Clinical Communications
Aplastic anemia and cytotoxic T To further investigate her vitamin B12 deficiency, upper and
lymphocyte antigen-4 haploinsufficiency
treated with bone marrow transplantation
Payal Makadia, MDa, Arvind Srinath, MDa,b,
Suneeta Madan-Khetarpal, MDb,c,
Marianne McGuire, MS, CGCd, Elena Infante, MS, CGCc,
Jing Zhang, PhDd, Raymond E. Felgar, MD, PhDe,
Amy W. Davis, MDe, Hey J. Chong, MD, PhDb,f, and
Randy M. Windreich, MDb,g,h
Clinical Implications

Cytotoxic T lymphocyte antigen-4 (CTLA-4) mutations
have been linked to cases of autoimmunity, recurrent
infections, and lymphocytic infiltration. We describe a
patient with aplastic anemia and immune deficiency
found to have a mutation in CTLA-4 who was
successfully treated with allogeneic matched unrelated-
donor bone marrow transplantation.
TO THE EDITOR:
Cytotoxic T lymphocyte antigen-4 (CTLA-4) mutations
result in severe immune dysregulation, with variable penetration
and presentation.1-4 We present the first reported case of aplastic
anemia found in a patient with a heterozygous mutation in the
CTLA-4 gene.
A 19-year-old previously well woman initially presented to her
gynecologist for menorrhagia. Routine blood work demonstrated
mild leukopenia and slightly low protein C activity level. She was
referred to a local hematologist where her pancytopenia was
confirmed (white blood cell count 2200 cells/mL; absolute
neutrophil count 1400 cells/mL; absolute lymphocyte count
400 cells/mL; hemoglobin 11.6 g/dL; platelet count 163,000/
mL). Further workup revealed iron deficiency, vitamin B12
deficiency, mild elevation in her C-reactive protein, and mildly
decreased IgG and IgA levels (Table I). She underwent a bone
marrow (BM) aspirate and biopsy that showed mildly hypo-
cellular marrow (approximately 40% cellular) with trilineage
hematopoiesis, scattered lymphoid aggregates, mildly increased
interstitial mast cells, and markedly reduced iron storage without
ringed sideroblasts.
At our institution, she was screened for autoimmune,
immunological, and hematologic disorders (Table I and Table E1,
available in this article’s Online Repository at www.jaci-inpractice.
org). Immunological workup revealed low CD4þ and CD8þ T
cells, very low natural killer (NK) cells, and nearly absent B cells;
persistent mildly low IgA and IgG levels (specifically IgG2 and
IgG4); and protective tetanus, diphtheria, and hepatitis B titers,
but only 1 of 14 pneumococcal titers was >1.3. Titers were
rechecked after administration of pneumococcal polysaccharide
vaccine (PPV23) and only 4 of 13 were considered protective,
which were lost after a year (Table E2, available in this article’s
Online Repository at www.jaci-inpractice.org).
lower endoscopies were performed, which showed patchy
duodenal villous blunting without apoptosis or inflammation,
mild inactive chronic gastritis, and focal minimally active colitis
with scattered epithelial apoptosis in the cecum and ascending
colon (Figure E1, available in this article’s Online Repository at
www.jaci-inpractice.org). Celiac serologies and genetic screens
were negative (Table E1, available in this article’s Online
Repository at www.jaci-inpractice.org).
Repeat BM evaluation approximately 1 year from the initial
showed an almost acellular marrow (<5% cellularity) that was
nearly devoid of erythroid, myeloid, and megakaryocytic ele-
ments, with only scattered lymphoid aggregates (<10% of
marrow space) consisting mostly of CD3þ T cells (Figure 1)
and without fibrosis. Flow cytometry showed predominantly
CD3þ T cells with few polyclonal B cells (no monoclonal B
cells present), near-absent B-cell progenitors, and few NK cells.
The CD4:CD8 ratio was normal. However, 42% of the CD3þ
T cells appeared to weakly coexpress CD20, which was sup-
ported by immunohistochemical staining for CD3, CD20,
and CD19 (Figure 1). Cytogenetic and fluorescence in situ
hybridization studies did not reveal a clonal cytogenetic
abnormality. As the patient experienced progressive BM failure
with an increased transfusion requirement, allogeneic matched
unrelated-donor bone marrow transplantation (allo-MUD
BMT) was pursued.
Pretransplantation studies discovered mild splenomegaly,
Epstein-Barr virus viremia (for which she received 1 dose of
rituximab before transplantation), and multiple, small pulmo-
nary nodules bilaterally. Lung wedge biopsy showed small foci of
lymphocytic pneumonitis and patchy bronchiolitis obliterans/
organizing pneumonia without granulomas, fungal organisms, or
viral inclusions (Figure E1, available in this article’s Online Re-
pository at www.jaci-inpractice.org). Culture grew Micrococcus
species.
An unrelated donor search was undertaken, and an
ABO-matched female donor was identified who was human
leukocyte antigen (HLA)-matched at HLA-A, -B, -C, and
-DRB1. Nonmyeloablative conditioning therapy consisted of
fludarabine 30 mg/m2/day on days 6 through 3, cyclo-
phosphamide 300 mg/m2/day on days 6 through 3, thy-
moglobulin 3.75 mg/m2/day on days 4 and 3, and 200 cGy
of total body irradiation on day 1,5 with cyclosporine A and
methotrexate for graft-versus-host disease (GVHD) prophylaxis.
Because of concerns for breakthrough Micrococcus infection
during the pre-engraftment period, granulocyte infusions were
given twice weekly until engraftment. Neutrophil and platelet
engraftment were achieved on day þ17 and day þ24,
respectively.
Post-transplant course was complicated by acute GVHD of
the skin and stomach, several bacterial infections, low-level
adenoviremia with adenovirus colitis, and acute kidney injury.
Stage 1 skin GVHD was diagnosed clinically on day þ24, which
resolved with topical therapy. Gastric GVHD was diagnosed
endoscopically 6 months after transplant. Cyclosporine was
changed to tacrolimus, and budesonide and prednisone were
added. She successfully weaned off of steroids and repeat
1
2 CLINICAL COMMUNICATIONS J ALLERGY CLIN IMMUNOL PRACT
MONTH 2017

TABLE I. Patient diagnostic evaluation, before transplantation transmembrane domain of the CTLA-4 protein. It is predicted
Lymphocyte subsets that this mutation may result in a loss of the CTLA-4 protein
Normal values
or production of a truncated protein.1,2 Interestingly, low levels
of CTLA-4 have been found in patients with aplastic anemia
Marker cells/cumm Percent cells/cumm Percent that may further support the association of aplastic anemia and
CD3 603-2990 49-84 291 85 CTLA-4 gene mutation in our patient.7 It is also of interest
CD4 441-2156 28-63 165 48 that BM examination showed increased numbers of dimly
CD8 125-1312 10-40 105 31 CD20-positive T cells. The exact significance of this finding is
CD19 107-698 6-27 8 2 unclear, though low level expression of CD20 on a usually
CD16/CD56 95-640 4-25 37 11 small subset of normal T cells has been well described. Inter-
CD4:CD8 ratio 0.71-3.23 1.56 estingly, in monkeys, it has been transiently induced on T cells
after immune stimulation and may reflect chronically activated
Immunoglobulin (mg/dL)
T cells.8 Also, most patients with CHAI exhibited profoundly
IgG (751-1560) 715 low B cells and a decrease in at least 1 immunoglobulin iso-
IgG-1 (342-1118) 528 type. This humoral immune deficiency may be caused by
IgG-2 (148-525) 119 impaired B-cell development due to T-cell infiltration of the
IgG-3 (21-114) 76 BM and chronic stimulation of B cells leading to an anergic/
IgG-4 (7-89) 4 exhausted state.1,2
IgA (87-453) 47 Currently, most patients with CHAI have had variable
IgM (40-274) 65 response rates to immunosuppression including corticosteroids,
IgE (<88) <1 sirolimus, rituximab, cyclosporine A, cyclophosphamide, myco-
phenolate mofetil, and abatacept (a soluble fusion protein of
Note: Lymphocyte proliferation assays demonstrate normal and robust CD45þ and
CTLA-4 linked to IgG1).1,2 Slatter et al4 described 8 patients
CD3þ T-cell response to phytohemagglutinin, but response to pokeweed was unable
to be assessed due to insufficient white blood cell counts. who underwent hematopoietic stem cell transplantation for life-
threatening, treatment-resistant immune dysregulation. All but
one were diagnosed retrospectively with CHAI. They received
well-matched, unrelated-donor grafts after a reduced-intensity
endoscopy demonstrated no GVHD. Two months later, she was conditioning. Six patients had 100% donor chimerism,
able to discontinue all immunosuppression. Whole exome whereas 2 had mixed donor chimerism of 85% in all cell
sequencing results later revealed a heterozygous c.515C>A lineages. Post-transplant course included GVHD in 4 patients
(p.S172X) mutation in the CTLA-4 gene. Her mother, who and complete resolution of enteropathy and/or cytopenias in
suffers from a nonspecific autoimmune enteropathy, was also 7 patients.4 Because of concurrent severe aplastic anemia and
found to possess the same heterozygous mutation. unspecified immunodeficiency, we selected a nonmyeloablative
At the time of this writing, the patient is 24 months after conditioning regimen before BMT to minimize long-term tox-
transplant. BM evaluations now show only mildly hypocellular icities. Slatter et al4 diagnosed GVHD in half of their cohort and
marrow (40% to 50% cellularity) with adequate trilineage subsequently proposed that greater immunosuppression may be
hematopoiesis and 100% donor chimerism in the BM, whole indicated for this population because of high levels of inflam-
blood, T-cell, B-cell, and myeloid fractions. She continues mation before transplant potentially promoting alloreactivity and
to have occasional mild lymphopenia (absolute lymphocyte increased rates of GVHD. Our patient had similarly developed
counts 700-910 cells/mL) and hypogammaglobulinemia mild acute GVHD but responded to our institution’s first-line
(IgG 423-687 mg/dL), for which she is receiving intravenous therapies. In addition, our patient’s persistent hypogammaglob-
immunoglobulin intermittently. ulinemia after transplant may have been related to the rituximab
The CTLA-4 receptor, expressed by regulatory T cells, is given shortly before transplant as published reports have found
important in maintaining immune tolerance.1-3,6 CTLA-4 defi- expansion of functionally immature B cells, impaired isotype
ciency can disrupt the balance of immune activation and sup- expression, and decreased memory B cells after rituximab
pression leading to alterations in the immune response.1,2 This therapy.9
autosomal dominant disease is characterized by CTLA-4 hap- We describe successful allo-MUD BMT in the first described
loinsufficiency in all affected patients and has been termed by case of CHAI with aplastic anemia achieving stable engraftment
Kuehn et al2 as CTLA-4 haploinsufficiency with autoimmune and resolution of pancytopenia. CHAI affects multiple aspects
infiltration (CHAI) disease. of the immune system, and more studies are warranted on the
Much like our patient, patients with CHAI disease present long-term outcomes of BMT in these patients.
with a constellation of findings including autoimmunity,
recurrent infections, hypogammaglobulinemia, cytopenias,
lymphocytic infiltration in various organs, and enteropathy. Acknowledgments
Many patients have tested positive for autoantibodies, though P.M., A.I.S., and H.J.C. were involved in reviewing the chart,
nondiagnostic for a specific autoimmune disorder, and may drafting, and critical revision of the manuscript. R.M.W. was
represent heightened immune activation. Although previous involved in reviewing the chart, drafting, critical revision of the
case studies describe various autoimmune cytopenias, we manuscript, and study supervision. A.W.D. and R.E.F. were
describe the first case of aplastic anemia that may be related to involved in providing pertinent histopathology pictures and
CTLA-4 deficiency.1,2,4 Our patient was found to have a novel cytogenetics, formatting of figures, and critical revision of the
nonsense mutation located on exon 3 likely affecting the manuscript. S.M.-K., M.M., E.I., and J.Z. were involved in
J ALLERGY CLIN IMMUNOL PRACT CLINICAL COMMUNICATIONS 3
VOLUME -, NUMBER -

FIGURE 1. Bone marrow findings. A, Hematoxylin and eosin (H&E) appearance of a nearly acellular marrow with focal lymphoid
aggregates composed of (B) CD3þ Tcells, half of which coexpress (C) CD20. D, Flow cytometric staining showed that 42% of the CD3þ
T cells were CD20-positive. Inset in (D) shows CD19 staining, demonstrating that most cells were not B cells.

interpretation of whole exome sequencing results and critical REFERENCES

1. Schubert D, Bode C, Kenefeck R, Hou TZ, Wing JB, Kennedy A, et al. Auto-
revision of the manuscript.
somal dominant immune dysregulation syndrome in humans with CTLA4
mutations. Nat Med 2014;20:1410-6.
a
Division of Gastroenterology, Children’s Hospital of Pittsburgh of UPMC, Pitts- 2. Kuehn HS, Ouyang W, Lo B, Deenick EK, Niemela JE, Avery DT, et al. Immune
burgh, Pa dysregulation in human subjects with heterozygous germline mutations in
b
Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pa CTLA4. Science 2014;345:1623-7.
c
Division of Medical Genetics, Children’s Hospital of Pittsburgh of UPMC, Pitts- 3. Waterhouse P, Penninger JM, Timms E, Wakeham A, Shahinian A, Lee KP, et al.
burgh, Pa Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4.
d
Division of Medical Genetics, Baylor Miraca Genetic Laboratories, Houston, Tex Science 1995;270:985-8.
e
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, 4. Slatter MA, Engelhardt KR, Burroughs LM, Arkwright PD, Nademi Z, Skoda-
Pa Smith S, et al. Hematopoietic stem cell transplantation for CTLA4 deficiency.
f
Division of Pulmonary Medicine, Allergy and Immunology, Children’s Hospital of J Allergy Clin Immunol 2016;138:615-619.e1.
Pittsburgh of UPMC, Pittsburgh, Pa 5. Bacigalupo A, Socie G, Lanino E, Prete A, Locatelli F, Locasciulli A, et al.
g
Division of Blood and Marrow Transplantation and Cellular Therapies, Children’s Fludarabine, cyclophosphamide, antithymocyte globulin, with or without low
Hospital of Pittsburgh of UPMC, Pittsburgh, Pa dose total body irradiation, for alternative donor transplants, in acquired severe
h
Division of Hematology/Oncology, Children’s Hospital of Pittsburgh of UPMC, aplastic anemia: a retrospective study from the EBMT-SAA Working Party.
Pittsburgh, Pa Haematologica 2010;95:976-82.
No funding was received for this work. 6. Qureshi OS, Zheng Y, Nakamura K, Attridge K, Manzotti C, Schmidt EM, et al.
Conflicts of interest: The authors declare that they have no relevant conflicts of Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic
interest. function of CTLA-4. Science 2011;332:600-3.
Received for publication December 16, 2016; revised February 22, 2017; accepted 7. Li B, Guo L, Zhang Y, Xiao Y, Wu M, Zhou L, et al. Molecular alterations in the
for publication March 8, 2017. TCR signaling pathway in patients with aplastic anemia. J Hematol Oncol 2016;
Available online -- 9:32.
Corresponding author: Randy M. Windreich, MD, Children’s Hospital of Pittsburgh 8. Rahemtullah A, Longtine JA, Harris NL, Dorn M, Zembowicz A, Quintanilla-
of UPMC, One Children’s Hospital Drive, 4401 Penn Avenue, Pittsburgh, Fend L, et al. CD20þ T-cell lymphoma: clinicopathologic analysis of 9 cases and
PA 15224. E-mail: randy.windreich@chp.edu. a review of the literature. Am J Surg Pathol 2008;32:1593-607.
2213-2198 9. Anolik JH, Friedberg JW, Zheng B, Barnard J, Owen T, Cushing E, et al. B cell
Ó 2017 American Academy of Allergy, Asthma & Immunology reconstitution after rituximab treatment of lymphoma recapitulates B cell
http://dx.doi.org/10.1016/j.jaip.2017.03.007 ontogeny. Clin Immunol 2007;122:139-45.
3.e1 CLINICAL COMMUNICATIONS J ALLERGY CLIN IMMUNOL PRACT
MONTH 2017

ONLINE REPOSITORY

FIGURE E1. Hematoxylin and eosin stain of biopsies. A, Duodenum: patchy areas of complete blunting with areas of normal architecture.
B, Gastric antrum: mild inactive chronic gastritis with reactive epithelial changes including mucin depletion. C, Colon: scattered epithelial
apoptoses (circle) with minimal architectural and inflammatory changes. D, Wedge lung biopsy: focal areas of lymphocytic pneumonitis
and bronchiolitis obliterans/organizing pneumonia.
J ALLERGY CLIN IMMUNOL PRACT CLINICAL COMMUNICATIONS 3.e2
VOLUME -, NUMBER -

TABLE E1. Patient diagnostic evaluation, before transplantation

Autoantibodies
Antinuclear antibodies  Anti-neutrophil antibodies 
Anti-double stranded DNA  GP Ia/Iia 
Rheumatoid factor  GP Ib/IX 
C3 (88-201 mg/dL) 101 GP IIb/IIIa +
C4 (10-40 mg/dL) 21 Direct Coombs /+
Intrinsic Factor Blocking Antibody  Anti-neutrophil cytoplasmic antibodies 
21-Hydroxylase Antibody  Anti-Saccharomyces cerevisiae antibodies 
Bone marrow failure syndromes
DEB assay  Trypsinogen Normal
PNH screen (CD55/CD59)  Hemoglobin F level Normal
Telomere length measurement Normal
Gastroenterology and nutrition
Tissue transglutaminase IgA/IgG  Vitamin B12 (211-911 pg/mL) 151
Anti-gliadin antibodies  Vitamin A, E, K Normal
Celiac screen Heterozygous DQ2.2 Folic acid Normal
Infectious disease
Adenovirus PCR  Herpes Zoster IgM/IgG 
CMV IgM/IgG  HIV 1/2 Antibody 
EBV serology + Parvovirus B19 PCR 
Hepatitis Antibody Panel (A, B, C)  Toxoplasma IgM/IgG 
Herpes Simplex Virus IgM/IgG 
Vaccination status
Tetanus IgG + Hepatitis A total antibodies +
Diphtheria IgG + Herpes zoster IgG 
Pneumococcal IgG antibodies  (see Table E2)
HLA molecular typing
Class I Class II

A 01:01 DRB1 01:01

A 25:01 DRB1 07:01
B 13:02 DQB1 02:02
B 44:02 DQB1 05:01
C 05:01 DPB1 05:01
C 06:02 DPB1 17:01
DQA1 01:01
DQA1 02:01
CMV, Cytomegalovirus; DEB, diepoxybutane; EBV, Epstein-Barr virus; HLA, human leukocyte antigen; PNH, paroxysmal nocturnal hemoglobinuria.

TABLE E2. Pneumococcal antibody titers, before transplantation

Baseline 1 mo after PPV23 1 y after PPV23
Serotype (mcg/mL) (mcg/mL) (mcg/mL)

1 (1) <0.3 2.4 1.0

3 (3) <0.3 3.1 0.7
4 (4) <0.3 0.4 <0.3
5 (5) 0.3 0.3 0.4
8 (8) <0.3 1.4 0.6
9 (9N) <0.3 <0.3 <0.3
12 (12F) <0.3 <0.3 <0.3
14 (14) <0.3 0.5 0.7
19 (19F) 1.6 1.5 1.5
23 (23F) 0.3 0.7 0.5
26 (6B) <0.3 <0.3 <0.3
51 (7F) <0.3 1.0 0.6
56 (18C) <0.3 <0.3 <0.3
68 (9V) <0.3 0.5 0.3
Bolded values indicate protective levels.
PPV23, Pneumococcal polysaccharide vaccine.