Monoclonal Invariant NKT (iNKT) Cell Mice Reveal a Role for Both Tissue of Origin and the TCR in Development of iNKT Functional Subsets

Abstract

Invariant NKT (iNKT) cell functional subsets are defined by key transcription factors and output of cytokines, such as IL-4, IFN-γ, IL-17, and IL-10. To examine how TCR specificity determines iNKT function, we used somatic cell nuclear transfer to generate three lines of mice cloned from iNKT nuclei. Each line uses the invariant Vα14Jα18 TCRα paired with unique Vβ7 or Vβ8.2 subunits. We examined tissue homing, expression of PLZF, T-bet, and RORγt, and cytokine profiles and found that, although monoclonal iNKT cells differentiated into all functional subsets, the NKT17 lineage was reduced or expanded depending on the TCR expressed. We examined iNKT thymic development in limited-dilution bone marrow chimeras and show that higher TCR avidity correlates with higher PLZF and reduced T-bet expression. iNKT functional subsets showed distinct tissue distribution patterns. Although each individual monoclonal TCR showed an inherent subset distribution preference that was evident across all tissues examined, the iNKT cytokine profile differed more by tissue of origin than by TCR specificity.

Figure 1. iNKT transnuclear mice have monoclonal iNKT cells with distinct antigenic specificities

(A) Splenocytes were isolated from the indicated TN mouse lines and stained with anti-Vβ7, CD1d (PBS57) tetramer, and anti-CD4. Results are representative of >50 mice per group. (B) Splenocytes were isolated from indicated C57BL/6 or TN mouse lines and stained with anti-CD3, anti-CD8, OCH tetramer, and α-Glc tetramer. Both tetramers were added simultaneously. Plots are gated on CD3⁺CD8⁻ T cells. (C) Thymocytes from C57BL/6 or TN mouse lines were isolated and stained with anti-CD3 and OCH tetramer. Cells were washed before being stained with CD1d (PBS57) tetramers, to assess displacement. (D) Quantification of OCH staining across multiple, independent experiments. **, p<0.01 compared to C57BL/6.

Figure 2. iNKT transnuclear mice have increased frequencies of iNKT cells

(A) iNKT cells were harvested from the spleen, mesenteric lymph nodes (mLN), skin-draining lymph nodes (sdLN), liver, adipose tissue, lungs, and thymus of C57BL/6 and Vα14 mice and stained with anti-CD3 and CD1d (PBS57) tetramer. Results are representative of 5 mice per group; averaged in (B). (B) Lymphocytes were harvested from the spleen, mesenteric lymph nodes (mLN), skin-draining lymph nodes (sdLN), liver, adipose tissue, lungs, and thymus of the indicated mice and counted. Cells were stained with anti-CD3 and CD1d (PBS57) tetramer and analyzed by flow cytometry. The percentage of iNKT cells from each organ was multiplied by the cell count, and added to obtain an estimated frequency of total iNKT cells in the body. Results shown are average of 5–8 mice per group. (C) Splenocytes from C57BL/6 and Vα14 mice were stimulated in vitro with PMA/Ionomycin for 4 hours in the presence of GolgiStop. Cells were stained with anti-CD3 and CD1d (PBS57) tetramer, before they were fixed, permeabilized, and stained with anti-IFNγ, anti-IL-4, and anti-IL-17. Results shown are gated on CD3+CD1d-tetramer+ cells. Results shown are representative of three independent experiments where n=3 biological replicates.

Figure 3. Conventional CD4, CD8 and Foxp3+ Tregs develop in hemizygous RAG-proficient iNKT TN mice

(A) Thymocytes from C57BL/6 and all TN lines were stained with anti-CD3, CD1d (PBS57) tetramer, anti-CD4, and anti-CD8. Results shown are gated on non-iNKT cells (CD3⁺ CD1d-tetramer⁻). (B) Thymocytes from C57BL/6 and all TN lines were stained with anti-CD3 and CD1d (PBS57) tetramer before being fixed, permeabilized, and stained with anti-Foxp3. Results shown are gated on non-iNKT cells. (C) Quantification of A–B where n=3 mice per group. (D) Lymph node cells from Vβ8.2 and Vβ8.2;RAG2^−/− mice were stimulated with the indicated concentrations of α-GalCer. IFNγ was measured by ELISA of 24 hour culture supernatants. Error bars are SD. (E) Each monoclonal TN line was crossed to RAG2^−/− background. Peripheral blood of mice of the indicated genotypes was stained with CD1d (PBS57) tetramer and anti-CD3. (F) Quantification of (E) averaged over 5 mice per group.

Figure 4. Monoclonal iNKT cells can produce all major cytokines

Cells from thymus (A) or spleen (B) of B6xBalb/c F1 mice of all TN lines were stimulated in vitro with PMA/Ionomycin for 4 hours, in the presence of GolgiStop. Cells were stained with anti-CD3 and CD1d (PBS57) tetramer, before being fixed, permeabilized, and stained with antibodies to IFNγ, IL-4, and IL-17. Results shown are gated on CD3⁺CD1d-tetramer⁺ cells. n=4 mice per group. Error bars are SD.

Figure 5. NKT1, NKT2 and NKT17 subsets are present in rigorously monoclonal mice

(A–B) Skin draining lymph node cells from all TN mice were stimulated in vitro with PMA/Ionomycin for 4 hours, in the presence of GolgiStop. Cells were stained with anti-CD3 and CD1d (PBS57) tetramer, before being fixed, permeabilized, and stained with antibodies to IFNγ, IL-4, and IL-17. Results shown are gated on CD3⁺CD1d-tetramer⁺ cells. TN mice are either RAG-proficient (top panels) or RAG-deficient (bottom panels). (C) Quantification of panels A–B, n=4 mice per RAG2^+/+ group and n=3 mice per RAG2^−/− group. Mice were 5–6 weeks of age and both sexes were included in each group.

Figure 6. TCR avidity correlates with PLZF expression during thymic development of monoclonal iNKT cells

Jα18^−/− mice were lethally irradiated, reconstituted with 95% Jα18^−/− bone marrow and 5% bone marrow of the indicated TN lines, and analyzed 8 weeks later. (A) Thymocytes from C57BL/6 and all TN lines were stained with anti-CD3 and CD1d (PBS57) tetramer, and analyzed by flow cytometry. Mean fluorescence intensities (MFI) of tetramer staining for gated populations are shown on plots. (B) Thymocytes from C57BL/6 and all TN lines were stained with anti-CD3 and CD1d (PBS57) tetramer before being fixed, permeabilized, and stained with antibodies to PLZF, T-bet, and RORγt. Results shown are gated on CD1d (PBS57) tetramer⁺ iNKT cells. Tetramer bright = top 20% of iNKT cells; tetramer dim = bottom 20% of iNKT cells as defined by intensity of PE signal. (C) Quantification of mice shown in panel A. (D) Quantification of mice shown in panel B. n=4 mice per group. Error bars are SD.

Figure 7. Limited dilution bone marrow chimeras reveal a minor role for TCR specificity in lineage preference of iNKT cells

Jα18^−/− mice were lethally irradiated, reconstituted with 95% Jα18^−/− bone marrow and 5% bone marrow of the indicated TN lines, and analyzed 8 weeks later. n=4 mice per group. (A) Skin draining lymph node cells from all TN mice were stimulated in vitro with PMA/Ionomycin for 4 hours, in the presence of GolgiStop. Cells were stained with anti-CD3 and CD1d (PBS57) tetramer, before being fixed, permeabilized, and stained with antibodies to IFNγ, IL-4, and IL-17. Results shown are gated on CD3⁺CD1d-tetramer⁺ cells. (B) Quantification of ICCS data from thymus, spleen, skin-draining lymph nodes and liver, normalized to Vα14 values for the same organ. Error bars are SD.

Figure 8. Tissue of origin plays a dominant role in cytokine profiles of monoclonal iNKT cells

Jα18^−/− mice were lethally irradiated, reconstituted with 95% Jα18^−/− bone marrow and 5% bone marrow of the indicated TN lines, and analyzed 8 weeks later. n=4 mice per group. (A) Skin draining lymph node cells from all TN mice were stained with anti-CD3 and CD1d (PBS57) tetramer, before being fixed, permeabilized, and stained with antibodies to PLZF, T-bet, and RORγt. Results shown are gated on CD3⁺CD1d-tetramer⁺ cells. (B) Quantification of transcription factor expression as shown in (A). n=4 mice per group. ANOVA was used to determine the relative contribution of TCR versus tissue of origin to explain the variation in transcription factor expression.