Hem1 controls T cell activation, memory, and the regulated release of immunosuppressive and proinflammatory cytokines

Abstract

Hematopoietic protein-1 (Hem1) is a component of the WASp family verprolin-homologous protein (WAVE) actin regulatory complex, which is activated downstream of multiple immune receptors. Mutations in the NCKAP1L gene encoding HEM1 have recently been found to result in severe primary immunodeficiency disease (PID), characterized by recurrent respiratory infections, hyperinflammation, autoimmunity, and high mortality. However, how loss of Hem1 results in PID is unclear. To define the importance of Hem1 specifically in T cells, we generated constitutive and T cell-specific Hem1-null mice. Hem1-deficient T cells exhibited an increased shift from naive to memory T cells and increased ratio of immunosuppressive regulatory to effector T cells. Loss of Hem1 resulted in hallmarks of T cell exhaustion, including T cell lymphopenia, decreased activation and proliferation, increased expression of PD-1 and Tim3, and increased IL-10 production. In vitro TCR stimulation of CD4+ T cells resulted in increased production of Th1 (IFN-γ), Th2 (IL-5, IL-13), Th17 (IL-17, IL-22), and Treg (IL-10) cytokines. This correlated with reduced F-actin, increased expression of CD107a, and increased granzyme release indicative of increased granule membrane fusion and exocytosis. These results suggest that Hem1 is critical for maintaining T cell activation, homeostasis, and regulated cytokine production following antigen encounter.

Keywords: Adaptive immunity; Cytoskeleton; Immunology; Inflammation; T cells.

Figure 1. Constitutive disruption of Hem1 disrupts T cell development.

Total thymocytes, splenocytes, and cells from axillary and inguinal lymph nodes (LNs) were isolated from Hem1^–/– mice and Hem1^+/– littermate controls. T cell populations were analyzed by flow cytometry. (A) Representative flow cytometric dot plots and histograms of thymocytes. Bar graphs show quantification of double-negative (DN), double-positive (DP), CD4⁺CD8^– (CD4), and CD8⁺CD4^– (CD8) cells in the thymus. (B) Representative flow cytometric dot plots and histograms of splenocytes. Bar graphs show quantification of CD4⁺ and CD8⁺ T cells. (C) Representative flow cytometric dot plots and histograms of cells from the axillary and inguinal LNs combined. Bar graphs show quantification of CD4⁺ and CD8⁺ T cells. n = 6/group, 11- to 16-week-old mice; each data point represents an individual mouse. Cells were first gated on FSC/SSC lymphocytes (Supplemental Figure 1A) and then FSC-height (FSC-H) and FSC-area (FSC-A) single cells. Data are representative of 2 or more independent experiments. Data were analyzed via unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2. Constitutive disruption of Hem1 results in increased proportion of Tregs and Tfh cells.

(A and B) Total thymocytes, splenocytes, and cells from axillary and inguinal lymph nodes (LNs) were isolated from Hem1^–/– mice and Hem1^+/– littermate controls. Representative flow cytometric dot plots and histograms (A), and bar graphs showing quantification of Tregs (CD4⁺CD25⁺FoxP3⁺) (B). Cells were first gated on FSC/SSC lymphocytes and then FSC-H/FSC-A single cells. n = 6/group, 11- to 16-week-old mice. (C) Total splenocytes were isolated from Hem1^–/– mice and Hem1^+/– littermate controls. Representative flow cytometric dot plots and histograms with gating strategy for Tfh cells (CD4⁺CXCR5⁺PD-1⁺). Bar graphs show quantification of Tfh cells. n = 8/group, 11- to 14-week-old mice; each dot represents an individual mouse. Data are representative of 2–3 independent experiments. Bar graphs represent mean ± SEM and were analyzed via unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. Treg, regulatory T cell; Tfh, T follicular helper.

Figure 3. Constitutive disruption of Hem1 results in decreased naive T cells with a concomitant increase in effector memory T cells.

Total splenocytes, cells from axillary and inguinal lymph nodes, and peripheral blood were isolated from Hem1^–/– mice and Hem1^+/– littermate controls. (A) Representative flow cytometric contour plots of splenocytes. Cells were first gated on FSC/SSC lymphocytes (Supplemental Figure 1A), FSC-H/FSC-A single cells, and then either CD4⁺ or CD8⁺ cells (Figure 1, B and C). Bar graphs and quantification of naive (CD44^–CD62L⁺), central memory (CD44⁺CD62L⁺), and effector memory (CD44⁺CD62L^–) T cells from splenocytes. Representative flow cytometric contour plots and bar graphs of T cells harvested from (B) lymph nodes and (C) peripheral blood. n = 4–6/group, 11- to 16-week-old mice; each data point represents an individual mouse. Data are representative of 2 or more independent experiments. Bar graphs represent mean ± SEM and were analyzed via unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CM, central memory; EM, effector memory.

Figure 4. T cell–specific conditional deletion of Hem1 disrupts T cell development.

Total thymocytes, splenocytes, and lymphocytes from the axillary and inguinal lymph nodes (LNs) were isolated from Hem1^fl/flCD4Cre mice and Hem1^fl/fl littermate controls. T cell populations were analyzed by flow cytometry. (A) Representative flow cytometric dot plots of thymocytes. Bar graphs show quantification of double-negative (DN), double-positive (DP), CD4⁺CD8^– (CD4), and CD8⁺CD4^– (CD8) cells in the thymus. (B) Representative flow cytometric dot plots of splenocytes. Bar graphs show quantification of CD4⁺ and CD8⁺ T cells. (C) Representative flow cytometric dot plot of cells from the axillary and inguinal LNs combined. Bar graphs show quantification of CD4⁺ and CD8⁺ T cells. n = 8–9/group, 10- to 40-week-old mice; each data point represents an individual mouse. Data are representative of 2 or more independent experiments. Cells were first gated on FSC-A/SSC-H lymphocytes and FSC-A, FSC-H single cells (Supplemental Figure 1B). Bar graphs represent mean ± SEM and were analyzed via unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ****P < 0.0001. CTL, control; cKO, conditional knockout.

Figure 5. Mice with T cell–specific conditional deletion of Hem1 have increased proportion of Tregs.

(A) Total thymocytes, splenocytes, and cells from axillary and inguinal lymph nodes (LNs) were isolated from Hem1^fl/flCD4Cre mice and Hem1^fl/fl littermate controls. Representative flow cytometric dot plots and histograms of gating strategy for Tregs. Cells were first gated on FSC/SSC lymphocytes and FSC-A/FSC-H single cells. (B) Bar graphs show quantification of Tregs (CD4⁺CD25⁺FoxP3⁺). n = 7–8/group, 10- to 12-week-old mice; each dot represents an individual mouse. Data are representative of 2 or more independent experiments. Data were analyzed via unpaired 2-tailed Student’s t test. **P < 0.01, ****P < 0.0001. Treg, regulatory T cell.

Figure 6. Conditional disruption of Hem1 in T cells results in decreased CD4⁺ T cell activation and proliferation.

Purified T cells from splenocytes harvested from Hem1^fl/flCD4Cre mice and Hem1^fl/fl littermate controls were stimulated with anti-CD3 and anti-CD28 antibody–coated beads. Cells were first gated on lymphocytes, single cells, and then either CD4⁺ or CD8⁺ cells. CD69 and CD25 histograms and bar graphs representing the frequency and MFI of (A) CD69⁺ and (B) CD25⁺ T cells after 24 hours of stimulation. (C) FSC-A histogram and bar graphs representing mean FSC after 24 hours of stimulation. (D) Enriched T cells were stained with CFSE and then stimulated for 48 hours. CFSE proliferation histograms and bar graphs of percentage divided cells and the division index. n = 4–8/group, 10- to 56-week-old mice; data representative of 3 or more independent experiments, each dot represents an individual mouse. Bar graphs represent mean ± SEM and were analyzed via unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. Rel, relative.

Figure 7. T cell–specific deletion of Hem1 results in increased expression of exhausted T cell markers and increased cell death.

Total splenocytes and cells from axillary and inguinal lymph nodes (LNs) were harvested from Hem1^fl/flCD4Cre mice and Hem1^fl/fl littermate controls and analyzed by flow cytometry. (A) Representative gating strategy and bar graphs representing the frequency of PD-1⁺Tim-3⁺ cells of CD4⁺CD44⁺ (left) and CD8⁺CD44⁺ (right) splenocytes. (B) Representative flow cytometric dot plots and histograms of cells from the axillary and inguinal LNs combined. (C) Bar graphs represent the frequency of apoptotic (Caspase3⁺Live/Dead^–) and dead (Caspase3⁺Live/Dead⁺) CD4⁺ (left) and CD8⁺ (right) T cells. n = 3–5/group, 39- to 54-week-old mice; each dot represents an individual mouse. Data representative of 2 independent experiments. Cells were first gated on FSC/SSC lymphocytes, FSC-H/FSC-A single cells, and then either CD4⁺ or CD8⁺ cells. Data were analyzed via unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01.

Figure 8. T cell–specific deletion of Hem1 results in increased mTORC signaling in CD4⁺ T cells. (A)

Immunoblot of indicated proteins from lysates of purified CD4⁺ T cells from Hem1^fl/flCD4Cre mice and Hem1^fl/fl littermate controls stimulated for 0, 10, and 30 minutes with anti-CD3 and anti-CD28 antibodies. Normalized intensity was determined using ImageJ software and bar graphs represent mean ± SEM. n = 2/group, 18- to 19-week-old mice. Data representative of 3 independent experiments. (B) Total splenocytes harvested Hem1^fl/flCD4Cre mice and Hem1^fl/fl littermate controls and stimulated for 0, 10, and 30 minutes with anti-CD3 and anti-CD28 antibodies. Flow cytometric analysis of intracellular signaling proteins p-AKT^S473 and p-S6R^S240/244 levels in CD4⁺ T cells. (C) Flow cytometric analysis of intracellular signaling protein p-AKT^S473 in naive (CD44^lo) and memory (CD44^hi) CD4⁺ T cells. Cells first gated on FSC/SSC lymphocytes, FSC-H/FSC-A single cells, and CD4⁺ T cells. See representative flow cytometric histograms in Supplemental Figure 5A. n = 4/group, 32- to 33-week-old mice. Data representative of 3 independent experiments, each dot represents an individual mouse. Data were analyzed via unpaired 2-tailed Student’s t test. *P < 0.05, **P < 0.01. NS, not significant; min, minutes; CTL, control; cKO, conditional knockout.

Figure 9. T cell–specific disruption of Hem1 results in altered cytokine production.

(A–D) Purified CD4⁺ T cells from Hem1^fl/flCD4Cre mice and Hem1^fl/fl littermate controls were stimulated with anti-CD3 and anti-CD28 antibodies for 72 hours. Concentrations of cytokines in supernatant were measured by multiplex immunoassay. Cytokines produced predominantly by Th1 cells (A), Th2 cells (B), Th17 cells (C), and Tregs (D). Data representative of 1 experiment. (E) Purified T cells from splenocytes harvested from Hem1^fl/flCD4Cre mice and Hem1^fl/fl littermate controls were stimulated with anti-CD3 and anti-CD28 antibodies for 72 hours followed by PMA and ionomycin stimulation for 5 hours. Bar graphs represent frequencies of CD4⁺ T cells expressing indicated cytokines measured by intracellular flow cytometry. Cells first gated on FSC/SSC lymphocytes, FSC-H/FSC-A single cells, and CD4⁺ T cells (Supplemental Figure 7A). Data representative of 2 or more independent experiments. n = 3–7/group, 11- to 15-week-old mice; each dot represents an individual mouse. Data were analyzed via unpaired 2-tailed Student’s t test except IL-17 in (C), which was analyzed via Mann-Whitney test because the data were not normally distributed. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 10. Disruption of Hem1 results in defective F-actin polymerization, actin cap formation, and dysfunctional cortical actin leading to increased exocytosis.

(A) Purified T cells from 10- to 14-week-old Hem1^fl/flpLCKCre mice and Hem1^fl/fl littermate controls were stimulated with anti-CD3 and anti-CD28 antibodies followed by PMA and ionomycin. Representative flow cytometric histograms showing F-actin fluorescence. Cells were first gated on FSC/SSC lymphocytes, FSC-H/FSC-A single cells, T cells, CD4⁺ cells, or CD8⁺ cells. Bar graphs represent relative F-actin MFI compared to baseline. Data representative of 2 independent experiments. (B) Fluorescence microscopy of purified T cells stimulated with anti-CD3/anti-CD28 Dynabeads and stained for actin. Representative images captured at ×100 original magnification showing F-actin/phalloidin (green), nucleus (blue), and Dynabeads (gray). Arrow indicates actin cap. Scale bar: 5 μm. Bar graph represents capped and noncapped T cells. Percentages of capped cells are annotated. Fisher exact test using a 2 × 2 contingency table. Data representative of 1 experiment. (C) Impaired intracellular calcium influx. Purified T cells from Hem1^fl/flCD4Cre and Hem1^fl/fl control mice were labeled with anti-CD4, anti-CD8, and the calcium binding dye Fluo-4 followed by flow cytometry. Cells were stimulated with anti-CD3ε followed by ionomycin (iono). Data are representative of 2 experiments (n = 6/group). (D) Splenocytes were harvested from 52-week-old Hem1^fl/flCD4Cre mice and Hem1^fl/fl LMCs and stimulated with PMA and ionomycin for 5 hours. CD107a surface localization assessed via flow cytometry. Histograms and bar graphs represent frequency and MFI of CD4⁺ and CD8⁺ cells. Data representative of 1 experiment. (E) Purified T cells were stimulated with anti-CD3/anti-CD28 antibody–coated beads for 72 hours. Supernatants were harvested and granzyme B levels measured by ELISA. Shown are bar graphs depicting levels of granzyme B after stimulation. Data are representative of 2 experiments. Each dot represents an individual mouse, n = 3–8/group. Data were analyzed via unpaired 2-tailed Student’s t test unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.