Calcium flux control by Pacs1-Wdr37 promotes lymphocyte quiescence and lymphoproliferative diseases

Abstract

Endoplasmic reticulum (ER) calcium (Ca²⁺ ) stores are critical to proteostasis, intracellular signaling, and cellular bioenergetics. Through forward genetic screening in mice, we identified two members of a new complex, Pacs1 and Wdr37, which are required for normal ER Ca²⁺ handling in lymphocytes. Deletion of Pacs1 or Wdr37 caused peripheral lymphopenia that was linked to blunted Ca²⁺ release from the ER after antigen receptor stimulation. Pacs1-deficient cells showed diminished inositol triphosphate receptor expression together with increased ER and oxidative stress. Mature Pacs1^-/- B cells proliferated and died in vivo under lymphocyte replete conditions, indicating spontaneous loss of cellular quiescence. Disruption of Pacs1-Wdr37 did not diminish adaptive immune responses, but potently suppressed lymphoproliferative disease models by forcing loss of quiescence. Thus, Pacs1-Wdr37 plays a critical role in stabilizing lymphocyte populations through ER Ca²⁺ handling and presents a new target for lymphoproliferative disease therapy.

Keywords: Pacs1; Wdr37; calcium homeostasis; lymphocyte quiescence; lymphoproliferative disease.

Figure 1. Pacs1 is required for normal numbers of circulating lymphocytes

1. Super‐pedigree mapping of two mutations in Pacs1 that were linked to peripheral B‐cell deficiency. Insert shows peripheral B cell deficiency in the endive (N = 22 REF, 28 HET, 10 VAR) and chicory (N = 24 REF, 15 HET, 3 VAR) pedigrees. Horizontal bars indicate mean values. Ordinary one‐way ANOVA with Tukey’s multiple comparisons test, ns = not significant, **P < 0.01, and ***P < 0.001. Protein domain model shows the encoded location of the ENU alleles.

2. A 1 bp insertion in Pacs1 using CRISPR/Cas9 leads to loss of Pacs1 protein (red arrowhead).

3. Peripheral blood immune cell counts from Pacs1 ^+/+ and Pacs1 ^−/− mice. Horizontal bars indicate mean values. Two‐tailed unpaired t test, *P < 0.05, **P < 0.01, and ***P < 0.001.

4. Absolute numbers of lymphocyte subpopulations in the bone marrow, thymus, and spleen. B cell development in the bone marrow was assessed by FACS analysis for surface expression of: B220⁺CD43⁺CD19⁻IgM⁻IgD⁻ (pre‐pro B); B220⁺CD43⁺CD19⁺IgM⁻IgD⁻ (pro B); B220⁺CD43⁻CD19⁺IgM⁻IgD⁻ (pre B); CD19⁺IgM⁺IgD⁻ (immature); CD19⁺IgM⁺IgD⁺ (mature). T cell development in the thymus was assessed by FACS analysis for surface expression of: CD4⁻CD8⁻ (double negative, DN); CD4⁺CD8⁺ (double positive, DP); CD4⁺CD8⁻ (CD4 single positive, SP); CD4⁻CD8⁺ (CD8 SP). Splenocytes were assessed by FACS analysis for surface expression of: B220⁺CD21⁺CD23⁺ (follicular B cells, FOB); B220⁺CD21⁺CD23^low (marginal zone B cells, MZB); CD4⁺ and CD8⁺ T cells; CD11b⁺ myeloid cells. Each symbol represents an individual mouse. Horizontal bars indicate mean values. Two‐tailed Mann–Whitney test, ns = not significant, *P < 0.05, and **P < 0.01.

5. Proportion of cell populations derived from Pacs1 ^+/+ ; CD45.1 and Pacs1 ^−/− ; CD45.2 donors during competitive bone marrow reconstitution. Populations were determined based on the same markers as in (D) with the added congenic markers CD45.1 and CD45.2. Each symbol represents an individual recipient. Results are representative of two independent transplant experiments. Two‐tailed unpaired t test, ns = not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.

6. Measurement of cell death with Annexin V staining in FOB and MZB cells from the spleens of Pacs1 ^+/+ and Pacs1 ^−/− mice. Results are representative of two independent experiments. Horizontal bars indicate mean values. Two‐tailed unpaired t test, ns = not significant, **P < 0.01.

Figure 2. Pacs1 deletion causes a defect in cytosolic Ca²⁺ flux after antigen receptor stimulation

1. Pacs1 ^+/+ and Pacs1 ^−/− splenocytes were labeled with Indo‐1 and stained for B220, CD21, and CD23 to identify FOB and MZB cells. Fluorescence was measured for 30 s to establish a baseline, and then cells were stimulated with the indicated amounts of anti‐IgM (black arrowhead). Cytosolic Ca²⁺ flux was monitored with FACS analysis by measuring the violet:blue fluorescence emission ratio of Indo‐1. Kinetic traces are displayed from five independent Pacs1 ^+/+ and Pacs1 ^−/− pairs and were normalized to baseline (Pacs1 ^+/+ gray traces, Pacs1 ^−/− pink traces). The mean Ca²⁺ flux for each genotype is overlaid in bold (Pacs1 ^+/+ black, Pacs1 ^−/− red).

2. Maximum Ca²⁺ flux (peak height) at each anti‐IgM concentration for FOB and MZB cells. Paired t test, ns = not significant, *P < 0.05, and ***P < 0.001.

3. Pacs1 ^+/+ and Pacs1 ^−/− FOB cells were labeled with Indo‐1 and stimulated in Ca²⁺ free buffer with 5 µg/ml anti‐IgM (black arrowhead) to assess ER Ca²⁺ efflux. Then, 2 mM Ca²⁺ was added back to assess SOCE. Kinetic traces normalized to baseline from three independent Pacs1 ^+/+ and Pacs1 ^−/− pairs are shown with the mean Ca²⁺ flux overlaid in bold.

4. Maximum Ca²⁺ flux after stimulation under Ca²⁺ free conditions and after Ca²⁺ was added back. Two‐tailed paired t test, ns = not significant, *P < 0.05.

Figure EV1. Ca²⁺ flux defects in Pacs1 ^−/− T cell populations

1. A

   Splenocytes from Pacs1 ^+/+ and Pacs1 ^−/− mice were stained for CD8 and labeled with Indo‐1. Cells were then stimulated with 10 µg anti‐CD3 (black arrowhead). Cytosolic Ca²⁺ flux was monitored by FACS analysis. Kinetic traces are displayed from three independent Pacs1 ^+/+ and Pacs1 ^−/− pairs and were normalized to baseline (Pacs1 ^+/+ gray traces, Pacs1 ^−/− pink traces). The mean Ca²⁺ flux for each genotype is overlaid in bold (Pacs1 ^+/+ black, Pacs1 ^−/− red).

2. B

   Maximum Ca²⁺ flux in CD8 T cells after anti‐CD3 stimulation. Symbols represent independent pairs of mice. Two‐tailed paired t test, *P < 0.05.

3. C

   Splenocytes from Pacs1 ^+/+ and Pacs1 ^−/− mice were also stained for CD4 during same experiments as (A) and stimulated with 10 µg anti‐CD3 (black arrowhead). Kinetic traces are displayed from three independent Pacs1 ^+/+ and Pacs1 ^−/− pairs and were normalized to baseline (Pacs1 ^+/+ gray traces, Pacs1 ^−/− pink traces). The mean Ca²⁺ flux for each genotype is overlaid in bold (Pacs1 ^+/+ black, Pacs1 ^−/− red).

4. D

   Maximum Ca²⁺ flux in CD4 T cells after anti‐CD3 stimulation. Symbols represent independent pairs of mice. Two‐tailed paired t test, *P < 0.05.

5. E, G

   Stimulation of (E) CD8 and (G) CD4 T cells with 10 µg anti‐CD3 under Ca²⁺‐free conditions (black arrowhead) followed by addition of 2 mM Ca²⁺.

6. F, H

   Peak of Ca²⁺ flux in (F) CD8 and (H) CD4 T cells under Ca²⁺‐free conditions and after addition of 2 mM Ca²⁺. Symbols represent independent pairs of mice. Two‐tailed paired t test, ns = not significant, *P < 0.05, **P < 0.01.

Figure EV2. Ca²⁺ flux defects in Pacs1 ^−/− B cells is independent of antigen receptor specificity

1. Total number of splenic B cell subpopulations in Pacs1 ^+/+ and Pacs1 ^−/− mice expressing the NP‐specific B1‐8 heavy chain. Symbols represent individual mice. Horizontal bars indicate mean values. Two‐tailed unpaired t test, ns = not significant, *P < 0.05, **P < 0.01.

2. Identification of NP‐specific FOB cells in spleens from Pacs1 ^+/+ ; IgH^{B1‐8 /+} and Pacs1 ^−/− ; IgH^{B1‐8 /+} mice using NP‐PE.

3. Ca²⁺ flux kinetic traces within the NP+ and NP− gates after treatment with NP‐PE and then with anti‐IgM from three independent experiments (Pacs1 ^+/+ ; IgH^{B1‐8 /+} are gray traces, Pacs1 ^−/− ; IgH^{B1‐8 /+} are red/pink traces). Traces are normalized to baseline.

4. Maximum Ca²⁺ flux after each stimulation within the NP+ and NP− gates. Symbols represent independent pairs of mice. Two‐tailed paired t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure EV3. Analysis of proximal BCR signaling in Pacs1 ^−/− B cells

B cells were purified from the spleens of Pacs1 ^+/+ and Pacs1 ^−/− mice and stimulated with 5 µg/ml of anti‐IgM for the indicated times. Phosphorylated and total amounts of Plcγ2, ERK, and AKT were analyzed by Western blot.

Figure 3. Wdr37 forms a mutually stabilizing complex with Pacs1

1. Super‐pedigree mapping of two mutations in Wdr37 that are linked to peripheral B cell deficiency. Insert shows peripheral B cell deficiency in radical (N = 12 REF, 18 HET, 4 VAR) and profound (N = 14 REF, 15 HET, 6 VAR) pedigrees. Horizontal bars indicate mean values. Ordinary one‐way ANOVA with Tukey’s multiple comparisons test, ns = not significant, ***P < 0.001.

2. Co‐immunoprecipitation of HA‐tagged Pacs1 by FLAG‐Wdr37 and HA‐Wdr37 by FLAG‐Pacs1 in co‐transfected 293T cells. EV, empty vector.

3. Western blot for Pacs1 and Wdr37 expression in peripheral blood cells from WT, Pacs1 ^−/−, and Wdr37 ^−/− mice.

4. B and T cell peripheral blood counts in Wdr37 ^−/− mice. Horizontal bars indicate mean values. Two‐tailed unpaired t test, ***P < 0.001.

5. Wdr37 ^+/+ and Wdr37 ^−/− splenocytes were labeled with Indo‐1, stained for cell surface markers to identify FOB cells, and stimulated with the indicated amounts of anti‐IgM (black arrowhead). Normalized traces from three (2.5 µg/ml anti‐IgM) or four independent experiments (10 and 5 µg/ml anti‐IgM) are shown (Wdr37 ^+/+ gray, Wdr37 ^−/− pink). Mean Ca²⁺ flux for each genotype is overlaid in bold (Wdr37 ^+/+ black, Wdr37 ^−/− red).

6. Maximum Ca²⁺ flux at each anti‐IgM concentration is shown. Two‐tailed paired t test, *P < 0.05, **P < 0.01.

7. Wdr37 ^+/+ and Wdr37 ^−/− FOB cells were labeled with Indo‐1 and stimulated in Ca²⁺ free buffer with 5 µg/ml anti‐IgM (black arrowhead) followed by addition of 2 mM Ca²⁺. Normalized traces from four independent experiments are shown with mean Ca²⁺ flux overlaid in bold.

8. Maximum Ca²⁺ flux after stimulation under Ca²⁺ free conditions and after Ca²⁺ was added back. Two‐tailed paired t test, ns = not significant, *P < 0.05.

Figure EV4. Pacs1 and Wdr37 stabilize each other’s expression

1. Immunoblot analysis of Pacs1‐dependent Wdr37 expression in lymphoid tissues from Pacs1 ^+/+ and Pacs1 ^−/− mice.

2. Gene model for 2 bp deletion from exon 4 of Wdr37 using CRISPR/Cas9 to generate Wdr37 ^−/− mice.

3. Immunoblot analysis of epitope‐tagged Pacs1 and Wdr37 in 293T cells expressing the indicated proteins after CXH treatment.

Figure 4. Pacs1 deletion induces ER stress, ROS, and heightened sensitivity to oxidative stress

1. Immunoblot of ER mass, ER stress, and autophagy markers in Pacs1 ^+/+ and Pacs1 ^−/− splenic B cells that were left unstimulated or stimulated overnight with 5 µg/ml IgM.

2. B cells were purified from Pacs1 ^+/+ and Pacs1 ^−/− spleens and OCR was measured using a Seahorse metabolic flux analyzer. OCR after rotenone/Antimycin A treatment was subtracted from baseline OCR to give basal mitochondrial respiratory rate. Data shown are the mean of 7–10 technical replicates from three independent experiments. Error bars indicate mean ± SD. Two‐tailed unpaired t test, *P < 0.05.

3. Determination of cellular ROS in Pacs1 ^+/+ and Pacs1 ^−/− FOB cells with CellRox Green analysis by flow cytometry. Quantification shows the mean fluorescence intensity (MFI) from three separate pairs of Pacs1 ^+/+ and Pacs1 ^−/− mice. Two‐tailed paired t test, **P < 0.01.

4. Splenocytes from Pacs1 ^+/+ and Pacs1 ^−/− mice were stained with cell surface antibodies to identify FOB cells and treated with 100 µM H₂O₂ for 35 min. Cells were then labelled with TMRE to monitor MMP by FACS analysis. Low TMRE fluorescence indicated susceptibility to H₂O₂ treatment. Data are presented as mean ± SD of 3–4 replicates, two‐tailed unpaired t test, ***P < 0.001. Results are representative of three independent experiments performed on different Pacs1 ^+/+ and Pacs1 ^−/− pairs.

Figure EV5. Pacs1 deletion warps mitochondrial Ca²⁺ flux after IP3R stimulation

1. Pacs1 ^+/+ and Pacs1 ^−/− splenocytes were stained to identify FOB cells then labeled with MitoTracker Green. Histogram shows representative intensity of MitoTracker fluorescence in FOB cells. Quantification shows the results of two pairs of Pacs1 ^+/+ and Pacs1 ^−/− mice. Each symbol represents the mean value from three technical replicates. Horizontal bars indicate mean values from the combined experiments.

2. Mitochondrial stress test of purified Pacs1 ^+/+ and Pacs1 ^−/− B cells. Symbols represent the mean of three independent experiments each with 7‐10 technical replicates. Error bars show SD between the combined experiments. Two‐tailed unpaired t test. *P < 0.05, ns = not significant.

3. Pacs1 ^+/+ and Pacs1 ^−/− NIH‐3T3 cells were infected with MSCV‐Mito‐Pericam. Mitochondrial Ca²⁺ flux was measured before and after treatment with 10 µM ATP with live cell imaging using the 488/405 nm excitation ratio. Each line shows the kinetic trace of an individual cell (Pacs1 ^+/+ gray, Pacs1 ^−/− pink) with the mean overlaid in bold (Pacs1 ^+/+ black, Pacs1 ^−/− red). N = 52 cells each for 3T3 and Pacs1 ^−/−. Results are representative of two independent experiments.

4. Maximum mitochondrial Ca²⁺ flux from the cells imaged in panel C. Symbols represent individual cells. Horizontal bars indicate mean values. Two‐tailed Mann–Whitney test, ***P < 0.001.

5. Pacs1 ^+/+ and Pacs1 ^−/− NIH‐3T3 cells were transfected with 2mtGCaMP6m and basal mitochondria Ca²⁺ content was measured. Symbols represent individual cells. N = 34 cells (3T3) and 37 cells (Pacs1 ^−/−). Results are combined from two independent experiments. Horizontal bars indicate mean values. Two‐tailed unpaired t test with Welch’s correction.

Figure 5. Pacs1 ^−/− B cells have reduced IP3R expression

1. Immunoblot of expression of all three IP3R isoforms and SERCA2 in primary splenic B cells from Pacs1 ^+/+ and Pacs1 ^−/− mice.

2. Real‐time quantitative PCR of IP3R and SERCA2 transcripts in B cells. Data are presented as mean ± SD from three independent Pacs1 ^+/+ and Pacs1 ^−/− pairs of mice.

3. Pacs1 ^−/− FOB cells were labeled with Indo‐1 and stimulated with 0.625 µM thapsigargin (black arrowhead) under Ca²⁺‐free conditions to measure intracellular Ca²⁺ stores. Kinetic traces of four independent experiments are shown (Pacs1 ^+/+ gray, Pacs1 ^−/− pink) with the mean overlaid in bold (Pacs1 ^+/+ black, Pacs1 ^−/− red).

4. Plateau of cytosolic Ca²⁺ flux from intracellular Ca²⁺ stores in (C) calculated by the mean value over the last 30 s of analysis with baseline subtracted. Two‐tailed paired t test, *P < 0.05.

5. AUC of cytosolic Ca²⁺ flux from intracellular Ca²⁺ stores in (C). Two‐tailed paired t test, ns = not significant.

Figure 6. Pacs1 deletion warps ER Ca²⁺ handling

1. Immunoblot of Pacs1, Wdr37, IP3Rs, and ER stress markers in the parental NIH‐3T3 cell line and three separate Pacs1 ^−/− clones.

2. Real‐time quantitative PCR of IP3R isoform expression WT and Pacs1 ^−/− 3T3 cells. Expression in the Pacs1 ^−/− cells was measured in three independent clones. Data are presented as mean ± SD.

3. Pacs1 ^+/+ and Pacs1 ^−/− NIH‐3T3 cells were transfected with cytosolic aequorin and Ca²⁺ flux was measured after treatment with 1 µM bradykinin. Representative kinetic tracing is shown.

4. Peak cytosolic Ca²⁺ concentration based on aequorin measurements as performed in (C). Symbols represent individual wells containing ~ 5 × 10⁵ transfected Pacs1 ^+/+ 3T3 or Pacs1 ^−/− 3T3 cells from one experiment. Horizontal bars indicate mean. Two‐tailed unpaired t test, **P < 0.01.

5. Pacs1 ^+/+ and Pacs1 ^−/− NIH‐3T3 cells (C1 and C2 from (A)) were transfected with ER‐GCamP6‐210. ER Ca²⁺ was measured before and after treatment with 10 µM ATP. Kinetic traces show the mean 488/405 nm excitation ratio of each cell line with error bars indicating SEM. N = 30 cells (3T3), 16 cells (C1), 25 cells (C2). Data are representative of 2 independent experiments.

6. ER Ca²⁺ release from the NIH‐3T3 cell lines imaged in (E). Symbols represent individual cells. Horizontal bars indicate mean value. One‐way ANOVA with Tukey’s test for multiple comparisons, **P < 0.01, ***P < 0.001.

Figure 7. Spontaneous proliferation and increased cell death of Pacs1 ^−/− B cells in vivo under lymphocyte‐replete conditions

1. A

   Pacs1 ^+/+ and Pacs1 ^−/− B cells were purified, labeled with CTV dye, and stimulated with the indicated mitogens. Cell proliferation was assessed after 72 h with FACS analysis based on CTV dilution.

2. B, C

   Pacs1 ^+/+ and Pacs1 ^−/− mice were immunized with alum‐ova and 1 week later with NP‐Ficoll. Anti‐ova IgG and anti‐NP IgM titers were measured at 14 and 7 days after immunization, respectively. Each symbol represents an individual mouse.

3. D

   Pacs1 ^+/+ and Pacs1^{ccy / ccy} mice were immunized with NP‐KLH. Low‐affinity (anti‐NP₃₀) and high‐affinity (anti‐NP₂) antibodies were measured 14 days after immunization.

4. E

   B cells were purified from Pacs1 ^+/+ and Pacs1 ^−/− mice and labeled with CTFR and CTV dyes, respectively. Labeled B cells were injected into unirradiated CD45.1 recipients at ~ 1:1 ratio. Proliferation and survival of adoptively transferred B cells were measured 8 days post‐transplant.

5. F

   Fraction of donor B cells that proliferated after adoptive transfer. Three independent experiments using three different Pacs1 ^+/+ and Pacs1^−/− donor pairs. Each symbol represents an individual recipient. Horizontal bars indicate median value. Two‐tailed unpaired t test, **P < 0.01, ***P < 0.001.

6. G

   Fraction of donor B cells that were Annexin V positive after adoptive transfer. Two independent experiments using two different Pacs1 ^+/+ and Pacs1 ^−/− donor pairs. Each symbol represents an individual recipient. Horizontal bars indicate median value. Two‐tailed unpaired t test, **P < 0.01, ***P < 0.001.

7. H, I

   Pacs1 ^+/+ and Pacs1 ^−/− mice were injected with EdU, and the fraction of EdU+ FOB and MZB cells was measured in the spleen at 1, 4, and 7 days post‐injection. Data are from one experiment.

Figure 8. Pacs1 deletion suppresses abnormal lymphocyte accumulation in models of lymphoproliferation

1. A

   Spleen size and FACS analysis of abnormally expanded B220⁺CD23⁺CD21^+/low FOB cells in Pacs1 ^+/− ; Bcl2^TG and Pacs1 ^−/− ; Bcl2^TG mice.

2. B

   Number of circulating B cells in the blood and FOB cells in the spleen of Pacs1 ^+/− ; Bcl2^TG and Pacs1 ^−/− ; Bcl2^TG mice. Horizontal bars indicate mean value. Two‐tailed Mann–Whitney test, *P < 0.05, **P < 0.01.

3. C

   B cells were purified from the spleens of Pacs1 ^+/− ; Bcl2^TG and Pacs1 ^−/− ; Bcl2^TG mice (CD45.2), labeled with CTFR and CTV proliferation dyes, respectively, and transplanted into unirradiated CD45.1 recipients. Donor B cells were measured in the spleen of recipient mice 7 days after B cell transfer based on CD45.2 expression and proliferation dye fluorescence.

4. D, E

   The fraction of proliferating (D) and recovered (E) donor cells from the experiment in (C). Symbols represent individual recipient mice, and data are from two independent adoptive transfer experiments. Two‐tailed unpaired t test, **P < 0.01, ***P < 0.001.

5. F

   Fraction of apoptotic B cells in the adoptively transferred B cell populations in the experiment in (C). Symbols represent individual recipient mice and data are from one adoptive transfer experiment. Two‐tailed unpaired t test, ***P < 0.001.

6. G

   Splenocytes from Pacs1 ^+/− ; Bcl2^TG and Pacs1 ^−/− ; Bcl2^TG mice were stained with cell surface antibodies to identify FOB cells and treated with 100 µM H₂O₂ for 35 min. Cells were then labeled with TMRE to monitor MMP. TMRE fluorescence was measured by FACS analysis. Data are presented as mean ± SD of 3–4 replicates, unpaired t test, ns = not significant, ***P < 0.001. Results are from one experiment.

7. H

   Lymph node size and flow cytometry analysis of lymphoproliferative CD3⁺B220⁺ cells in Pacs1 ^+/+ ; Fas^{lpr / lpr} and Pacs1 ^−/− ; Fas^{lpr / lpr} mice.

8. I

   Enumeration of CD3⁺B220⁺ cells in the peripheral blood and lymph nodes of Fas^{lpr / lpr} dependent on Pacs1 expression. Symbols represent individual mice. Horizontal bars indicate median value. Two‐tailed Mann–Whitney test, **P < 0.01.