# Loss-of-phosphorylation of IKZF1 results in gain-of-function associated with immune dysregulation

Akihiro Hoshino, Benoît Heid Picard, Sophia Polychronopoulou, Charikleia

Kelaidi, Saba Azarnoush, Sven Kracker, Frédéric Rieux-Laucat, David

Boutboul, Sylvain Latour

<figure><img src="image_1.png" /></figure>

## To cite this version:

Akihiro Hoshino, Benoît Heid Picard, Sophia Polychronopoulou, Charikleia Kelaidi, Saba Azarnoush, et al.. Loss-of-phosphorylation of IKZF1 results in gain-of-function associated with immune dysregulation. Journal of Allergy and Clinical Immunology, 2024, 154 (1), pp.229-236.e2. 10.1016/j.jaci.2024.01.029. hal-04738369

HAL Id: hal-04738369

https://hal.science/hal-04738369v1

Submitted on 19 Nov 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

# Loss-of-phosphorylation of *IKZF1* results in gain-of-function associated with immune dysregulation

Akihiro Hoshino, MD, PhD,ª Benoit Heid Picard, MD,ª,ᵇ Sophia Polychronopoulou, MD, PhD,ᶜ

Charikleia Kelaidi, MD, PhD,ᶜ Saba Azarnoush, MD,ᵈ Sven Kracker, PhD,ᵇ,ᵉ Frédéric Rieux-Laucat, PhD,ᵇ,ᵉ

David Boutboul, MD, PhD,ª and Sylvain Latour, PhDª,ᵇ

Paris, France, and Athens, Greece

**Background:** Immune dysregulation often presents as autoimmunity, inflammation, and/or lymphoproliferation. Several germline genetic defects have been associated with immune dysregulation; they include heterozygous gain-of-function (GOF) mutations in *IKZF1*, an essential transcription factor for hematopoiesis containing zinc finger domains (ZFs). However, in a large percentage of patients, the genetic origin of their immunedysregulation remains undetermined.

**Objective:** A family with 2 members presenting immune dysregulation signs was studied to identify the genetic cause of their disease.

**Methods:** Whole exome sequencing, analysis of immunologic parameters, and functional assays (including Western blotting, electrophoretic mobility shift assay during the cell cycle, and T<sub>H</sub> cell differentiation) were performed.

**Results:** The 2 patients carried a novel heterozygous mutation in *IKZF1* (*IKZF1*<sup>T398M</sup>). *IKZF1* heterozygous mutations have previously been shown to be responsible for several distinct human immunologic diseases by directly affecting the ability of ZFs to bind to DNA or to dimerize. Herein, we showed that the *IKZF1*<sup>T398M</sup>, which is outside the ZFs, caused impaired phosphorylation of *IKZF1*, resulting in enhanced DNA-binding ability at the S phase of the cell cycle, reduction of the G1-S phase transition, and decreased proliferation. Confirming these data, similar functional alterations were observed with *IKZF1*<sup>T398A</sup>, but not with *IKZF1*<sup>T398D</sup>, mimicking dephosphorylation and phosphorylation, respectively. In T lymphocytes, expression of *IKZF1*<sup>T398M</sup> led to T<sub>H</sub> cell

differentiation skewed toward T<sub>H2</sub> cells. Thus, our data indicate that *IKZF1*<sup>T398M</sup> behaves as a GOF variant underlying immune dysregulation.

**Conclusion:** Disturbed *IKZF1* phosphorylation represents a novel GOF mechanism (GOF by loss of phosphorylation (termed as GOF-LOP) associated with immune dysregulation, highlighting the regulatory role of *IKZF1* during cell cycle progression through phosphorylation. (J Allergy Clin Immunol 2024;154:229-36.)

**Key words:** Immune dysregulation, inborn errors of immunity, genetic, mutation, IKAROS, *IKZF1*, phosphorylation, gain-of-function, T<sub>H2</sub> cell differentiation

## INTRODUCTION

IKAROS family zinc finger 1 (*IKZF1*) also known as IKAROS, which is encoded by *IKZF1*, is an essential transcription factor in hematopoietic development.<sup>1-3</sup> It is characterized by 2 sets of C2H2 zinc finger (ZFs) domains consisting of 4 N-terminal ZFs (ZF1-ZF4) that mediate DNA-binding and 2 C-terminal ZFs (ZF5 and ZF6, respectively) involved in dimerization. *IKZF1* dimer regulates the expression of a number of genes by interacting with the polycomb repressive and the nucleosome remodeling and histone deacetylase complexes.<sup>4</sup> Several inborn errors of immunity have been associated with genetic alterations in *IKZF1*, clinically ranging from common variable immunodeficiency to severe combined immunodeficiency associated with lymphoid malignancy.<sup>5-10</sup> Alterations include loss-of-function (LOF) and dominant-negative heterozygous variants that target ZF2 and ZF3, causing impaired DNA binding, or variants in ZF5 and ZF6 that impede dimerization. Recently, we extended the genotype and phenotype of *IKZF1*-associated diseases by identification of gain-of-function (GOF) heterozygous variants (R183H/C) in the ZF3, resulting in enhanced binding to DNA associated with an inappropriate lymphoid development at the later stages including low regulatory T (Treg) cell numbers and T<sub>H2</sub> cell skewing.<sup>6</sup> Carriers of these variants were clinically characterized by inflammation symptoms, autoimmunity, allergies, and plasma cell proliferation.

In the present study, we report a family with immune dysregulation symptoms associated with a novel GOF variant affecting the phosphorylation site threonine 398.

<figure><img src="image_4.png" /></figure>

<footnote>From ªthe Laboratory of Lymphocyte Activation and Susceptibility to EBV Infection, ¨the Laboratory of Human Lymphohematopoiesis, and fthe Laboratory of Immunogenetics of Pediatric Autoimmunity, INSERM UMR 1163, Imagine Institute, Paris; bthe Université de Paris-Cité; ´the Department of Pediatric Hematology-Oncology, Aghia Sophia Children’s Hospital, Goudi-Athens; and dthe Department of Pediatric Immunohematology, Hôpital Robert-Debré, Assistance Publique-Hôpitaux de Paris.</footnote>

<footnote>Received for publication April 12, 2023; revised January 21, 2024; accepted for publication January 25, 2024.</footnote>

<footnote>Available online March 2, 2024.</footnote>

<footnote>Corresponding author: Sylvain Latour, PhD, Laboratory of Lymphocyte Activation and Susceptibility to EBV, Equipe Labellisée Ligue Contre le Cancer, Imagine-Inserm UMR 1163; 24 boulevard du Montparnasse, 75015 Paris, France. E-mail: sylvain.latour@inserm.fr.</footnote>

<footnote>The CrossMark symbol notifies online readers when updates have been made to the article such as errata or minor corrections</footnote>

<footnote>0091-6749</footnote>

<footnote>© 2024 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</footnote>

<footnote>https://doi.org/10.1016/j.jaci.2024.01.029</footnote>
Abbreviations used

GOF: Gain-of-function

IKZF1: IKAROS family zinc finger 1

LOF: Loss-of-function

MS: Multiple sclerosis

T<sub>FH</sub>: Follicular helper T

Treg: Regulatory T

WT: Wild-type

ZF: Zinc finger

RESULTS AND DISCUSSION

From the age of 2 years, patient 1 has presented with recurrent
Evans syndrome following infectious episodes characterized by
immune thrombocytopenia, autoimmune neutropenia, a positive
direct antiglobulin test result, presence of antiplatelet IgG
antibodies and nodal lymphoproliferation, with a follow-up at
age 6 years requiring corticosteroids and intravenous immuno-
globulins. He also experienced chronic crusted lesions consistent
with staphylococcal skin infection or atopic dermatitis folliculitis
and had intermittent oral aphthous ulcers. His mother (patient 2)
had a history of multiple sclerosis (MS) that was diagnosed at age
33 years (for details see in Methods section of the Online
Repository at www.jacionline.org).

The 2 patients exhibited normal B- and T-cell counts with
normal serum immunoglobulin levels. Their T cells showed an
increased CD4/CD8 ratio, reduction of effector Treg cell
numbers, and increased CD45RA<sup>-</sup>CXCR5<sup>+</sup> T follicular helper
(T<sub>FH</sub>) cell and CCR4<sup>+</sup>CXCR3<sup>-</sup> T<sub>H2</sub> cells counts (Fig 1, A and
Table I). In patient 1, CXCR5<sup>+</sup>PD1<sup>+</sup> T<sub>FH</sub> cell numbers were
also increased. CD4<sup>+</sup> T cells from patient 1 and patient 2 dis-
played decreased IL-2 and IFN-γ levels and increased IL-4 pro-
duction after PMA and ionomycin stimulation (Fig 1, B and C),
consistent with the skewing of CXCR5<sup>-</sup>CCR6<sup>-</sup>CD4<sup>+</sup> memory
T cells toward CCR4<sup>+</sup>CXCR3<sup>-</sup> T<sub>H2</sub> cells, likely reflecting unbal-
anced T<sub>H1</sub> cell/T<sub>H2</sub> cell differentiation. T-cell proliferation from
PBMCs in response to CD3, CD3+CD28, and PHA was dimin-
ished (Fig 1, D). Autoimmune lymphoproliferation syndrome
was excluded owing to the absence of elevated double-negative
αβ<sup>+</sup> T-cell and normal vitamin B<sub>12</sub> levels.

In both patients, whole exome sequencing identified a hetero-
zygous *IKZF1* missense variant, g.7:50400260C>T, c.1193C>G, T,
p.T398M, that was absent in the father (Fig 2, A and B). The
asymptomatic maternal grandmother had the same variant, indi-
cating incomplete penetrance, as was also observed for the
*IKZF1*<sup>R183H</sup> GOF variant⁶ and several *IKZF1* LOF/haploinsuffi-
cient variants.⁸ The *IKZF1*<sup>T398M</sup> variant had a Combined
Annotation-Dependent Depletion score of 33, which is consid-
ered highly deleterious, and a very low minor allele frequency
(1 in 152,176 in the Genome Annotation Database and 0 in
34,650 in our institute's exome database). The threonine 398
change to methionine is located in a region that is highly
conserved through species and enriched in threonine and serine
residues known to be phosphorylated (Fig 2, C).<sup>11,12</sup> We thus
considered the possibility that this variant could be responsible
for the immune symptoms in the 2 patients.

Interestingly, the immunologic (ie, decreased Treg cell and
increased T<sub>FH</sub> cell counts) and clinical phenotypes of the patients
appeared to be close to those previously ascribed to patients with

the GOF mutant IKZF1<sup>R183H/C</sup>.<sup>6</sup> In particular, Treg cell defect or
dysfunction could be associated with the development of neuroin-
flammatory or autoimmune disorders, including MS, as recently
described.<sup>13-15</sup> However, it would be interesting to explore popu-
lations of patients with MS for IKZF1 GOF variants. To test
whether impaired IL-2 production and T<sub>H1</sub> cell–T<sub>H2</sub> cell imbal-
ance in patients was the direct consequence of the T398M muta-
tion in IKZF1 (IKZF1<sup>T398M</sup>), IKZF1<sup>T398M</sup> was ectopically
expressed in CD4<sup>+</sup> T cells from healthy controls by using a len-
tiviral vector, and T cells were analyzed for IL-2 and IL-4 produc-
tion after activation (Fig 2, D). IKZF1<sup>T398M</sup> expression CD4<sup>+</sup>
T cells resulted in a decrease in IL-2 production and significant
increase in IL-4 production (compared with wild-type [WT]
IKZF1), similar to that previously reported for IKZF1<sup>R183H/C</sup> vari-
ants<sup>6</sup> (Fig 2, D). Therefore, these observations further support the
idea that IKZF1<sup>T398M</sup> may act as a GOF mutant such as
IKZF1<sup>R183H/C</sup>, although the T398M is not located in the DNA-
binding ZF1 to ZF4 region.

In HEK-293T cells, IKZF1<sup>T398M</sup> had the same nuclear expres-
sion as WT IKZF1 (IKZF1<sup>WT</sup>) and 2 mutants, IKZF1<sup>R183H</sup> and
IKZF1<sup>R162Q</sup>, which were previously reported to behave as GOF
and LOF mutants, respectively<sup>5,8</sup> (data not shown). The ability
of IKZF1<sup>T398M</sup> to dimerize with IKZF1<sup>WT</sup> was also preserved
(Fig 2, E), as was nuclear localization to pericentromeric hetero-
chromatin (PC-HC) regions with foci formation similar to that of
IKZF1<sup>WT</sup> and IKZF1<sup>R183H</sup>, whereas IKZF1<sup>R162Q</sup> did not form
foci as expected and previously shown<sup>5</sup> (Fig 2, F). IKZF1 expres-
sion in T cells of the 2 patients showed no difference from that in
the control or father T cells (data not shown).

IKZF1 undergoes phosphorylation and dephosphorylation by
casein kinase 2 and protein phosphatase 1.<sup>12,16,17</sup> Notably, the
mouse equivalent of threonine 398 has been shown to be phos-
phorylated by casein kinase 2,<sup>11</sup> thus suggesting that its replace-
ment by a methionine could impair IKZF1phosphorylation.
Phosphorylation of IKZF1<sup>T398M</sup> was thus evaluated by immuno-
blotting in HEK-293T cells and compared with phosphorylation
of IKZF1<sup>WT</sup>, IKZF1<sup>R183H</sup> and IKZF1<sup>R162Q</sup> (Fig 2, G). Treatment
with calyculin A, a well-known potent inhibitor of protein phos-
phatase 2A and protein phosphatase 1,<sup>18</sup> led to marked changes in
the molecular weight of IKZF1<sup>WT</sup>, resolving in several bands
shifting from 63 kDa (the expected size of IKZF1) to 75 kDa.
These bands likely correspond to a gradient of phosphorylation
at different sites and/or other posttranslational modifications
following phosphorylation. After immunoprecipitation with
anti-IKZF1 antibody, bands of the same molecular weights
were revealed by using an anti-phospho-serine/threonine anti-
body, including the 63-kDa product. These bands were not de-
tected in lysates from untreated cells, except for a weak 63-kDa
product corresponding to the basal IKZF1<sup>WT</sup> phosphorylation.
Therefore, these data indicate that IKZF1 is markedly phosphor-
ylated subsequent to calyculin A treatment. Under the same con-
ditions, phosphorylation of the IKZF1<sup>T398M</sup> was markedly
diminished when compared with those of IKZF1<sup>WT</sup>, IKZF1<sup>R183H</sup>,
and IKZF1<sup>R162Q</sup> (under conditions both with and without calycu-
lin A). The T398M mutation thus causes impaired phosphoryla-
tion of IKZF1.

Phosphorylation of IKAROS/IKZF1 is dynamically regulated
during the cell cycle.<sup>11,16</sup> IKZF1 is in a dephosphorylated state at
the late G1 phase, becoming highly phosphorylated in the S phase
and with phosphorylation remaining stable during mitosis. Impor-
tantly, dephosphorylation of IKZF1 increased its binding to DNA
<figure><img src="image_5.png" /><figcaption>**FIG 1.** Aberrant T-cell differentiation and subsets in 2 patients with immune dysregulation symptoms. A, Dot plots from flow cytometric analyses of peripheral blood of 1 healthy control (Ctrl) and patients (P1, P2) showing T (CD3⁺CD19⁻) and B (CD3⁻CD19⁺) cells after gating on lymphocytes (top), FOXP3⁺ Treg cells after gating on CD3⁺CD4⁺ cells (middle up), CXCR5⁺PD-1⁺ T<sub>FH</sub> cells after gating on CD3⁺CD4⁺CD45RA⁻ cells (middle down) and CCR4⁺CXCR3⁻ T<sub>H2</sub> cells or CCR4⁻CXCR3⁺ T<sub>H1</sub> cells after gating on CD3⁺CD4⁺CD45RA⁻CXCR5⁻CCR6⁻ cells corresponding to CD4⁺ memory T cells (bottom). Numbers correspond to percentage of cells in the gates. B and C, Histograms from flow cytometric analyses of intracellular IL-2 (B) and IFN-γ and IL-4 (C) production by T-cell blasts after overnight stimulation with PMA and ionomycin of controls (Ctrl1, Ctrl2), P1 and P2. Numbers correspond to percentage of positive cells. D, Histograms from flow cytometric analyses of cell trace violet dye (CTV) dilutions showing T-cell proliferation from PBMCs of 1 Ctrl (the father), P1, and P2 in response to anti-CD3, anti-CD3/CD28 beads or phytohaemagglutinin (PHA).</figcaption></figure>

and its ability to restrict cell cycle progression. Hence, the impact
of phosphorylation and dephosphorylation on the ability of human
IKZF1<sup>WT</sup> and IKZF1<sup>T398M</sup> to bind to DNA was evaluated during
the cell cycle at the G1 phase or at the S phase by electrophoretic
mobility shift assay with the IKZF1 consensus-binding sequence
4 (IK-bs4) as a DNA probe (Fig 3, A and B). The GOF mutant
IKZF1<sup>R183H</sup> and additional mutants mimicking dephosphorylation
(IKZF1<sup>T398A</sup>) or phosphorylation (IKZF1<sup>T398D</sup>) were also assessed
in the same assay. As previously reported, the DNA-binding capac-
ity of IKZF1 (all mutants) is higher during the G1 phase or under
unsynchronized conditions versus during the S phase or after caly-
culin A treatment (although protein levels do not change).<sup>11,16</sup> One
plausible explanation is that during the phase S an important
portion of IKZF1 proteins are associated with the host DNA
decreasing the amount of “free” IKZF1 proteins available to

bind to the probe for electrophoretic mobility shift assay (whereas
the global expression of IKZF1 [“free and “nonfree”] is similar be-
tween the different conditions).

However, under unsynchronized conditions, more intense
shifts were observed with IKZF1<sup>T398M</sup>, IKZF1<sup>T398A</sup>, and
IKZF1<sup>R183H</sup> than with IKZF1<sup>WT</sup> and IKZF1<sup>T398D</sup>. This increased
shift intensity resulted from increased the DNA-binding ability of
IKZF1<sup>T398M</sup> and IKZF1<sup>T398A</sup> at the S phase, whereas the level of
IKZF1<sup>R183H</sup> binding to DNA was higher at the G1 phase. Further-
more, the promoter activity of HES1, a known target of IKZF1
that is more highly repressed at the S phase than at the G1 phase
(Fig 3, C), was relatively more repressed by IKZF1<sup>T398M</sup> and
IKZF1<sup>T398A</sup> during the S phase, whereas relative repression by
IKZF1<sup>R183H</sup> occurred mostly at the G1 phase (Fig 3,
D). However, IKZF1<sup>T398D</sup> (mimicking phosphorylation) showed
<table><caption>TABLE I. Immunologic features of the 2 patients</caption><thead><tr><th rowspan="2">Immunologic features</th><th colspan="2">Patient</th><th rowspan="2">Normal values</th></tr><tr><th>P1</th><th>P2</th></tr></thead><tbody><tr><td>T-cell lineage (%)</td><td></td><td></td><td></td></tr><tr><td>T cells (CD3<sup>+</sup>/lymphocytes)</td><td>77.6 (1280)</td><td>77.8</td><td>62.4-87.2 (1200-2600)</td></tr><tr><td>CD4/CD8 cell ratio</td><td>2.1</td><td><strong>4.3*</strong></td><td>1.0-2.8</td></tr><tr><td>CD4 T cells (CD4<sup>+</sup>/CD3<sup>+</sup>)</td><td>58.1 (744)</td><td><strong>77.5*</strong></td><td>50.0-69.8 (650-1500)</td></tr><tr><td>CD4 T cells, naive (CD45RA<sup>+</sup>CCR7<sup>+</sup>/CD3<sup>+</sup>CD4<sup>+</sup>)</td><td>58.0</td><td>69.1</td><td>16.2-80.2</td></tr><tr><td>CD4 T cells, T<sub>CM</sub> (CD45RA<sup>-</sup>CCR7<sup>+</sup>/CD3<sup>+</sup>CD4<sup>+</sup>)</td><td>29.7</td><td>22.8</td><td>22.1-65.3</td></tr><tr><td>CD4 T cells, T<sub>EM</sub> (CD45RA<sup>-</sup>CCR7<sup>-</sup>/CD3<sup>+</sup>CD4<sup>+</sup>)</td><td>9.7</td><td>6.3</td><td>4.9-33.2</td></tr><tr><td>CD4 T cells, T<sub>EMRA</sub> (CD45RA<sup>+</sup>CCR7<sup>-</sup>/CD3<sup>+</sup>CD4<sup>+</sup>)</td><td>2.7</td><td>1.8</td><td>0.0-3.2</td></tr><tr><td>CD8 T cells (CD8<sup>+</sup>/CD3<sup>+</sup>)</td><td>27.8 (356)</td><td><strong>17.9†</strong></td><td>24.4-42.8 (370-1100)</td></tr><tr><td>CD8 T cells, naive (CD45RA<sup>+</sup>CCR7<sup>+</sup>/CD3<sup>+</sup>CD8<sup>+</sup>)</td><td>74.7</td><td>55.4</td><td>24.8-85.5</td></tr><tr><td>CD8 T cells, T<sub>CM</sub> (CD45RA<sup>-</sup>CCR7<sup>+</sup>/CD3<sup>+</sup>CD8<sup>+</sup>)</td><td><strong>4.1†</strong></td><td>7.8</td><td>6.5-44.8</td></tr><tr><td>CD8 T cells, T<sub>EM</sub> (CD45RA<sup>-</sup>CCR7<sup>-</sup>/CD3<sup>+</sup>CD8<sup>+</sup>)</td><td>13.0</td><td>17.8</td><td>4.8-40.0</td></tr><tr><td>CD8 T cells, T<sub>EMRA</sub> (CD45RA<sup>+</sup>CCR7<sup>-</sup>/CD3<sup>+</sup>CD8<sup>+</sup>)</td><td>8.2</td><td>19.0</td><td>3.0-33.9</td></tr><tr><td>Follicular T<sub>H</sub> cells (CD45RA<sup>-</sup>CXCR5<sup>+</sup>/CD3<sup>+</sup>CD4<sup>+</sup>)</td><td><strong>13.7*</strong></td><td><strong>11.8*</strong></td><td>1.7-10.5</td></tr><tr><td>Regulatory T cells (FOXP3<sup>+</sup>CD25<sup>+</sup>/CD3<sup>+</sup>CD4<sup>+</sup>)</td><td><strong>2.9†</strong></td><td><strong>2.9†</strong></td><td>3.1-9.2</td></tr><tr><td>B-cell lineage (%)</td><td></td><td></td><td></td></tr><tr><td>B cells (CD19<sup>+</sup>/lymphocytes)</td><td>8.8 (<strong>145†</strong>)</td><td>5.4</td><td>6.1-18.7 (270-640)</td></tr><tr><td>Naive B cells (CD27<sup>-</sup>IgD<sup>+</sup>/CD19<sup>+</sup>)</td><td>77.1</td><td>51.3</td><td>43.3-91.5</td></tr><tr><td>Non-switched memory B cells (CD27<sup>+</sup>IgD<sup>+</sup>/CD19<sup>+</sup>)</td><td>3.8</td><td>9.9</td><td>2.7-20.2</td></tr><tr><td>Switched memory B cells (CD27<sup>+</sup>IgD<sup>-</sup>/CD19<sup>+</sup>)</td><td>5.5</td><td>21.6</td><td>3.3-30.1</td></tr><tr><td>Exhausted B cells (CD21<sup>-</sup>CD38<sup>-</sup>/CD19<sup>+</sup>)</td><td>11.1</td><td>12.1</td><td></td></tr><tr><td>NK cell lineage (%)</td><td></td><td></td><td></td></tr><tr><td>NK cells (CD3<sup>-</sup>CD16<sup>+</sup>CD56<sup>+</sup>/lymphocytes)</td><td>8.8 (145)</td><td>11.4</td><td>1.3-17.5 (100-680)</td></tr><tr><td>Immunoglobulin level</td><td></td><td></td><td></td></tr><tr><td>IgG (mg/dL)</td><td>717</td><td>ND</td><td>700-1,600</td></tr><tr><td>IgA (mg/dL)</td><td>100</td><td>ND</td><td>70-400</td></tr><tr><td>IgM (mg/dL)</td><td>116</td><td>ND</td><td>40-230</td></tr><tr><td>IgE (IU/mL)</td><td>88</td><td>ND</td><td>&lt;173</td></tr><tr><td>IgG1 (mg/dL)</td><td>515</td><td>ND</td><td>351-962</td></tr><tr><td>IgG2 (mg/dL)</td><td>325</td><td>ND</td><td>239-838</td></tr><tr><td>IgG3 (mg/dL)</td><td>26</td><td>ND</td><td>8.5-140</td></tr><tr><td>IgG4 (mg/dL)</td><td>25</td><td>ND</td><td>4.5-117</td></tr></tbody></table>

Values above or below reference ranges are marked by asterisks or daggers, respectively. Values in parentheses are absolute numbers.

ND, Not determined; NK, natural killer; T<sub>CM</sub>, central memory T; T<sub>EM</sub>, effector memory T; T<sub>EMRA</sub>, terminally differentiated RA<sup>+</sup> T.

patterns similar to those in IKZF1<sup>WT</sup> (Fig 3, A-D). Therefore, IKZF1<sup>T398M</sup> and IKZF1<sup>T398A</sup> behave as GOF variants as the result of increased or residual ability to bind to DNA during the S phase due to impaired phosphorylation. Of note, no decreased DNA binding of IKZF1<sup>T398D</sup> (irrespective of the cell cycle) was observed compared with that of IKZF1<sup>WT</sup>, suggesting that T398D did not behave as a full phosphomimetic mutation.

Interestingly, phosphorylation of IKZF1 in mice was also shown to regulate IKZF1 expression through the ubiquitin proteasome pathway.¹⁷ Hence, we examined protein stability of IKZF1<sup>T398M</sup> in HEK-293T cells after or not after treatment with cycloheximide, a protein synthesis inhibitor (Fig 4, A and B). Compared with IKZF1<sup>WT</sup>, IKZF1<sup>T398D</sup>, IKZF1<sup>R183H</sup>, and IKZF1<sup>R162Q</sup> proteins, IKZF1<sup>T398M</sup> and IKZF1<sup>T398A</sup> proteins were less expressed under the condition without treatment, likely caused by impaired phosphorylation. Treatment with cycloheximide led to significantly higher relative protein stability of IKZF1<sup>T398M</sup> and IKZF1<sup>T398A</sup>, supporting the idea that those 2 mutants are more stable and/or less subject to degradation. As a control of degradation, calyculin A, which induced a strong phosphorylation, caused fast degradation of IKZF1<sup>WT</sup> (Fig 4, A). Therefore, these data suggest that IKZF1<sup>T398M</sup> is associated with increased protein stability, which may contribute to its

GOF effect. However, in primary resting (noncycling) T cells from the 2 patients, IKZF1 protein level was found to be comparable to that in the control T cells (data not shown).

Lastly, the effect of IKZF1<sup>T398M</sup> on the cell cycle was examined in HEK-293T cells (Fig 4, C and D). Consistent with the findings of mouse studies, expression of IKZF1<sup>WT</sup> had a tendency to increase the percentage of HEK cells in the G1 phase, whereas the percentage of cells in the S phase was decreased versus that with HEK cells transfected with an empty vector. The proportions of cells in the G1 phase were even more significantly enhanced in cells expressing IKZF1<sup>T398M</sup>, IKZF1<sup>T398A</sup>, and IKZF1<sup>R183H</sup> to the detriment of cells in the S phase, which were even fewer. In contrast, IKZF1<sup>R162Q</sup> expression resulted in a slight but significant accumulation of cells in the S phase. Expression of IKZF1<sup>T398D</sup> had an effect on the cell cycle that was quite similar to that of expression of IKZF1<sup>WT</sup>, which was as expected for a mutation (T398D) that mimics phosphorylation. Enhanced G1-phase cell cycle arrest by IKZF1<sup>T398M</sup> and IKZF1<sup>R183H</sup> was confirmed by their greater ability to suppress cell proliferation than that of IKZF1<sup>WT</sup> (Fig 4, E). This suppression was consistent with the decreased T-cell proliferation observed in patient 1 and patient 2 (Fig 1, D). Therefore, these data confirm that IKZF1<sup>T398M</sup> and IKZF1<sup>R183H</sup> behave as GOF variants by virtue of their enhanced capacity to inhibit the entry in the S phase.
<figure><img src="image_6.png" /><figcaption>**FIG 2.** Identification of a heterozygous *IKZF1*<sup>T384M</sup> variant in 2 patients leading to impaired IKZF1 phosphorylation. **A**, Pedigree of the family. **B**, IKZF1 structure with serine (S)/threonine (T) sites. ZFs (gray boxes) with T398M, R162Q, and R183H haploinsufficient (HI) and GOF variants. **C**, IKZF1 alignments from different species surrounding the T398 (*red*). Asterisks indicate phosphorylation sites. **D**, Graph bars (means ± SDs) from fluorescence-activated cell sorting data of frequencies of IL-2, IFN-γ, and IL-4-positive CD4<sup>+</sup> naive control T cells transduced with IKZF1<sup>WT</sup> or IKZF1<sup>T398M</sup>, or an empty vector (n = 3) stimulated with PMA and ionomycin. Two-tailed paired *t* tests, \* *P* < .05. **E**, Coimmunoprecipitation (IP) of FLAG-tagged IKAROS/IKZF1<sup>WT</sup> and HA-tagged IKZF1<sup>WT</sup>, T398M, R183H or R162Q expressed in HEK-293T cells with anti-FLAG (IP: FLAG) or control mouse IgG (IP: IgG). Upper immunoblots with anti-HA. Middle and lower immunoblots of whole cell lysates (WCL) with anti-FLAG and anti-HA respectively. **F**, IKZF1 localization in NIH-3T3 cells expressing same mutants as in (*E*). Staining with anti-IKZF1 (*red*) and 4',6-diamino-2-phenylindole (DAPI [*blue*]). Scale bars = 10 µm. **G**, Upper anti-phospho-serine/threonine (anti-P-Ser/Thr) immunoblots of immunoprecipitations (IP) with anti-IKZF1/IKAROS of HEK-293T cell lysates expressing same mutants as in (*E*), or not (empty vector) after treatment or not with calyculin A. Middle and lower anti-IKZF1, anti-P-Ser/Thr, and anti-ACTIN immunoblots of WCL. Arrow, arrowheads and asterisk on the right indicate phosphorylated forms of IKZF1 with the expected size at 63 kDa (arrow), unexpected sizes (arrowheads), and a nonspecific band (asterisk).</figcaption></figure>

Presumably, this effect is directly dependent on the increased DNA-
binding capacity of these mutants during the G1-S phase transition as
IKZF1 function is enhanced following its dephosphorylation.
Enhanced regulation of the G1-S phase transition by IKZF1<sup>T398M</sup>
and IKZF1<sup>R183H</sup> may contribute to the immune dysregulation effects
in the patients. Cell cycle arrest is well known to be coupled in many
tissues with terminal differentiation, including T<sub>H</sub> cell differentiation.
T<sub>H</sub>2 cell differentiation associated with IL-4 expression requires a
limited number of cell cycles and divisions, whereas T<sub>H</sub>1 cell differ-
entiation associated with IFN-&gamma; expression is more progressive,
requiring successive cell cycles.<sup>19</sup> This is consistent with the increase
in cell cycle arrest and skewing toward T<sub>H</sub>2 cell differentiation by
IKZF1<sup>T398M</sup> (in this study) and IKZF1<sup>R183H/C</sup> mutants.<sup>6</sup> Increased

cell cycle arrest may allow or favor silencing of T-bet and IFN-γ
gene expression by IKZF1, as shown in T<sub>H</sub>2 cell differentiation in
mice.<sup>20</sup>

The present study demonstrates that IKZF1 T398M is a novel
GOF variant associated with impaired phosphorylation, leading
to stronger DNA binding at the S phase and G1-S phase transition
arrest. The clinical manifestations and lymphocyte phenotype of
the patients with IKZF1<sup>T398M</sup> seem to partially overlap with those
of patients with IKZF1<sup>R183H/C,<sup>6</sup></sup> although the extent to which they
differ from those of patients with IKZF1<sup>R183H/C</sup> is difficult to say.
As for patients with IKZF1<sup>R183H/C,<sup>6</sup></sup> the immune dysregulation
symptoms are very different from one patient to another, with pa-
tient 1 having developed an Evans syndrome whereas his mother
<figure><img src="image_7.png" /><figcaption>**FIG 3.** IKZF1<sup>T398M</sup> behaves as a GOF mutation. **A**, Immunoblot of IKZF1/IKAROS expression (upper panel) and electrophoretic mobility shift assay (EMSA) with a IK Bs4 probe (middle panel, black and white image; lower panel, pseudocolor image). Nuclear extracts were prepared from HEK-293T cells transiently expressing IKZF1<sup>WT</sup>, IKZF1<sup>T398M</sup>, IKZF1<sup>T398D</sup>, IKZF1<sup>T398A</sup>, IKZF1<sup>R183H</sup> (IKZF1<sup>GOF</sup>) or IKZF1<sup>R162Q</sup> (IKZF1<sup>H1</sup>). Cells were unsynchronized or synchronized at the G1 or S phases. Unsynchronized (US) HEK-293T cells expressing IKZF1<sup>WT</sup> were also treated with calyculin A (+calyc. A). **B**, Graph bars of EMSA quantifications (n = 4). Relative intensity with IKZF1<sup>WT</sup> without synchronization was defined as 1. **C** and **D**, Luciferase reporter assays using a HES1 promoter construct expressed in HEK-293T and cells unsynchronized or synchronized as in (A), (n = 4). **C**, Relative luciferase activity under the unsynchronized (unsynchr.) conditions defined as 1. **D**, HEK-293T cells were cotransfected with an empty vector, IKZF1<sup>WT</sup> or the different mutants of IKZF1 as indicated in (A). Relative luciferase activity with the empty vector was defined as 1. **B** and **C**, bars are presented as means ± SDs. Comparison between groups were analyzed with 1-way ANOVA with the Dunnett *post hoc* multiple-comparisons test. \*P < .05; \*\*P < .01; \*\*\*P < .001.</figcaption></figure>

(patient 2) has MS. Additional genetic and/or environmental factors, including incomplete penetrance, likely explain this variability. Incomplete penetrance together with large clinical spectrum is a common feature of many autosomal dominant diseases. Identification of additional patients could help to better define the clinical phenotypes associated with IKZF1 T398M. However, to mechanistically distinguish the IKZF1<sup>T398M</sup> mutant from “classical” IKZF1<sup>R183H/C</sup> GOF mutants (that are within the ZF3 of DNA binding site), we propose that it be referred to as “GOF by loss-of-phosphorylation” (termed as GOF-LOP).

Whereas IKZF1 is phosphorylated at multiple sites<sup>11,12</sup> (see Fig 1, B), the T398M variant resulted in a marked decrease in phosphorylation, suggesting that pan-phosphorylation of other serine/threonine residues depends on T398 phosphorylation. Because the threonine 398 is outside the DNA binding site (encompassing ZF1-ZF4), its inhibitory effect on DNA binding when phosphorylated is indirect. Phosphorylation of residues in

the region from 383 to 404 has been previously proposed to result in conformational changes reducing the accessibility of the DNA binding.<sup>11</sup> In conclusion, our observations identify a new mechanism leading to the GOF of IKZF1 and the first evidence that IKZF1 function is regulated by post-translational modifications in humans, leading to disease when impeded.

**Data and reagent availability statement**

The data sets and reagents generated during the current study are available from the corresponding author on reasonable request.

**DISCLOSURE STATEMENT**

Supported by the Agence Nationale de la Recherche, France (to A.H. and D.B.); the Ligue Contre le Cancer-Équipe Labélisée,
<figure><img src="image_8.png" /><figcaption>**FIG 4.** IKZF1<sup>T398M</sup> is associated with increased protein stability and G1-S phase transition arrest. **A-E**, All experiments from HEK-293T cells that transiently expressed IKZF1, IKZF1<sup>WT</sup>, IKZF1<sup>T398M</sup>, IKZF1<sup>T398D</sup>, IKZF1<sup>T398A</sup>, IKZF1<sup>R183H</sup>, IKZF1<sup>R162Q</sup>, or transfected with an empty vector. **A**, Immunoblots of IKZF1 (upper panel) and ACTIN expression (lower panel) as a loading control. Cells after treatment with (+) and without (-) cycloheximide for 48 hours. Cells expressing IKZF1<sup>WT</sup> were also treated with calyculin A (+calyculin A). Cell lysates prepared from sorted and expanded mCherry<sup>+</sup> cells. **B**, Graph bars of relative IKZF1 protein stability (n = 3). IKAROS/IKZF1 expression was normalized with ACTIN expression, and ratios of normalized IKZF1 expression with and without treatment of cycloheximide are shown. **C** and **D**, Dot plots (**C**) and graph bars (**D**) from flow cytometry data of cell cycle analysis (n = 4). Cells expressing 5-ethynyl-2'-deoxyuridine (EdU) stained by 4',6-diamino-2-phenylindole (DAPI) for DNA content. Cells were gated on mCherry<sup>+</sup> cells. **E**, Graph bars of proliferation assays by IncuCyte measurements (n = 3) of mCherry<sup>+</sup> cells. **B**, **D**, and **E**, Error bars represent means ± SDs. One-way ANOVA with the Dunnett *post hoc* multiple-comparisons test. \*P < .05; \*\*P < .01; \*\*\*P < .001.</figcaption></figure>

France [to S. L.]); the Institut National de la Santé et de la Recherche Médicale, France, the Agence Nationale de Recherche, France (grant ANR-18-CE15-0025-01 [to S.L.] and grant ANR-10-IAHU-01 [to Institut Imagine]); the Institut National du Cancer, France (grant PLBIO-2020 and grant PEDIAC INCa_15670 (both to S.L.)), the Société Francaise de Lutte contre les Cancers et Leucémies de l'Enfant et de l'Adolescent, AREMIG, France (to S.L.); and the Fédération Enfants et Santé, France (to S.L.). Exome sequencing was funded by the Rare Diseases Foundation, France (to S.L.).

Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

We acknowledge the patients and their family for their contribution and the healthy donors for blood gifts. S. L. is a senior scientist at the Centre National de la Recherche Scientifique-CNRS (France).

Key messages

* A mutation in *IKZF1* causing impaired phosphorylation (IKZF1<sup>T398M</sup>) behaving as a GOF mutation was identified in 2 patients with immune dysregulation symptoms.

* GOF by loss of phosphorylation (termed as GOF-LOP) in IKZF1 extends the genotype and phenotype spectrum of IKZF1-associated immune diseases.

REFERENCES

1. Cortes M, Wong E, Koipally J, Georgopoulos K. Control of lymphocyte development by the Ikaros gene family. Curr Opin Immunol 1999;11:167-71.

2. Heizmann B, Kastner P, Chan S. The Ikaros family in lymphocyte development. Curr Opin Immunol 2018;51:14-23.
3. Georgopoulos K, Bigby M, Wang JH, Molnar A, Wu P, Winandy S, et al. The Ikaros gene is required for the development of all lymphoid lineages. Cell 1994; 79:143-56.

4. Georgopoulos K. Haematopoietic cell-fate decisions, chromatin regulation and ikaros. Nat Rev Immunol 2002;2:162-74.

5. Boutboul D, Kuehn HS, Van de Wyngaert Z, Niemela JE, Callebaut I, Stoddard J, et al. Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency. J Clin Invest 2018;128:3071-87.

6. Hoshino A, Boutboul D, Zhang Y, Kuehn HS, Hadjadj J, Ozdemir N, et al. Gain-of-function IKZF1 variants in humans cause immune dysregulation associated with abnormal T/B cell late differentiation. Sci Immunol 2022;7:eabi7160.

7. Hoshino A, Okada S, Yoshida K, Nishida N, Okuno Y, Ueno H, et al. Abnormal hematopoiesis and autoimmunity in human subjects with germline IKZF1 mutations. J Allergy Clin Immunol 2017;140:223-31.

8. Kuehn HS, Boisson B, Cunningham-Rundles C, Reichenbach J, Stray-Pedersen A, Gelfand EW, et al. Loss of B cells in patients with heterozygous mutations in IKAROS. N Engl J Med 2016;374:1032-43.

9. Kuehn HS, Niemela JE, Stoddard J, Mannurita SC, Shahin T, Goel S, et al. Germ-line IKAROS dimerization haploinsufficiency causes hematologic cytopenias and malignancies. Blood 2021;137:349-63.

10. Yoshida N, Sakaguchi H, Muramatsu H, Okuno Y, Song C, Dovat S, et al. Germ-line IKAROS mutation associated with primary immunodeficiency that progressed to T-cell acute lymphoblastic leukemia. Leukemia 2017;31:1221-3.

11. Gomez-del Arco P, Maki K, Georgopoulos K. Phosphorylation controls Ikaros's ability to negatively regulate the G(1)-S transition. Mol Cell Biol 2004;24:2797-807.

12. Gurel Z, Ronni T, Ho S, Kuchar J, Payne KJ, Turk CW, et al. Recruitment of ikaros to pericentromeric heterochromatin is regulated by phosphorylation. J Biol Chem 2008;283:8291-300.

13. Schindler MK, Pittaluga S, Enose-Akahata Y, Su HC, Rao VK, Rump A, et al. Haploinsufficiency of immune checkpoint receptor CTLA4 induces a distinct neu- roinflammatory disorder. J Clin Invest 2020;130:5551-61.

14. Mangott TC, Vanden Driessche K, Norga KK, Moes N, De Bruyne M, Haerynck F, et al. Central nervous system manifestations of LRBA deficiency: case report of two siblings and literature review. BMC Pediatr 2023;23:353.

15. Consonni F, Ciullini Mannurita S, Gambineri E. Atypical Presentations of IPEX: expect the unexpected. Front Pediatr 2021;9:643094.

16. Li Z, Song C, Ouyang H, Lai L, Payne KJ, Dovat S. Cell cycle-specific function of Ikaros in human leukemia. Pediatr Blood Cancer 2012;59:69-76.

17. Popescu M, Gurel Z, Ronni T, Song C, Hung KY, Payne KJ, et al. Ikaros stability and pericentromeric localization are regulated by protein phosphatase 1. J Biol Chem 2009;284:13869-80.

18. Favre B, Turowski P, Hemmings BA. Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycin. J Biol Chem 1997;272:13856-63.

19. Bird JJ, Brown DR, Mullen AC, Moskowitz NH, Mahowald MA, Sider JR, et al. Helper T cell differentiation is controlled by the cell cycle. Immunity 1998;9:229-37.

20. Thomas RM, Chen C, Chunder N, Ma L, Taylor J, Pearce EJ, et al. Ikaros silences T-bet expression and interferon-gamma production during T helper 2 differentiation. J Biol Chem 2010;285:2545-53.
# METHODS

## Study approval

This study was conducted in accordance with the Declaration of Helsinki and local legislation and ethical guidelines and was approved by the Comité de Protection des Personnes de l'Ile de France II and the French advisory committee. Informed consent was obtained from the patients and parents.

## Clinical history of the patients

Patient 1 is a 10-year-old boy who was diagnosed with Evans syndrome following ENT infection at the age of 2 years. He presented with immune thrombocytopenia, autoimmune neutropenia, a positive direct antiglobulin test result (without evidence of hemolysis), and nodal lymphoproliferation with axillary and inguinal enlarged lymph nodes (40-mm-diameter). No biopsy was performed. The bone marrow aspiration findings were normal. Between the ages of 2 and 4 years, his blood cell counts were normal. At age 4 years, thrombocytopenia was again observed following ENT infection. Bone marrow aspiration showed abundant cells of the megakaryocytic lineage within a rich bone marrow. Antinuclear antibodies and antineutrophil cytoplasm antibodies were not detected. The results of a direct antiglobulin test and detection of antiplatelet IgG antibody were positive. The result of testing for blood EBV viral load was negative. The patient subsequently presented with several episodes of severe thrombocytopenia and neutropenia, mostly at the time of infectious episodes, requiring corticosteroids and intravenous immunoglobulins, which led to prolonged partial remission of the cytopenias. There was no hemorrhagic syndrome. Weight and height growth were normal. The patient's medical history was also positive for chronic crusted lesions on the buttocks and legs, consistent with staphylococcal skin infection or atopic dermatitis folliculitis. He had also intermittent oral aphthous ulcers. Lymphoproliferation with enlarged lymph nodes and direct antiglobulin test positivity persisted, with a follow-up of 6 years. Three café-au-lait macules were noted on subsequent examinations. His mother (patient 2) had a history of MS diagnosed at age 33 years. No other disease was reported in patient 2.

## Genetic analysis

For whole exome sequencing, exome capture was carried out by using an exome enrichment kit (TruSeq [Illumina, San Diego, Calif]), and massively parallel sequencing was performed by using a HiSeq2500 sequencing system (Illumina) with 100-bp paired-end reads. Data were processed with an in-house-constructed analysis pipeline, which aligned the reads using BWA31, counted variant allele numbers using SAMtools, and annotated genes using ANNOVAR33, as previously described. E1 Sanger sequencing was performed to validate the results.

## Plasmid constructs and transfection

The following IKZF1 expression vectors were generated, as described elsewhere: pcDNA3.1 (Invitrogen, Waltham, Mass), pCMV3-HA, pFLAG-CMV2 (OriGene, Rockville, Md), and pLVX-eF1a-IRES-mCherry (Clontech, Mountain View, Calif) containing IKZF1 WT and mutated cDNAs (for IKZF1<sup>R162Q</sup> and IKZF1<sup>R183H</sup>), E1,E2 cDNAs for IKZF1<sup>T398M</sup>, IKZF1<sup>T398D</sup> and IKZF1<sup>T398A</sup> were obtained by mutagenesis using the Q5 Site-

Directed Mutagenesis Kit (NewEngland BioLabs, Ipswich, Mass) and verified by sequencing. The following oligos were used: the forward primer 5'-CAAGACTCCATGGACACCG AGAG-3' and reverse primer 5'-GCAGCTGTTGCTCGGGGA-3' for IKZF1<sup>T398M</sup>, the forward primer 5'-CCAAGACTCCGCG GACACCGA-3' and reverse primer 5'-CAGCTGTTGCTCGGG GAC-3' for IKZF1<sup>T398A</sup>, and the forward primer 5'-CCAA- GACTCCGACGACACCGAGAGCAACAACG -3' and reverse primer 5'-CAGCTGTTGCTCGGGGAC-3' for IKZF1<sup>T398D</sup>. For transfection into HEK-293T or NIH-3T3 cells, lipofectamine 2000 (Thermo Fisher Scientific, Waltham, Mass) was used. For transfection of T cells, viral supernatants were produced in HEK-293T cells from pLVX-eF1a-IRES-mCherry plasmids. T cells were infected at day 1 of anti-CD3/CD28 bead stimulation with viral particles as reported before. E2

## Immunoblotting, immunoprecipitation, and confocal microscopic analysis

Immunoblotting, immunoprecipitation, and confocal microscopic analysis were performed as previously described. E2 For immunoblotting, the following antibodies were used: anti-IKZF1 (ab191394 [Abcam, Waltham, Mass]), anti-phosphoserine/threonine (ab117253, Abcam), anti-ACTIN (A2066 [Sigma-Aldrich, Burlington, Mass]), anti-GAPDH (2118 [Cell Signaling Technology, Danvers, Mass]), antiproliferating cell nuclear antigen (PCNA, New Kensington, Pa) (2586, Cell Signaling Technology), anti-FLAG antibody (F3165, Sigma-Aldrich), and antihemagglutinin antibody (H6908, Sigma-Aldrich). For degradation and stability protein assay, cells were treated with cycloheximide (40 µg/mL [Sigma-Aldrich]). Calyculin A (10 nM [Sigma-Aldrich]) was used to inhibit the protein phosphatases PP1 and PP2A. Cell lysates were prepared, and immunoprecipitations were carried out under conditions previously reported. E3 For immunoprecipitation experiments, Protein G Sepharose 4 Fast Flow (GE Healthcare, Chicago, Ill) and mouse anti-FLAG antibody (catalog no. F3165 [Sigma-Aldrich]) or mouse anti-IKZF1 antibody (MABE912 [Merck Millipore, Burlington, Mass]) were used. For confocal microscopic analyses, cells were stained with anti-IKZF1 antibody (ab191394 [Abcam]) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Invitrogen). Images were acquired with a Leica SP8 STED confocal microscope (Leica Microsystems, Wetzlar, Germany).

## Electrophoresis mobility shift assay (EMSA) and luciferase reporter assay

EMSA and luciferase reporter assay were performed as previously described. E2 For EMSA, a DY682 infrared dye-labeled IKbs4 probe (forward primer 5'-TGACAGGGAATACACATTC CCAAAAGC-3' and reverse primer 5'-GCTTTTGGGAATGTGTATTCCCTGTCA-3') was used. DNA-protein complexes were analyzed by using an Odyssey CLx infrared scanner (LI-COR, Lincoln, Neb). Anti-IKZF1 antibody (ab191394 [Abcam]) was used for the EMSA supershift assay. For luciferase reporter assay, pGL3 firefly luciferase reporter vector driven by the HES1 (–872 to +299 bp) promoter was used. Luciferase activity was measured by using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wis) and a multilabel reader (VICTO X4 [PerkinElmer, Waltham, Mass]).
## Cell cycle analysis, synchronization, and proliferation

For cell cycle analysis, HEK-293T cells were transfected with pLVX-IKZF1-IRES-mCherry. Cells were stained with a Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen) and 4',6-diamidino-2-phenylindole. Stained cells were analyzed by means of flow cytometry with gating on mCherry-positive cells. For synchronization, cells were treated with hydroxyurea (1 mM [Sigma-Aldrich]) for 24 hours to arrest cells in the G1 phase and released for 6 hours to accumulate cells in the S phase. Proliferation of transfected HEK-293T cells was measured using IncuCyte ZOOM (Essen BioScience, Ann Arbor, Mich).

Briefly, mCherry-positive cells were sorted and plated. Cell confluence was measured every 3 hours. Direct cell proliferation of PBMCs was monitored by first labeling the cells with the Cell Trace Violet dye (Violet Proliferation Dye 450 [BD Biosciences, Franklin Lakes, NJ]) and cells were cultured respectively for 6 days with coated anti-CD3 antibody (clone OKT3 functional grade [2 µg/mL] [eBiosciences, San Diego, Calif]) or anti-CD3⁺CD28-coated beads (Invitrogen) or PHA at final dilution of 1/400 (Thermofisher France).

## Flow cytometric analysis

For lymphocyte phenotyping, the following antibodies were used to stain the cell surface: anti-CD3 (SK7), anti-CD4 (RPA-T4), anti-CD8 (RPA-T8), anti-CD19 (SJ25C1), anti-CD27 (LG.3A10), anti-CD38 (HIT2), and anti-CD197/CCR7 (3D12) (BD Biosciences); anti-CD21 (BL13), anti-CD45RA (ALB11), and anti-IgD (IA6-2) (Beckman Coulter, Brea, Calif); anti-CD25 (BC96), and anti-CD56 (HCD56) (Sony Biotechnology, San Jose, Calif); anti-CD45RO (UCHL1) (Dako, Glostrup, Denmark); anti-CD16 (3G8), anti-CD183/CXCR3 (G025H7), anti-CD185/CXCR5 (J252D4), anti-CD196/CCR6 (G034E3), and CD279/PD-1 (EH12.2H7) (BioLegend, San Diego, Calif); anti-CD38 (IB6) (Miltenyi Biotec, Bergisch Gladbach); and anti-CD194/CCR4 (205410) (R&D Systems, Minneapolis, Minn). For intracellular staining, cells were fixed, permeabilized, and stained

with the following antibodies: anti-FOXP3 (259D/C7) (BD Pharmingen, Franklin Lakes, NJ); anti-IL-2 (MQ1-17H12), and anti-IFN-γ (4S.B3) (BioLegend); and anti-IL-4 (8D4-8) (eBioScience). Stained cells were analyzed with BD LSRII Fortessa (BD Biosciences), and data were processed with FlowJo software (Tree Star Inc, Ashland, Ore).

## Lentiviral gene transfer

Purified CD4⁺ naive T or HEK-293T cells were transduced with pLVX-eF1a-IRES-mCherry encoding IKZF1 by lentivirus-mediated gene transfer as previously described.⁵² All cell lines were verified after transduction and found to express similar levels of IKZF1 or mCherry expression. For cytokine expression assay, mCherry-positive cells were sorted 3 days after infection and cultured with anti-CD3 mAb-coated plates for 7 days. Cells were restimulated with PMA (100 ng/mL [Sigma-Aldrich]) and ionomycin (1 µg/mL [Sigma-Aldrich]) overnight in the presence of brefeldin A and monensin.

## Statistics

All values were presented as means plus or minus SDs. Comparisons between groups were analyzed with 2-tailed paired *t* tests and 1-way ANOVA with the Dunnett *post hoc* multiple comparisons test or Holm-Šidak multiple comparisons test using GraphPad Prism 8 software (GraphPad Software Inc, San Diego, Calif). Differences were considered significant when *P* values were less than .05.

## REFERENCES

E1. Boutboul D, Kuehn HS, Van de Wyngaert Z, Niemela JE, Callebaut I, Stoddard J, et al. Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency. J Clin Invest 2018;128:3071-87.

E2. Hoshino A, Boutboul D, Zhang Y, Kuehn HS, Hadjadj J, Ozdemir N, et al. Gain-of-function IKZF1 variants in humans cause immune dysregulation associated with abnormal T/B cell late differentiation. Sci Immunol 2022;7:eabi7160.

E3. Rodriguez R, Fournier B, Cordeiro DJ, Winter S, Izawa K, Martin E, et al. Concomitant PIK3CD and TNFRSF9 deficiencies cause chronic active Epstein Barr virus infection of T cells. J Exp Med 2019;216:2800-18.