Contrasting roles for IKK regulated inflammatory signalling pathways for development and maintenance of type 1 and adaptive γδ T cells

Reviewer #1 (Public review):

Summary:

The NF-kB signaling pathway plays a critical role in the development and survival of conventional alpha beta T cells. Gamma delta T cells are evolutionarily conserved T cells that occupy a unique niche in the host immune system and that develop and function in a manner distinct from conventional alpha beta T cells. Specifically, unlike the case for conventional alpha beta T cells, a large portion of gamma delta T cells acquire functionality during thymic development, after which they emigrate from the thymus and populate a variety of mucosal tissues. Exactly how gamma delta T cells are functionally programmed remains unclear. In this manuscript, Islam et al. use a wide variety of mouse genetic models to examine the influence of the NF-kB signaling pathway on gamma delta T cell development and survival. They find that the inhibitor of kappa B kinase complex (IKK) is critical to the development of gamma delta T1 subsets, but not adaptive/naïve gamma delta T cells. In contrast, IKK-dependent NF-kB activation is required for their long-term survival. They find that caspase 8-deficiency renders gamma delta T cells sensitive to RIPK1-mediated necroptosis, and they conclude that IKK repression of RIPK1 is required for the long-term survival of gamma delta T1 and adaptive/naïve gamma delta T cells subsets. These data will be invaluable in comparing and contrasting the signaling pathways critical for the development/survival of both alpha beta and gamma delta T cells.

The conclusions of the paper are mostly well-supported by the data, but some aspects need to be clarified.

(1) The authors appear to be excluding a significant fraction of the TCRlow gamma delta T cells from their analysis in Figure 1A. Since this population is generally enriched in CD25+ gamma delta T cells, this gating strategy could significantly impact their analysis due to the exclusion of progenitor gamma delta T cell populations.

(2) The overall phenotype of the IKKDeltaTCd2 mice is not described in any great detail. For example, it is not clear if these mice possess altered thymocyte or peripheral T cell populations beyond that of gamma delta T cells. Given that gamma delta T cell development has been demonstrated to be influenced by gamma delta T cells (i.e, trans-conditioning), this information could have aided in the interpretation of the data. Related to this, it would have been helpful if the authors provided a comparison of the frequencies of each of the relevant subsets, in addition to the numbers.

(3) The manner in which the peripheral gamma delta T cell compartment was analyzed is somewhat unclear. The authors appear to have assessed both spleen and lymph node separately. The authors show representative data from only one of these organs (usually the lymph node) and show one analysis of peripheral gamma delta T cell numbers, where they appear to have summed up the individual spleen and lymph node gamma delta T cell counts. Since gamma deltaT17 and gamma deltaT1 are distributed somewhat differently in these compartments (lymph node is enriched in gamma deltaT17, while spleen is enriched in gamma deltaT1), combining these data does not seem warranted. The authors should have provided representative plots for both organs and calculated and analyzed the gamma delta T cell numbers for both organs separately in each of these analyses.

(4) The authors make extensive use of surrogate markers in their analysis. While the markers that they choose are widely used, there is a possibility that the expression of some of these markers may be altered in some of their genetic mutants. This could skew their analysis and conclusions. A better approach would have been to employ either nuclear stains (Tbx21, RORgammaT) or intracellular cytokine staining to definitively identify functional gamma deltaT1 or gamma deltaT17 subsets.

(5) The analysis and conclusion of the data in Figure 3A is not convincing. Because the data are graphed on log scale, the magnitude of the rescue by kinase dead RIPK1 appears somewhat overstated. A rough calculation suggests that in type 1 game delta T cells, there is ~ 99% decrease in gamma delta T cells in the Cre+WT strain and a ~90% decrease in the Cre+KD+ strain. Similarly, it looks as if the numbers for adaptive gamma delta T cells are a 95% decrease and an 85% decrease, respectively. Comparing these data to the data in Figure 5, which clearly show that kinase dead RIPK1 can completely rescue the Caspase 8 phenotype, the conclusion that gamma delta T cells require IKK activity to repress RIPK1-dependent pathways does not appear to be well-supported. In fact, the data seem more in line with a conclusion that IKK has a significant impact on gamma delta T cell survival in the periphery that cannot be fully explained by invoking Caspase8-dependent apoptosis or necroptosis. Indeed, while the authors seem to ultimately come to this latter conclusion in the Discussion, they clearly state in the Abstract that "IKK repression of RIPK1 is required for survival of peripheral but not thymic gamma delta T cells." Clarification of these conclusions and seeming inconsistencies would greatly strengthen the manuscript. With respect to the actual analysis in Figure 3A, it appears that the authors used a succession of non-parametric t-tests here without any correction. It may be helpful to determine if another analysis, such as ANOVA, may be more appropriate.

(6) The conclusion that the alternative pathway is redundant for the development and persistence of the major gamma delta T cell subsets is at odds with a previous report demonstrating that Relb is required for gamma delta T17 development (Powolny-Budnicka, I., et al., Immunity 34: 364-374, 2011). This paper also reported the involvement of RelA in gamma delta T17 development. The present manuscript would be greatly improved by the inclusion of a discussion of these results.

(7) The data in Figures 1C and 3A are somewhat confusing in that while both are from the lymph nodes of IKKdeltaTCD2 mice, the data appear to be quite different (In Figure 3A, the frequency of gamma delta T cells increases and there is a near complete loss of the CD27+ subset. In Figure 1A, the frequency of gamma delta T cells is drastically decreased, and there is only a slight loss of the CD27+ subset.)

Reviewer #2 (Public review):

This study presents a comprehensive genetic dissection of the role of IKK signaling in the development and maintenance of lymphoid gd T cells. By employing a variety of conditional and mutant mouse models, the authors demonstrate that IKK-dependent NF-κB activation is essential for the generation of type 1 gd T cells, while adaptive gd T cells require this pathway primarily for long-term survival. The use of multiple complementary genetic strategies, including IKK deletion and modulation of RIPK1 and CASPASE8 activity, provides robust mechanistic insight into subset-specific regulation of gd T cell homeostasis. Overall, the study provides mechanistic insight for IKK-dependent regulation of gd T cell development and peripheral maintenance. However, additional experiments can be performed to improve this manuscript and its interpretations.

Specific Concerns:

(1) All approaches used confer changes to the entire T cell compartment. Therefore, the authors are unable to resolve whether the observations are mediated by direct and/or indirect effects (e.g., disorganized lymphoid architecture impacting maintenance/survival/homing).

(2) Assessment of factors that impact T cell numbers in the periphery is necessary. Are there observable changes to the proliferation, survival, and migration of gd T cell subsets?

(3) TCRd chain usage, especially among type 3 gd T cells, should be assessed.

(4) The functional consequences of IKK signaling on gd T cells were largely unaddressed. Cytokine analyses were performed only in the RIPK1D138N Casp8∆TCD2 model, leaving open the question of how canonical NF-κB-dependent signaling impacts the long-term functionality of gd T cells.

(5) The authors suggest that Caspase 8 is required for the development and maintenance of type 3 gd T cells. While the authors discussed the limitations of assessing adult mice in interpreting the data, it seems like a relatively straightforward experiment to perform.

(6) While analyses of Casp8∆TCD2 RIPK1D138N mice suggest that loss of adaptive and type 1 gamma delta T cells in Casp8∆TCD2 animals is due to necroptosis, the contribution of RIPK3 kinase activity remains unexamined. RIPK3 activity determines whether cells die via necroptosis or apoptosis in RIPK1/Caspase8-dependent signaling, and inclusion of this analysis would strengthen mechanistic insights.

(7) Canonical NF-κB signaling through cRel alone was not evaluated, leaving a gap in the understanding of transcriptional pathways required for gd T cell subsets.

Reviewer #3 (Public review):

Summary

The regulation of NF-κB signaling is complex and central to the differentiation and homeostasis of αβT cells, essential to adaptive immunity. γδ T cells are a distinct population that responds to stress/injury-induced cues by producing inflammatory cytokines, representing an important bridge between innate and adaptive immunity. This study from Islam et al. demonstrates that the IKK complex, a central regulator of NF-κB signaling, plays distinct and essential roles in the differentiation and maintenance of γδ T cells. The authors use elegant murine genetic models to generate clear data that disentangle these requirements in vivo.

Although NF-κB activity was found to be dispensable for specification of γδ T cell progenitors and the generation of adaptive γδ T cells, it was required for both the ontogeny of type 1 γδ T cells and the survival of mature adaptive γδ T cells. Subunit-specific analyses revealed parallels with αβ T cells: RELA was necessary for type 1 γδ T cell development, while maintenance of adaptive γδ T cells relied upon redundancy between REL subunits, with cREL and p50 compensating in the absence of RELA but not vice versa. These findings reflect distinct biological requirements for ontogeny versus maintenance, likely driven by differences in receptor signaling, such as TCR and TNFRSF family members. Moreover, IKK also maintained γδ T cell survival through repression of RIPK1-mediated cell death, echoing its dual role in αβ T cells, where it both prevents TNF-induced apoptosis and provides NF-κB-dependent survival signals.

Strengths:

The multiple, unique murine genetic models employed for detailed analysis of in vivo γδ T cell differentiation and homeostasis are a major strength of this paper. NF-κB signaling processes are devilishly complex. The conditional mutants generated for this study disentangle the requirements for the various IKK-regulated pathways in γδ T cell differentiation, cell survival, and homeostasis. Data are clearly presented and suitably interpreted, with a helpful synthesis provided in the Discussion. These data will provide a definitive account of the requirements for NF-κB signaling in γδ T cells and provide new genetic models for the community to further study the upstream signals.

Weaknesses:

The paper would benefit greatly from a graphical abstract that could summarize the key findings, making the key findings accessible to the general immunology or biochemistry reader. Ideally, this graphic would distinguish the requirements for NF-κB signals sustaining thymic γδ T cell differentiation from peripheral maintenance, taking into account the various subsets and signaling pathways required. In addition, the authors should consider adding further literature comparing the requirements for NF-κB /necroptosis pathways in regulating other non-conventional T cell populations, such as iNKT, MAIT, or FOXP3+ Treg cells. These data might help position the requirements described here for γδ T cells compared to other subsets, with respect to homeostatic cues and transcriptional states.

Last and least, there are multiple grammatical errors throughout the manuscript, and it would benefit from further editing. Likewise, there are some minor errors in figures (e.g., Figure 3A, add percentage for plot from IKKDT.RIPK1D138N mouse; Figure 7, "Adative").