RIPK3-/-, DKO, TKO and RIPK1kd mice were a kind gift from Dr. William Kaiser (UTHSCSA) (4, 7). C57BL/6, B6.129 S4 IFNγ tn3.Ilky (“GREAT” mice which report IFNγ with cytoplasmic production of eYFP) (17) and B6.KN2 Cre mice which report IL-4 production by surface expression of the human CD2 protein (18) (detected by anti-human CD2 antibodies in flow cytometry) were housed and bred under specific pathogen-free (SPF) conditions and all mice were maintained at the Laboratory Animal Resources (LAR) animal facility of UT Health San Antonio. All experiments were performed using IACUC approved protocols. Age- and sex-matched female or male mice were used in all experiments, primarily aged 6-14 weeks, with a few experiments using mice up to 28 weeks, as noted in the legends.

6μm-8μm sections of optimal cutting temperature (OCT compound, Fisher Healthcare)-embedded murine thymi were cut using a cryostat NX (Thermo Scientific) and fixed in 95% alcohol formalin. Tissue was stained with hematoxylin and eosin, washed with 95% alcohol, and coverslipped using Cytoseal. 3-5 images per section, multiple sections per thymus, from multiple animals were imaged with a Zeiss microscope as noted in figure legends.

3x10⁶ murine splenocytes or peritoneal washout cells per sample were treated with ACK lysis buffer (Lonza), non-specific labeling was blocked with anti-mouse CD16/32 (clone 93), and dead cells were excluded by staining with Live/Dead Fixable Zombie viability dye (BD Biosciences). Further surface labeling was performed using antibodies (BD Pharmingen, Biolegend, R&D Systems, or eBiosciences) outlined in Supplementary Table 1 .

Doublet exclusion and live-cell discrimination was performed on all cell populations prior to sub-gating. Samples were acquired on a BD FACS Celesta (Becton Dickinson) and Cytek Aurora (Cytek) at the UT Health San Antonio flow cytometry core and analyzed using FlowJo software (10.5.3). CD1d-PBS57 and 5-OP-RU-loaded MR1 tetramers were provided by the NIH Tetramer Core Facility. Cell numbers of certain lymphocyte populations were determined by counting total leukocytes per whole organ using trypan blue diluted single cell suspensions on a hemocytometer. Total organ cell numbers were combined with flow cytometry-derived percentages of live cells, and percentages of serially gated cell populations of interest to calculate absolute numbers of each relevant population.

OMIQ cytometry analysis software was used for dimensionality reduction analysis. Dimensionality reduction using UMAPs was performed by excluding doublets and dead cells. iNKT_FH cells were then identified as CD1d tet⁺, TCRβ^int, CD4⁺, CXCR5⁺, and PD-1⁺. UMAPs of splenic iNKT_FH cells from naïve and immunized mice (NP-KLH+αGC or NPαGC) used mean fluorescent intensity parameters of ICOS, PD-1, Tim3, and Lag3 with default settings.

RNA was isolated according to manufacturer’s instructions (Direct-zol RNA microprep; Catalog #R2062). In short, splenocytes and thymocytes were lysed in QIAZol buffer. A volume of 95-100% ethanol equal to the volume of QIAZol was added and the mixture was transferred into a zymo spin column. After a few wash steps, DNase treatment was performed to remove genomic DNA. RNA was then eluted in nuclease free water.

cDNA was synthesized using the MultiScribe cDNA synthesis kit according to manufacturer’s instruction. Up to 2μg of RNA was reverse transcribed in a buffer mixture consisting of dNTPs, MultiScribe Reverse Transcriptase, Random Primers, and RNase inhibitor. cDNA was used to amplify NK1.1 mRNA transcripts, specifically NKRP1A, NKRP1B and NKRP1C. PCR master mix consisted of dNTPs, PCR buffer, MgCl₂, Taq Polymerase with appropriate forward primers and reverse primers outlined in Supplementary Table 2 . Polymerase Chain Reaction) protocol is as follows: 25°C for 10 mins -> 37°C for 120 mins -> 85°C for 5 mins. PCR products were resolved on a 1.5% agarose gel containing 1% Gel Green (Biotium, Catalog #: 41004 - 41005-T) and imaged using a BioRad GelDoc system.

To assess cytokine production, WT, RIPK3^-/-, DKO, TKO, and RIPK1kd mice were immunized intravenously (IV) with 0.5μg of αGalactosylCeramide (αGC; Avanti Polar Lipids) in 200μL of PBS + 0.1% BSA and organs harvested 4 hours later for flow cytometry assessment. To measure iNKT_FH responses, WT, RIPK3^-/-, DKO, TKO and RIPK1kd mice were immunized IV with 0.5μg/μL NP-KLH and 0.5μg/200μL αGC or 0.5μg/200μL of NPαGC and organs harvested 5 days later.

2x10⁶ splenocytes from B6.129 S4 IFNγ tn3.Ilky (“GREAT” mice IFNγ eYFP reporter) mice or B6.KN2 Cre mice were pretreated in vitro with 39.5μM RIPK inhibitor, Necrostatin-1 (Cat# N9037, Sigma-Aldrich) for 60 minutes. Control untreated and pretreated cells were then stimulated with various concentrations of αGC (10ng/mL, 50ng/mL and 100ng/mL). To observe peak cytokine responses, splenocytes from GREAT mice and B6.KN2 mice were analyzed by flow cytometry 4 and 12 hours after stimulation, respectively. PMA/Ionomycin (2μg/mL), αCD3 (50μg/mL), and αCD28 (25μg/mL) were used as positive controls.

Statistical significance was assessed by GraphPad PRISM 7 using a 1-way ANOVA as indicated. Variance is similar between all groups compared, and data points were excluded from consideration if identified by the ROUT method using Graphpad PRISM software. Box heights and circle, triangle, box centers indicate group means and/or individual values. Error bars indicate s.e.m.

To study the in vivo role of RIPK1 in innate T cell homeostasis, we used flow cytometry to quantify three innate T cell populations and their distribution in secondary lymphoid organs in WT, RIPK3^-/-, DKO, TKO, and RIPK1kd mice ( Supplementary Figure 1 ). Because of their large likely-autoimmune CD3⁺B220⁺ population, DKO mice had a significantly lower frequency of total T cells (TCRβ⁺ T cells) in the spleen when compared to WT mice and mice of other genotypes ( Supplementary Figure 1A ). However, the absolute number of total T cells in the spleen of DKO and TKO mice was comparable to RIPK3^-/-, RIPK1kd, and control WT mice ( Supplementary Figure 1B ). Nevertheless, to minimize distortion of frequencies of lymphocyte populations, the autoimmune CD3⁺ B220⁺ cells were excluded from analysis during flow cytometry gating.

To determine the impact of RIPK1 deficiency on innate immune T cells, we first looked at the distribution and frequency of innate-like Mucosal Associated Invariant T (MAIT) cells in the secondary lymphoid organs by flow cytometry (gating strategy in Supplementary Figure 1E, F ). TKO mice had a significantly expanded frequency of splenic MAIT cells, identified using the 5-OP-RU loaded MR1 tetramer, compared to WT controls ( Figures 1A, B ), but this difference did not extend so far as to be a specific increase in MAIT cell number ( Figure 1C ). We also did not observe any significant difference in the frequency or cell number of MAIT cells in the lymph nodes from WT, RIPK3-/-, TKO, and RIPK1kd mice ( Figures 1D–F ). Surprisingly, although there were no differences in frequency, DKO mice did show an isolated expansion of MAIT cell numbers in LNs ( Figure 1F ). In short, alterations in MAIT cell frequencies and numbers in DKO or TKO mice do not seem to reflect any consistent trends. Next, we examined another innate T cell population, γδT cells, in mice lacking Casp8 and/or RIPK1. Using flow cytometry (gating strategy in Supplementary Figure 1G, H ), we found that TKO mice had a significantly higher frequency of γδT cells in the spleen than WT and trending differences from all other controls but again this did not extend to include changes in splenic γδT cell numbers ( Figures 1G–I ). Similarly, γδT cell frequencies and numbers in the lymph nodes of DKO and TKO mice did not significantly differ from WT mice or other controls ( Figures 1J–L ). These isolated increases in frequencies (but not numbers) of MAIT and γδT cells in the spleens of TKO mice most likely reflect losses of other peripheral populations, and do not support a general RIPK1-dependent defect in these two innate T cell populations.

We next used the PBS57-loaded CD1d tetramer to identify iNKT cells in peripheral organs; spleen, and liver ( Figures 1M-R ) (gating strategy in Supplementary Figure 1I, J ). An unloaded CD1d tetramer was used to determine gating. DKO and TKO mice exhibited significantly lower frequencies and numbers of splenic iNKT cells compared to WT, RIPK3^-/-, and RIPK1kd mice ( Figures 1N, O ). To confirm that the changes in frequency of iNKT cells in the spleen were not simply a reflection of overall impaired proliferation or cell death, we measured the frequency and number of total T cells, as well as the frequency of dead lymphocytes in the spleen ( Supplementary Figure 1 ). There was a modest drop in total splenic T cell frequency in DKO mice ( Supplementary Figure 1A ), but there were no changes in the number of total splenic T cells across all genotypes of mice tested ( Supplementary Figure 1B ). There were also no differences in percentage of dead lymphocytes in the spleen of any strains tested, but DKO mice had a modest increased in the number of dead lymphocytes in the spleen when compared to all other genotypes tested ( Supplementary Figure 1C, D ). The liver, a major reservoir for iNKT cells, mirrored the spleen with a significantly lower frequency of iNKT cells in DKO and TKO compared to WT ( Figure 1P, Q ), but the changes in hepatic iNKT frequency were not reflected by changes in cell numbers ( Figure 1R ). This suggests the iNKT changes in the liver reflected alterations in another cell population, and not a direct effect on iNKT cells themselves. In summary, since both DKO and TKO mice display a lower frequency and number of iNKT cells in the liver and/or spleen, these effects are consistent with a role for Casp8 (deficient in both DKO and TKO) in mediating iNKT cell development or peripheral persistence.

We next used flow cytometry to examine whether changes in iNKT cell numbers reflected preferential alterations of certain iNKT cell subsets in the periphery and spleen of DKO, TKO (gating strategies in Supplementary Figure 2A ). We did not observe any significant differences in iNKT1, iNKT2 or iNKT17 frequencies or number in spleen of TKO mice compared to WT controls ( Figures 2A–G ). On the other hand, DKO mice had a significantly lower frequency and moderately lower number of iNKT2 cells compared to the same population in TKO mice ( Figures 2C, F ). In fact, DKO mice had detectable reductions in the number of all subsets of iNKT cells (NKT1, NKT2, and NK17), but these changes did not reach significance. However, these generalized iNKT cell losses in the DKO mice are consistent with the trend towards increased cell death evident in all splenic T cells in DKO mice and suggest the absence of RIPK1 (missing in TKO but not DKO) may rescue this downward trend in the TKO mice ( Supplementary Figure 1B ).

Nur77 expression is widely used as a surrogate for TcR signaling in T cells (19) and is necessary for the development and differentiation of iNKT cells into phenotypic subsets. Therefore, we next measured Nur77 expression in splenic iNKT subsets. Nur77 expression was significantly reduced in all splenic iNKT subsets from TKO mice when compared to WT mice and other genotypes ( Figures 2H–J ). There was a visible decrease in Nur77 in all iNKT subsets in DKO mice as well, but it only reached significance in the NKT1 subset ( Figures 2H–J ). To ensure that the observed changes in the Nur77 expression were not due to impaired proliferation of iNKT cells but a consequence of RIPK1/Casp8 deficiency, we measured Ki67 levels in splenic iNKT subsets from all mouse strains. In fact, Ki67 expression was increased in all three subsets of iNKT cells from DKO mice, suggesting lack of proliferation was not a factor in the loss of these iNKT cell subsets ( Figures 2K–M ).

To comprehensively assess the systemic requirements of RIPK1/Casp8 signaling to regulate iNKT cells, we analyzed the frequency and distribution of iNKT subsets in liver, another peripheral organ and a reservoir for iNKT cells (gating strategies in Supplementary Figure 2B ). iNKT1, iNKT2 and iNKT17 cells did not significantly differ in frequency or number in the livers of all genotypes of mice analyzed ( Figures 2N–Q ), consistent with findings from the spleen. In conclusion, global RIPK3/Casp8 (in DKO) or RIPK1/Casp8/RIPK3 deficiencies (in TKO) do not skew splenic iNKT subset distribution. While the numbers of iNKT cells in spleens of DKO mice are visibly reduced, these changes are across all subsets and do not reach statistical significance. Thus, both DKO and TKO mice have relatively normal iNKT1, iNKT2, and iNKT17 frequency in the spleen. Thus, neither RIPK1 or Casp8 regulate peripheral iNKT subset frequency or distribution in the spleen and liver.

The reduced frequency of total iNKT cells in the periphery of TKO mice could develop from a proliferation defect or a developmental defect. Since Figures 1 and 2 have largely ruled out a proliferative defect, we next asked if reduction of total iNKT cells in the periphery of TKO mice could be a direct consequence of changes in iNKT development in the thymus. To this end, we used flow cytometry to identify and measure developing iNKT cell subsets in the thymus (gating strategies in Supplementary Figure 3A ). TKO mice had a significantly reduced frequency and number of thymic iNKT cells when compared to all other genotypes ( Figures 3A, B ). We used CD44 and NK1.1 conventional markers to identify the iNKT developmental stages 0 + 1, 2, and 3 ( Figure 3C ). We did not observe any significant differences in the frequency of CD44^- and NK1.1^- stage 0 + 1 iNKT cells between any of the genotypes tested ( Figures 3C–E ). TKO mice had a significantly higher frequency of CD44⁺ NK1.1^- stage 2 iNKT cells than all other genotypes ( Figure 3C–E ). Concomitantly and surprisingly, TKO mice lacked virtually all CD44⁺ NK1.1⁺ stage 3 iNKT cells ( Figures 3C–E ). All other groups had normal WT levels of stage 3 iNKT cells ( Figure 3E ).

To determine if the accumulation of Stage 2 iNKT cells in the thymus of TKO mice reflects an impediment to thymic emigration, we measured CCR7 expression on thymic iNKT subsets (20). CCR7 expression was similar among all genotypes of mice during all stages of thymic iNKT development ( Figure 3F ). CD69 is another thymic emigration and egress marker for iNKT cells and is specifically required for the development of mature NKT2 cells (21). Therefore, we also measured the expression of CD69 on the surface of various developmental stages of thymic iNKT cells. Stage 0, 1 and 3 iNKT cells showed no differences in the expression of CD69 between any of the genotypes tested ( Figure 3G ). However, Stage 2 iNKT cells from the TKO mice expressed significantly higher levels of CD69 when compared to other genotypes ( Figure 3G ).

We next looked at intracellular Nur77 expression by Stage 0 + 1 and Stage 2 iNKT cells, since levels of Nur77 expression can serve as a surrogate for TcR engagement (22, 23). DKO, TKO, and RIPK1kd mice all had lower Nur77 expression compared to WT mice ( Figure 3H ), although only the changes observed in TKO and RIPK1kd mice reached significance. Differences in the RIPK1kd mice compared to WT mice are consistent with a possible role for the kinase domain in transducing TcR signals ( Figure 3H ) and could explain differences in the RIPK1-deficient TKO as well. We also measured Ki67 levels in all iNKT developmental stages to ensure that the changes we observed in numerous factors affecting iNKT cell development and egress were not due to impaired proliferation. Ki67 levels did not differ significantly in any iNKT developmental stages among all genotypes of mice tested ( Figure 3I ).

To consider a contributory role for thymic architectural changes in mediating the developmental defects observed in iNKT cells, we used H&E staining to assess the thymic architecture of WT, RIPK3^-/-, DKO, TKO and RIPK1kd mice. We observed no visible architectural changes in the thymic cortex or medulla ( Supplementary Figure 3B ).

Since NK1.1+ was the primary marker used to identify stage 3 iNKT cells, and it was completely absent in the thymus, we next examined NK1.1 expression on other cell types to determine if there was a general impairment in NK1.1 protein expression. Interestingly, NK1.1 protein expression was also profoundly lacking on TcRβ- cells, most likely NK cells, in the spleen, liver, and thymus of TKO mice as compared to WT mice ( Figure 4A ). Frequencies of DX5 (CD49b) cells were not significantly reduced in the spleens of TKO mice, suggesting RIPK1 deficiency and subsequent NK1.1 deficiency did not impact the development of NK cells as profoundly as iNKT cells ( Figures 4B–D ). We further confirmed the absence of NK1.1 protein by assessing the expression of NK1.1 mRNA in DKO and TKO thymus and spleen. Both the thymus and spleen of DKO and TKO mice express all isoforms of the NK1.1 mRNA (nkrp1a, nkrp1b and nkrp1c) in the spleen and the thymus ( Figures 4E, F ). However, expression of nkrp1c, the isoform specifically recognized by the anti-NK1.1 antibody clone PK136, is dramatically reduced in the thymus of TKO mice ( Figure 4E ). Levels of expression of mRNA from the nkrp1b isoform are more moderately reduced in both thymus and spleen of TKO mice ( Figures 4E, F ). Reductions in nkrp1b and nkrp1c are consistent with reductions detected in iNKT cell number in these organs ( Figure 4F ).

In summary, against a backdrop of RIPK3 and Casp8 deficiency, RIPK1 deficiency uniquely reduced total thymic iNKT cell frequency. An accumulation of NK1.1 negative stage 2 thymocytes only in TKO mice lacking RIPK1, but not DKO mice, also suggests RIPK1 is required for NK1.1 expression and stage 2 to stage 3 transition of developing iNKT cells. In the absence of RIPK1, we observed no changes in iNKT cell expression of CCR7 or Ki67, minimizing the possibility of migration or proliferation defects. However, reductions in intracellular Nur77 in thymocytes from TKO mice compared to controls suggests that reduced signal transduction downstream of the TcR may be contributing to the arrested development and accumulation of stage 2 iNKT cells in the TKO mice.

Emerging research postulates that during development, iNKT cells attain a common precursor state that differentiates into the various effector subsets (24). iNKT precursors (iNKTp) express high levels of both PLZF and CCR7 (25). To determine the role of RIPK1 in iNKTp development and emigration, we measured the frequency of iNKTp cells in the thymus of mice lacking Casp8/RIPK3 (DKO) or Casp8/RIPK3 as well as RIPK1 (TKO). TKO mice had a significantly higher frequency of iNKTp when compared to all other genotypes ( Figures 5A, B ) (flow cytometry gating strategies in Supplementary Figure 4A ). However, this change in frequency was not reflected as a change in iNKTp cell numbers for any mice tested ( Figure 5C ). Concomitantly, iNKTp cells from TKO mice expressed significantly higher levels of PLZF (MFI) compared to WT mice and DKO mice ( Figure 5D, E ). To determine if the absence of RIPK1 affects fate determining transcription factor expression in iNKTp cells, we measured RoRγT expression in iNKTp cells. RoRγT was expressed at similar low levels as determined by MFI in iNKTp cells across all genotypes tested except TKO mice ( Figures 5F, G ). A higher frequency of iNKTp cells from TKO mice were positive for RoRγT than WT controls but did not reach statistical significance ( Figures 5F, G ). Thus, in the absence of RIPK1 (in TKO mice), iNKTp cells accumulate in the thymus and trend towards increased RoRγT expression.

We next considered the effects of RIPK1 deficiency on individual thymic subsets, defining iNKT1, iNKT2, and iNKT17 cells by their relative expression of PLZF and RoRγT ( Supplementary Figure 4B ). Compared to WT and other controls, TKO mice had significantly reduced frequency of NKT1 cells, and concomitant increases in frequencies of NKT2 and NKT17 cells ( Figures 5H–J ). iNKT1 and iNKT17 cells were also decreased in number in TKO mice compared to WT mice and other control mice ( Figure 5K, M ). We did not observe any changes in iNKT2 cell numbers across all genotypes tested ( Figure 5L ).

We next considered Nur77 expression, and noticed it was significantly reduced in iNKT2 of DKO and TKO when compared to WT cells ( Figure 5N ). Unexpectedly, Nur77 was also reduced in both iNKT2 and iNKT17 cells from RIPK1kd mice as compared to WT controls ( Figures 5O, P ), which implies there may be a kinase-dependent role for RIPK1 in maintaining the Nur77 expression levels in these two subsets. We then measured Ki67 levels as a surrogate for cellular proliferation. iNKT1 and iNKT2 cells from DKO mice expressed lower levels of Ki67, but iNKT17 cells expressed higher levels of Ki67 as compared to WT mice ( Figures 5Q–S ). Therefore, in the absence of RIPK1 (i.e. TKO mice), iNKTp cells accumulate in the thymus and express more RoRγT. This increase in thymic iNKTp frequency also coincides with a parallel increase in the frequency of thymic iNKT2 and iNKT17 cells, which is consistent with a developmental relationship between these populations. TKO mice also reveal that RIPK1 is also required to maintain thymic iNKT1 cell frequency and number. Other changes in thymic frequency and Ki67 expression are more likely to be mediated by Casp8 because they are present in both DKO and TKO mice.

It is now clear that RIPK1 plays a role in iNKT cell development, but it remained to be determined if RIPK1 also mediates effector functions of peripheral iNKT cells which are induced as a consequence of TcR engagement. To determine if the lack of RIPK1 and/or Casp8 affects iNKT cell cytokine effector function, we immunized WT, RIPK3^-/-, DKO, TKO and RIPK1kd mice with the iNKT agonist glycolipid αGalactosylceramide (αGC). αGC does not directly activate any cells other than iNKT cells. Four hours after immunization, we analyzed the frequency of splenic IFNγ⁺ iNKT1 cells (gating strategy in Supplementary Figure 5A ). Immunization with αGC induced an increased frequency of IFNγ ⁺ iNKT cells in WT animals as compared to naive WT animals ( Figures 6A, B ). iNKT cells in αGC-immunized RIPK3^-/- and RIPK1kd mice also mounted an IFNγ response comparable to immunized WT mice ( Figures 6A, B ). In contrast, αGC immunized DKO and TKO mice failed to induce IFNγ production by as many of their iNKT1 cells as were induced by immunization of WT mice ( Figures 6A, B ). The shared deficiency in Casp8 between the DKO and TKO mice implicates Casp8 in facilitating the in vivo IFNγ⁺production by iNKT1 cells elicited by αGC.

To identify whether a specific blockade of RIPK1 impairs cytokine production in iNKT cells, we in vitro stimulated splenocytes from GREAT (IFNγ reporter) mice with αGC alone or in the presence of RIPK1 phosphorylation-inhibitor Necrostatin-1 (Nec-1) (gating strategy in Supplementary Figure 5B ). PMA/ionomycin served as a positive control stimulus because it induces RIPK1-mediated cytokine production (26). αGC induced IFNγ production by iNKT cells, and Nec-1 pretreatment prevented the increase in IFNγ production by iNKT cells following αGC or control PMA/ionomycin stimulation ( Figures 6C, D ). This suggests that the IFNγ cytokine effector function of iNKT cells depends on a necrostatin-inhibitable kinase, most likely RIPK1.

We next looked at the production of a second effector cytokine, IL-4, another iNKT cell effector cytokine. Upon αGC stimulation, splenic iNKT2 cells rapidly release large quantities of IL-4. To determine if there is a role for RIPK1 in IL-4 production by iNKT cells, we immunized WT mice with αGC and measured the IL-4 response by iNKT cells (gating strategy in Supplementary Figure 5C ). As expected, αGC induced high levels of IL-4 production by WT iNKT2 cells as compared to naïve WT iNKT cells ( Figures 6E, F ). iNKT2 cells from DKO and TKO mice were equally capable of producing IL-4 as iNKT cells from WT animals ( Figures 6E, F ). In fact, IL-4-producing iNKT cells from all genotypes tested were equally responsive to αGC stimulation ( Figures 6E, F ). To determine if RIPK1 inhibition alone impairs IL-4 production by iNKT cells, we stimulated splenocytes from KN2.Cre mice in vitro with αGC or positive control αCD3/αCD28 with/without Nec-1 pretreatment. Two timepoints were needed because RIPK1 inhibition was only evident for αGC induced responses at 4 hour early timepoint ( Figures 6G, H ), but αCD3/αCD28 induced peak IL-4 responses (measured as expression of huCD2 in reporter mice) by 12 hours ( Figures 6I, J ). αGC stimulation induces IL-4 production by iNKT cells in vitro, which can be significantly inhibited by in vitro pretreatment with Nec-1 ( Figures 6G, H ) (gating strategies in Supplementary Figure 5D ). αCD3/αCD28 induced robust expression of IL-4 by WT iNKT cells which was significantly inhibited by pretreatment with Nec-1 ( Figures 6I, J ) (gating strategies in Supplementary Figure 5E ). Therefore, RIPK1 likely plays a role specifically in early αGC-induction of IL-4 in vitro.

The production of the proinflammatory cytokine IL-17 is mainly attributed to but not limited to, RoRγT expressing iNKT cells after receptor ligation (27, 28). To consider a possible role for RIPK1 in IL-17 production, we stimulated DKO, TKO, and control mice with αGC and measured intracellular IL-17 using flow cytometry (gating strategies in Supplementary Figure 5C ). As expected, αGC immunization induced IL-17 production from iNKT17 cells in WT mice ( Figures 6K, L ). αGC immunization of RIPK3^-/- and RIPK1kd mice expanded IL-17⁺ iNKT17 frequency to the same increased level as WT immunized mice ( Figures 6K, L ). However, iNKT cells from αGC immunized DKO and TKO mice had lower frequency of IL-17⁺ iNKT17 cells when compared to WT immunized mice ( Figures 6K, L ), although only the difference in the TKO mice was statistically significant. This is consistent with IL-17 cytokine effector function depending on Casp8/RIPK1. Given the in vivo evidence for Casp8-dependence of IFNγ and IL-17 responses to iNKT activation, and the in vitro evidence for Casp8/RIPK1-dependence of IFNγ and IL-4 responses to iNKT activation, it is most likely that iNKT cell effector function in response to αGC generally requires both Casp8 and RIPK1.

To extend these studies to consider the role of Casp8 and RIPK1 in mediating another iNKT effector function, we looked at the induced expansion of iNKT_FH cells. We have previously demonstrated that immunization of mice with NP-KLH plus αGC activates iNKT cells to express CXCR5, bcl-6, and PD-1, rending them capable of providing helper signals to activated B cells (29). To determine if RIPK1 is required for innate iNKT_FH cell expansion, we immunized mice with NP-KLH plus αGC. Seven days after immunization we used flow cytometry to measure the frequency of splenic iNKT cells and iNKT_FH cells from immunized WT, RIPK3^-/-, DKO, TKO and RIPK1kd mice and their naïve counterparts (gating strategy in Supplementary Figure 6 ). NP-KLH plus αGC immunized animals had only a modestly higher frequency of TcRβ+ CD1d-tet+ iNKT cells compared to their respective naïve controls ( Figures 7A, B ). NP-KLH plus αGC immunization expanded iNKT_FH cells in WT, RIPK3^-/-, and RIPK1kd mice compared to their respective naïve controls, but because of large variability and high iNKT_FH background frequencies, did not show any increases in iNKT_FH cells in DKO and TKO mice ( Figures 7C, D ).

To assess the activation/exhaustion status of iNKT_FH cells in mice lacking RIPK1/Casp8 in response to NP-KLH and αGC, we measured expression of a combination of exhaustion and activation markers; PD-1, Lag3, Tim3 and ICOS. We used UMAPs to visualize naïve and activated populations of iNKT_FH cells in an unbiased format across all genotypes of mice. The Umap analysis considers the surface expression by MFI of PD-1, CXCR5, ICOS, Tim3 and Lag3 proteins, and then reveals populations of cells which cluster together as a consequence of similar marker expression. The Umap analysis identified clear populations of naive and activated iNKT_FH cells in WT, RIPK3-/- and RIPK1kd mice. However, in DKO and TKO mice, the naïve iNKT_FH population overlapped extensively with the activated iNKT_FH population ( Figure 7E ), suggesting these iNKT_FH cells may already exist in the naïve DKO and TKO mice. This is consistent with the increased frequencies of iNKT_FH cells observed in the un-immunized mice ( Figures 7C, D ). In summary, iNKT_FH cell expansion in response to NP-KLH plus αGC is independent of RIPK1/Casp8. Naïve DKO and TKO mice may harbor preactivated iNKT_FH cells in their spleens consistent with an ongoing autoimmune response because of their shared lack of Casp8.