Abstract
Shwachman-Diamond syndrome (SDS) is one of the more common inherited bone marrow failure syndromes, characterized by neutropenia, occasional thrombocytopenia and anemia. Bone marrow evaluation reveals an increased number of monocytes and mature B cells along with decreased granulocytes. However, little is known about the subpopulations of peripheral blood cells and few previous publications have been based on a small number of patients. Here we report a comprehensive immunophenotypic analysis from a cohort of 37 SDS patients that display impairment mostly in the myeloid compartment with a deficiency also in the number of B cells and CD4/CD8 double negative T cells.
Keywords: Shwachman-Diamond syndrome, Bone Marrow Failure, Immunophenotype, Neutropenia, Double Negative T cells
Introduction
Shwachman-Diamond syndrome (SDS) is a recessively inherited disease, mostly caused by mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene1. This gene encodes SBDS, a protein involved in the 80S ribosome assembly2. Mutations in EFL1, DNAJC21 and SRP54 are associated with abnormalities seen in SDS3. These genes may constitute a pathway involved in proteostasis, as SBDS directly interacts with EFL1, DNACJ21 stabilizes the 80S ribosome, and SRP54 facilitates the trafficking of the nascent polypeptide.
In SDS, perturbed hematopoiesis results in neutropenia and occasional thrombocytopenia and/or anemia4. Approximately15% of SDS patients develop myelodysplastic syndrome (MDS) with a high risk of progression to acute myeloid leukemia5. Although SDS-associated bone marrow failure affects the myeloid lineages6, little is known about the lymphoid compartment. Immunophenotypic analysis of bone marrow (BM) samples obtained from a cohort of 19 patients reported a significantly lower percentage of BM CD34+ cells, increased number of monocytes, decreased number of granulocytes, and increased maturation of B cells. Importantly, no differences in the percentage of lymphoid and erythroid progenitors were detected6. One study on the immunophenotypic analysis of blood cells was conducted on a small cohort of nine patients, with ages range between 2 and 18 years. The investigators found a decreased number of circulating NK cells and abnormalities of B- and T-lymphocyte in terms of cell number, lymphocyte proliferation response and serum immunoglobulin levels. Furthermore, three of nine patients displayed a reversed CD4/CD8 ratio7. Unfortunately due to the rarity of SDS8, these data lacked statistical power and showed few leukocyte populations or subpopulations. The aim of our study is to characterize the peripheral blood immunophenotype in individuals with SDS. We report a comprehensive analysis of most leukocyte populations and subpopulations obtained from 37 SDS patients. In order to compare results with appropriate controls, we used an extended cohort of 617 age-matched control subjects.
Design and Methods
Patients
Medical records of 37patients regularly followed by Immunology or Transfusion Medicine sections of the Azienda Ospedaliero Universitaria Integrata (AOUI) in Verona or the Cystic Fibrosis Center of Ancona between 2009 and 2018 were used for the study. All blood samples were analyzed after informed written consent was obtained (IRB approvals 658 CESC and CERM 2018–82). All patients included in this study were diagnosed with SDS based on clinical criteria and genotype (biallelic mutations in SBDS), screened at time of diagnosis in the absence of MDS. The 617 age-related controls were obtained from the electronic data warehouse of AOUI Verona.
Flow Cytometry
Sample preparation was performed according to the Clinical and Laboratory Standard Institute H42-A2 “Enumeration of Immunologically Defined Cell Populations by Flow Cytometry, 2nd Edition” guidelines. Immunophenotyping was performed by ten-color Navios flow cytometer (Beckman Coulter, Pasadena, CA). The UK Neqas External Quality Assessment “Leucocyte Immunophenotyping” internal quality control (IQC) was used9 as detailed in the supplementary material.
Statistics
Missing data (present in 5–40% of cell subpopulations of B cells and T lymphocytes) were substituted by mean or median (when normality distribution did not subsist) imputation. Differences in cell counts between cases and controls were assessed by a multiple linear regression model in which the SDS condition was used as binary variable, and age and sex were added as confounders. The statistical software SAS (SAS Institute Inc., Cary, NC) was used. The data are presented as mean ± standard deviation. Regression coefficient and 95% confidence interval were also analyzed.
Results and discussion
The SBDS genotypes and clinical characteristics of the SDS patients are provided in Table 1 and supplemental Table S1. Of the 37 SDS patients, their ages ranged between 0.5 and 38 years (mean 13.2 ± 10.6) and was comparable to the cohort of 617 control subjects (mean 7.3 ± 6.3). None of the patients were receiving steroid or recombinant human granulocyte colony-stimulating factor (filgrastim) therapy. Besides the lower number of circulating neutrophils (control 3,504 ± 1,229 (SD), SDS 1,083 ± 745), we found that SDS patients display a reduction of eosinophils (control 258 ± 152, SDS 28 ±42). These findings are consistent with the previously reported arrest of myeloid maturation in SDS cells both in vitro and in vivo6,11 (Table 2). Eosinophils can cooperate with neutrophils in resolving infections by phagocytosing bacteria, yeast, and parasites12 and releasing mitochondrial DNA traps in response to pathogenic bacteria13. Of note, patients presenting recurrent infections show even less neutrophils and eosinophils than patients without infections (supplemental Table S2). Thus, together with neutropenia, eosinophil deficiency could account for recurrent infections which are sometimes observed in children affected by SDS4.
Table 1.
Clinical characteristics of SDS patients enrolled in this study
| UPN | Gender | Age at diagnosis (years) |
Age at analysis (years) |
PMN/ μl |
EOS/ μl |
DN T cells/μl |
Recurrent infections | Phenotype |
|---|---|---|---|---|---|---|---|---|
| 1 | M | 0.42 | 20.06 | 1231 | 19 | N/A | No | FTT, PI, Bone malformations (valgus knees), HbF>2%, Hepatomegaly |
| 7 | M | 1.26 | 14.43 | 672 | 84 | N/A | Otitis | FTT, PI, Bone malformations (metaphyseal changes in the ribs), Hepatomegaly, CD |
| 13 | M | 0.97 | 13.00 | 1132 | 22 | 180 | No | FTT, PI, Bone malformations (femur dysostosis) |
| 15 | F | 1.36 | 17.94 | 1454 | 86 | 87 | Pneumonia, URTI | FTT, PI, HbF>2%, Hepatomegaly |
| 26 | M | 0.46 | 10.35 | 58 | 0 | 180 | Sepsis, Skin abscesses | PI, Severe bone marrow hypoplasia, Thrombocytopenia, CD |
| 33 | F | 0.31 | 18.88 | 852 | 20 | 15 | Otitis, Skin abscesses | FTT, PI, Bone malformations (vertebral deformities), Anemia, Thrombocytopenia |
| 37 | F | 2.39 | 5.73 | 1834 | 10 | 236 | No | FTT, PI, Hepatomegaly |
| 39 | M | 0.51 | 37.10 | 1100 | 71 | 99 | Otitis, Sepsis | FTT, PI, Bone malformations (valgus knees), HbF>2%, Hepatomegaly, CD |
| 41 | F | 3.43 | 6.13 | 1730 | 59 | 84 | Otitis | FTT, PI, Bone malformations (vertebral deformities), Hepatomegaly, Thrombocytopenia |
| 47 | M | 1.09 | 3.98 | 544 | 12 | 85 | Otitis, Pneumonia, UTI | PI, Bone malformations (valgus knees), HbF>2%, Hepatomegaly |
| 52 | M | 2.31 | 16.02 | 908 | 76 | 226 | No | PI, Bone malformations (ribs, femur dysostosis), HbF>2%, Hepatomegaly |
| 56 | F | 0.76 | 7.60 | 1838 | 7 | 320 | No | PI, HbF>2% |
| 57 | F | 32.83 | 38.09 | 480 | 9 | 67 | Skin abscesses | FTT, PI, Bone malformations (valgus knees, vertebral deformities), HbF>2%, Thrombocytopenia, CD |
| 59 | M | 0.16 | 0.91 | 720 | 22 | 144 | No | FTT, PI, Bone malformations (metaphyseal changes in the ribs), HbF>2%, Hepatomegaly, Thrombocytopenia |
| 61 | M | 0.5 | 0.5 | 588 | 11 | 79 | Pneumonia | PI, HbF>2%, Hepatomegaly |
| 63 | M | 0.74 | 10.34 | 2369 | 251 | 79 | Otitis, Sepsis | FTT, PI, Bone malformations (coxa vara, metaphyseal changes in the ribs), HbF>2%, Hepatomegaly |
| 64 | M | 0.58 | 22.95 | 976 | 29 | N/A | No | FTT, PI, Bone malformations (bilateral humerus dysostosis), Anemia, CD |
| 65 | M | 0.94 | 13.59 | 1591 | 49 | N/A | No | FTT, PI, Bone malformations (Perthes disease), Epilepsy |
| 66 | M | 2.13 | 2.13 | 543 | 7 | N/A | No | FTT, PI |
| 68 | M | 10.63 | 20.19 | 856 | 54 | N/A | Dermatitis, Pneumonia | FTT, PI, Bone malformations (hip dysplasia), Thrombocytopenia, CD |
| 69 | F | 0.25 | 0.50 | 771 | 0 | 131 | No | FTT, PI, HbF>2%, Anemia |
| 71 | F | 0.75 | 27.42 | 1330 | 53 | N/A | Otitis | FTT, PI, Hepatomegaly, Anemia, Thrombocytopenia |
| 72 | M | 0.33 | 20.28 | 480 | 9 | 38 | Otitis, Pneumonia, UTI | FTT, PI, Bone malformations (bilateral femur dysostosis), HbF>2%, Hepatomegaly, Cognitive disorders |
| 73 | F | 0.53 | 0.89 | 521 | 9 | 122 | No | FTT, PI, HbF>2%, Thrombocytemia |
| 74 | M | 1.74 | 7.75 | 1170 | 10 | 172 | No | FTT, PI, HbF>2%, Thrombocytemia, Hepatomegaly, CD |
| 75 | F | 0.50 | 1.41 | 1029 | 7 | N/A | No | PI, Bone malformations (metaphyseal changes in the ribs), hepatic duct metaplasia |
| 76 | M | 0.5 | 1.52 | 465 | 0 | N/A | Bronchiolitis, Dermatitis, URTI |
FTT, PI, Bone malformations (ankle valgus), HbF>2%, Hepatomegaly, Thrombocytopenia |
| 80 | M | 0.79 | 1.09 | 1222 | 0 | N/A | No | FTT, PI, Bone malformations (metaphyseal changes in the ribs), Anemia |
| 81 | M | 0.51 | 0.79 | 640 | 0 | N/A | Bronchiolitis, Sepsis, Skin abscesses | FTT, PI, Bone malformations (femur dysostosis), HbF>2%, Hepatomegaly, Anemia |
| 87 | M | 10.68 | 14.56 | 657 | 11 | 200 | No | FTT, PI, Bone malformations (valgus knees), Hepatomegaly, Anemia, Thrombocytopenia, CD |
| 93 | M | 1.57 | 3.26 | 4468 | 142 | N/A | No | FTT, PI |
| 99 | M | 1.15 | 37.63 | 389 | 0 | 24 | Otitis, URTI | FTT, PI, Severe osteoporosis, HbF>2%, Thrombocytopenia |
| 104 | M | 6.60 | 7.00 | 646 | 17 | 119 | No | FTT, PI, Bone malformations (tibia vara), HbF>2%, Anemia, Thrombocytopenia, CD |
| 106 | M | 1.06 | 31.00 | 1212 | 70 | N/A | No | FTT, PI, Bone malformations (femur dysostosis), Hepatomegaly, Anemia |
| 108 | M | 2.39 | 13.77 | 971 | 0 | N/A | No | FTT, PS, Bone malformations (upper and lower limbs dysostosis), Hepatomegaly, Anemia, CD |
| 110 | M | 0.62 | 17.62 | 321 | 8 | 60 | Pneumonia, Sepsis | FTT, PI, Bone malformations (upper and lower limbs dysostosis), HbF>2%, Hepatomegaly, Anemia, Thrombocytopenia |
| 111 | M | 1.11 | 21.42 | 1858 | 22 | N/A | No | FTT, PI, Bone malformations (spinal column), HbF>2%, Hepatomegaly, Anemia, Thrombocytopenia |
In this study we compared 37 SDS patients (27 males, 10 females) to 617 control subjects (393 males, 224 females). Ages range SDS 0.5–38 years (mean 13.20 ± 10.58). Ages range control subjects 0.5–38 years (mean 7.3 ± 6.3). Recurrent infection events are indicated. Values for PMN, EOS and DN T cell counts indicate absolute number. Abbreviations: UPN, unique patient number; F, female; M, male; PMN, neutrophils; EOS, eosinophils; DN T cells, double negative T cells; UTI, urinary tract infetion; URTI, upper respiratory tract infection; FTT, failure to thrive; PI, pancreatic insufficiency; PS, pancreatic sufficiency; HbF, fetal hemoglobin; CD, cognitive disorder. N/A, not available.
Table 2.
Comprehensive analysis of the peripheral blood immunophenotype of SDS patients
| Cell type | Cell Markers | Condition | Mean (cells/ul) |
SD | Regression Coefficient |
95% C.I. |
p value§ |
p value# |
|---|---|---|---|---|---|---|---|---|
| Neutrophils | Control | 3504 | 1229 | −2583.41 | −2926.93, −2239.89 | <0.0001 | <0.0001 | |
| SDS | 1083 | 745 | ||||||
| Eosinophils | Control | 258 | 152 | −212.94 | −247.00, −178.88 | <0.0001 | <0.0001 | |
| SDS | 28 | 42 | ||||||
| Monocytes | Control | 403 | 138 | 78.11 | 31.21, 125.02 | 0.003 | 0.001 | |
| SDS | 504 | 298 | ||||||
| Lymphocytes | Control | 3201 | 1110 | −182.78 | −462.14, 96.59 | n.s. | n.s. | |
| SDS | 2773 | 1372 | ||||||
| T cells | CD3+ CD4+ | Control | 1308 | 551 | −67.28 | −228.77, 94.20 | n.s. | n.s. |
| SDS | 1200 | 709 | ||||||
| CD3+ CD4+ CD38+ | Control | 700 | 359 | −185.63 | −472.08, 100.83 | n.s. | n.s. | |
| SDS | 574 | 839 | ||||||
| CD3+ CD4+ CD38+ HLA-DR+ | Control | 27 | 40 | −2.46 | −13.60, 8.70 | n.s. | n.s. | |
| SDS | 22 | 7 | ||||||
| CD3+ C8+ | Control | 748 | 246 | 80.53 | 18.03, 143.03 | 0.019 | 0.024 | |
| SDS | 756 | 356 | ||||||
| CD3+ CD8+ CD38+ | Control | 245 | 133 | −117.23 | −213.51, −20.96 | 0.028 | 0.032 | |
| SDS | 162 | 208 | ||||||
| CD3+ CD4- CD8- | Control | 222 | 100 | −51.04 | −78.13, −23.95 | <0.0001 | <0.0001 | |
| SDS | 100 | 76 | ||||||
| Ratio CD4/CD8 | Control | 1.8 | 0.5 | −0.22 | −0.40, −0.039 | n.s. | n.s. | |
| SDS | 1.7 | 0.7 | ||||||
| NK | CD3- CD19- CD56+ CD16+ | Control | 319 | 163 | −33.96 | −82.70, 14.77 | n.s. | n.s. |
| SDS | 278 | 262 | ||||||
| B cells | Total CD19+ | Control | 596 | 323 | −87.18 | −168.22, −6.13 | 0.048 | 0.031 |
| SDS | 440 | 398 | ||||||
| CD19+ CD27+ | Control | 82 | 42 | −22.55 | −43.00, −2.11 | 0.045 | 0.011 | |
| SDS | 54 | 78 | ||||||
| CD19+ CD23+ | Control | 332 | 245 | −42.63 | −140.80, 55.53 | n.s. | n.s. | |
| SDS | 268 | 211 | ||||||
| CD19+ CD5+ CD23+ CD27- | Control | 113 | 100 | −43.96 | −99.78, 11.86 | n.s. | n.s. | |
| SDS | 82 | 93 | ||||||
| CD19+ CD5+ CD23+ CD27+ | Control | 7 | 6 | −3.05 | −6.89 0.77 | n.s. | n.s. | |
| SDS | 5 | 6 | ||||||
| CD19+ CD5+ CD23- CD27- | Control | 44 | 58 | −17.60 | −53.28, 18.08 | n.s. | n.s. | |
| SDS | 48 | 71 | ||||||
| CD19+ CD5+ CD23- CD27+ | Control | 9 | 7 | −3.92 | −8.09, 0.25 | n.s. | n.s. | |
| SDS | 6 | 6 | ||||||
| CD19+ CD5- CD23+ CD27- | Control | 181 | 83 | −2.23 | −49.35, 44.88 | n.s. | n.s. | |
| SDS | 175 | 125 | ||||||
| CD19+ CD5- CD23+ CD27+ | Control | 14 | 8 | −4.92 | −9.34, −0.51 | n.s. | n.s. | |
| SDS | 8 | 11 | ||||||
| CD19+ CD5- CD23- CD27- | Control | 92 | 66 | −8.90 | −44.93, 27.13 | n.s. | n.s. | |
| SDS | 94 | 104 | ||||||
| CD19+ CD5- CD23- CD27+ | Control | 48 | 21 | −15.63 | −29.67, −1.58 | 0.042 | n.s. | |
| SDS | 31 | 41 |
Differences in cell number between SDS (37 patients as reported in Table 1) and controls (617 control subjects with no hematological issues) were investigated by using multiple linear regression models in which the case/control status was used as predictor and its effect was corrected for age and sex. Standard deviation (SD), regression coefficient and 95% confidence interval (95% C.I.) are indicated. The p value tests the hypothesis that the SDS/control status coefficient is equal to zero (no change in number of cells between SDS patients and controls).
p value calculated using imputed data.
p value in the absence of imputed data. n.s., not significant.
As shown in Table 2, we found a statistically significant increase (25%) in circulating monocytes in SDS patients (control 403± 138, SDS 504± 298), suggesting that myeloid differentiation is shifted towards monocyte commitmentin bone marrow. This is consistent with previously reported data6. B cell maturation and function have been poorly investigated in SDS. Bone marrow from SDS patients display an enrichment in mature B cells and plasma cells6. However, some patients present fewer circulating B cells in peripheral blood with impaired immunological functions7. Here, we report that the B cell number is significantly lower in SDS (control 596 ± 323, SDS 440 ± 398). Our data suggest that decreased B cell number is mainly due to the CD27+ B memory compartment. Contrary to a previous report7, no significant difference was observed in the number of NK or CD4+ T cells in patients with SDS compared to controls. We did not observe a reversed CD4/CD8 ratio in T cell populations of patients (controls 1.8 ± 0.5, SDS 1.7 ± 0.7). CD8+ T cell number was not different from that in SDS (control 748 ± 246, SDS 756 ± 356), but there was a lower number of CD38+ CD8+ T cells. These data suggest a functional impairment in T cell activation, consistent with a previous report7. Importantly, we observed a significant decrease in CD4/CD8 double negative (DN) T cells (control 222 ± 100, SDS 100 ± 76). DN T cells have been proposed to regulate innate immunity by acting as both pro-inflammatory (DN T cells) and anti-inflammatory (DN Tregs) cells14. Because DN Tregs play an important role in the development of tolerance after transplantation15,16, a deficiency of DN T cells in patients should be monitored in patients with SDS undergoing stem cell transplantation. Finally, through logistic regression analysis, in which we used the SDS/control status as a dependent variable, we generated an exploratory diagnostic model of SDS, which confirm the hypotheses suggested in Table 2, cross-correcting the statistical associations (supplemental Table S3). According to this model, the diagnosis of SDS may be advanced from the estimated odds ratio of altered counts of eosinophils, neutrophils, monocytes and B-cells.
In conclusion, individuals with SDS possess differences in both myeloid and lymphoid compartments. Notably, granule-rich neutrophils and eosinophils were markedly decreased. An increase in circulating monocyte number could contribute, along with the moderate degree of neutropenia, to the low frequency of infections observed in only 46% of patients with SDS (Table 1). The risk of infection lies in contrast to that observed in severe congenital neutropenia. Our data suggest that patients presenting with lower number of DN T cells together with severe neutropenia face worse recurrent infections (Table 1). This might point to subtle changes in innate and acquired immunity and provide a clue as to how SBDS deficiency plays a role in tissue development.
Supplementary Material
Acknowledgments
In loving memory of Dr. Peter Durie. We are grateful to Francesco Pasquali (University of Insubria, Italy) for cytogenetic data, to Emily Pintani and Tiziana Cestari (AOUI Verona, Italy) for the excellent technical support.
VB and MC conceived the overall idea; VB, AV, MC and SJC interpreted the data;VB and SJC wrote the manuscript; VB critically reviewed the manuscript; AV, RO, and EN coordinated flow cytometry assays and performed immunophenotypic analysis; GD performed statistical analysis; SC, BF and MC recruited and clinically followed the patients.
Funding
This work was supported by the Italian Ministry of Health (Grant GR-2016–02363570 to VB), the Associazione Italiana Sindrome di Shwachman (AISS) (Grants #AISS2017 to AV and MC), and CURE Childhood Cancer and National Institutes of Health R01-HL128173 (to SJC).
Abbreviations Key
- AOUI
Azienda Ospedaliera Universitaria Integrata
- SDS
Shwachman-Diamond syndrome
- SBDS
Shwachman-Bodian-Diamond syndrome gene
- EFL1
Elongation Factor Like GTPase 1
- DNAJC21
DnaJ Heat Shock Protein Family (Hsp40) Member C21
- SRP54
Signal Recognition Particle 54
- MDS
Myelodysplastic syndrome
- DN T cells
Double Negative T cells
- SD
Standard Deviation
- UPN
Unique Patient Number
- SS
Side Scatter
- FS
Forward Scatter
Footnotes
Conflict of Interest Statement
Authors disclose no competing interests.
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