ORIGINAL ARTICLE Year : 2023  |  Volume : 12  |  Issue : 1  |  Page : 71-77

Detection of senescent CD8+ T-lymphocyte in newly diagnosed and relapsed/refractory multiple myeloma using CD28 and CD57

Zahraa Mahdi Sareeh1, Yassmin Ali Abdul-Kareem2, Ali Mohammed Jawad3
1 Department of Pathology, Basra General Hospital, Basra, Iraq
2 Hematology Center in Medical City, Baghdad, Iraq
3 Department of Medicine, College of Medicine, University of Baghdad, Baghdad, Iraq

Date of Submission10-Jan-2023Date of Acceptance24-Feb-2023Date of Web Publication30-May-2023

Correspondence Address:
Dr. Zahraa Mahdi Sareeh
Department of Pathology, Basra General Hospital, Basra
Iraq

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ijh.ijh_6_23

BACKGROUND: Multiple myeloma is a clonal B-cell malignancy characterized by proliferation of plasma cells that secrete a complete and/or partial monoclonal immunoglobulin protein. It induces dysfunction of cytotoxic T cells that may be responsible for immune evasion and therapeutical failure of immunotherapies. T cells during senescence tend to lose co-stimulatory molecules such as CD27 and CD28 while expressing killer cell lectin-like receptor subfamily G (KLRG-1) and CD57. Therefore, enhanced knowledge about the actual status of T cells in myeloma patients is needed.
Objectives: The aims of this study were to detect the senescent cytotoxic T-cell (CD8+) in patients with refractory/resistance multiple myeloma, and to compare it with newly diagnosed multiple myeloma patients, and their implications for cellular therapies.
MATERIALS AND METHODS: This is a cross sectional study performed, from January to October 2021. Sixty multiple myeloma patients were sequentially chosen, thirty of them were newly diagnosed patients and another thirty were relapse/refractory who progress after receiving 2 lines of chemotherapy containing bortezomib and immunomodulators. And/or patients progressing after autologous stem cell transplantation. Seventeen apparently healthy age and gender matched adults were enrolled as a control group. Multicolor flow cytometry was utilized for the analyses of surface molecules CD 28 and CD57 on CD 8 positive T-lymphocyte using peripheral blood, subsequently the percentage of T cells senescent were estimate in CD 8+ T cells with CD28- and CD57+.
Results: The mean percentage of senescent CD8 positive T lymphocyte (negative CD28 and positive CD57) were significantly higher in multiple myeloma patients than control (P<0.001). There was no statistically significant difference in percentage of CD8+ T cells between newly diagnosed multiple myeloma and relapsed /refractory cases. However higher percentage of senescent T cells was found in relapsed cases. No significant correlation was found between percentage of CD8 +T cells and patients age, or duration of disease.
Conclusion: Percentage of senescent CD 8+ T cells with CD28- and CD 57+were significantly higher in MM patients compared to healthy controls. Percentage of senescent CD 8+ T cells with CD28- and CD 57+were lower in newly diagnosed MM patients compared to relapsed patients but the difference was statistically not significant

Keywords: CD28, CD57, myeloma, senescent, T-lymphocytes

How to cite this article:
Sareeh ZM, Abdul-Kareem YA, Jawad AM. Detection of senescent CD8+ T-lymphocyte in newly diagnosed and relapsed/refractory multiple myeloma using CD28 and CD57. Iraqi J Hematol 2023;12:71-7
How to cite this URL:
Sareeh ZM, Abdul-Kareem YA, Jawad AM. Detection of senescent CD8+ T-lymphocyte in newly diagnosed and relapsed/refractory multiple myeloma using CD28 and CD57. Iraqi J Hematol [serial online] 2023 [cited 2023 Jun 10];12:71-7. Available from: https://www.ijhonline.org/text.asp?2023/12/1/71/377854   Introduction 

Multiple myeloma (MM) is a malignancy of plasma-cell, characterized by the expansion of malignant plasma cells in the bone marrow (BM), leading to destruction and loss in the major organ functions.[1]

It constitutes 1% of all malignancies and around 10% of all hematologic malignancies.[2]

Virtually, all patients with MM develop from a premalignant asymptomatic stage called monoclonal gammopathy of undetermined significance. It is present in over 3% of the population above the age of 50, and the average rate of progression to MM is 1%/year.[2],[3]

Patients with MM have a significant reduce in the function of T-cells, in with changes in T-cell immune response and loss of antigen-specific T-cell function associated with disease progression. In MM, several mechanisms involved associated reductions in T-cell responsiveness including T-cell exhaustion, energy, and/or senescence.[4]

Immunosenescence is the main feature of T-cells' dysfunctional activity in MM characterized by maintained functional activity but limited proliferative capacity, with characteristic phenotypic changes including positive CD57/CD160/killer cell lectin-like receptor (KLRG1+), negative CD28, with low programmed death 1 (PD1), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression, suggesting that in MM the T-cells have a molecular signature of senescence instead of exhaustion phenotype.[5]

  Patients, Materials, and Methods 

This study is a case‒control study done in Baghdad Teaching Hospital, Hematology Department in Medical City, Baghdad, Iraq, from January to October 2021. From all patients, written informed consent was taken, and the collection of data was done according to a questionnaire form which is collected directly from patients and their data records.

The study was approved by review ethical committee of Pathology Scientific Council of Iraqi Board for Medical Specializations.

A sample of 60 adult patients with multiple myeloma has been chosen, 30 with newly diagnosed MM, and others 30 with relapsed/refractory MM patients diagnosed by workup of both consultant clinical hematologists and hematopathologists dependent on International Myeloma Working Group criteria for MM diagnosis, which was updated in 2016.[6]

Eighteen of them had relapsed after receiving therapeutic protocol containing bortezomib and/or immune-modulating drugs, whereas the remaining twelve had relapsed after autologous stem cell transplant (ASCT).

Seventeen healthy individuals' age and gender-matched were involved in the study as the control group (six males and eleven females) the shortage in markers supply during study II was behind his limited number.

Materials include fluorochrome-conjugated monoclonal antibodies for gating and surface staining of CD8 and CD28 obtained from BioLegend USD and from Biosciences (BD Pharmingen™, USA), for CD57. Monoclonal antibodies for surface staining and gating which include: (anti-CD8: Brilliant Violet 421™.‒ anti-CD28: phycoerythrin and anti-CD57: allophycocyanin.[7],[8],[9]

Blood sampling

Under the aseptic technique, 3 ml of venous blood from the antecubital fossa was taken from each patient included in this study. These 3 ml of blood were added to K3-EDTA tubes and taken for flow cytometric evaluation of the expression surface CD28, and CD57 in CD8+, and estimation percentage of CD28−, CD57+ cells out of CD8+ T-lymphocytes.[10],[11],[12] The sample analysis, percentage of expression, and data acquisition were achieved by using eight color FCM (BD FACSCanto™ II) USA and device software based on (BD FACSDiva).

Principles of the procedure of estimating senescent T-lymphocyte

Incubate whole blood with fluorochrome-conjugated monoclonal antibody reagents at room temperature in a dark place, the antibodies will bind specifically to the appropriate leukocyte antigens on the cell surface.

Gating on events with low SSC and CD8 T-lymphocyte, then the percentage of senescent T-cell estimated (CD28 − and CD57+).

Stain-lyse-wash method was used to detect the surface markers.

Data acquisition and sample analysis

Data acquisition and sample analysis were performed in a BD FACSCanto™ II (8-color, Becton Dickinson, USA), using BD FACSDiva software.

The gating strategy for cytotoxic CD8+ T-cell depends on FSC/SSC and using dot plots of CD8 expression versus intracellular complexity (SSC) SSC/CD8. A total of 100,000 events were acquired at the gate. Both CD28 and CD57 antigens are surface markers and are considered to be a positive expression in T-cell CD8+ if ≥20% [Figure 1].

Figure 1: Dot plot showing: (a) CD8 versus side scatter. (b). The expression of CD28 and CD57. SSC-H = Side scatter, PE-A = Phycoerythrin

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Statistical analysis

The studied sample data were entered and analyzed using the Statistical Package for the Social Sciences (SPSS) version 26 Released 2015.(IBM SPSS Staticsfor Windows,Armonk,NY:IBM Crop). Descriptive statistics were presented as frequencies, proportions (%), means, and standard deviations. Analytic statistics such as the Chi-square test were used to estimate the association between two categorical variables, ANOVA test, and student's t-test were used to find the association between categorical variables and continuous variables, and significant results in ANOVA test are analyzed by Tukey's post hoc test to detect which groups have a significant results. A Pearson's correlation test was used to find the correlation between two continuous variables, where a correlation coefficient between 0.2 and 0.29 mean weak correlation, 0.3–0.39 mean moderate correlation, , 0.4–0. 69 mean strong correlation, and when a correlation coefficient ≥0.7 mean very strong correlation. The P ≤ 0.05 was considered to be statistically significant.

  Results 

Steady-state samples were collected from 30 patients who are newly diagnosed with MM, 30 patients with relapsed MM, and 17 normal subjects; the demographic characteristics of the studied groups are shown in [Table 1], the samples were homogenous regarding age and gender (P = 0.063, 0.74).

The hematological parameters (hemoglobin [Hb], white blood cell [WBC], and platelet) showed a significant difference between patient groups (P ≤ 0.001, 0.035, and 0.021 respectively), [Table 2].

Table 2: Difference in hematological and biochemical tests between patients groups

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There was no significant difference in biochemical tests (blood urea and serum creatinine, serum calcium, and albumin level) between newly diagnosed and relapsed cases (P = 0.27, 0.93, 0.2, and 0.13 respectively), [Table 2].

The mean percentage of CD28−/57+ CD8+ T-cells was significantly different between the studied groups (P < 0.001) with the highest mean found in relapsed cases and lowest mean found in normal group, [Table 3].

Table 3: Difference in percentage of CD28-/57+senescent CD8+T-cells among studied groups

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The post hoc analysis of [Table 3] shows a significant difference in the percentage of the senescent CD8+ T cells between newly diagnosed MM cases and control group, and between relapsed MM cases and control group, but shows no statistically significant difference between new and relapsed MM cases, [Table 4].

In new cases of MM, there was no correlation between the percentage of senescent CD8+ T-cells and age (r = 0.23, P = 0.204), and also there was no significant correlation between BM plasma cell % and percentage of senescent CD8+ T-cells (r = 0.25, P = 0.17). In relapsed cases of MM, there was no correlation between the percentage of CD8+ T-cells and age (r = 0.23, P = 0.21), and there was no significant correlation between BM plasma % and percentage of + CD8+ T cells (r = −0.16, P = 0.38), and no correlation with disease duration [r = 0.37, P = 0.84, [Table 5]].

Table 5: Correlation between studied marker, age, and bone marrow plasma percentage in cases of multiple myeloma group

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  Discussion 

MM is a malignancy of clonal plasma cells, recently the outcomes for patients with MM have considerably improved due to introduce of approval novel agents and high-dose therapy followed by ASCT. However, MM remains incurable, with a high frequency of disease progression and relapsing.

In this study, the mean age of newly diagnosed MM patients was 65.1 ± 9.1 with a range of 47–80 years This result was comparable with other Iraqi studies done by Alwan[10] Badi et al.[11] and with a Saudi Arabian study,[12] and the mean age of relapsed MM patients included was 68.9 ± 6.2 with a range of 59–80 years. These results were comparable with American studies of Katodritou et al.[13] and Kumar et al.[14] but it differs from the Indian study of Jasrotia et al.[15]

The mean of Hb in newly diagnosed patients was 9.3 ± 1.2 g/dl which is comparable to what was reported by other studies of El-Naby et al.[16] and Kumar et al.[17]

However, the mean level of Hb in relapsed MM patients was 8.3 ± 0.9. This is lower than other studies done by Kumar et al.[14] and Richardson[18] and this may be due to the late presentation of our patients and delay in the diagnosis of relapses.

The mean level of WBCs count and platelets count is lower in relapse cases than in newly diagnose although both of them are still within the normal range. In newly diagnosed cases, the mean of WBCs count was 5.7 ± 1.7 × 109/L, and mean of the platelets count was 190.4 ± 69.2 × 109/L. These were consistent with what was reported by Alwan[10] Yassin[19] Sultan et al.[20]

While in relapsed cases, mean WBCs was 4.8 ± 1.6 × 109/L and mean platelets count was 154.6 ± 45.8 × 109/L which is comparable to what is reported by Richardson[18] Lonial et al.[21]

The percentage of senescent CD8+ T-lymphocytes was found to be higher in MM cases compared to the normal control group. This result is supported by another study done by Suen et al.[22] who evaluated the dysfunctional activity of clonal T-cells in MM cases and confirmed that immunosenescence is the main phenotype of CD8+ T-lymphocytes. These senescent T-lymphocytes carry effector phenotype characterized by positive KLRG-CD57+/CD160+/CD28 − and low PD1 and CTLA-4 expression, suggesting that T-cells in MM have a molecular signature of senescence.[22] The importance of this senescent phenotype is the possibility of a link with therapy failure, and further studies are needed to clarify the effect of the senescent phenotype on treatment decisions.

In addition, Zelle-Rieser et al.[23] evaluated the expression of inhibitory molecules PD-1, CTLA-4, 2B4, CD160, and senescence markers CD57, and CD28 on T-lymphocyte of untreated and treated MM cases in both the BM and peripheral blood. He defined that T-cells from the MM cases were impaired in the BM more than in the peripheral blood. He also established that at the tumor site (BM); the proliferation of CD8+ T cells in MM cases was more lower when compared to the healthy control group. These CD8+ T-cells carry some molecular markers related to T-cell exhaustion such as PD1 and CTLA-4, and also negative for CD28 and are positive for CD57; a senescence phenotype, the percentage of senescent CD57+ CD28 − CD8+ T cells were more lower in treated myeloma cases in related to untreated cases associated with a reduction in both proliferative capacity and function.[23] However, the current study did not include treated MM patients who are in remission, it included groups of newly diagnosed MM and patients who are relapsed MM or refractory MM patients and all these patients are having active MM. This can explain the expansion of senescent T-cells in both groups which seems to parallel disease activity rather than refractoriness of disease.

The comparison between newly diagnosed and relapsed MM showed no significant difference in the mean percentage of CD28−/57+ in CD8+ T-cells, with the highest mean percentage found in relapsed cases. This result was seen in another study done by Lee et al.[24] who found a subset of relapsed patients with a significant increase in the markers of senescence in the peripheral CD8+ T cells.[24]

Furthermore, Raitakari et al.[25] showed progressive elevation in the percentage of CD8+ T-lymphocytes with positive CD57 from patients with Stage I to those with Stage III MM. Concurrently, decrease in the percentage of lymphocytes expressing CD28. While Chung et al.[26] showed that patients with relapsed disease, post-ASCT show elevation in the percentage of CD8+ CD28 − CD57+ cells when compared to healthy donors' age-matched or cases in a complete remission post-ASCT.

The mean percentage of senescent T-cells was not different between post-ASCT-relapsed subgroup (who are 12 patients) and chemotherapy-resistant subgroup (who are 18 patients), both subgroups showed a high percentage of senescent T-cells. Sample size can explain this result in which further study including a larger number of patients is needed. Whatever the study was done by Chung et al.[26] showed that relapsing cases have a higher population of CD8+ CD28 − T-cells after transplant by 3 months or more, but before detection of clinical relapsed disease, representing their applicability in recognizing patients at higher risk of relapse, so the senescent phenotype of T-lymphocyte may help in predicting early relapsing diseases, and support the introduction of immunotherapy to stimulate antitumor immunity post-ASCT.

Correlation between the mean percentage of senescent CD8+ T-cells with patients' age, % plasma cells in BM aspirate, and duration of disease showed nonsignificant results, this result disagreed with a study done by Lee et al.[24] who found older patients, and those treated for a longer period before relapsing found to have higher percentage of senescent T-cell, this can be explained by the sample size or characteristics of the population in the area involved in the study.

In the study of Dosani et al.,[27] he reported that smoldering MM cases had lymphocytes with lower expression of negative CD57 (with an increase in positive CD57 expression) in patients who were more likely to progress to MM, proposing that CD57 expression may be associated with increasing disease stage.[27]

Till now there is no clear clinical implication of the effects of the senescent phenotype of CD8+ T-lymphocyte but further studies may disclose the exact etiology and significance of senescent T-cell in MM and if reversal of this phenomenon may add to treatment lines of MM.

Other factors may contribute to a high percentage of T-cells senescent in newly cases, both aging and human cytomegaly virus infection are believed to affect CD8+ T-cells phenotype and may lead to immunosenescence of these cells.[28],[29]

The percentage of senescent T-cells is higher in the elderly, with the most phenotypical changes occurring in the CD8+ T-cells subset isolated from the elderly, with a progressive loss of CD28 expression, and increase expression of CD57 in CD8+ T-cells.[28],[29],[30]

The studies reported by Strioga[31] and Mojumdar.[32] found a high correlation between human cytomegalovirus (HCMV) and T-cells senescence. HCMV infection gives rise to the higher percentage of long-term memory cells carrying mature phenotype with low CD27/CD28, high CD57 expression, and often perforin positive with T-cells.[31],[32]

In an Iraqi study, the detection of anti-cytomegalovirus antibody was conducted on 472 healthy blood donors using enzyme-linked immunosorbent assay test. The study found that 31.36% of the donors had+ ve anti-CMV Immunoglobulin G (IgG) Nanakaly and Hussen,[33] whereas another Iraqi study found that 46.6% of blood donors out of 120 samples had + ve anti-CMV IgG antibody Yasir and Majhol.[34]

Both studies showed that our population has a significant percentage of infection with CMV, which may affect the percentage of senescent T-cell, However, more sensitive methods, for example, polymerase chain reaction (PCR) is needed for better correlation.[33],[34]

  Conclusion 

The percentage of senescent CD8+ T-cells with CD28 − and CD57+ was significantly higher in MM cases related to healthy control group. The percentage of senescent CD8+ T cells with CD28 − and CD57+ was lower in recently diagnosed MM cases related to relapsed cases but the difference was statistically not significant.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

  References 
1.Cohen YC, Zada M, Wang SY, Bornstein C, David E, Moshe A, et al. Identification of resistance pathways and therapeutic targets in relapsed multiple myeloma patients through single-cell sequencing. Nat Med 2021;27:491-503.  
    2.Chou T. Multiple myeloma: Recent progress in diagnosis and treatment. J Clin Exp Hematop 2012;52:149-59.  
    3.Weiss BM, Abadie J, Verma P, Howard RS, Kuehl WM. A monoclonal gammopathy precedes multiple myeloma in most patients. Blood 2009;113:5418-22.  
    4.Cohen AD, Raje N, Fowler JA, Mezzi K, Scott EC, Dhodapkar MV. How to train your T cells: Overcoming immune dysfunction in multiple myeloma. Clin Cancer Res 2020;26:1541-54.  
    5.Udd KA, Spektor TM, Berenson JR. Monitoring multiple myeloma. Clin Adv Hematol Oncol 2017;15:951-61.  
    6.Pei Lin M. Multiple myeloma and other plasma cell neoplasms. In: His ED, editor. Hematopathology: A Volume in the Series: Foundations in Diagnostic Pathology. 3rd ed. Elsevier Health Sciences; 2017. p. 642-63.  
    7.BioLegend of Brilliant Violet 421 Anti-Mouse CD8a Antibody anti-CD8a 53-6.7.Cat. No 301036-. Available from: https://www.biolegend.com/en-us/products/brilliant-violet-421-anti-mouse-cd8a-antibody-7138?GroupID=BLG6765. [Last accessed on 2021 Oct 30].  
    8.BioLegend CD28 PE Cat. No 302908 – Microsoft Bing Search. Available from: https://www.bing.com/search?q=+biolegend+CD28+PE++302908 and go=Search and qs=ds and form=QBRE. [Last accessed on 2021 Oct 30].  
    9.BD Biosciences APC Mouse Anti-Human CD57. 560845 USA. Available from: https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research-reagents/single-color-antibodies-ruo/apc-mouse-anti-human-cd57.560845 [Last accessed on 2021 Oct 30].  
    10.Alwan AF. Survival of patients with multiple myeloma diagnosed at the national center of hematology in Baghdad. Iraqi J Cancer Med Genet 2018.  
    11.Badi AI, Al-Allawi NA, Yassin AK, Safar BM, Abdulla BK, Shamoon RP, et al. Health-related quality of life in multiple myeloma in Kurdistan Iraq. Iraqi J Hematol 2020;9:101.  
  [Full text]  12.Almueilo SH. Renal failure in patients with multiple myeloma. Saudi J Kidney Dis Transpl 2015;26:482-8.  
[PUBMED]  [Full text]  13.Katodritou E, Kyrtsonis MC, Delimpasi S, Kyriakou D, Symeonidis A, Spanoudakis E, et al. Real-world data on Len/Dex combination at second-line therapy of multiple myeloma: Treatment at biochemical relapse is a significant prognostic factor for progression-free survival. Ann Hematol 2018;97:1671-82.  
    14.Kumar S, Gertz MA, Dispenzieri A, Lacy MQ, Geyer SM, Iturria NL, et al. Response rate, durability of response, and survival after thalidomide therapy for relapsed multiple myeloma. Mayo Clin Proc 2003;78:34-9.  
    15.Jasrotia S, Gupta R, Sharma A, Halder A, Kumar L. Cytokine profile in multiple myeloma. Cytokine 2020;136:155271.  
    16.El-Naby AY, Gawaly AM, Elshweikh SA. CKS1B/CDKN2C (P18) amplification/deletion as prognostic markers in multiple myeloma patients. Egypt J Haematol 2016;41:87.  
    17.Kumar M, Panigrahi A, Dolai TK, De R, Mandal PK, Chakrabarti P. VTD in newly diagnosed myeloma: An institutional experience. Egypt J Haematol 2015;40:175.  
    18.Richardson PG, Barlogie B, Berenson J, Singhal S, Jagannath S, Irwin DH, et al. Extended follow-up of a phase II trial in relapsed, refractory multiple myeloma: Final time-to-event results from the SUMMIT trial. Cancer 2006;106:1316-9.  
    19.Yassin AK. Clinical and laboratory profiles of 109 patients diagnosed as multiple myeloma in Erbil city. J Fac Med Baghdad 2013;55:121-4.  
    20.Sultan S, Irfan SM, Parveen S, Ali H, Basharat M. Multiple myeloma: A retrospective analysis of 61 patients from a tertiary care center. Asian Pac J Cancer Prev 2016;17:1833-5.  
    21.Lonial S, Waller EK, Richardson PG, Jagannath S, Orlowski RZ, Giver CR, et al. Risk factors and kinetics of thrombocytopenia associated with bortezomib for relapsed, refractory multiple myeloma. Blood 2005;106:3777-84.  
    22.Suen H, Brown R, Yang S, Weatherburn C, Ho PJ, Woodland N, et al. Multiple myeloma causes clonal T-cell immunosenescence: Identification of potential novel targets for promoting tumour immunity and implications for checkpoint blockade. Leukemia 2016;30:1716-24.  
    23.Zelle-Rieser C, Thangavadivel S, Biedermann R, Brunner A, Stoitzner P, Willenbacher E, et al. T cells in multiple myeloma display features of exhaustion and senescence at the tumor site. J Hematol Oncol 2016;9:116.  
    24.Lee LX, Toskic D, Ma X, Zhou P, Kugelmass A, Fogaren T, et al. Senescent CD8+T cells in myeloma patients: Implications for cellular therapies. Blood 2018;132:5688.  
    25.Raitakari M, Brown RD, Sze D, Yuen E, Barrow L, Nelson M, et al. T-cell expansions in patients with multiple myeloma have a phenotype of cytotoxic T cells. Br J Haematol 2000;110:203-9.  
    26.Chung DJ, Pronschinske KB, Shyer JA, Sharma S, Leung S, Curran SA, et al. T-cell exhaustion in multiple myeloma relapse after autotransplant: Optimal timing of immunotherapy. Cancer Immunol Res 2016;4:61-71.  
    27.Dosani T, Mailankody S, Korde N, Manasanch E, Bhutani M, Tageja N, et al. Host-related immunodeficiency in the development of multiple myeloma. Leuk Lymphoma 2018;59:1127-32.  
    28.Verma K, Ogonek J, Varanasi PR, Luther S, Bünting I, Thomay K, et al. Human CD8+CD57- TEMRA cells: Too young to be called “old.” PLoS One 2017;12:e0177405.  
    29.Elwenspoek MM, Sias K, Hengesch X, Schaan VK, Leenen FA, Adams P, et al. T Cell Immunosenescence after early life adversity: Association with cytomegalovirus infection. Front Immunol 2017;8:1263.  
    30.Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ, Crotty LE, et al. Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+T cells. Blood 2003;101:2711-20.  
    31.Strioga M, Pasukoniene V, Characiejus D. CD8+CD28- and CD8+CD57+T cells and their role in health and disease. Immunology 2011;134:17-32.  
    32.Mojumdar K, Vajpayee M, Chauhan NK, Singh A, Singh R, Kurapati S. Altered T cell differentiation associated with loss of CD27 and CD28 in HIV infected Indian individuals. Cytometry B Clin Cytom 2012;82:43-53.  
    33.Nanakaly HT, Hussen BM. Seroprevalence of cytomegalovirus among voluntary blood donors in Erbil province, North Iraq. Zanco J Pure Appl Sci 2019;31:1-6.  
    34.Yasir SJ, Majhol RB. Screening of anti-cytomegalovirus IgG antibodies in blood donors in Al-Najaf governorate. Kufa Med J 2008. Available from: https://www.iasj.net/iasj/article/52414. [Last accessed on 2021 Dec 11].  
    
  [Figure 1]
 
 
  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]