Natural Helper cells derive from lymphoid progenitors

Abstract

Natural Helper (NH) cells are recently discovered innate immune cells that confer protective type 2 immunity during helminth infection and mediate influenza induced airway hypersensitivity. Little is known about the ontogeny of NH cells. We now report NH cells derive from bone marrow lymphoid progenitors. Using RAG-1Cre/ROSA26^YFP mice, we show that the majority of NH cells are marked with a history of RAG-1 expression, implying lymphoid developmental origin. The development of NH cells depends on the cytokine receptor Flt3, which is required for the efficient generation of bone marrow lymphoid progenitors. Finally, we demonstrate that lymphoid progenitors, but not myeloid-erythroid progenitors, give rise to NH cells in vivo. This work therefore expands the lymphocyte family, currently comprising T, B and NK cells, to include NH cells as another type of innate lymphocyte that derives from bone marrow lymphoid progenitors.

Introduction

The T helper type 2 (Th2) cytokines IL-4, IL-5 and IL-13 play an important role in the pathophysiology of allergic diseases and in protective immunity against helminth infection (1). The innate immune pathways responsible for the induction, maintenance and amplification of Th2 cytokine responses during allergic diseases and parasite infection remain poorly understood. Recent studies have discovered novel populations of innate immune cells that promote Th2 cytokine responses (2-6). MPP^type2 cells produce IL-4 in response to IL-25, and give rise to myeloid cells and mast cells in vitro (6). Other innate effector populations termed natural helper cells, nuocytes, and innate type 2 helper cells, appear to be terminally differentiated cell types which produce IL-5 and IL-13 in response to IL-25 or IL-33 (2-5). These novel innate immune cells promote type 2 immunity during helminth infection and mediate influenza-induced airway hypersensitivity (2-6).

We have investigated the developmental origin of type 2 innate effector cells. We found that more than half of Natural Helper (NH) cells were marked with a history of RAG-1 expression, suggesting lymphoid origin. Development of NH cells relies on the cytokine receptor Flt3 that is required for the efficient generation of lymphoid progenitors. Finally, adoptive transfer of purified hematopoietic progenitors established that NH cells are generated by lymphoid but not myeloid-erythroid progenitors. Together, these results indicate that NH cells predominantly originate from bone marrow lymphocyte progenitors.

Materials and Methods

Mice

B6.Ly5^SJL (CD45.1), RAG-2^-/-, and nu/nu mice were purchased from the National Institute of Health (NIH) or the Jackson animal facility. RAG-1Cre/ROSA26^YFP mice (7) and Flt3^-/- mice (8) were bred in accordance with the Institutional Animal Care and Use Committee (IACUC) policies at the University of Pennsylvania.

Isolation of Hematopoietic Cells from the Lung

Mice were exsanguinated, and lungs were perfused by injecting 10 ml of PBS into the right ventricle of the heart. Lungs were carefully cut into small fragments and digested in HBSS containing 0.025mg/ml Liberase D (Roche Diagnostic) and 10 U/ml DNase 1 (Roche Diagnostic). Cells were filtered using a cell strainer.

Flow cytometry

All antibodies (Abs) used here are purchased from eBioscience unless otherwise specified. Abs in the Lineage cocktail include anti-FcεR (MAR-1), anti-B220 (RA3-6B2), anti-CD19 (1D3), anti-Mac-1 (M1/70), anti-Gr-1 (8C5), anti-CD11c (HL3), anti-NK1.1 (PK136), anti–Ter-119 (Ter-119), anti-CD3 (2C11), anti-CD8α (53.6-7), anti-CD8β(53-5.8), anti-TCRβ (H57), and anti-γδTCR (GL-3). Additional Abs used included anti-CD45.2 (104), anti-CD45.1 (A20), anti-Kit (2B8), anti-Sca1 (D7), anti-CD4 (GK1.5), anti-Flt3 (A2F10), anti-IL-7Rα (A7R34), anti-CD25 (PC61.5), anti-Thy1.2 (53-2.1), and anti-T1/ST2 (DJ8, MD Biosciences).

Bone marrow transplantation and intrathymic injections

For bone marrow transfer, bone marrow cells were depleted of Thy1^high cells by magnetic beads. Thy1-depleted donor cells or sorted progenitors (CD45.2) were mixed with 2 × 10⁵ competitor bone marrow cells (CD45.1), and together injected intravenously into lethally irradiated (9.5Gy) recipient mice (CD45.1) through retro-orbital injections. Recipient mice were examined at different time points post-transplant.

For intrathymic transfer, 5000 sorted NH cells or control bone marrow Flt3^highLineage^-Kit⁺ cells (CD45.2) were transferred intrathymically into sublethally irradiated (6.5Gy) mice (CD45.1). Mice were examined 12 days and again 21 days after injection.

Cell Culture

Sorted NH cells were cultured in MEM-alpha medium with 20% FCS containing 10ng/ml of IL-7, IL-2 or IL-33 for 7 days. Cytokine production was determined by intracellular staining using Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD Bioscience).

Statistics

Intergroup comparisons were performed using Student's t-test. The differences were considered significant with a p-value less than 0.05.

Results and Discussion

Characterization of Natural Helper cells in lung

Lung resident NH cells mediate influenza-induced airway hypersensitivity (2). The lung NH cells in C57BL/6 mice are negative for lineage markers (Lin^-), and express T1/ST2 (9) and Thy1 (Fig. 1A). They also express the stem cell antigen Sca-1 and the cytokine receptors IL-7Rα, CD25, and Kit, but not Flt3 (Supplemental Fig. S1A). Like the previously characterized NH cells in Fat Associated Lymphoid Clusters (FALC) (3), lung NH cells express a high level of GATA-3, a transcription factor essential for Th2 cell differentiation (Supplemental Fig. S1B). These cells are present in RAG2^-/- mice and athymic nude mice, but are diminished in IL-7R^-/- mice (Supplemental Fig. S1C). Lung NH cells expand in the presence of IL-2 and IL-7, and they produce type 2 cytokines IL-5 and IL-13 in response to IL-33 stimulation (Supplemental Fig. S1D, S1E). Unlike the T1/ST2⁺ NH cells, the lung resident T1/ST2^- Lin^-Thy1^high cells do not expand in vitro with the same cytokines that support NH cell growth and activation, distinguishing them from functional NH cells (Supplemental Fig. S1E). Thus, the phenotype of Lin^-Thy1^hiT1/ST2⁺ was used to identify NH cells. Together, lung resident NH cells display the same phenotype, gene expression and type 2 cytokine-producing activity of previously described NH cells in the FALC (3).

Figure 1. NH cells are marked with history of RAG expression.

(A) Identification of lung NH cells by flow cytometry. Plots are gated on CD45⁺ cells in the lung. (B) Expression of RAG-1 in thymic DN3 cells and lung NH cells was determined by quantitative PCR. Shown are the relative mRNA levels normalized to GAPDH. (C) Lung NH cells, CD3⁺ T cells, spleen CD3^-NK1.1⁺ NK cells, and bone marrow Gr-1⁺ CD11b⁺cells from RAG1-Cre/ROSA26^YFP mice were analyzed for YFP expression by flow cytometry. (D) The percentages of YFP⁺ cells were quantified. N= 3 mice per group.

MPP^type2 cells are another newly described tissue-resident type 2 innate immune cells (6). MPP^type2 cells give rise to myeloid cells and mast cells in vitro. Lung NH cells, however, did not expand or differentiate in myeloid differentiation conditions, which distinguished NH cells from MPP^type2 cells (Supplemental Fig. 2A). NH cells also phenotypically resemble thymic DN2 cells or circulating T cell progenitors (CTP) (10). However, they did not differentiate into T cells when co-cultured with OP9-DL1 stromal cells (Supplemental Fig. 2B), or after intrathymic transfer (Supplemental Fig. 2C), thus excluding the possibility that NH cells are extrathymic T cell progenitors.

Defective NH cell development upon ablation of lymphocyte progenitors

To investigate the ontogeny of NH cells, we used the RAG-1Cre/ROSA26^YFP mice in which the YFP⁺ cells either express RAG-1 or derive from RAG-1 expressing progenitors. NH cells themselves do not express RAG-1 (Fig. 1B). However, more than half of the NH cells express YFP, indicating a history of RAG-1 expression (Fig. 1C, 1D). These results suggest that NH cells develop from bone marrow lymphoid progenitors in which RAG expression has initiated. Such progenitors include common lymphoid progenitors (CLPs) and developmentally more primitive lymphoid-primed multipotent progenitors (LMPPs), that are efficient progenitors of lymphocytes although they retain a degree of myeloid potential (11, 12).

To understand whether lymphoid progenitors are required for the generation of NH cells, we asked whether NH cell development is dependent on the cytokine receptor Flt3. Previous studies have established that Flt3 is expressed at high levels on bone marrow lymphoid progenitors, and is necessary for the efficient generation of CLPs and LMPPs (13-17). Using mixed bone marrow chimeras, we first confirmed that Flt3 signaling was important for the generation of the lymphoid progenitors LMPPs and CLPs (Fig. 2A, 2B), which was previously suggested in Flt3-ligand deficient mice (13, 15-17). Since we could not use Flt3 itself to define lymphoid progenitors derived from the Flt3^-/- progenitors, we instead used L-selectin (CD62L). L-selectin has been suggested to distinguish lymphoid progenitors from myeloid progenitors within the bone marrow (18), and here we show that L-selectin is a valid marker to replace Flt3 in defining CLPs (Supplemental Fig. S2D). More than 90% of bone marrow Lin^-Sca-1^loKit^loIL-7R⁺L-selectin^hi cells expressed a high level of Flt3 consistent with the phenotype of CLPs, whereas L-selectin^neg cells were almost entirely negative for Flt3 (Supplemental Fig. S2D). Conversely, L-selectin^hi cells constitute the majority (more than 90%) of total CLPs (Lin^-Sca-1^loKit^loIL-7Rα⁺Flt3^hi) (data not shown). Thus, we were able to identify CLPs from Flt3^-/- donor progenitors as Lin^-Sca-1^loKit^loIL-7Rα⁺L-selectin^hi cells. Flt3^-/- bone marrow cells gave rise to greatly reduced number of CLPs in competition with WT bone marrow cells (Fig. 2A, 2B), confirming the requirement of Flt3 signaling for the efficient generation of these lymphoid progenitors (13, 15-17). The reconstitution of Mac-1⁺Gr-1⁺ granulocytes remained intact despite the absence of Flt3, indicating that Flt3 signaling is dispensable for myeloid development (Fig. 2A, 2B). Like T cells and NK cells, NH cell development was also dependent on Flt3 (Fig. 2A, 2B). Frequencies of donor-derived NH cells were reduced more than 10-fold in the absence of Flt3. We cultured NH cells in vitro, and confirmed that Flt3 KO progenitors gave rise to approximately 10 times fewer IL-5 and IL-13 producing functional NH cells (Fig. 2C). These results indicate that Flt3 signaling is important for the development of NH cells. NH cells themselves do not express Flt3, and their proliferation and activation are unaffected by Flt3-ligand (Supplemental Fig. S1A and data not shown); thus, Flt3 signaling is required at earlier stages of NH cell development. We reason that NH cells, like T cells and NK cells, likely derive from Flt3/Flt3-ligand dependent hematopoietic progenitors, most likely LMPP and CLP (13, 15-17).

Figure 2. NH cell development depends on the cytokine receptor Flt3.

(A) 6 X 10⁵ Thy.1 depleted test donor BM cells (CD45.2) and 3 X 10⁵ competitor WT BM cells (CD45.1) were transferred intravenously into lethally irradiated mice (CD45.1). Plots shown are the reconstitution of the hematopoietic populations examined at 16 weeks post-transplant. (B) The percentages of donor-derived populations from Flt3^-/- donor cells were compared with those of WT test donor derived populations. (C) Total phenotypic NH cells from chimeric mice were sorted and cultured with IL-2, IL-7 and IL-33 for 7 days. The cells were stained with CD45.2 and CD45.1, followed by intracellular staining of IL-5 and IL-13. Shown are the percentages of donor-derived IL-5 and IL-13 producing functional NH cells. Data represent 2 independent experiments, 5 mice per group.

NH cells derive from lymphoid progenitors

We next directly tested the capability of purified populations of hematopoietic progenitors to generate NH cells in vivo. Sorted bone marrow HSCs (Lin^-Sca-1⁺Kit^hiFlt3^-), LMPPs (Lin^-Sca-1⁺Kit^hiFlt3^hi), CLPs (Lin^-Sca-1^loKit^loIL-7R⁺Flt3^hi) and myeloid-erythroid progenitors (Lin^-Sca-1^-Kit⁺) were competed with a radioprotective dose of CD45.1 host-type competitor WT bone marrow cells. At 7 days post-transplant, none of these progenitors gave rise to NH cells (data not shown). At 14 days post-transplant, however, NH cells developed from HSCs and Flt3^hi LMPPs and CLPs, a finding consistent with the Flt3-dependency of NH cell development (Fig. 3A, 3B). No detectable NH cells were generated by myeloid-erythroid progenitors (Fig. 3A, 3B). Lymphoid progenitors gave rise to functional NH cells which produced IL-5 and IL-13 producing NH cells; whereas myeloid-erythroid progenitors did not give rise to type 2 cytokine-producing functional NH cells (Fig. 3C). Lymphoid progenitor derived NH cells expressed a high level of GATA3, a major Th2-associated transcription factor (Fig. 3D). They also expressed mRNA for IL-4, another type 2 cytokine, but did not produce IL-4 protein (data not shown). Expression of CD3ε was not detected in the lymphoid progenitor derived NH cells, distinguishing them from T cells (Fig. 3D). RAG-2^-/- CLPs and LMPPs also efficiently give rise to NH cells, confirming that NH cells in these assays are innate lymphocytes (Fig. 3E). Because the myeloid-erythroid progenitors are approximately 10-fold more abundant in bone marrow (19), we repeated these experiments using 20 times as many myeloid-erythroid progenitors as LMPP and CLP. Again, donor-derived NH cells were not detected from myeloid-erythroid progenitors but were easily detected from LMPP and CLP (Fig. 3E). Interestingly, LMPPs were more efficient than CLPs in generating NH cells (Fig. 3B, 3C, 3E), a difference that is similar to the previously noted difference in the efficiency with which these progenitor populations generate T cells in vivo (15, 20). It remains to be determined whether NH cells can directly derive from LMPP as has been suggested for T cells (15), or whether LMPP are more efficient progenitors of NH cells because they efficiently generate CLP (20). However, the results from this experiment show that lymphocyte progenitor populations LMPP and CLP, but not myeloid-erythroid progenitor populations, possess the potential to efficiently develop into NH cells in vivo.

Figure 3. NH cell develop from lymphoid progenitors.

(A) 5 X 10³ progenitors (CD45.2) isolated by cell sorting were injected into lethally irradiated mice (CD45.1) together with 2 X 10⁵ WT competitor BM cells (CD45.1). Lung NH cells were examined at 2 weeks post-transplant. Plots are gated on donor-derived cells (CD45.2⁺). (B) The number of NH cells deriving from purified progenitor populations was quantified. Data represent 2 independent experiments, 3-4 mice per group. (C) 2 X 10⁴ purified progenitors (CD45.2) were transferred into lethally irradiated mice (CD45.1) together with 2 X 10⁵ WT competitor cells (CD45.1). Total phenotypic NH cells from chimeric mice were sorted and cultured with IL-2, IL-7 and IL-33 for 7 days. The top panels depict IL-5 and IL-13 production by functional NH cells. The bottom panels show the frequency of CD45.2⁺ functional NH cells derived from the indicated progenitor populations. Data represent 2 independent experiments, 4 mice per group. (D) CLP-derived NH cells from 4 recipient mice were pooled and cultured with IL-2, IL-7 and IL-33 for 7 days. mRNA was extracted, and gene expression examined using quantitative PCR. The relative mRNA levels were normalized to GAPDH. (E) 2 X 10³ LMPP, CLPs or 4 X 10⁴ Lineage^- Sca-1^- Kit⁺ progenitors from Rag2^-/- mice were transferred with 2 X 10⁵ WT competitor BM cells. The number of lung NH cells deriving from each progenitor population is shown.

In summary, we have established that NH cells are a new type of innate lymphocyte that derives from lymphoid progenitors. We demonstrate that the development of NH cells depends on Flt3 signaling which is required for the efficient generation of lymphoid progenitors. The majority of NH cells are marked with a history of RAG-1 expression, and derive from bone marrow lymphoid but not myeloiderythroid progenitors in vivo. While we cannot exclude the possibility that a rare subset of myeloiderythroid progenitors might possess the potential to give rise to NH cells, our results from multiple lines of evidence suggest a predominantly lymphoid origin of NH cells. The possibly overlapping yet distinct molecular pathways that orchestrate development of NH cells and other innate and adaptive lymphocytes are an important area for future research.