classification, as defined by pathologists' review. Histological quantification confirmed loss of podocyte, endothelial, and tubular markers, correlating with DKD progression. Altered expression patterns of prominin-1, protein-tyrosine phosphatase receptor type O, and coronin 2B were validated, in agreement with reported literature., Conclusions: The nPOD-K cohort provides a unique open resource opportunity to not only validate putative drug targets but also better understand DKD pathophysiology. A broad range of pathogenesis can be visualized in each case, providing a simulated timeline of DKD progression. We conclude that organ donor-derived tissues serve as high-quality samples, provide a comprehensive view of tissue pathology, and address the need for human kidney tissues available for research., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Society of Nephrology.)

Current classification of chronic kidney disease (CKD) into stages using indirect systemic measures (estimated glomerular filtration rate (eGFR) and albuminuria) is agnostic to the heterogeneity of underlying molecular processes in the kidney thereby limiting precision medicine approaches. To generate a novel CKD categorization that directly reflects within kidney disease drivers we analyzed publicly available transcriptomic data from kidney biopsy tissue. A Self-Organizing Maps unsupervised artificial neural network machine-learning algorithm was used to stratify a total of 369 patients with CKD and 46 living kidney donors as healthy controls. Unbiased stratification of the discovery cohort resulted in identification of four novel molecular categories of disease termed CKD-Blue, CKD-Gold, CKD-Olive, CKD-Plum that were replicated in independent CKD and diabetic kidney disease datasets and can be further tested on any external data at kidneyclass.org. Each molecular category spanned across CKD stages and histopathological diagnoses and represented transcriptional activation of distinct biological pathways. Disease progression rates were highly significantly different between the molecular categories. CKD-Gold displayed rapid progression, with significant eGFR-adjusted Cox regression hazard ratio of 5.6 [1.01-31.3] for kidney failure and hazard ratio of 4.7 [1.3-16.5] for composite of kidney failure or a 40% or more eGFR decline. Urine proteomics revealed distinct patterns between the molecular categories, and a 25-protein signature was identified to distinguish CKD-Gold from other molecular categories. Thus, patient stratification based on kidney tissue omics offers a gateway to non-invasive biomarker-driven categorization and the potential for future clinical implementation, as a key step towards precision medicine in CKD., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

To enable accurate, high-throughput and longer-term studies of the immunopathogenesis of type 1 diabetes (T1D), we established three in-vitro islet-immune injury models by culturing spheroids derived from primary human islets with proinflammatory cytokines, activated peripheral blood mononuclear cells or HLA-A2-restricted preproinsulin-specific cytotoxic T lymphocytes. In all models, β-cell function declined as manifested by increased basal and decreased glucose-stimulated insulin release (GSIS), and decreased intracellular insulin content. Additional hallmarks of T1D progression such as loss of the first-phase insulin response (FFIR), increased proinsulin-to-insulin ratios, HLA-class I expression, and inflammatory cytokine release were also observed. Using these models, we show that liraglutide, a glucagon-like peptide 1 receptor agonist, prevented loss of GSIS under T1D-relevant stress, by preserving the FFIR and decreasing immune cell infiltration and cytokine secretion. Our results corroborate that liraglutide mediates an anti-inflammatory effect that aids in protecting β-cells from the immune-mediated attack that leads to T1D., Competing Interests: Declaration of Competing Interest J.M.C., A.N., C.R., M.K., A.B., F.F. and A.T. are or were employees of InSphero AG, a company commercializing islet microtissues and relates services. B.Y. is a member of the management team of InSphero AG. M.G.v.H. is on the scientific advisory board of InSphero AG. C.B.G, F.F.K., J.W., N.P., and M.G.v.H. are employees of Novo Nordisk A/S, a company commercializing liraglutide and other GLP-1 RAs., (Copyright © 2022. Published by Elsevier Inc.)

Purpose of Review: Dissect the field of antigen-specific immunotherapy (ASIT) in type 1 diabetes (T1D), highlighting the major barriers currently blocking clinical translation., Recent Findings: ASIT remains a promising approach in T1D to re-establish the proper balance in the immune system to avoid the autoimmune-mediated attack or destruction of beta-cells in the pancreas. Despite some encouraging preclinical results, ASIT has not yet successfully translated into clinical utility, predominantly due to the lack of validated and clinically useful biomarkers., Summary: To restore immune tolerance towards self-antigens, ASIT aims to establish a favourable balance between T effector cells and T regulatory cells. Whilst most ASITs, including systemic or oral administration of relevant antigens, have appeared safe in T1D, meaningful and durable preservation of functional beta-cell mass has not been proven clinically. Development, including clinical translation, remains negatively impacted by lack of predictive biomarkers with confirmed correlation between assay readout and clinical outcomes. To be able to address the high unmet medical need in T1D, we propose continued reinforced research to identify such biomarkers, as well efforts to ensure alignment in terms of trial design and conduct., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Type 1 diabetes is an autoimmune disease in which insulin-secreting β-cells are destroyed, leading to a life-long dependency on exogenous insulin. There are no approved disease-modifying therapies available, and future immunotherapies would need to avoid generalized immune suppression. We developed a novel plasmid expressing preproinsulin2 and a combination of immune-modulatory cytokines (transforming growth factor-beta-1, interleukin [IL] 10 and IL-2) capable of near-complete prevention of autoimmune diabetes in non-obese diabetic mice. Efficacy depended on preproinsulin2, suggesting antigen-specific tolerization, and on the cytokine combination encoded. Diabetes suppression was achieved following either intramuscular or subcutaneous injections. Intramuscular plasmid treatment promoted increased peripheral levels of endogenous IL-10 and modulated myeloid cell types without inducing global immunosuppression. To prepare for first-in-human studies, the plasmid was modified to allow for selection without the use of antibiotic resistance; this modification had no impact on efficacy. This pre-clinical study demonstrates that this multi-component, plasmid-based antigen-specific immunotherapy holds potential for inducing self-tolerance in persons at risk of developing type 1 diabetes. Importantly, the study also informs on relevant cytokine and immune cell biomarkers that may facilitate clinical trials. This therapy is currently being tested for safety and tolerability in a phase 1 trial (ClinicalTrials.gov Identifier: NCT04279613)., (© 2021 by the American Diabetes Association.)

Type 1 diabetes is an autoimmune disease in which insulin-secreting β-cells are destroyed, leading to a life-long dependency on exogenous insulin. There are no approved disease-modifying therapies available, and future immunotherapies would need to avoid generalized immune suppression. We developed a novel plasmid expressing preproinsulin2 and a combination of immune-modulatory cytokines (transforming growth factor-beta-1, interleukin [IL] 10 and IL-2) capable of near-complete prevention of autoimmune diabetes in non-obese diabetic mice. Efficacy depended on preproinsulin2, suggesting antigen-specific tolerization, and on the cytokine combination encoded. Diabetes suppression was achieved following either intramuscular or subcutaneous injections. Intramuscular plasmid treatment promoted increased peripheral levels of endogenous IL-10 and modulated myeloid cell types without inducing global immunosuppression. To prepare for first-in-human studies, the plasmid was modified to allow for selection without the use of antibiotic resistance; this modification had no impact on efficacy. This pre-clinical study demonstrates that this multi-component, plasmid-based antigen-specific immunotherapy holds potential for inducing self-tolerance in persons at risk of developing type 1 diabetes. Importantly, the study also informs on relevant cytokine and immune cell biomarkers that may facilitate clinical trials. This therapy is currently being tested for safety and tolerability in a phase 1 trial (ClinicalTrials.gov Identifier: NCT04279613)., (© 2021 by the American Diabetes Association.)

Antigen-specific immunotherapy (ASI) holds great promise for type 1 diabetes (T1D). Preclinical success for this approach has been demonstrated in vivo , however, clinical translation is still pending. Reasons explaining the slow progress to approve ASI are complex and span all stages of research and development, in both academic and industry environments. The basic four hurdles comprise a lack of translatability of pre-clinical research to human trials; an absence of robust prognostic and predictive biomarkers for therapeutic outcome; a need for a clear regulatory path addressing ASI modalities; and the limited acceptance to develop therapies intervening at the pre-symptomatic stages of disease. The core theme to address these challenges is collaboration-early, transparent, and engaged interactions between academic labs, pharmaceutical research and clinical development teams, advocacy groups, and regulatory agencies to drive a fundamental shift in how we think and treat T1D., Competing Interests: JC is an employee of Novartis. JW is an employee of Novo Nordisk. RT has filed provisional patents surrounding technology and is commercializing immunotherapy for targeting DCs for antigen-specific tolerance. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Thomas, Carballido, Wesley and Ahmed.)

Objectives: The detection of a peripheral immune cell signature that specifically reflects autoimmunity in type 1 diabetes would enable the prediction and staging of disease on an individual basis. However, defining such a signature is technically challenging. Reliable interpretation of immune cell-related biomarkers depends on their inherent variability and, to understand this variability, longitudinal analyses are required., Methods: We performed a longitudinal observational study in which 40 individuals with elevated genetic risk of type 1 diabetes and persistent islet autoantibodies provided a blood sample every 4-6 weeks for > 1 year., Results: Peripheral immune cell composition (T cells, NK cells and monocytes) was assessed using well-validated flow cytometry panels and demonstrated that, while non-antigen-specific immune cell subsets were stable over time, autoantigen-reactive T-cell frequencies were highly variable in and between individuals. Neither the frequency nor phenotype of non-antigen-specific subsets or autoreactive CD8 + T cells associated with clinical onset of T1D., Conclusion: The findings from the T ype 1 D iabetes Longitudinal BI omarker T rial underscore the inherent challenge of evaluating changes in peripheral immune cell populations as surrogates of organ-specific disease activity. The variability of peripheral antigen-specific T cells precludes their use as a prognostic marker and clearly demonstrates that a reliable prognostic cell signature remains elusive., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Insulin-like growth factors (IGFs), specifically IGF1 and IGF2, promote glucose metabolism, with their availability regulated by IGF-binding proteins (IGFBPs). We hypothesized that IGF1 and IGF2 levels, or their bioavailability, are reduced during type 1 diabetes development. Total serum IGF1, IGF2, and IGFBP1-7 levels were measured in an age-matched, cross-sectional cohort at varying stages of progression to type 1 diabetes. IGF1 and IGF2 levels were significantly lower in autoantibody (AAb) + compared with AAb - relatives of subjects with type 1 diabetes. Most high-affinity IGFBPs were unchanged in individuals with pre-type 1 diabetes, suggesting that total IGF levels may reflect bioactivity. We also measured serum IGFs from a cohort of fasted subjects with type 1 diabetes. IGF1 levels significantly decreased with disease duration, in parallel with declining β-cell function. Additionally, plasma IGF levels were assessed in an AAb + cohort monthly for a year. IGF1 and IGF2 showed longitudinal stability in single AAb + subjects, but IGF1 levels decreased over time in subjects with multiple AAb and those who progressed to type 1 diabetes, particularly postdiagnosis. In sum, IGFs are dysregulated both before and after the clinical diagnosis of type 1 diabetes and may serve as novel biomarkers to improve disease prediction., (© 2019 by the American Diabetes Association.)

Immune analytes have been widely tested in efforts to understand the heterogeneity of disease progression, risk, and therapeutic responses in type 1 diabetes (T1D). The future clinical utility of such analytes as biomarkers depends on their technical and biological variability, as well as their correlation with clinical outcomes. To assess the variability of a panel of 91 immune analytes, we conducted a prospective study of adults with T1D (<3 years from diagnosis), at 9-10 visits over 1 year. Autoantibodies and frequencies of T-cell, natural killer cell, and myeloid subsets were evaluated; autoreactive T-cell frequencies and function were also measured. We calculated an intraclass correlation coefficient (ICC) for each marker, which is a relative measure of between- and within-subject variability. Of the 91 analytes tested, we identified 35 with high between- and low within-subject variability, indicating their potential ability to be used to stratify subjects. We also provide extensive data regarding technical variability for 64 of the 91 analytes. To pilot the concept that ICC can be used to identify analytes that reflect biological outcomes, the association between each immune analyte and C-peptide was also evaluated using partial least squares modeling. CD8 effector memory T-cell (CD8 EM) frequency exhibited a high ICC and a positive correlation with C-peptide, which was also seen in an independent dataset of recent-onset T1D subjects. More work is needed to better understand the mechanisms underlying this relationship. Here we find that there are a limited number of technically reproducible immune analytes that also have a high ICC. We propose the use of ICC to define within- and between-subject variability and measurement of technical variability for future biomarker identification studies. Employing such a method is critical for selection of analytes to be tested in the context of future clinical trials aiming to understand heterogeneity in disease progression and response to therapy., (Copyright © 2019 Speake, Bahnson, Wesley, Perdue, Friedrich, Pham, Lanxon-Cookson, Kwok, Sehested Hansen, von Herrath and Greenbaum.)

Background: Oral insulin as a preventive strategy and/or treatment of type 1 diabetes has been the target of much research. Producing oral insulins is a complex and challenging task, with numerous pitfalls, due to physiological, physical, and biochemical barriers. Our aim was to determine the impact of oral insulin on the delicate gut microbiota composition., Methods: Female nonobese diabetic mice were given oral porcine insulin 2 times a week from 5 weeks of age for 4 weeks, and then subsequently once a week for 21 weeks, or until euthanized. The mice were divided into groups on a gluten-reduced diet or a standard diet. Gut microbiota composition was analysed based on faecal samples, and the type 1 diabetes incidence of the mice was monitored., Results: We observed no influence of the oral porcine insulin on the gut microbiota composition of mice on a gluten-reduced or a standard diet at 9 weeks of age. Also, the administration of oral insulin did not influence the incidence of type 1 diabetes at 30 weeks of age., Conclusions: Oral porcine insulin does not alter the gut microbiota composition of nonobese diabetic mice on either a gluten-reduced diet or standard diet. Also, the oral porcine insulin did not influence the incidence of type 1 diabetes in the groups., (Copyright © 2018 John Wiley & Sons, Ltd.)

Background: Type 1 diabetes (T1D) is thought to be an autoimmune disease driven by anti-islet antigen responses and mediated by T-cells. Recent published data suggests that T-cell reactivity to modified peptides, effectively neoantigens, may promote T1D. These findings have given more credence to the concept that T1D may not be solely an error of immune recognition but may be propagated by errors in protein processing or in modifications to endogenous peptides occurring as result of hyperglycemia, endoplasmic reticulum (ER) stress, or general beta cell dysfunction. In the current study, we hypothesized that diabetes-associated epitopes bound human leukocyte antigen (HLA) class I poorly and that post-translational modifications (PTM) to key sequences within the insulin-B chain enhanced peptide binding to HLA class I, conferring the CD8+ T-cell reactivity associated with T1D., Results: We first identified, through the Immune Epitope Database (IEDB; www.iedb.org ), 138 published HLA class I-restricted diabetes-associated epitopes reported to elicit positive T-cell responses in humans. The peptide binding affinity for their respective restricting allele(s) was evaluated in vitro. Overall, 75% of the epitopes bound with a half maximal inhibitory concentration (IC50) of 8250 nM or better, establishing a reference affinity threshold for HLA class I-restricted diabetes epitopes. These studies demonstrated that epitopes from diabetes-associated antigens bound HLA with a lower affinity than those of microbial origin (binding threshold of 500 nM for 85% of the epitopes). Further predictions suggested that diabetes epitopes also bind HLA class I with lower affinity than epitopes associated with other autoimmune diseases. Therefore, we measured the effect of common PTM (citrullination, chlorination, deamidation, and oxidation) on HLA-A*02:01 binding of insulin-B-derived peptides, compared to native peptides. We found that these modifications increased binding for 44% of the insulin-B epitopes, but only 15% of the control peptides., Conclusions: These results demonstrate that insulin-derived epitopes, commonly associated with T1D, generally bind HLA class I poorly, but can be subject to PTM that improve their binding capacity and may, in part, be responsible for T-cell activation in T1D and subsequent beta cell death.

The standard of care (SoC) for Type 1 diabetes (T1D) today is much the same as it was in the early 1920s, simply with more insulin options-fast-acting, slow-acting, injectable, and inhalable insulins. However, these well-tolerated treatments only manage the symptoms and complications, but do nothing to halt the underlying immune response. There is an unmet need for better treatment options for T1D that address all aspects of the disease. For decades, we have successfully treated T1D in preclinical animal models with immune-modifying therapies that have not demonstrated comparable efficacy in humans. The path to bringing such options to the clinic will depend on the implementation and standard inclusion of biomarkers of immune and therapeutic efficacy in T1D clinical trials, and dictate if we can create a new SoC that treats the underlying autoimmunity as well as the symptoms it causes., (Copyright © 2015. Published by Elsevier Inc.)

Previous cross-sectional analyses demonstrated that CD8(+) and CD4(+) T-cell reactivity to islet-specific antigens was more prevalent in T1D subjects than in healthy donors (HD). Here, we examined T1D-associated epitope-specific CD4(+) T-cell cytokine production and autoreactive CD8(+) T-cell frequency on a monthly basis for one year in 10 HD, 33 subjects with T1D, and 15 subjects with T2D. Autoreactive CD4(+) T-cells from both T1D and T2D subjects produced more IFN-γ when stimulated than cells from HD. In contrast, higher frequencies of islet antigen-specific CD8(+) T-cells were detected only in T1D. These observations support the hypothesis that general beta-cell stress drives autoreactive CD4(+) T-cell activity while islet over-expression of MHC class I commonly seen in T1D mediates amplification of CD8(+) T-cells and more rapid beta-cell loss. In conclusion, CD4(+) T-cell autoreactivity appears to be present in both T1D and T2D while autoreactive CD8(+) T-cells are unique to T1D. Thus, autoreactive CD8(+) cells may serve as a more T1D-specific biomarker., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Type 1 diabetes (T1D) results from autoimmune destruction of the pancreatic β-cells. Current T1D therapies are exclusively focused on regulating glycemia rather than the underlying immune response. A handful of trials have sought to alter the clinical course of T1D using various broad immune-suppressors, e.g., cyclosporine A and azathioprine.(1-3) The effect on β-cell preservation was significant, however, these therapies were associated with unacceptable side-effects. In contrast, more recent immunomodulators, such as anti-CD3 and antigenic therapies such as DiaPep277, provide a more targeted immunomodulation and have been generally well-tolerated and safe; however, as a monotherapy there appear to be limitations in terms of therapeutic benefit. Therefore, we argue that this new generation of immune-modifying agents will likely work best as part of a combination therapy. This review will summarize current immune-modulating therapies under investigation and discuss how to move the field of immunotherapy in T1D forward.

Sipuleucel-T, the first autologous active cellular immunotherapy approved by the United States Food and Drug Administration, is designed to stimulate an immune response to prostate cancer. Sipuleucel-T is manufactured by culturing a patient's peripheral blood mononuclear cells (including antigen presenting cells) with a recombinant protein comprising a tumor-associated antigen (prostatic acid phosphatase) and granulocyte-macrophage colony stimulating factor. Treatment consists of 3 infusions at approximately 2-week intervals, resulting in a prime-boost pattern of immune activation, a robust antigen-specific cellular and humoral immune response, and, consequently, a survival benefit in subjects with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer. Adverse events are generally mild to moderate and resolve within 2 d. Serious adverse events occur at a low rate. As the first autologous cellular immunotherapy to demonstrate a survival benefit, sipuleucel-T is a novel oncologic therapeutic that warrants the reassessment of the current prostate cancer treatment paradigm.

Type 1 diabetes (T1D) results from the immune-mediated destruction of the insulin-producing β-islet cells in the pancreas. The genetic and environmental mechanisms promoting the development of this disease remain poorly understood. We have explored the cellular requirements for T1D development in DO11.10xRIPmOVA (DORmO) mice, which carry a TCR transgene specific for an MHC class II-restricted epitope from OVA and express membrane-bound OVA in the pancreas under the control of the rat insulin promoter. We found that DORmO.RAG2(-/-) mice do not develop insulitis and are completely protected from diabetes, demonstrating that endogenous lymphocyte receptor rearrangement is required for disease development. Diabetes in DORmO mice is preceded by the development of OVA-specific autoantibodies and is delayed in B cell-deficient DORmO.JhD(-/-) mice, demonstrating that B cells contribute to disease progression. In addition, transfer of CD8(+) T cells from diabetic animals into DORmO.RAG2(-/-) mice promoted insulitis by OVA-specific CD4(+) T cells. Finally, although diabetes develops in DORmO mice in the presence of a significant population of Foxp3(+) OVA-specific regulatory T cells, boosting regulatory T cell numbers by injecting IL-2 immune complexes dampens autoantibody production and prevents development of insulitis and overt diabetes. These results help define the events leading to diabetes in DORmO mice and provide new insights into the cellular interactions required for disease development in an Ag-specific model of T1D.

Immunity to the murine cytomegalovirus (MCMV) is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Valpha14 invariant natural killer T cell response to MCMV has not been examined. We found that Valpha14i NK T cells become activated and produce significant levels of IFN-gamma, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Valpha14i NK T cells into MCMV-infected CD1d(-/-) mice demonstrate that CD1d is dispensable for Valpha14i NK T cell activation. In contrast, both IFN-alpha/beta and IL-12 are required for optimal activation. Valpha14i NK T cell-derived IFN-gamma is partially dependent on IFN-alpha/beta but highly dependent on IL-12. Valpha14i NK T cells contribute to the immune response to MCMV and amplify NK cell-derived IFN-gamma. Importantly, mortality is increased in CD1d(-/-) mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Valpha14i NK T cells that act as effector T cells during bacterial infection, but have NK cell-like behavior during the innate immune response to MCMV infection.

Valpha14 invariant (Valpha14i) NK T cell development is unique from mainstream T cell selection, and the polygenic factors that influence NK T cell ontogeny are still unclear. In this study, we report the absence of Valpha14i NK T cells in B6.IFN-alphabetaR1-/- male mice, whereas both the conventional T and NK cell populations are relatively unaffected. The lack of Valpha14i NK T cells in the B6.IFN-alphabetaR1-/- males is not due to an insufficient level of CD1d1 or a defect in CD1d1-Ag presentation, but it is intrinsic to the male Valpha14i NK T cells. This surprising defect displays >or=99% penetrance in the male population, whereas female mice remain unaffected, indicating the deficiency is not X linked. Analysis of the Valpha14i NK T cell compartment in B6.Tyk2-/-, B6.STAT1-/-, 129.IFN-alphabetaR1-/-, and B6.IFN-alphabetaR1-/+ mice demonstrate that the deficiency is linked to the Y chromosome, but independent of IFN-alphabeta. This is the first study demonstrating that Y-linked genes can exclusively impact Valpha14i NK T development and further highlight the unique ontogeny of these innate T cells.

Activated NK T cells are known to rapidly stimulate NK cells and, subsequently, CD8(+) T cells and B cells. In this report, we first demonstrate that the downstream effects induced by alpha-galactosylceramide activated NK T cells on NK cells are mainly dependent on IFN-gamma. We found that NK T cell activation of NK cells requires a functional IFN-gamma signaling in macrophages and dendritic cells but not in B cells, NK cells, or NK T cells. NK T cell activation is dendritic cell-dependent whereas NK T cell activation of NK cells is indirect and in part mediated by macrophages. Interestingly, in this context, macrophage participation in the CD1d Ag presentation of alpha-galactosylceramide to NK T cells is not necessary. These data indicate that NK T cell-dependent activation of macrophages is required for optimal NK T cell-induced stimulation of NK cells.