|Year : 2019 | Volume
| Issue : 2 | Page : 207-214
The effect of age on the CD16-positive cell count in human male blood
Elsayed S Hamed, Esam O Kamel, Mohammad A Kasem
Medical Histology and Cell Biology Department, Faculty of Medicine, Al-Azhar University, Assiut, Egypt
|Date of Submission||14-Jun-2019|
|Date of Decision||16-Jun-2019|
|Date of Acceptance||27-Jun-2019|
|Date of Web Publication||23-Oct-2019|
Esam O Kamel
Medical Histology and Cell Biology Department, Faculty of Medicine, Al-Azhar University, Assiut 71622
Source of Support: None, Conflict of Interest: None
Background Immuno-senescence is a process that affects all cell components of the immune system. Age-associated changes have been demonstrated in different aspects of the innate immunity system, including natural killer cells, neutrophils, and monocytes.
Aim To identify and compare the effect of aging on the number of CD16+ve cells in the blood among different age groups of Egyptian human males.
Patients and methods A total of 40 male donors (under legal consents) were classified into four groups. Each group involved 10 individuals. Group 1 individuals aged from 5 to 10 years, group 2 individuals aged from 15 to 25 years, group 3 individuals aged 30–40 years, whereas group 4 individuals aged from 50 to 60 years. Samples were obtained from each donor and divided into two parts. One part was subjected to complete blood counting and the other part was investigated by immunohistochemistry to detect CD16+ve cells. The collected data were tabulated and statistically analyzed by analysis of variance test using SPSS program.
Results Our results showed a high significant difference among age groups regarding CD16+ve cell count, with higher count in group 4.
Conclusion We can conclude that the number of CD16-positive cells is directly proportional to the advancement of age in human males. This increase possibly represents an immune response against the abnormal products of aging.
Keywords: blood, CD16+ve cells, human male
|How to cite this article:|
Hamed ES, Kamel EO, Kasem MA. The effect of age on the CD16-positive cell count in human male blood. Al-Azhar Assiut Med J 2019;17:207-14
|How to cite this URL:|
Hamed ES, Kamel EO, Kasem MA. The effect of age on the CD16-positive cell count in human male blood. Al-Azhar Assiut Med J [serial online] 2019 [cited 2020 May 30];17:207-14. Available from: http://www.azmj.eg.net/text.asp?2019/17/2/207/269767
| Introduction|| |
Aging is a complex phenomenon characterized by progressive loss of tissue homeostasis with decline of normal cellular functions and capacity for replication ,. Several trials have been conducted to explain how the process of aging may exist. A recent theory has explained aging as an extremely complex, multifactorial process . Therefore, the different theories of aging should not be considered as mutually exclusive but complementary to each other in the explanation of some or all the features of the normal aging process .
CD16, also known as FcγRIII, is a cluster of differentiation molecule found in blood on the surface of natural killer cells (NK cells), neutrophils, and monocytes . CD16 has been identified as Fc receptors, FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which participate in signal transduction . Being the most well-researched membrane receptor implicated in triggering lysis by NK cells, CD16 is a molecule of the immunoglobulin superfamily involved in antibody-dependent cellular cytotoxicity . CD16 is expressed by blood monocytes, neutrophils, and NK cells. These cells represent the first line of defense against infectious and malignant diseases.
NK cells are lymphocytes that are critical for cellular and innate immunity. They comprise ∼10–20% of lymphocytes in the human peripheral blood ,. Phenotypically, NK cells are known to express various levels of CD56 and CD16, and lack expression of T cell marker CD3 and the B cell marker CD19. NK cells have the capacity to induce cell lysis or apoptosis in virus-infected cells and tumor cells; the underlying mechanism for this function is known to be regulated by a balance between various activating and inhibitory receptors ,.
Changes in peripheral blood NK cell count, percentage of NK cell subtypes, NK cell receptor expression, and NK cell activity have been reported in various disease conditions and also in healthy populations ,.
Sex can affect the distribution of lymphocytes. A few studies have shown that men have a higher percentage of NK cells than women ,. However, NK cell cytotoxicity was shown to be higher in women ,. In terms of ethnic factors, the number and subset distribution of NK cells among different races (Chinese, White, and African) have been studied by Feng et al.  who showed that the absolute NK cell count in Chinese patients was significantly higher than that in Whites. Therefore, NK cell proportions and functions are diverse depending on age, sex, and ethnicity, suggesting the importance of establishing local reference ranges in healthy volunteers.
Monocytes are nongranular leucocytes developed and matured in bone marrow, transit in blood, and migrate into tissues where they function as macrophages . There are three known types of monocytes in human blood : the classical monocytes are characterized by expressing high levels of CD14 cell surface receptor, the nonclassical monocytes show low level expression of CD14 and additional co-expression of the CD16 receptor , and the intermediate type with high level expression of CD14 and low level expression of CD16.
Based on CD16 expression, human monocytes can be distinguished into two major subsets (CD16− and CD16+). Moreover, they can be further subdivided via additional surface markers like CD56 . Monocytes that express low level of CD56 are usually CD16− and they increase in number in inflammatory conditions like rheumatoid arthritis . The CD16+ and CD16− monocyte populations express similar levels of CD32, but CD64 is preferentially expressed on the CD16− subtype .
Neutrophils are polymorphonuclear leukocytes that are developed in the bone marrow from myeloid colonies and live for a short time in the circulation. They represent the most abundant leukocyte population in the blood . They are phagocytic cells that constitute the first cellular component of innate immune defense . They quickly migrate to sites of injury and inflammation where they remove pathogens through professional phagocytosis. For identification of human neutrophil populations, CD11b, CD14, CD15, CD16, and CD62L are used individually or in some combination , as all previously mentioned CDs are expressed on the surface of human neutrophils.
In this study, we analyzed CD16+ve cell count in a healthy population and established reference ranges relative to age. Furthermore, we analyzed the association between CD16+ve cells and other blood cellular components in different age groups.
| Patients and methods|| |
A total of 40 male Egyptians from Al Hussein Hospital, Cairo, under their (or their parents) written consents and the consent of the ethical review board of the Faculty of Medicine, Al-Azhar University, were randomly chosen and classified into four groups according to their age. The age groups were designed according to the availability of volunteers. Each group consisted of 10 males as follow:
- Group 1: aged 5–10 years (n=7).
- Group 2: aged 15–25 years (n=20).
- Group 3: aged 30–40 years (n=35).
- Group 4: aged 50–60 years (n=55).
- Collecting blood samples: two samples were collected from each individual; one for complete blood count (CBC) and another sample for immunohistochemical study of CD16-positive cells.
- Sample for CBC.
- A 3 ml peripheral blood sample was obtained through venipuncture from the superficial veins of the forearm and collected in vacutainer tubes with sodium heparin and EDTA. Then counting of blood cells was done to confirm that the CBCs are within normal.
- Sample for centrifugation and buffy coat separation
- A 5 ml fresh peripheral blood sample was obtained through venipuncture from each individual and collected in vacutainer tubes (Al-Gomhoria Company, Assiut, Egypt) with sodium heparin and EDTA.
- Centrifugation and separation of the buffy coat was done by using Eppendorf centrifuge 5430R as follows:
- Lymphocyte separation medium ‘Ficoll’ (code#17-829E; Lonza Company, Basel, Switzerland).
- PBS (code#17-512F; Lonza Company, Basel, Switzerland).
- Procedures (according to the manufacturer manual from Lonza Company, Basel, Switzerland):
- Blood sample diluted with 5-ml PBS solution.
- A 3-ml Ficoll solution was added to a 15-ml conical tube (Falcon tube) (Al-Gomhoria Company).
- Carefully, a layer of 10-ml diluted blood was suspended over 3-ml Ficoll in the 15-ml conical tube.
- Sample was centrifuged at 400g for 30–40 min at 18–20°C.
- The layer of mononuclear cells was transferred to a sterile Falcon tube using a sterile pipette.
- Three volumes of PBS solution were added to the mononuclear cells in the Falcon tube.
- The sample was centrifuged at 400g for 20 min at 20°C.
- The supernatant was removed.
- A sample of 100 μl was taken from the remaining pellet.
- Sample mounting
- By using a micropipette, 10 μl of the pellet was mounted on a coated charged slide.
- After mounting, the sample was dried at room temperature for 10 min.
- Sample was fixed in cold acetone for 5 min, and then processed by immunohistochemistry.
- Immunohistochemical study
Immunohistochemical study (according to the manufacturer manual) was done by using CD16 monoclonal antibody (code# sc-20052; Santa Cruz Biotechnology, Santa Cruz, California, USA).
Tonsillar tissue samples were obtained from patients undergone tonsillectomy at Al-Housain University Hospital under their written consents. Samples were fixed, processed, and embedded in paraffin and used as positive control for the CD16 monoclonal antibody.
Corneal tissue samples were obtained from patients undergoing corneal transplantation under their written consents at Al-Housain University Hospital. Samples were fixed, processed, and embedded in the same manner as the tonsil and used as negative control for the CD16 monoclonal antibody.
Additionally, tissue sections from the tonsils were cut and processed by immunohistochemistry without adding CD16 antibody, which were also used as a negative control.
Identification of CD16-positive cell morphological values among age groups
This was done via computerized image analysis by using Image Pro Plus version 6 software (Media Cybernetics 2006, Maryland, United States). We measured the positive cell area, diameter, and perimeter of CD16-positive cells.
| Results|| |
The tonsillar positive control tissue showed high staining intensity of CD16-positive nuclei visualized with DAB and counterstained with hematoxylin ([Figure 1]).
|Figure 1 A photomicrograph of a section from a tonsil used as a positive control for CD16 monoclonal antibody and counterstained by hematoxylin showing positive cells (black arrows) (×400).|
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The tonsillar negative control tissue (without adding CD16 antibody) was completely negative ([Figure 2]). The corneal negative control tissue showed completely negative expression of CD16 ([Figure 3]).
|Figure 2 A photomicrograph of a section from the tonsil processed by immunohistochemistry without adding CD16 and counterstained by hematoxylin showing no positive reactions of cells (×400).|
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|Figure 3 A photomicrograph of a cornea processed by immunohistochemistry using CD16 monoclonal antibody and counterstained by hematoxylin showing negative reaction of cells (×400).|
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The cells were examined for CD16-positive cells by using a light microscope. The positive cells were counted in different groups by hemocytometer. The data were analyzed by Microsoft Excel to study the possible statistical correlations among the four groups.
The collected data were revised, tabulated, and analyzed using SPSS version 22.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Quantitative variables were expressed as mean±SD. Normal distribution of the quantitative variables was tested by Shapiro–Wilk test. The effect of variables was observed and compared together by one-way analysis of variance. The significance of the mean difference between the groups was done by Tukey’s post-hoc test. All results were considered statistically significant at the level of P value less than 0.05.
The young age group showed the lowest levels of CD16-positive cells count ([Table 1] and [Figure 4]).
|Table 1 Showing group 1 cases with their ages in years and CD16+ve cell count per mm3|
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|Figure 4 A photomicrograph showing immunohistochemical expression of CD16-positive cells in group 1 that show 3 CD16+ve cells (×400).|
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The young adult group showed slightly higher levels of CD16-positive cells compared WITH group 1 ([Table 2] and [Figure 5]).
|Table 2 Group 2 cases with their ages in years and CD16+ve cell count per mm3|
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|Figure 5 A photomicrograph showing immunohistochemical expression of CD16-positive cells in group 2 that show 4 CD16+ve cells (×400).|
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In the adult group, the expression of CD16-positive cell count was higher than both group 1 and 2 ([Table 3] and [Figure 6]).
|Table 3 Group 3 cases with their ages in years and CD16+ve cell count per mm3|
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|Figure 6 A photomicrograph showing immunohistochemical expression of CD16-positive cells in group 3 that show 5 CD16+ve cells (×400).|
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The oldest group expressed the highest levels of CD16-positive cells compared with other groups ([Table 4] and [Figure 7]).
|Table 4 Group 4 cases with their ages in years and CD16+ve cell count per mm3|
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|Figure 7 A photomicrograph showing immunohistochemical expression of CD16-positive cells in group 4 that show 6 CD16+ve cells (×400).|
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CD16-positive cell morphological values among age groups
The relation between CD16-positive cell count (mean±SD) among age groups was investigated by F-test (analysis of variance) analysis, which showed high statistically significant difference among age groups, with higher count in group 4 ([Table 5]).
Computerized image analysis was done by Image Pro Plus version 6 software (Media Cybernetics 2006). We measured the positive cell area, diameter, and perimeter. We found that all the morphological values were within standard normal histological parameters of CD16-positive cells ([Table 6] and Charts 1-4).
|Table 6 The table shows area, diameter, and perimeter of CD16+ve cells by using image analysis on 120 samples, comparing data among different age groups|
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| Discussion|| |
This study was designed to find any possible relationship between aging and the parameters of the CD16-bearing cells in the human normal male blood. We tried to estimate the numerical standards of these cells in normal (healthy) Egyptian males, which may help in understanding the nature of these immune cells in different ages.
The expression of FcγRIIIA (CD16) by immune cells is vital for antibody-dependent cellular cytotoxicity against cells coated with antibody such as abnormal or virus-infected cells ,. In the human blood, there are three cells that may express CD16, which are monocytes, neutrophils, and NK cells .
Experimentally, it was found that mice deficient in FcγRIV (the murine homolog of human CD16) exhibit defects in several models of antibody-dependent cellular cytotoxicity . Another study showed that the CD16+ monocytes of leukemia patients have a potent antibody-dependent cellular cytotoxic activity and suggested its therapeutic use for cancer and infectious diseases .
It is clinically established that aging is associated with increased susceptibility to infections and malignancy. Development of immune senescence is an important cause which affects all cell compartments of the immune system ,,,. Additionally, both T and B lymphocyte subpopulations are diversely affected by age .
If we compare the size parameters of the CD16-positive cells among the four groups ([Table 6] and Charts 2–4), we will generally find that there is a slight increase in the mean area, diameter, and perimeter with aging. The CD16-positive cells appear gradually larger from group 1 to group 4. Although the numbers are generally close to each other, the difference was constantly seen in the three parameters of size among the four groups.
This may be possibly explained by the nature of the CD16 expression in the blood cells, as it is normally expressed by monocytes, neutrophils, and NK cells . As the three cells have variable diameters and sizes in different conditions, the expression of CD16 appears to occur more frequently on larger cells than in smaller cells with aging. The number of positive ‘monocytes and large activated NK cells’ may be relatively increased in older ages. However, this explanation may be better confirmed in the future studies with more selective CD markers to achieve accurate deferential results.
Our results showed a high significant difference among age groups regarding CD16-bearing cell count. The CD16-positive cell count was directly proportional to age. The positive cells increased in ascending manner from group 1 (which showed the lowest positive cell count) to group 4 (which showed the maximum positive cell count) ([Table 1],[Table 2],[Table 3],[Table 4],[Table 5] and Chart 1).
A previous study  reported increasing numbers of monocytes, neutrophils, and NK cells with the advance of age, which are concomitant with our results. The study explored the effects of ageing on the immune system by flow cytometry and also reported a progressive decrease in the percentage of total lymphocytes. The number of NK cells increased in elderly patients but declined from infancy to adulthood. The percentages of T cells increased with age from infant to adulthood and then declined.
Previous studies showed similar results to our work ,, as they reported a higher number of the CD16-positive NK cells in the peripheral blood of elderly patients than in young adults.
Apoil et al.  conducted a study about the effect of age and sex in 253 healthy blood donors aged from 19 to 67 years. They had investigated the frequencies of 38 subpopulations of lymphocytes. Their study showed a significant decrease of the frequencies of T cells among both CD4+ and CD8+ T lymphocytes with aging.
One of the most accepted theories about aging suggests that the production of free radicals is the major factor of the aging process. Free radicals lead to an inflammatory process, which further promotes their release . This may explain our results, as CD16-bearing cells possibly respond to this inflammatory phenomenon aiming to eliminate the abnormal products of aging.
This explanation looks reasonable, as it is documented that failure of the immune response may lead to cancer. Le Garff-Tavernier et al.  reported that older people with a low NK cell count have a risk of mortality three times higher than those with high NK cell counts, in the first 2 years of follow-up. Accordingly, the increase in the NK cell population with age may be interpreted as an important defense factor for promoting longevity.
| Conclusion|| |
From this study, we can conclude that the number of CD16-positive cells is directly proportional with the advance of age in human males. This increase possibly represents an immune response against the abnormal products of aging.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Campisi J. Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 2005; 120:513–522.
Reid FT, Atzmon G, Gheorghe C, Liang HQ, Lowes C, Greally JM, Barzilai N. Tissue-specific dysregulation of DNA methylation in aging. Aging Cell 2010; 9:506–518.
Wensink MJ, van Heemst D, Rozing MP, Westendorp RG. The maintenance gap: a new theoretical perspective on the evolution of aging. Biogerontology 2012; 13:197–201.
Matteo T, Zamboni V, Ferrini A, Cesari M. The aging process and potential interventions to extend life expectancy. Clin Interv Aging 2007; 2:401–412.
Janeway CA, Travers P, Walport M, Shlomchik MJ. Immunobiology: the immune system in health and disease. 5th edition. New York, NY: Garland Science; 2001.
Vivier E, Morin P, O’Brien C, Druker B, Schlossman SF, Anderson P. Tyrosine phosphorylation of the Fc gamma RIII (CD16): zeta complex in human natural killer cells.Induction by antibody-dependent cytotoxicity but not by natural killing. J Immunol 1991; 146:206–210.
Hazeldine J, Lord JM. The impact of ageing on natural killer cell function and potential consequences for health in older adults. Ageing Res Rev 2013; 12:1069–1078.
Hazeldine J, Hampson P, Lord JM. Reduced release and binding of perforin at the immunological synapse underlies the age-related decline in natural killer cell cytotoxicity. Aging Cell 2012; 11:751–759.
Moretta L, Biassoni R, Bottino C, Cantoni C, Pende D, Mingari MC, Moretta A. Human NK cells and their receptors. Microbes Infect 2002; 4:1539–1544.
Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL et al.
Innate or adaptive immunity?The example of natural killer cells. Science 2011; 331:44–49.
Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 2005; 106:376–383.
Mandal A, Viswanathan C. Natural killer cells: in health and disease. Hematol Oncol Stem Cell Ther 2015; 8:47–55.
Santagostino A, Garbaccio G, Pistorio A, Bolis V, Camisasca G, Pagliaro P, Girotto M. An Italian national multicenter study for the definition of reference ranges for normal values of peripheral blood lymphocyte subsets in healthy adults. Haematologica 1999; 84:499–504.
Jentsch-Ullrich K, Koenigsmann M, Mohren M, Franke A. Lymphocyte subsets’ reference ranges in an age-and gender-balanced population of 100 healthy adults, a monocentric German study. Clin Immunol 2005; 116:192–197.
Morikawa K, Morikawa S, Nakano A, Furuya H, Takagi C, Oseko F, Note S. Natural killer cell activity against a variety of target cell lines in normal persons: NK-target sensitivity and effect of age and sex on NK levels. Jpn J Med 1986; 25:46–51.
Al-Attar A, Presnell SR, Peterson CA, Thomas DT, Lutz CT. The effect of sex on immune cells in healthy aging: elderly women have more robust natural killer lymphocytes than do elderly men. Mech Ageing Dev 2016; 156:25–33.
Feng YM, Zhang RJ, Zhu H, Peng H, Zhou XP, Hong KX et al.
Comparison of the quantities and subset distributions of natural killer cells among different races. Chin Med J (Engl) 2010; 123:3272–3276.
Nichols BA, Dorothy Ford Bainton DF, Farquhar MG. Differentiation of monocytes: origin, nature, and fate of their azurophil granules. J Cell Biol 1971; 50:498–515.
Ziegler-Heitbrock L. Nomenclature of monocytes and dendritic cells in blood. Blood 2010; 116:74–80.
Ziegler-Heitbrock L. The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol 2007; 81:584–592.
Sconocchia G. Phenotype and function of a CD56+ peripheral blood monocyte. Leukemia 2004; 19:69–76.
Krasselt M, Baerwald C, Wagner U, Rossol M. CD56+ monocytes have a dysregulated cytokine response to lipopolysaccharide and accumulate in rheumatoid arthritis and immunosenescence. Arthritis Res Ther 2012; 15:139–149.
Wong KL. Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets. Blood 2011; 118:e16–e31.
Mócsai A. Diverse novel functions of neutrophils in immunity, inflammation, and beyond. J Exp Med 2013; 210:1283–1299.
Bzowska M, Hamczyk M, Skalniak A, Guzik K. Rapid decrease of CD16 (FcγRIII) expression on heat-shocked neutrophils and their recognition by macrophages. J Biomed Biotechnol 2011; 2011:14.
Pillay J, Kamp VM, vanHoffen E, Visser T, Tak T, Lammers JW. A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1. J Clin Invest 2013; 122:327–336.
Yeap WH, Wong KL, Shimasaki N, Teo ECY, Quek JKS, Yong HX et al.
CD16 is indispensable for antibody dependent cellular cytotoxicity by human monocytes. Sci Rep 2016; 6:34310.
Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol 2008; 9:503–510.
Nimmerjahn F, Lux A, Albert H, Woigk M, Lehmann C, Dudziak D et al.
FcγRIV deletion reveals its central role for IgG2a and IgG2b activity in vivo. Proc Natl Acad Sci USA 2010; 107:19396–19401.
Valiathan R, Ashman M, Asthana D. Effects of ageing on the immune system: infants to elderly. Scand J Immunol 2016; 83:255–266.
Fulop T, Larbi A, Kotb R, de Angelis F, Pawelec G. Aging, immunity, and cancer. Discov Med 2011; 11:537–550.
Le Garff-Tavernier M, Béziat V, Decocq J, Siguret V, Gandjbakhch F, Pautas E et al.
Human NK cells display major phenotypic and functional changes over the life span. Aging Cell 2010; 9:527–535.
Gounder SS, Abdullah BJJ, Radzuanb NEL, Zain FDB, Sait NBM, Chua C, Subramani B. Effect of aging on NK cell population and their proliferation at ex vivo culture condition. Anal Cell Pathol (Amst) 2018; 2018:7871814.
Harrison D, Phillips JH, Lanier LL. Involvement of a metalloprotease in spontaneous and phorbol ester-induced release of natural killer cell-associated FcgRIII (CD16-II). J Immunol 1991; 147:3459–3465.
Phan MT, Chun S, Kim SH, Ali AK, Lee SH, Kim S et al.
Natural killer cell subsets and receptor expression in peripheral blood mononuclear cells of a healthy Korean population: reference range, influence of age and sex, and correlation between NK cell receptors and cytotoxicity. Hum Immunol 2016; 78:103–112.
Apoil PA, Puissant-Lubrano B, Congy-Jolivet N, Peres M, Tkaczuk J, Roubinet F, Blancher A. Influence of age, sex and HCMV serostatus on blood lymphocyte subpopulations in healthy adult. Cell Immunol 2017; 314:42–53.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]