# Differentiation of subsets of NKT cells

> **NIH NIH R01** · LA JOLLA INSTITUTE FOR IMMUNOLOGY · 2020 · $539,188

## Abstract

Abstract
Invariant natural killer T cells (iNKT cells) are a highly conserved lymphocyte population in mice and humans
that express an invariant TCR α chain. These lymphocytes make rapid, innate-like responses, and as a
consequence, there are a number of commercial and clinical efforts to activate these cells in vivo, or expand
them in vitro, in order to combat cancer, provide adjuvant or stimulating effects for vaccines, and to prevent
immune-mediated diseases. It is surprising that in some cases iNKT cells stimulate Th1 or Th2 immunity and
inflammation, while in other instances they are anti-inflammatory. The diversity of their effects on the immune
response could be related to the selective activation of functional subsets of iNKT cells, called NKT1, NKT2
and NKT17 cells. These subsets are analogous to the well known Th1, Th2 and Th17 cells, but they
differentiate in the thymus without exogenous antigenic stimulation. Our preliminary population-based and
single cell RNA-Seq data show that the transcriptomes of the NKT1, NKT2 and NKT17 subsets are highly
divergent, despite their similar specificity. The experiments in this application are designed to identify the
differentiation steps leading to the subsets and the relationships between them, and their stability. To do this,
in Aim 1 we will analyze the developmental potential of individual subsets, including iNKT cells with an
intermediate or possibly transitional phenotype, using both organ cultures and by intrathymic injection.
Additionally, we will use cytokine transcript fate-mapping mice to trace lineage relationships. In aims 2 and 3,
we will determine what are the cell intrinsic and extrinsic factors that drive the differentiation of the iNKT cell
subsets. We will analyze nonproductive β rearrangements by next generation sequencing to provide a clonal
marker, in order to determine if a single iNKT cell clone can give rise to different functional subsets.
Additionally, using different types of mice in which expression of a single TCR β is enforced, we will determine
if variations in the β chain can drive preferential subset differentiation. In aim 4, we will challenge mice with
antigen, infection or chronic inflammation to determine how stable the thymus generated NKT17 subset is
when confronted with inflammation or antigenic challenge. Additionally, we will analyze the transcriptomes of
NKT1, NKT2 and NKT17 cells in spleen and liver, to assess the stability of the thymus-generated gene
programs. Because iNKT cells do not recirculate, the transcriptome data also will provide information on the
imprint of long-term residence in different organs on the iNKT cell subsets. Different populations of innate-like
lymphocytes, which in addition to iNKT cells; include γδ T cells and innate lymphoid cells (ILCs), acquire during
their differentiation gene programs that enforce polarized patterns of cytokine secretion. Undoubtedly there will
be common themes between these cell types, and therefore ou...

## Key facts

- **NIH application ID:** 9927558
- **Project number:** 5R01AI071922-28
- **Recipient organization:** LA JOLLA INSTITUTE FOR IMMUNOLOGY
- **Principal Investigator:** MITCHELL KRONENBERG
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $539,188
- **Award type:** 5
- **Project period:** 1991-04-01 → 2021-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9927558

## Citation

> US National Institutes of Health, RePORTER application 9927558, Differentiation of subsets of NKT cells (5R01AI071922-28). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9927558. Licensed CC0.

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