Introduction
Identification of the genetic causes of human immunodeficiencies has revealed the roles of many factors critical for human lymphocyte development and function. Combined immunodeficiencies (CIDs) listed by the IUIS Expert Committee on Primary Immunodeficiencies [
1], include a wide spectrum of gene defects underlying susceptibility to bacterial, viral and fungal infections. The most profound of these, collectively termed severe combined immunodeficiency (SCID), are disorders with few to absent autologous T cells and absent cellular and humoral immune function [
1‐
4]. In contrast, many CID gene defects do not abrogate development or release into the periphery of T and B cells, but instead disrupt pathways critical for their effector and regulatory roles; examples are ORAI-I, STIM-1 [
5], and MHC class II deficiency [
6]. While over 14 different SCID genes are known [
7], many patients with CID without T cell lymphopenia have as yet unidentified genetic defects. Whole exome sequencing (WES) may identify molecular causes of CID.
Studies in knockout mice and human malignancies and immunodeficiencies have delineated the intracellular signaling pathways activated by engagement of lymphocyte antigen receptors and G-protein coupled receptors [
8]. NF-κB, a central mediator of activation signals, translocates from the cytoplasm into the nucleus to initiate transcription of genes that bring about lymphocyte maturation, activation and proliferation [
9]. NF-κB activation and signaling is in turn controlled by multiple mechanisms, one of which is the signalosome formed from assembly of CARMA1 (also called CARD11), BCL-10 and MALT1 into the “CBM” signaling complex [
10,
11]. While the precise molecular mechanisms are still not completely clear, stimulation through the T cell and B cell receptors causes phosphorylation of CARMA1, recruitment of MALT1 and BCL-10, and oligomerization of components of the CBM complex [
12‐
14]. This in turn activates the IκB kinase complex through TNF receptor-associated factor 6 (TRAF6)-mediated ubiquitination of NF-κB essential modulator (NEMO) [
15‐
17], leading to phosphorylation and proteasomal degradation of the inhibitor IκBα and release of NF-κB. Thus, it is not surprising that defects in NEMO, CARMA1 and MALT1 have been found to cause human CID [
18‐
23].
We describe a new patient in whom CID and immune dysregulation due to
MALT1 compound heterozygous mutations was successfully treated by allogeneic hematopoietic cell transplantation (HCT). This case in a non-consanguineous family, combined with 2 prior reports [
22‐
24], broadens the spectrum of MALT1 deficiency disease and suggests an effective treatment.
Methods
Patient
After informed consent, as approved by the University of California San Francisco Committee on Human Research, the patient, his parents and 2 healthy siblings were studied with whole exome sequencing and immunological assessments.
DNA Studies
Genomic DNA from the patient, obtained prior to HCT, and from his parents and siblings was subjected to WES. Analysis tools were similar to [
25], with modifications detailed in the
Supplementary methods. DNA variants were confirmed by Sanger sequencing. With parental consent, residual dried blood spots obtained in the newborn nursery were recovered from the California Department of Public Health Newborn Screening Program, and T cell receptor excision circles (TRECs) were analyzed as described [
26].
Cell Separations and Reagents
After HCT from an unrelated donor differing at a single HLA-C locus, the patient developed mixed chimerism of the hematopoietic system. Patient alleles were HLA-C *08:01, *03:04; donor alleles were *08:01, *07:02. Staining cells with monoclonal human IgM antibody (clone ID: TRA2G9) recognizing antigens encoded by C*01/*03/*04:01/*14:02, but not C*07/*08 [
27‐
29], followed by PE-anti-human IgM (clone MHM-88), permitted separation of autologous patient lymphocytes from those of the donor by flow cytometry. For specific antibodies see
Supplementary methods.
PCR and Western Blotting
RNA was isolated from sorted autologous patient PBMCs obtained post-HCT (RNeasy kit, Qiagen), and expression of
MALT1 transcripts (primers in Suppl Table
1) was detected by PCR (Superscript III system, Life Technologies) followed by Sanger sequencing. The sorted cells were also lysed with 1 % NP-40 and analyzed by Western blotting using antibodies recognizing MALT1 (EP603Y, Abcam) and BCL-10 (H-197, Santa Cruz Biotechnologies).
Intracellular Signaling Assays
For phosphorylation assays PBMCs or Epstein-Barr virus (EBV) transformed B cells were stimulated with 400 nM PMA and 250 ng/ml ionomycin at 37 °C, for 10 min. For cytokine assays PBMCs were stimulated for 6 h with PMA plus ionomycin; 200 ng/ml superantigen staphylococcal enterotoxin E (SEE, Toxin Technology, Inc.) plus 4 ug/ml anti-CD28 clone 9.3; or 1:500 anti-CD3 clone Leu-4 ascites plus 4 ug/ml anti-CD28. The cells were then fixed, permeabilized (Invitrogen Solutions A and B, Life Technologies) and incubated with either phospho-specific unconjugated antibodies followed by anti-rabbit-PE, or anti-mouse-FITC labeled secondary antibodies, or antibodies against IL-2 or IFN-γ. Fluorescent antibodies to relevant surface markers were included.
Lentivirus Transduction
MALT1 cDNA (Genecopia) was ligated into lentiviral vector MP-283: pSicoR-BstXI-EF1a-puro-T2A-mCherry, (kindly provided by Michael McManus, Lentiviral RNAi Core, UCSF). MP-283-MALT1 lentiviral supernatant prepared by transient transfection of 293 cells in DMEM medium with 10 % fetal calf serum (FCS) was used to transduce EBV cells in plates pre-coated with Rectronectin (Takara, Japan). After two 24 h infections, the EBV cells were washed and expanded in RPMI 1640 medium with GlutaMax (Invitrogen), 20 % FCS, penicillin, and streptomicin. MP-283 lentivirus prepared as above without the insert was used in parallel. Transduced cells were detected by mCherry fluorescence.
Discussion
Our patient with profound CID and dysregulation adds to the 2 prior reports and extends our understanding of MALT1-associated disease and its therapy [
22,
23]. Like the prior cases (Table
2), our patient had functionally impaired T and B cells leading to recurrent bacterial and viral infections from early life, notably with CMV, which in our patient was not controlled by antiviral therapy and required post-HCT donor T cell infusions for resolution. While HCT was recently postulated as treatment for MALT1-deficient CID [
24], our report is the first of a MALT1-deficient patient cured by HCT. Two prior siblings died in childhood and a surviving teenager suffers significant multi-organ disease, including T cell inflammation of the skin and bowel similar to that in our patient.
MALT1 mutant patients reported to date had variable B cell numbers, serum immunoglobulin levels and ability to make specific antibodies (Table
2). Immune dysregulation consisting of prominent rash and suspected inflammatory bowel disease was shared between our patient and the living girl with W580S mutation [
23]. In contrast to that patient, however, ours had normal B cell numbers, no IgE elevation and (like the deceased children with S89I mutation) absent protective antibody production.
Our patient’s
MALT1 compound heterozygous mutations resulted in undetectable protein and MALT1 function. As with the previous reports, after introduction of wild type
MALT1 cDNA, our patient’s mutant cells had MALT1 expression and NF-κB signaling reconstituted. Our case also highlights how
MALT1 mutations may lead to immune dysregulation and autoimmunity. After HCT, almost all Tregs and all active Tregs in our patient’s PBMCs were donor-derived (Fig.
2b), accounting for the pre-HCT failure of autologous T cells to control auto-reactive attack on the skin and possibly the intestinal tract. The mixed chimerism exhibited by our patient post-HCT allowed us to evaluate both wild type and MALT1 deficient lymphocytes that had developed from hematopoietic progenitors in vivo. Moreover, our patient’s disease resolved even though he received a non-myeloablative preparative regimen (due to his high risk status with ongoing CMV infection) that did not result in a substantial circulating naïve T cell population. Whether the donor T cells originated from limited thymic development from CD34 progenitors vs. expanded donor T cells, or a combination of both, cannot be determined. However, the patient’s successful outcome indicates that only 15 % of donor T cells were sufficient to reconstitute functional immunity and immune regulation.
The two heterozygous mutations in our patient abrogated MALT1 protein expression. Whereas the p.Y353fs*18 transcript was, as expected, degraded by nonsense-mediated mRNA decay, full-length transcripts skipping exon 10, due to the splice acceptor site defect, were also not detectable. MALT1 protein normally shuttles between the cytoplasm and nucleus with the aid of a nuclear export signal (NES1) [
31,
32] and a regulatory region for NES1 encoded in exon 10, as well as second NES (NES2) in the C-terminal region of the protein [
33,
34]. If a transcript missing exon 10 had been stable in our patient, the resulting protein might have been trapped in the nucleus. However, no nuclear or cytoplasmic protein from autologous patient cells was detected.
The high degree of consanguinity in the previously reported patients may indicate that loci other than MALT1 modified their immune phenotype as well as contributing to delayed bone age, fractures, short stature, and dysmorphia in the surviving patient and poor growth in the deceased siblings; these features were absent in our outbred patient who had compound heterozygosity at the MALT1 locus and has regained normal weight and stature following HCT.
Malt1−/− mice have defects in TCR activation and cytokine production similar to those observed in humans with MALT1 deficiency [
10,
35].
Malt1−/− mice demonstrate diverse B cell defects, as have humans, with NF-κB activity in B cells reduced in one report [
36], but only marginally altered after immunoglobulin receptor engagement in another [
35]. Mice deficient in CBM complex proteins have reduced thymic Treg cells [
37,
38], but this has not been described in mice lacking Malt1. While the recently discovered human patients with
CARD11 mutations had Treg deficiency, similar to the mouse model [
20,
21], and Tregs were either normal or not studied in the previously described patients with
MALT1 mutations, our patient’s autologous PBMCs had nearly absent Tregs and very low levels of Foxp3 expression. Thus, although Malt1 appears not to be required for development of Tregs in mice, it may be important in humans, in keeping with our patient’s resolution of dysregulated immune phenotypes following HCT that provided donor-derived Tregs.
When determining the underlying cause of immune deficiencies without lymphopenia and with normal TREC numbers, signaling molecules downstream of the antigen receptors have become candidates for analysis. As shown by our patient and others with defects in MALT1, as well as patients lacking CARMA1
, NF-κB essential modulator, and IKK2 (IKBKB) [
39], proteins important in antigen receptor and NF-κB signaling should be investigated in patients with combined immunodeficiency.
Acknowledgments
We thank the patient and his family for their participation, the clinicians who provided skillful care, and Yanning Wang, Karly Kondratowicz and Misako Stillion for expert technical assistance. We thank Dr. Jar-How Lee (Thermo Fisher Scientific) for kindly providing a monoclonal antibody against HLA-C*3. This work was supported by NIH R01 AI078248, R01 AI105776 (JMP, SEB); U54 AI 082973 (MJC, JMP); the UCSF Jeffrey Modell Foundation Diagnostic Center for Primary Immunodeficiencies; Tata Consultancy Services (SEB, US, RS) and the Howard Hughes Medical Institute (AW). H.W. was supported by the Arthritis Foundation.