PROOF COVER SHEET

Author(s): Rabab El Hawary, Safa Meshaal, Diana Nagy, Ingy Fikry, Radwa Alkady, Dalia Abdel Aziz, Nermeen
Galal, Jeanette Boutros, Aisha El Marsafy, and Reem Jan

Article title: Study of naı̈ve and memory cells in a cohort of Egyptian chronic granulomatous disease patients

Article no: LRST_A_996818

Enclosures: 1) Query sheet

2) Article proofs

Dear Author,
Please check these proofs carefully. It is the responsibility of the corresponding author to check against the original
manuscript and approve or amend these proofs. A second proof is not normally provided. Informa Healthcare cannot be
held responsible for uncorrected errors, even if introduced during the composition process. The journal reserves the right
to charge for excessive author alterations, or for changes requested after the proofing stage has concluded.

The following queries have arisen during the editing of your manuscript and are marked in the margins of the proofs.
Unless advised otherwise, submit all corrections using the CATS online correction form. Once you have added all your
corrections, please ensure you press the ‘‘Submit All Corrections’’ button.

Please review the table of contributors below and confirm that the first and last names are structured correctly and that
the authors are listed in the correct order of contribution.

Contrib. Prefix Given name(s) Surname Suffix

No.
1 Rabab El Hawary
2 Safa Meshaal
3 Diana Nagy
4 Ingy Fikry
5 Radwa Alkady
6 Dalia Abdel Aziz
7 Nermeen Galal
8 Jeanette Boutros
9 Aisha El Marsafy
10 Reem Jan

AUTHOR QUERIES

Q1: Please specify the significance of asterisks appearing in table 2.

 1 61
2 http://informahealthcare.com/rst 62
3 ISSN: 1079-9893 (print), 1532-4281 (electronic) 63
4 64
J Recept Signal Transduct Res, Early Online: 1–6
5 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10799893.2014.996818 65
6 66
7 67
8 RESEARCH ARTICLE 68
9 69
10 70
11 Study of naı̈ve and memory cells in a cohort of Egyptian chronic 71
12 granulomatous disease patients 72
13 73
14 Rabab El Hawary1, Safa Meshaal1, Diana Nagy1, Ingy Fikry1, Radwa Alkady2, Dalia Abdel Aziz2, Nermeen Galal2, 74
15 Jeanette Boutros2, Aisha El Marsafy2, and Reem Jan1 75
16 76
17 1 2
Clinical and Chemical Pathology Department and Pediatrics Department, Faculty of Medicine, Cairo University, Cairo, Egypt 77
18 78
19 79
20 Abstract Keywords 80
21 Context: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder caused CD27, CD45RA, CD45RO, Chronic 81
22
by inherited defects in the NADPH oxidase complex which may be involved in important granulomatous disease, memory cells, 82
pathways that connect innate and adaptive immunity. Objectives: Characterize the naive and naı̈ve cells
23 memory compartment of B and T lymphocytes in patients with CGD. Methods: Twenty CGD 83
24 patients and twenty healthy controls matched for age and sex were enrolled in this study. Flow 84
History
25 cytometric assessment of the naı̈ve and memory compartments of peripheral blood 85
lymphocytes was done using cell surface markers CD45RO, CD45RA, CD27, CD3 and CD19. Received 29 August 2014
26 86
Results: There were 15 (79%) autosomal recessive CGD patients (8 females (53%) and 7 males Revised 6 December 2014
27 Accepted 6 December 2014 87
(47%), 100% positive parental consanguinity) and four (21%) X-linked CGD patients. On
28 comparing the 3 groups; AR CGD, X-linked CGD and controls, there was a positive statistical Published online 2 2 2 88
29 significant difference for the percentage and absolute count of CD19 + CD27+ memory B cell 89
30 (p ¼ 0.028 and p ¼ 0.047 respectively), CD45RA cells (with p values of p ¼ 0.000 and 0.033, 90
31 respectively), the naı̈ve compartment CD3 + CD45RA+ cells percentage and absolute counts 91
32
(p ¼ 0.005, 0.01respectively), CD3 + CD27 + cells percentage and absolute counts (p ¼ 0.001, 92
0.012 respectively), CD3 + CD45RA + CD27+ cells percentage and absolute counts (p ¼ 0.015,
33 0.005, respectively). The significance was mainly attributed to the decrease in the X-linked 93
34 group than control group. Conclusion: There was an altered naı̈ve and memory B profile in CGD 94
35 patients, this may increase susceptibility of the patients to opportunistic infections and 95
36 autoimmune disorders. T-cell alterations have to be interpreted cautiously especially in the 96
37
presence of infections. 97
38 98
39 99
40 100
41 Introduction to opportunistic infections and autoimmune disorders have 101
42 also been reported in CGD patients (5). 102
Chronic granulomatous disease (CGD) is a primary immuno-
43 T lymphocytes are classified into naı̈ve or antigen 103
deficiency disorder caused by inherited defects in the NADPH
44 experienced populations (memory) based on markers as 104
oxidase complex (1). This enzyme complex is used by
45 CD45, a protein tyrosine phosphatase regulating src-family 105
phagocytic cells to generate microbicidal superoxide and its
46 kinases expressed on all hematopoietic cells. Based on 106
metabolites hydrogen peroxide, hydroxyl anion, and hypo-
47 alternative splicing of exons that comprise the extracellular 107
chloric acid (2). Decreased superoxide production represents
48 domain it can have many isoforms (CD45RA, CD45RB, 108
the major pathophysiologic mechanism during CGD, not only
49 CD45RC and CD45RO) which can be identified specifically 109
because NADPH oxidase is a critical host defense mechan-
50 by the binding of monoclonal antibodies and are differentially 110
ism, but also because it may be associated with important
51 expressed depending on the cell’s activation status (6). 111
pathways that connect innate and adaptive immunity (3).
52 CD45RA is expressed on naı̈ve T cells. After antigen 112
Autoimmune diseases resembling systemic lupus erythema-
53 experience, central and effector memory T cells gain expres- 113
tosus and inflammatory bowel disease are also experienced by
54 sion of CD45RO and lose expression of CD45RA (7). CD27 114
CGD patients and their relatives (4). Increased susceptibility
55 and CD28 are also highly expressed by the naive T cell 115
56 population while CD27 is considered an immunophenotypic 116
57 marker identifying peripheral blood memory B cells (8). 117
58 There is an established O generating NADPH oxidase in 118
Address for correspondence: Dr Rabab El Hawary, Clinical and
59 Chemical Pathology Department, Faculty of Medicine, Cairo B cells, however differing from that in professional phago- 119
60 University, Cairo, Egypt. E-mail: rabab_elhawary@hotmail.com cytes; for instance, oxidants generated at low levels can act as 120
 2 R. El Hawary et al. J Recept Signal Transduct Res, Early Online: 1–6

121 signaling molecules, and B cells produce only a small amount Kendall, FL) and CXP Software version 2.2. The absolute 181
122 of superoxide even when fully stimulated by various stimuli. numbers of each lymphocyte subset was calculated from the 182
123 B lymphocytes use their slowly generated O 2 and H2O2 for percentage of positive cells multiplied by the absolute PBL 183
124 other purposes like acting as second messenger in signal count. 184
125 transduction pathways (9). 185
126 This study was done to characterize the naive and memory 186
Statistical analysis
127 compartments of B and T lymphocytes in patients with CGD 187
128 and establish correlation with their clinical phenotype. Data were statistically described in terms of mean ± standard 188
129 deviation (±SD), median and range, or frequencies (number of 189
130 Subjects and methods cases) and percentages when appropriate. Comparison 190
131 between cases and controls was done using the Student t-test 191
The study included 20 patients with CGD (CGD group) and 20
132 for independent samples in comparing 2 groups when normally 192
healthy age- and sex-matched group (control group). The
133 distributed and the Mann–Whitney U-test for independent 193
patients were recruited from the Primary Immunodeficiency
134
clinic at Cairo University Specialized Pediatric Hospital, from samples when not normally distributed. Comparison between 194
135 more than two groups was done using one way analysis of 195
2012 to 2014. The study was approved by the Clinical
136 variance (ANOVA) test with Bonferroni 2-group comparisons. 196
Pathology Department‘s ethical committee. All patients were
137 p values less than 0.05 was considered statistically significant. 197
diagnosed as CGD according to the European Society
138 All statistical calculations were done using computer program 198
of Immunodeficiency Diseases (ESID’s) diagnostic
139 SPSS (Statistical Package for the Social Science; SPSS Inc., 199
criteria, mainly presenting with deep seated infection (liver,
140 Chicago, IL) release 15 for Microsoft Windows (2006). 200
perirectal or lung abscess; adenitis; or osteomyelitis) due to
141 201
Staphylococcus, Serratia Marcescens, Candida or Aspergillus,
142 202
and diffuse granulomata in respiratory, gastrointestinal or Results
143 203
urogenital tracts.
144
Patients were subjected to thorough history taking and This study was conducted on twenty CGD patients and 20 204
145
meticulous examination. All samples were withdrawn after healthy controls matched for age and sex. All patients were 205
146
patients’ guardians’ consents were obtained. Control samples diagnosed based on their clinical picture (multiple abscesses, 206
147
were leftover samples from routine complete blood count recurrent infections with catalase positive microorganisms) as 207
148
(CBC) done for elective procedures. well as by the flow cytometric dihydrorhodaminefluorescent 208
149 (DHR) test for functional assessment of the NADPH oxidase 209
150 enzyme function. 210
151 Flow cytometry There were 15 CGD patients with autosomal recessive 211
152 Anticoagulated (EDTA) peripheral blood specimens were pattern (AR) 79%, 8 females (53%) and 7 males (47%), 100% 212
153 obtained by phlebotomy and analyzed within 24 hours of parents consanguinity with all mothers showing normal SI on 213
154 sampling. DHR test (Some patients were confirmed by detecting 214
155 affected protein by flow cytometry, data not shown). Four 215
156 Dihydrorhodaminefluorescent assay of granulocytes in CGD male CGD patients were classified as X-linked pattern (21%), 216
157 patients based on DHR profile and confirmed by maternal carrier 217
158 patterns. One male patient was not classified, he had positive 218
159
In brief, cells were incubated with dihydrorhodamine (Sigma- consanguinity but his mother was not available for testing. 219
160
Aldrich, St. Louis, MO) for 15 min, followed by stimulation Age of patients ranged from 40 days to 12 years (mean 220
161
with phorbolmyristate acetate (Abcam, Cambridge, MA) for 5.91year ±4.6 SD), descriptive data of the patients are 221
162
30 min, then washed and RBCs were lysed for 10 min summarized in Table 1. 222
163
(Versalyse, Beckman Coulter, Paris, France). A stimulation Serum immunoglobulin G (IgG) was elevated in 10/12 223
164
index (SI) was calculated for gated neutrophils by dividing the patients. A study of the naı̈ve and memory compartments of 224
165
mean fluorescent intensity (MFI) of stimulated cells by the peripheral blood lymphocytes was done by measurement of 225
166
MFI of unstimulated cells. SI of 70 was considered the cutoff CD45RO, CD45RA, CD27 on gated lymphocytes as well as 226
167
for our laboratory. on gated B cells (CD19+ cells) and T cells (CD3+ cells). 227
168 Absolute count for each subset was calculated from absolute 228
Assessment of the immune cells naı̈ve and memory
169 lymphocytic count in the complete blood counts. The mean 229
compartment
170 percentages and counts for all the lymphocyte populations are 230
171 In all experiments, B cell subsets were identified by shown in Table 2. 231
172 expression of CD19 (PE CY5) and T cell subsets by There was no statistically significant differences in the 232
173 antiCD3 (ECD). Other Abs included were anti-CD45RO percentages and absolute count of lymphocytes, CD19+, 233
174 (FITC), anti CD45RA (PE) and CD27 (PE CY7). CD45RO+, CD3+CD45RO+ between the three groups. 234
175 All monoclonal antibodies were obtained from Beckman There was no statistical significant difference in the absolute 235
176 Coulter (Paris, France), and used as recommended by the count of CD27+ cells, however CD27+cell percentage was 236
177 manufacturers. Lymphocytes were identified by the forward statistical significance between AR CGD, X linked CGD and 237
178 and side scatter, and were set as target gate. controls (p ¼ 0.001) (X-linked versus control p ¼ 0.001, X 238
179 Approximately 10 000 labeled cells were analyzed through linked versus AR p ¼ 0.01) with least expression among the 239
180 a CYTOMICS FC 500 Flow Cytometer (Beckman Coulter, X-linked group. 240
 DOI: 10.3109/10799893.2014.996818

Table 1. Descriptive data of the CGD patients.

Age sex consanguinity DHR SI CGD pattern Affected siblings First presenting symptom Other symptoms IgG (mg/dl)
1 12 y M + 1.5 Not specified Negative Pneumonia Brain abscess –
2 6y M + 1.8 X-linked Negative Right Upper Limb abscesses Pneumonia, brain and lung abscesses 2787 (608–1229)
3 3 mon M + 2.6 X-linked Positive Recurrent cutaneous abscesses Hepatomegaly, brain abscess
4 14 y M – 1.5 X-linked 1 abortion Cervical abscess Liver abscess
5 18 mon M + 3.6 X-linked 2 Sibling deaths Pneumonia Hepatosplenomegaly, ankle osteomylitis 1460 (407–1009)
6 40 days M + 2.3 Autosomal 1 Sibling death Pneumonia Hepatosplenomegaly, lung abscess
7 7y F + 3.1 Autosomal Negative LN abscess Pneumonia, splenomegaly 1911 (608–1229)
8 10 y M + 4.2 Autosomal Negative Pneumonia Hepatosplenomegaly, wrist osteomylitis 1180 (768–1632)
9z 12.5 y F + 2.3 Autosomal Affected brother Recurrent cutaneous abscesses Liver abscess 1632 (768–1632)
10 12 y F + 3.8 Autosomal Negative Liver abscess Pneumonia, hepatomegaly 2834 (768–1632)
11z 2y M + 6 Autosomal Affected Sister Accidently discovered
12 3y F + 4.8 Autosomal Negative Pneumonia Lung and bone abscesses 1955 (423–1090)
13 18 mon M + 4.4 Autosomal 1 Sibling death Pneumonia Perianal and bone abscesses 1278 (407–1009)
14 8y M + 6.8 Autosomal 4 Abortions Otitis-draining ears Submandibular abscess 2058 (584–1509)
15 18 mon M + 2 Autosomal BCGoma Perianal abscesses 1230 (407–1009)
16* 8y F + 6.7 Autosomal 1 Affected sibling Draining ears Liver abscess, boils
17* 10 y F + 6.5 Autosomal 1 Affected sibling Purulent rhinitis Recurrent ulcers
18y 3.5 y M + 2.8 Autosomal 1 Affected sibling pneumonia Abscess, lymphadenitis, HLH 1408 (608–1229)
19y 1y F + 1.6 Autosomal 1 Affected sibling pneumonia hepatosplenomegaly
20 4.5 y F + 1.4 Autosomal 2 Abortions, 2 cousins deaths Upper respiratory tract infection Pneumonia, hepatosplenomegaly, 1500 (444–1187)
upper and lower limb abscesses,
inguinal LNs

Legend: y ¼ Year, mon ¼ Month, M ¼ Male, F ¼ Female, DHR ¼ Dihydrorhodamine assay, SI ¼ Stimulation index, IgG ¼ Immunoglobulin G, HLH ¼ Hemophagocytic Lymphohistiocytosis, LN ¼ lymph nodes
y,*,z identify siblings.
Memory cells in Egyptian CGD patients
3
 4

Table 2. Comparison of the immunophenotyping of CGD cases and controls.

Autosomal recessive CGD (n ¼ 15) X-linked CGD (n ¼ 4) Control Cases (n ¼ 20)

R. El Hawary et al.

p Value
p Value (independent
(ANOVA) sample test)
Between CGD cases
Mean SD Mean SD Mean SD 3 groups Mean SD versus controls
Gated on lymphocytes
Total lymphocytes % 37.4 14.8 30 8.3 42.06 9.64 0.184 34.88 14.22 0.078
Total lymphocytes absolute count 3143.2 1361.8 3201.3 1632.5 3592 1360.5 0.651 3322 1376 0.269
CD3% 66.6 10.6 39.5 17.1 64.63 7.93 0.000** 60.6 15.92 0.327
CD3 absolute count 2026.3 833.7 1081.7 351.3 2294 824 0.034* 1770 847 0.069
CD19% 18 9.2 12.5 6.7 20.19 8.98 0.309 17.76 9.49 0.430
Q1 CD19 absolute count 657.2 550.37 320.8 79.4 763 504 0.295 582 489 0.285
CD45RA% 68.1 8.11 39.6 21.7 71.91 8.53 0.000** 62.15 16.09 0.025*
CD45RA absolute count 2208 1036 1003.3 213.15 2635 1189 0.033* 1895 1037 0.056
CD45RO% 26.07 8.38 19.42 7.88 21.55 7.59 0.183 24.66 8.36 0.212
CD45RO absolute count 784.86 397.62 613.7 315.07 699 199 0.558 733.6 376 0.728
CD27% 50.43 10.09 30.57 18.98 57.2 10.3 0.001** 47.47 15.18 0.029*
CD27 absolute count 2289.4 2780 759.9 313.4 2086.5 915.7 0.364 1911 2455.9 0.77
CD3 + CD45RA+% 39.9 11.8 22.02 15.81 44.49 10.16 0.005** 35.52 14.26 0.033*
CD3 + CD45RA+ absolute count 1201.4 535.8 513.74 92.15 1626.2 764.4 0.010** 1014 559 0.011*
CD3 + CD45RO+% 24.6 7.8 16.9 10.18 19.32 6.9 0.084 23.24 8.5 0.13
CD3 + CD45RO+ absolute count 762.3 428.6 553.15 349.8 619 145 0.336 702 405 0.41
CD3 + CD27+% 48.08 10.4 28.47 19.2 54.2 9.7 0.001** 43.65 14.32 0.011*
CD3 + CD27+ absolute count 1493.3 663.1 689.01 319.42 1987.5 889.8 0.012* 1279 685.5 0.013*
CD19 + CD27+% 1.39 0.75 0.63 0.33 2.33 1.67 0.028* 2.32 4.92 0.99
CD19 + CD27+ absolute count 47 34.8 18.67 12.79 77.2 56.4 0.047* 59.3 87.23 0.465
CD19 + CD45RA+% 18 10.01 12.37 6.44 20.23 8.98 0.315 17.69 10.03 0.421
CD19 + CD45RA+ absolute count 668.09 589.7 322.22 94.5 764.6 504.7 0.321 590.8 522.8 0.318
Gated on CD3+cells
CD45RA% 58.5 11.4 46.77 23.3 66.04 12.39 0.035* 55.39 14.67 0.022*
CD45RO% 37.2 12.33 45.85 21.9 30.44 10.64 0.079 39.73 14.51 0.032*
CD27% 73.4 13.3 64.6 25.3 84.12 10.94 0.022* 71.8 15.6 0.008**
CD45RA + CD27+% 54.5 13.6 48.2 27.9 69.15 14.41 0.015* 52.63 16.54 0.003**
CD45RA + CD27+ absolute count 1107.7 518.07 476.13 195.04 1643.8 785.3 0.005** 936.5 536 0.004**
Gated on CD19 + cells
CD27% 8.32 3.9 5.7 1.9 11.92 8.38 0.131 10.7 13.8 0.75
J Recept Signal Transduct Res, Early Online: 1–6
 DOI: 10.3109/10799893.2014.996818 Memory cells in Egyptian CGD patients 5

241 In spite of the normal B cell compartment (CD19+) in the defect depending on finding that X-linked carriers had 301
242 CGD patients in comparison with the control group and significantly high correlation between percentage of 302
243 the high level of IgG as determinant of the B cell function (in CD27 + B cells and percent of granulocytes with normal 303
244 response to existing infections), the CD19 + CD27+ memory NADPH activity. Thus the Oxidative deficiencies might also 304
245 cells were lower in the cases showing a statistical significant alter B-cell differentiation in CGD patients and/or the traffic 305
246 difference (p ¼ 0.028 for cell percentage and p ¼ 0.047 for of B cells between blood and tissues. Other mechanisms as 306
247 absolute counts), between the X-linked CGD, AR CGD and decrease recruitment of naı̈ve B cells to the germinal centers, 307
248 controls, this difference was attributed mainly to X-linked related to altered anatomical integrity of lymphoid tissue or 308
249 group that showed least expression versus the control group. interruption of memory B cells expansion in favor of plasma 309
250 There was a highly statistical significant difference cells due to altered cytokine and chemokine environment. 310
251 between CD3 percent and absolute counts between the However, the humoral immunity as determined by IgG 311
252 3 groups (p ¼ 0.000 and 0.034). CD3 cells were lower in level was elevated in CGD patients similar to other studies 312
253 the cases; the difference in percentage was likely attributed to and is explained by recurrent infections in those patients 313
254 X-linked group versus control and versus AR group (1,10). Our study revealed a statistical significant decrease 314
255 (p ¼ 0.000 and p ¼ 0.000, respectively), however in absolute in expression of naı̈ve CD45RA+ (p ¼ 0.022) and CD27+ 315
256 counts was attributed mainly to X-linked group versus the (p ¼ 0.008) T-lymphocytes and a significant increase 316
257 controls (p ¼ 0.03). (p ¼ 0.032) in the percentage of memory CD45RO+ 317
258 Also a significant difference was found in the percentages T-lymphocytes in CGD patients compared to the healthy 318
259 and absolute count of CD45RA cells, the naı̈ve T cell control group. The defective NADPH oxidase in CGD 319
260 compartment CD3 + CD45RA+, CD3 + CD27+, CD3 + patients would lead to diminished T-cell suppression and 320
261 CD45RA + CD27+ cells, as all naı̈ve T cell compartments increased conversion of naı̈ve T-cells (CD45RA+) to memory 321
262 were lower in cases versus the control with the least T-cells (CD45RO+). The difference between the three groups 322
263 expression in the X-linked group. revealed a significant difference between the X-linked group 323
264 and the control group. 324
265 Our study was in agreement to Najera et al. (11) study 325
Discussion
266 which stated that the presence of infection in children 326
267 The aim of this study was to identify the alteration in decreased the peripheral blood CD4+CD45RA+ cells as a 327
268 peripheral blood B and T cell compartments in patients with response to an antigenic stimulus by an infective organism 328
269 CGD. We characterized memory B cells by CD27 expression -the naı̈ve cells were transformed into memory cells. 329
270 and memory and naı̈ve T cells by CD45RO, CD45RA and CD Winkelstein et al. (4) demonstrated that patients with the 330
271 27 markers. X-linked recessive form of the disease appear to have a more 331
272 In our study, CD19 B cell count was normal in the CGD serious clinical phenotype than patients with the autosomal 332
273 patients in comparison with the healthy controls. This was recessive forms of the disease. This explained why the 333
274 contradictory to the results of Moir et al. (10) whose data significant difference in our study was mostly attributed to the 334
275 revealed significantly lower CD19 B-cell count in the CGD. X linked group. 335
276 This could be attributed to immune-suppressive therapies in Kraaij et al. (12) hypothesized that reactive oxygen species 336
277 the CGD group in their study as comparison of B cell became deficiency in CGD patients might lead to decreased 337
278 non-significant on exclusion of CGD patients taking doses of T regulatory cells (Tregs) induction. This finding was 338
279 prednisone 0.1 mg/kg per day or methotrexate from the confirmed genetically: rat or human CGD Mph with mutated 339
280 analysis. p47phox or gp91phox displayed hampered Tregs induction 340
281 There was paucity of CD19 + CD27+ memory cells in and T-cell suppression. Tregs can suppress activation and 341
282 CGD patients versus the control group that did not mount to proliferation of effector T cells and thereby diminish immune 342
283 statistical significance, the contradictory findings between our responses. The decrease in Tregs in CGD patients might 343
284 study and other studies might be the result of the different explain the consumption of the naı̈ve T-cell compartment and 344
285 background and mode of inheritance as in our study most of the increase in the memory compartment. However, there was 345
286 the patients were AR due to high level of consanguineous difficulty in judging the T cell compartment in CGD patients 346
287 marriages, while in western and European studies most due to the presence of infections all the time. 347
288 patients were from the X-linked group; this could be clearly As the allogenic hematopoietic stem cell transplantation 348
289 deduced from our results; as in our patients the X-linked CGD (HSCT) is the only curative treatment for CGD, study of the 349
290 group showed statistical significant difference when com- immune system dysfunction in those patients is needed, as it 350
291 pared to the control. affects the transplantation outcome and prognosis. The results 351
292 Mohsenzadegan et al. (5) concluded that certain NADPH of the present study show that in the CGD patients, most of 352
293 components play a role in the signaling system and activation the T cell compartment is constituted from the memory 353
294 of some transcription factors in B cells; the signaling through T cells (CD3 + CD45RO+) with paucity in the naı̈ve T cells. 354
295 the CD40/CD40L system was a crucial element in the This reflects the repeated stimulations (mainly from infec- 355
296 generation of memory B cells and may be adversely affected tions) challenging the immune system in those patients. This 356
297 in patients with CGD. would be relevant in the setting of adjusting proper 357
298 Our results were also in agreement to Bleesing et al. (1), conditioning regimen for CGD patients as it was reported 358
299 who concluded that a profound reduction in B cells expressing that memory T cells are implicated in the BMT failure in 359
300 CD27 in patients with CGD was linked to NADPH oxidase immunized patients (13). 360
 6 R. El Hawary et al. J Recept Signal Transduct Res, Early Online: 1–6

361 Also the inflammatory status, the CGD patients suffer 4. Winkelstein JA, Marino MC, Johnston RB, et al. Chronic granu- 421
lomatous disease: report on a national registry of 368 patients.
362 from and proved by the present study, adds to the high risk Medicine 2000;79:155–69.
422
363 of organ toxicity and serious complications of the HSCT. 5. Mohsenzadegan M, Fattahi F, Fattahi F, et al. Altered pattern of 423
364 A reduced intensity conditioning regimen became an alter- naı̈ve and memory B cells and B1 cells in patients with chronic 424
365 native choice (14). With this method, graft rejection is more granulomatous disease. Iran J Allergy Asthma Immunol 2014;13: 425
157–65.
366 likely, but the risk of graft versus host disease (GVHD), 6. Trowbridge IS, Thomas ML. CD45: an emerging role as a protein 426
367 particularly acute GHVD and regimen-related toxicity, tyrosine phosphatase required for lymphocyte activation and 427
368 appears to be reduced (15). development. Annu Rev Immunol 1994;12:85–116. 428
369 Assessment of the naive and memory T cell compartment 7. Cossarizza A, Ortolani C, Paganelli R, et al. CD45 isoforms 429
expression on CD4+ and CD8+ T cells throughout life, from
370 in CGD patients may be needed while adjusting the proper newborns to centenarians: implications for T cell memory. Mech 430
371 conditioning regimen used before BMT. Ageing Dev 1996;86:173–95. 431
372 8. Agematsu K, Hokibara S, Nagumo H, Komiyama A. CD27: a 432
373 memory B cell marker. Immunol Today 2001;21:204–6. 433
Conclusion 9. Batot G, Paclet MH, Doussière J, et al. Biochemical and
374 immunochemical properties of B lymphocyte cytochrome b558. 434
375
Increased susceptibility of CGD patients to opportunistic 435
Biochim Biophys Acta 1998;1406:188–202.
376
infections and autoimmune disorders could be partly 10. Moir S, De Ravin SS, Santich BH, et al. Humans with chronic 436
377
explained by the altered phenotype of B lymphocytes in granulomatous disease maintain humoral immunologic memory
437
these patients. despite low frequencies of circulating memory B cells. Blood 2012;
378 120:4850–8. 438
379 11. Najera O, Gonzalez C, Toledo G, et al. CD45RA and CD45RO 439
380 Declaration of interest isoforms in infected malnourished and infected well-nourished 440
children. Clin Exp Immunol 2001;126:461–5.
381 This work was funded by Cairo University. There is no conflict of 441
12. Kraaij MD, Savage NDL, van der Kooij SW, et al. Induction of
382 interest. regulatory T cells by macrophages is dependent on production of 442
383 reactive oxygen species. PNAS 2010;12:17686–91. 443
384 References 13. Mattsson J, Ringdén O, Storb R. Graft failure after allogeneic 444
hematopoietic cell transplantation. Biol Blood Marrow Transplant
385 1. Bleesing JJ, Souto-Carneiro MM, Savage WJ, et al. Patients with 445
2008;14:165–70.
386 chronic granulomatous disease have a reduced peripheral blood 14. Ramzi M, Rezvani A, Haghighinejad H. Allogeneic hematopoietic 446
387 memory B cell compartment. J Immunol 2006;176:7096–103. stem cell transplant for high-risk adult patients with chronic 447
388 2. Segal BH, Leto TL, Gallin JI, et al. Genetic, biochemical, and granulomatous disease: first case report from Iran. Exp Clin 448
clinical features of chronic granulomatous disease. Medicine 2000; Tranplant 2014;12:490–3.
389 79:70–200. 15. Güngör T, Halter J, Klink A, et al. Successful low toxicity 449
390 3. Salmen S, Corte D, Goncalves L, et al. CD40/CD40L expression in hematopoietic stem cell transplantation for high-risk adult chronic 450
391 leukocytes from chronic granulomatous disease patients. APMIS granulomatous disease patients. Transplantation 2005;79: 451
392 2007;115:939–47. 1596–606. 452
393 453
394 454
395 455
396 456
397 457
398 458
399 459
400 460
401 461
402 462
403 463
404 464
405 465
406 466
407 467
408 468
409 469
410 470
411 471
412 472
413 473
414 474
415 475
416 476
417 477
418 478
419 479
420 480