Precision Medicine
Flow Cytometric Assays

Flow Cytometry


T-lymphocyte Immunophenotyping Assays

T-lymphocytes or T-cells are integral to the adaptive immune system. Their origins lie in the bone marrow, but maturation occurs in the thymus where they undergo thymopoiesis – a process involving negative and positive selection [1]. During thymopoiesis, bone marrow progenitors lacking the CD4+ and CD8+ co-receptor undergoes T-cell receptor (TCR) rearrangement to form CD4+CD8+ double positive thymocytes. Double positive thymocytes must undergo further selection to become single positive thymocytes expressing either the CD4 or CD8 receptor to ultimately become naïve T-cells [1]. Naïve T-cells can give rise to multiple T-cell subsets. There are different subsets of T-cells, each with specialised roles within the immune system. The four primary T-cell subsets and their respective functions are:
Naïve T-cells that respond to new antigens,
Effector T-cells that co-ordinate and execute the immune response,  
Memory T-cells that result from previous antigen encounters and contribute to long-term immunity and,
T-regulatory cells that suppress the immune system [1, 2, 3, 4].
T-cells known for their diverse antigen recognition capacity, high antigen specificity, potent effector activity and enabling long-lasting immunologic memory. PeploBio offers robust marker panels for the characterisation of all key T-cell subsets.

Naïve T-cells

Naïve T-cells are undifferentiated T-cells that are key for the adaptive immune system to confront novel infections [1]. Naïve T-cells circulate the blood and lymphatic systems, surveying for signs of pathogens or foreign substances. The Naive T-cell TCR have vast capacity for antigen recognition totalling up to 100 million specificities [1, 3, 4]. Naïve T-cells presented with antigens from antigen presenting cells (APCs) e.g., dendritic cells, undergo activation and differentiation into effector T-cells or memory T-cells [3,4]. The bioanalytical team at PeploBio detect and characterise these cells through markers for the “naïve” phenotype. Classically, CD4+ Naïve T-cells have been identified by their CD45RA+, CD62L+, CD27+ and CD11adim immunophenotype [3]. Recent thymic emigrants (RTEs) are proportion of naïve T-cells with least differentiated phenotype. RTEs are the naïve T-cell subset with highest TCR diversity but are functionally immature, with low proliferative capacity and cytokine production. Most RTEs express CR2 and IL8, however expression of PTK7 and CD31 is highest in the CD4+ population and CD103 in the CD8+ [4].

Effector T-cells

Cytotoxic T-cells

Cytotoxic T-lymphocytes (TCLs) or CD8+ T-cells are directly involved in the destruction of intracellular pathogens or abnormal cells. TCLs respond to MHC class I on APCs via their TCR and can induce cell death using a number of mechanisms, but all ultimately leads to apoptotic or necrotic cell death. The preferred mode of destruction by TCLs is apoptosis [5]. TCLs release perforins, a pore-forming protein, onto the targeted cell membrane followed by granzymes to trigger apoptosis in the cytosol. Alternatively, apoptosis can also be achieved directly, via signalling through the Fas ligand (FasL) on the TCL [5]. With the assistance of CD4+ T-helper (Th) cells to aid in CTLs activation and function, CTLs can mount a more potent and effective immune response [5]. Naïve CD8+ T-cell interaction with a peptide-MHC class I in conjunction with costimulatory molecules CD80/CD86 leads to their activation and differentiation into a few specialised cytotoxic effector cells. Phenotypic markers for common CD8+ T-cell subsets are as follows: TC1: CD183+; TC2: CD194+, CD294+; TC9: CD196+; and TC17: CD161+, CD194+, CD196+, IL23R+ [6].

Helper T-cells

Helper T-cells (Th) or CD4+ T-cells orchestrate the immune response against a range of pathogens and diseases. To confront a broad range of conditions, naïve CD4+ differentiation into several lineages of Th-cells, each with a customised repertoire of cytokines to aid in their specific functions. The three core conditions that must be met for naïve CD4+ undergo clonal expansion and differentiation into each Th subsets are:
TCR stimulation upon foreign antigen and MHC class II interaction,
Co-stimulatory signals on surface of the APC and
Exposure to cytokines, secreted primarily by the APC [7, 8].
In consensus with the scientific community, PeploBio utilizes the expression of cell signature cytokines and master transcription factors for the identification of distinct Th-cell subsets.

T helper type 1 cells

T helper type 1 cells (Th1) are specialised for fighting viruses and intracellular bacteria primarily through cell mediated cell cytolytic activity. Th1 cells promote B-cell IgG2a antibody production. Th1 cells can be recognized by their distinctive cytokine profile, which includes INFγ, TGFα, TGFb and STAT4, and their lineage specific transcription factor, T-bet [6-9].

T helper type 2 cells

T helper type 2 cells (Th2) are specialised for neutralising extracellular parasites and helminths via B-cell activation, immunoglobulin production and eosinophil recruitment. In particular, Th2 cells induce IgG1 and IgE class-switching. Th2 cells can be recognized by their distinctive cytokine profile, which includes IL4, IL5, IL10, IL13 and STAT6, and their lineage specific transcription factor, GATA-3 [6-9].

T helper type 9 cells

The T helper type 9 (Th9) subtype is less characterised compared to Th1 and Th2. Their function overlaps with Th2, aiding in fighting helminths but are distinct from Th2 cells as they preferentially produce IL9 [9, 10]. In addition to IRF4, PU.1 have been identified as an important intracellular marker of Th9 cells [6, 10].

T helper type 17 cells

T helper type 17 cells (Th17) are named for their ability to produce 3 (IL17A, IL17E and IL17F) of the 6 members of the IL17 family, though IL21 and IL22 have now also been added to their signature cytokine repertoire. Having been relatively recently identified as a distinct subset of Th cells, Th17 cell function is still subject to much research and debate. RORγt is a known lineage specific transcription factor of Th17 cells and they are thought to be involved in fighting fungi and extracellular bacteria [6,7,9].

Follicular helper T-cells

Follicular helper T-cells (TFH) a subset of Th cell expressing markers CXCR5, PD1, ICOS, CD25, CD69, CD95, CD57, OX40 and CD40L. TFH cell populations have been located in lymphoid tissue, in particular, in follicular regions, B-cell zones and germinal centres. TFH cells express B-cell promoting cytokines IL10, IL21 and CXCL13 and are involved germinal centre reactions [9, 11].

Memory T-cells

During the primary immune response, a heterogenous population of effector cells are generated. When most of the offending antigen is cleared from the body, the population of effector T-cells begin to die, leaving behind a small population of memory cells [10]. These cells continue to live even in the absence of the peptide-MHC ligand (CD4+ T-cells: MHC class II, CD8+ T-cells: MHC class I) complex that initiated the primary immune response and are capable of self-renewal [10]. Memory cells retain their high specificity to the antigens they encountered during the initial infection and upon re-exposure, are able to mount a rapid and specific immune response, eliminating the pathogen before it can cause a full-blown infection.

Memory cells contribute to the establishment of herd immunity within a population due to their long lasting ‘memory’ of the infection in a sufficient proportion of individuals in the community, limiting further spread. Vaccines strategies in R&D leverage the concept of memory cells by exposing the immune system to attenuated, inactivated or components of a pathogen, to stimulate the formation of memory cells. This primes the immune system to respond rapidly and effectively if the individual encounters the actual pathogen in the future.

PeploBio offers flow cytometry panels for the detection and characterisation of effector memory cells and central memory cells.  

Effector memory T-cells

Effector memory (TEM) cells express specific homing receptors on their cell surface. These receptors help to navigate TEM cell migration to sites of inflammation in non-lymphoid organs. TEM cells provide a rapid and immediate response to re-infection in peripheral tissues without the need for further differentiation, making them critical for the first line of defence. TEM cells are known to produce key cytokines to aid in host defence in response to TCR stimulation [10].

CD4+ TEM phenotype is typically recognised as CD4+, CD8-, CD45RA-, CD45RO+, CD95(FAS)+ and CD197-. CD4+ TEM expresses intracellular markers IFNg, IL2, TNFα, GZMA, GZMB and perforin when stimulated [6].

On the other hand, CD8+ TEM have the immunophenotype CD4-, CD8+, CD45RA+, CD45RO+, CD95(FAS)+ and CD197-. Like their CD4+ counterpart, CD8+ TEM also produce IFNg, IL2, TNFα and GZMA when stimulated. CD8+ TEM cells express intracellular markers ACTN1, FOX01, EOMES, FOXP1, ID2, ID3, PRDM1, TBX21, TCF7 and ZEB2 [6].  

Central memory T-cells

Like TEM cells, central memory T cells (TCM cells) are responsible for providing long-lasting immunity. But unlike TEM cells, TCM cells need to be reactivated to differentiate into effector T-cells upon re-exposure to the antigen. This differentiation is typically faster and more robust than that of naive T cells. TCM cells do not produce cytokines in response to TCR stimulation, except for IL2. This ability is only acquired later after they undergo rapid proliferation. The expression of CD62L and CCR7 on the surface of TCM cells indicate their migration in the lymphoid organs [12].

CD4+ TCM are identified by the immunophenotype CD4+, CD8-, CD45RA-, CD45RO+, CD95(FAS)+ and CD197+. Some intracellular markers expressed by CD4+ TCM include EOMES, FOXP1, LEF1, PRDM1, TBX21, TCF7 and ZEB2. Upon stimulation these cells express cytokines IFNg, IL2 and TNFα [6].

Phenotypic markers expressed by CD8+ TCM are CD4-, CD8+, CD45RA-, CD45RO+, CD95(FAS)+ and CD197+. CD8+ TCM produce IFNg, IL2, TNFα and GZMA (but not GZMB) when stimulated. Similar to CD4+ TCM, CD8+ TCM express a variety of intracellular markers such as ACTN1, FOX01, EOMES, FOXP1, ID2, ID3, IL6ST, LASS6, LEF1, KLF7, TAF4B, TBX21, ZEB2, PRDM1, TBX21 and TCF7 [6].  

Regulatory T-cells

B-cells and T-cells are central to the host’s defence against pathogens and foreign substances. They are also the primary culprits in driving abnormal immune responses against the host e.g., in auto-immune disease or allergic reactions [13]. Regulatory T-cells (Treg) are a unique subset of T-cells, key to the development of immunological tolerance of the adaptive immune system to self-antigens [13-15].

Two mechanisms are used for achieving self-tolerance: cell intrinsic (recessive) or cell extrinsic (dominant). Several cell intrinsic methods are used when T-cells are exposed to self-antigens e.g., receptor editing to replace self-reacting receptors, clonal deletion where self-reactive cells undergo apoptosis during cell development in the thymus or raising the threshold for receptor activation. Treg cells are part of the cell extrinsic mechanism, where cells actively monitor and supress other abnormal immune cells, in particular, other types of T-cells [13].  

At PeploBio, we offer a robust panel of markers for the characterisation of Treg cells in biological fluids and tissue extracts.
Immunophenotype: CD3+, CD4+ or CD8+, CD25+, FOXP3+, CD127+ [6, 13-15]
Extended markers: CTLA4, CD45RA, GITR, IL4, TGFb [6, 13-15]

T-lymphocyte subset differentiation assays

At PeploBio we also offer flow cytometry assays on whole blood that may be used individually or in conjunction with other assays to aid the immunological assessment of normal and immunocompromised individuals.

A CD4/CD8/CD3 or a CD3/CD8/CD45/CD4 flow cytometry assay can help detect T-cell subsets, T-helper cells (Th) and cytotoxic T-cells (TCLs). Surface expression of CD3 and CD4 differentiates Th cells (CD3+ CD4+) from contaminating monocytes (CD3- CD4+). Whereas CTLs are a subset of CD3+ T-cells that express CD8. CD45 is a pan marker of leucocytes and inclusion of CD45 alongside CD3, CD4, and CD8 markers when identifying Th cells and CTLs provides a more accurate and specific method for distinguishing between these T-cell subpopulations and ensures that the cells analysed are indeed leukocytes.

Clinically, the counts and percentages of CD3+CD8+ (CTLs) and CD3+CD4+ (Th) cells can be monitored in a variety of diseases to assess the immune status of a patient to gain insights into the progression or management of these diseases. Some examples for potential clinical uses of this assay are:
  • Immunodeficiency disorders e.g., acquired immunodeficiency syndrome (AIDS) [16]

  • Autoimmune disease e.g., systemic lupus erythematosus (SLE) and multiple sclerosis (MS) [17, 18]

  • Infectious disease e.g., tuberculosis (TB) and viral hepatitis [19, 20]

  • Organ transplant i.e., to assesses effectiveness of immunosuppressive therapy and risk of rejection [21].

Like the 6-colour TBNK assay, this assay is particularly useful in aiding the monitoring of HIV progression to determine the stage of the disease and the need for antiretroviral therapy (ART).

Our team performs these assays using CE-IVD marked BD Multitest CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC or BD Tritest CD4 FITC/CD8 PE/CD3 PerCP to assure their use in clinical studies [22, 23]. A simple CE-IVD marked BD Tritest for CD3 FITC/CD4 PE/CD45 PerCP can identify mature human T-cells (CD3+) and Th cells (CD3+CD4+) [24]. Each of these assays can be performed with or without Trucount tubes which can determine absolute counts. Speak to a member of our talented bioanalytical team to discuss the specific needs of your study.

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[2] Caccamo N, Joosten SA, Ottenhoff TH, Dieli F. Atypical human effector/memory CD4+ T cells with a naive-like phenotype. Frontiers in immunology. 2018 Dec 3; 9:2832.
[3] Larbi A, Fulop T. From “truly naïve” to “exhausted senescent” T cells: when markers predict functionality. Cytometry Part A. 2014 Jan;85(1):25-35.
[4] Van den Broek T, Borghans JA, Van Wijk F. The full spectrum of human naive T cells. Nature Reviews Immunology. 2018 Jun;18(6):363-73.
[5] Barry M, Bleackley RC. Cytotoxic T lymphocytes: all roads lead to death. Nature Reviews Immunology. 2002 Jun 1;2(6):401-9.
[6] Mousset CM, Hobo W, Woestenenk R, Preijers F, Dolstra H, van der Waart AB. Comprehensive phenotyping of T cells using flow cytometry. Cytometry Part A. 2019 Jun;95(6):647-54
[7] Schorer M, Kuchroo VK, Joller N. Role of co-stimulatory molecules in T helper cell differentiation. Co-signal Molecules in T Cell Activation: Immune Regulation in Health and Disease. 2019:153-77.
[8] Murphy K, Weaver C. (2017) Janeway's immunobiology (9th edition). New York and London: Garland Science.
[9] Wan YY, Flavell RA. How diverse—CD4 effector T cells and their functions. Journal of molecular cell biology. 2009 Oct 1;1(1):20-36.
[10] Végran F, Apetoh L, Ghiringhelli F. Th9 cells: a novel CD4 T-cell subset in the immune war against cancer. Cancer research. 2015 Feb 1;75(3):475-9
[11] Laurent C, Fazilleau N, Brousset P. A novel subset of T-helper cells: follicular T-helper cells and their markers. Haematologica. 2010 Mar;95(3):356
[12] Pepper M, Jenkins MK. Origins of CD4+ effector and central memory T cells. Nature immunology. 2011 Jun;12(6):467-71.
[13] Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T-cells and immune tolerance. cell. 2008 May 30;133(5):775-87.
[14] Santegoets SJ, Dijkgraaf EM, Battaglia A, Beckhove P, Britten CM, Gallimore A, Godkin A, Gouttefangeas C, de Gruijl TD, Koenen HJ, Scheffold A. Monitoring regulatory T-cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry. Cancer Immunology, Immunotherapy. 2015 Oct; 64:1271-86.
[15] Manuszak C, Brainard M, Thrash E, Hodi FS, Severgnini M. Standardized 11-color flow cytometry panel for the functional phenotyping of human T regulatory cells. Journal of Biological Methods. 2020;7(2).
[16] Giorgi JV, Liu Z, Hultin LE, Cumberland WG, Hennessey K, Detels R. Elevated levels of CD38+ CD8+ T cells in HIV infection add to the prognostic value of low CD4+ T cell levels: results of 6 years of follow-up. JAIDS Journal of Acquired Immune Deficiency Syndromes. 1993 Aug 1;6(8):904-12.
[17] Smolen JS, Chused TM, Leiserson WM, Reeves JP, Alling D, Steinberg AD. Heterogeneity of immunoregulatory T-cell subsets in systemic lupus erythematosus: correlation with clinical features. The American Journal of Medicine. 1982 May 1;72(5):783-90.
[18] Munschauer FE, Stewart C, Jacobs L, Kaba S, Ghorishi Z, Greenberg SJ, Cookfair D. Circulating CD3+ CD4+ CD+ T lymphocytes in multiple sclerosis. Journal of clinical immunology. 1993 Mar;13:113-8.
[19] Sabhapandit D, Hazarika P, Phukan AC, Lynrah KG, Elantamilan D. Comparison of CD4 and CD8 counts and ratio in HIV negative pulmonary tuberculosis patients with normal healthy controls. Int J Res Med Sci. 2017 Oct;5(10):4567-73.
[20] Petrova M, Muhtarova M, Nikolova M, Magaev S, Taskov H, Nikolovska D, Krastev Z. Chronic Epstein-Barr virus-related hepatitis in immunocompetent patients. World Journal of Gastroenterology: WJG. 2006 Sep 9;12(35):5711.
[21] Wong T, Nouri‐Aria KT, Devlin J, Portmann B, Williams R. Tolerance and latent cellular rejection in long‐term liver transplant recipients. Hepatology. 1998 Aug;28(2):443-9.
[22] BD Bioscience; BD Multitest CD3/CD8/CD45/CD4; 2017 [cited Nov 2023]. Available from:
[23] BD Bioscience; BD Tritest CD4/8/3; 2014 [cited Nov 2023]. Available from:
[24] BD Bioscience; BD Tritest CD3/CD4/CD45; 2014 [cited Nov 2023]. Available from:

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