Distinct and Overlapping Effector Functions of Expanded Human CD4+, CD8α+ and CD4-CD8α- Invariant Natural Killer T Cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4+, CD8α+ and CD4−CD8α− double-negative (DN) subsets. CD4+ iNKT cells expanded more readily than CD8α+ and DN iNKT cells upon mitogen stimulation. CD8α+ and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d+ cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4+ and CD8α+ fractions, respectively. Only CD4+ iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α+, DN or CD4+ iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease

Human Invariant NKT Cell Subsets Differentially Promote Differentiation, Antibody Production, and T Cell Stimulation by B Cells In Vitro

Invariant NK T (iNKT) cells can provide help for B cell activation and Ab production. Because B cells are also capable of cytokine production, Ag presentation, and T cell activation, we hypothesized that iNKT cells will also influence these activities. Furthermore, subsets of iNKT cells based on CD4 and CD8 expression that have distinct functional activities may differentially affect B cell functions. We investigated the effects of coculturing expanded human CD4+, CD8α+, and CD4−CD8α− double-negative (DN) iNKT cells with autologous peripheral B cells in vitro. All iNKT cell subsets induced IgM, IgA, and IgG release by B cells without needing the iNKT cell agonist ligand α-galactosylceramide. Additionally, CD4+ iNKT cells induced expansions of cells with phenotypes of regulatory B cells. When cocultured with α-galactosylceramide–pulsed B cells, CD4+ and DN iNKT cells secreted Th1 and Th2 cytokines but at 10–1000-fold lower levels than when cultured with dendritic cells. CD4+ iNKT cells reciprocally induced IL-4 and IL-10 production by B cells. DN iNKT cells expressed the cytotoxic degranulation marker CD107a upon exposure to B cells. Remarkably, whereas iNKT cell subsets could induce CD40 and CD86 expression by B cells, iNKT cell–matured B cells were unable to drive proliferation of autologous and alloreactive conventional T cells, as seen with B cells cultured in the absence of iNKT cells. Therefore, human CD4+, CD8α+, and DN iNKT cells can differentially promote and regulate the induction of Ab and T cell responses by B cells

Activation of human invariant natural killer T cells with a thioglycoside analogue of α-galactosylceramide

Activation of CD1d-restricted invariant NKT (iNKT) cells with the glycolipid α-galactosylceramide (α-GalCer) confers protection against disease in murine models, however, clinical trials in humans have had limited impact. We synthesized a novel thioglycoside analogue of α-GalCer, denoted α-S-GalCer, and tested its efficacy for stimulating human iNKT cells in vitro. α-S-GalCer stimulated cytokine release by iNKT cells in a CD1d-dependent manner and primed CD1d+ target cells for lysis. α-S-GalCer-stimulated iNKT cells induced maturation of monocyte-derived dendritic cells into antigen-presenting cells that released IL-12 and small amounts of IL-10. The nature and potency of α-S-GalCer and α-GalCer in human iNKT cell activation were similar. However, in contrast to α-GalCer, α-S-GalCer did not activate murine iNKT cells in vivo. Because of its enhanced stability in biological systems, α-S-GalCer may be superior to α-GalCer as a parent compound for developing adjuvant therapies for humans

Single and dual production of IFN-γ and IL-4 production by iNKT subsets.

<p><b>A,</b> Flow cytometry plots showing IFN-γ and IL-4 expression by iNKT cells after 4 hours of co-culture with α-GalCer pulsed HeLa-CD1d cells. 6B11⁺ cells were electronically gated followed by gating on CD4⁺, DN and CD8α⁺ cells. Plots are representative of experiments involving iNKT cell lines from 3 healthy donors. <b>B,</b> IFN-γ and IL-4 production by iNKT cell subsets stimulated with HeLa-CD1d cells pulsed with various concentrations of α-GalCer. Graphs show mean (±SEM) percentages of cells expressing IFN-γ (left) and IL-4 (right) out of iNKT cell lines from 4 healthy donors. <b>C,</b> Mean (±SEM) percentages of CD4⁺, DN and CD8α⁺ iNKT cells that expressed IFN-γ only, IFN-γ and IL-4 and IL-4 only after stimulation with HeLa-CD1d cells pulsed with 100 ng/ml α-GalCer (n = 2). <b>D,</b> Ratios of IFN-γ/IL-4-expressing CD4⁺, DN and CD8α⁺ iNKT cells after stimulation with α-GalCer-pulsed HeLa-CD1d cells (n = 2).</p

Cytokine secretion by iNKT cell subsets.

<p>Highly-purified populations of CD4⁺, DN and CD8α⁺ iNKT cells were cultured in medium alone, with iDC or HeLa-CD1d cells in the absence or presence of 100 ng/ml α-GalCer, or with PMA and ionomycin for 24 hours. Levels of IFN-γ, TNFα, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17a and IL-22 in cell supernatants were measured using the FlowCytomix Multiplex kit. Results show mean (±SEM) cytokine levels in experiments involving iNKT cell lines from 3 healthy donors. <i>p</i> values in shaded boxes show Kruskal-Wallis comparisons of cytokine levels released by CD4⁺, CD8α⁺, and DN iNKT cells; *<i>p</i><0.05 comparing CD4⁺ and CD8α⁺ iNKT cells using post hoc Dunn's test.</p

Antitumor cytotoxicity by CD4⁺, CD8α⁺, and DN iNKT cells.

<p><b>A,</b> Expression of cell surface CD107a by iNKT cell subsets after co-culturing with CD1d⁻ K562 cells, CD1d-transfected HeLa cells or CD1d⁺ Jurkat cells, which were first pulsed for 18 hours with 0–1000 ng/ml α-GalCer. Results show mean (±SEM) percentage expression by iNKT cell subsets within 4 iNKT cell lines (3 for Jurkat). <i>p</i> values in shaded boxes show Kruskal-Wallis comparisons of CD107a expression by CD4⁺, CD8α⁺, and DN iNKT cells; asterisks indicate significant differences between CD4⁺ and CD8α⁺ iNKT cells using post hoc Dunn's multiple comparison tests; *<i>p</i><0.05; **<i>p</i><0.01. <b>B,</b> Surface CD107a expression by iNKT subsets in response to primary B-CLL cells pulsed with 100 ng/ml of α-GalCer. <b>C,</b> Cytolytic killing of CD1d-transfected C1R cells pulsed with 100 ng/ml α-GalCer or vehicle by highly-purified CD4⁺, DN or CD8α⁺ iNKT cells. Target cells were labelled with CFSE before addition of iNKT cells and death was then analysed by staining with 7-AAD. Data are expressed as means (±SEM) of experiments involving iNKT cell lines from 3 healthy donors. <i>p</i><0.05 (Kruskal-Wallis); *<i>p</i><0.05 comparing CD4⁺ and CD8α⁺ iNKT cells (post hoc Dunn's test).</p

A Bayesian reanalysis of the Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial

Background Timing of initiation of kidney-replacement therapy (KRT) in critically ill patients remains controversial. The Standard versus Accelerated Initiation of Renal-Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial compared two strategies of KRT initiation (accelerated versus standard) in critically ill patients with acute kidney injury and found neutral results for 90-day all-cause mortality. Probabilistic exploration of the trial endpoints may enable greater understanding of the trial findings. We aimed to perform a reanalysis using a Bayesian framework. Methods We performed a secondary analysis of all 2927 patients randomized in multi-national STARRT-AKI trial, performed at 168 centers in 15 countries. The primary endpoint, 90-day all-cause mortality, was evaluated using hierarchical Bayesian logistic regression. A spectrum of priors includes optimistic, neutral, and pessimistic priors, along with priors informed from earlier clinical trials. Secondary endpoints (KRT-free days and hospital-free days) were assessed using zero–one inflated beta regression. Results The posterior probability of benefit comparing an accelerated versus a standard KRT initiation strategy for the primary endpoint suggested no important difference, regardless of the prior used (absolute difference of 0.13% [95% credible interval [CrI] − 3.30%; 3.40%], − 0.39% [95% CrI − 3.46%; 3.00%], and 0.64% [95% CrI − 2.53%; 3.88%] for neutral, optimistic, and pessimistic priors, respectively). There was a very low probability that the effect size was equal or larger than a consensus-defined minimal clinically important difference. Patients allocated to the accelerated strategy had a lower number of KRT-free days (median absolute difference of − 3.55 days [95% CrI − 6.38; − 0.48]), with a probability that the accelerated strategy was associated with more KRT-free days of 0.008. Hospital-free days were similar between strategies, with the accelerated strategy having a median absolute difference of 0.48 more hospital-free days (95% CrI − 1.87; 2.72) compared with the standard strategy and the probability that the accelerated strategy had more hospital-free days was 0.66. Conclusions In a Bayesian reanalysis of the STARRT-AKI trial, we found very low probability that an accelerated strategy has clinically important benefits compared with the standard strategy. Patients receiving the accelerated strategy probably have fewer days alive and KRT-free. These findings do not support the adoption of an accelerated strategy of KRT initiation

Regional Practice Variation and Outcomes in the Standard Versus Accelerated Initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) Trial: A Post Hoc Secondary Analysis.

ObjectivesAmong patients with severe acute kidney injury (AKI) admitted to the ICU in high-income countries, regional practice variations for fluid balance (FB) management, timing, and choice of renal replacement therapy (RRT) modality may be significant.DesignSecondary post hoc analysis of the STandard vs. Accelerated initiation of Renal Replacement Therapy in Acute Kidney Injury (STARRT-AKI) trial (ClinicalTrials.gov number NCT02568722).SettingOne hundred-fifty-three ICUs in 13 countries.PatientsAltogether 2693 critically ill patients with AKI, of whom 994 were North American, 1143 European, and 556 from Australia and New Zealand (ANZ).InterventionsNone.Measurements and main resultsTotal mean FB to a maximum of 14 days was +7199 mL in North America, +5641 mL in Europe, and +2211 mL in ANZ (p p p p p p p p = 0.007).ConclusionsAmong STARRT-AKI trial centers, significant regional practice variation exists regarding FB, timing of initiation of RRT, and initial use of continuous RRT. After adjustment, such practice variation was associated with lower ICU and hospital stay and 90-day mortality among ANZ patients compared with other regions