B Cells

T Cells

 
 
 

 

LT-HSC (Long-Term Hematopoietic Stem Cell)

LT-HSCs are pluripotent cells which give rise to all blood cell populations of lymphoid, myeloid, and erythroid lineages and persist throughout the entire lifespan. They have the potential to self-renew sustaining the stem cell pool or differentiate into ST-HSCs and other multi-, oligo-, and unipotent progenitors which give rise to terminally differentiated cells.

 

Mouse Markers:

Lin- (Ter119-, CD3-, B220- or CD19-, CD11b-, Gr-1-, CD11c- , NK1.1- )

CD117 (c-kit)hi

Sca-1 (Ly-6A)+

CD34-

CD135 (Flt3)-

CD150 (SLAM)+

ST-HSC (Short-Term Hematopoietic Stem Cell)

ST-HSCs are multipotent progenitors with a short-term renewal potential as compared to LT-HSCs. It is believed that ST-HSCs differentiate from the daughter cell during an asymmetrical division of LT-HSCs. ST-HSCs can give rise to other multi-, oligo-, and unipotent progenitors which give rise to terminally differentiated cells of lymphoid, myeloid and erythroid lineages.

 

Mouse Markers:

Lin- (Ter119-, CD3-, B220- or CD19-, CD11b-, Gr-1-, CD11c- , NK1.1- )

CD117 (c-kit)hi

Sca-1 (Ly-6A)+

CD34+

CD135 (Flt3)-

CD150 (SLAM)-

MPP (Multipotent Progenitor)

MPPs are multipotent progenitors derived from ST-HSCs. They can give rise to another multipotent progenitor, LMPPs, or oligopotent and unipotent progenitors which give rise to terminally differentiated cells.


Mouse Markers:

Lin- (Ter119-, CD3-, B220- or CD19-, CD11b-, Gr-1-, CD11c- , NK1.1- )

CD117 (c-kit)hi

Sca-1 (Ly-6A)+

CD34+

CD135 (Flt3)low

CD150 (SLAM)-

LMPP (Lymphoid-Primed Multipotent Progenitor)

LMPPs are responsible for the generation of lymphocytes and pDCs through subsequent differentiation steps including CLPs and pre-pDCs respectively. LMPPs were originally thought to be a homogeneous population of precursors containing multipotent cells with the equal potential to differentiate into several lymphoid lineages. However, a recent study has demonstrated that LMPP is a heterogeneous population of cells comprising unipotent clones with a non-overlapping lineage differentiation potential(21).

 

Mouse Markers:

Lin- (Ter119-, CD3-, B220- or CD19-, CD11b-, Gr-1-, CD11c- , NK1.1- )

CD117 (c-kit)hi

Sca-1 (Ly-6A)+

CD34+

CD135 (Flt3)hi

CD150 (SLAM)-

CD127 (IL-7Rα)-

CLP (Common Lymphoid Progenitor)

CLPs are multipotent progenitors derived from Lymphoid Multipotent Progenitors (LMPPs). They give rise to all lymphoid lineages including T cells, B cells and NK cells.

 

Mouse Markers:

Lin- (Ter119-, CD3-, B220- or CD19-, CD11b-, Gr-1- ,CD11c- , NK1.1- )

CD117 (c-kit)int/low

CD135 (Flt3)+

CD127 (IL-7Rα)+

Pre-pro B cell

Pre-pro B cells are differentiated from CLPs and are the earliest B cell progenitor identified in the bone marrow.


Mouse Markers:

CD117 (c-kit)-

CD127 (IL-7Rα)+

CD19-

CD24low/-

CD43-

CD45R (B220)low

IgM-

IgD-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, SPI1 (PU.1)+

Pro-B cell

Pro-B cells are differentiated from Pro-pre B cells. Upregulation of RAG1/RAG2 recombinases induces rearrangement of the D and J segments of the µ-heavy chain in these cells. Meanwhile, TdTs (Terminal Deoxynucleotidyl Transferases) add N- and P- nucleotides to the end of the D and J segments to diversify the specificity of newly forming B cell receptor.


Mouse Markers:

CD117 (c-kit)-

CD127 (IL-7Rα)+

CD19+/-

CD24low

CD43+

CD45R (B220)low

IgM-

IgD-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, SPI1 (PU.1)+

Late Pro-B cell

Late Pro-B cells undergo rearrangement of the V segment of the µ-heavy chain. Upon V segment rearrangement, it joins the DJ segments to form a mature µ-heavy chain. Late Pro-B cells which have successfully rearranged a heavy chain that is capable of pairing with surrogate light chains (VpreB and λ5) to form a pre-BCR (B cell receptor) are allowed to progress to the next stage.

 

Mouse Markers:

CD117 (c-kit)-

CD127 (IL-7Rα)+

CD19+

CD20+

CD24+

CD43+

CD45R (B220)low

IgM-

IgD-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, SPI1 (PU.1)+

Pre-B cell

Upon upregulation of the pre-BCR on the surface of Pre-B cells, the expression of RAG1/2 recombinases is downregulated and Pre-B cells undergo several divisions before they enter the Late Pre-B cell stage.


Mouse Markers:

CD117 (c-kit)-

CD127 (IL-7Rα)+

CD19+

CD20+

CD24+

CD43low

CD45R (B220)+

Surface IgM-

Cytoplasmic μ heavy chain+

IgD-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

Late Pre-B cell

After a second wave of RAG1 and RAG2 recombinase upregulation, Pre-B cells undergo V and J segment rearrangement and diversification to form λ and κ light chains. Upon successful pairing of light chain with the µ-heavy chain, the B cell receptor associates with Ig-α and Ig-β molecules to form a fully functioning receptor.


Mouse Markers:

CD117 (c-kit)-

CD127 (IL-7Rα)+

CD19+

CD20+

CD24+

CD43-

CD45R (B220)+

IgM-

IgD-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

Immature B cell

Immature B cells expressing a newly formed IgM/BCR (B Cell Receptor) undergo a negative selection process in which B cells expressing BCRs with a high affinity for self-antigens are eliminated or undergo additional receptor editing to change the antigen specificity. Immature B cells egress the bone marrow to continue their differentiation in blood and other lymphoid organs.


Mouse Markers:

CD127 (IL-7Rα)+

CD19+

CD20+

CD23-

CD24+

CD43-

CD45R (B220)+

IgM+

IgD-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

T1 B cell (Transitional Type 1 B cell)

T1 B cells differentiate from immature B cells which have successfully passed negative selection. Transitional B cell maturation occurs primarily in the spleen, with T1 B cells present in the red pulp. Upon encountering antigen, T1 B cells differentiate to T2 B cells which are able to give rise to follicular, marginal zone, germinal center, memory, and plasma B cells.


Mouse Markers:

CD127 (IL-7Rα)+

CD19+

CD20+

CD22+

CD23-

CD24+

CD45R (B220)+

IgMhi

IgDlow/-

CD93+

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

T2 B cell (Transitional Type 2 B cell)

T2 B cells can be identified in spleen follicles. T2 B cells can differentiate further into either circulating B cells which cluster into GCs or non-circulating B cells that populate the marginal zones.


Mouse Markers:

CD127 (IL-7Rα)+

CD19+

CD20+

CD22+

CD23+

CD24+

CD45R (B220)+

IgMhi

IgD+

CD93+

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

Mature B cell

Mature B lymphocytes develop sequentially from transitional type 1 (T1) and type 2 (T2) precursors in the spleen. They can be distinguished from their progenitors by expression of both IgM and IgD, as well as a lack of Immature B cell marker CD24. Mature B cells can further differentiate into germinal center and marginal zone B cells.


Mouse Markers:

CD127 (IL-7Rα)+

CD19+

CD20+

CD22+

CD23+

CD24low/-

CD45R (B220)hi

IgM+

IgD+

CD93-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

FO B cell (Follicular B cell)

Follicular B cells can be found within structures called follicles (which contain germinal centers) in secondary and tertiary lymphoid organs such as the spleen, Peyer's patches, and lymph nodes. Unlike marginal zone B cells, Follicular B cells circulate throughout the body. They are capable of becoming either Memory B cells or Plasma cells. In most instances, these cells require T cells for full activation.


Mouse Markers:

CD19+

CD20+

CD21/35low

CD22+

CD23+

CD38+

CD45R (B220)hi

IgMlow

IgD+

CD93-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

GC B cell (Germinal Center B cell)

Upon antigen encounter and interaction with T follicular cells, B cells cluster into germinal centers within follicles. B cell maturation in GCs is associated with somatic hypermutation of antibody V regions allowing the cells to generate antibodies with high avidity BCRs. Antigen selected GC B cells leave the germinal center to differentiate further into memory or plasma B cells.


Mouse Markers:

CD19+

CD20+

CD38+

CD45R (B220)+

IgM+

IgD+/-

IgA+

IgG+

GL7+

CD95+

PNA+

 

Transcription Factors: Pax-5+ , XBP-1+, IRF4+, Bcl-6+

MZ B cell (Marginal Zone B cell)

Marginal Zone (MZ) B cells are non-circulating B cells which can be identified in the marginal zone of lymphoid organs such as the spleen. They typically have a lower threshold of activation compared to follicular B cells and can develop into plasma cells with the help of T cells.


Mouse Markers:

CD19+

CD20+

CD21/35+

CD22+

CD23+

CD45R (B220)hi

IgM+

IgDlow

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

Memory B cell

Memory B cells are found predominantly in the marginal zone of the spleen, the sub-capsular sinus of the lymph nodes, and under the intestinal epithelium in Peyer's patches and crypt epithelium of the tonsils. Memory B cells develop after infection and retain memory of the antigen allowing for a more rapid immune response upon reintroduction of the antigen.


Mouse Markers:

CD19+

CD20+

CD27+

CD38+/-

CD45R (B220)hi

IgM+

IgD-

IgA+

IgG+

CD138-

 

Transcription Factors: Pax-5+ , POU2AF1 (OBF1)+, SPI-B+

Plasma B cell

Plasma B cells are terminally differentiated B cells which circulate throughout the body and produce large volumes of antibodies. They can be identified by high levels of expression of CD27 and CD138.


Mouse Markers:

CD19low

CD20low

CD21/35-

CD23-

CD27hi

CD38low

CD45R (B220)low

IgM-

IgD-

CD138+

CD184 (CXCR4)hi

 

Transcription Factors: Blimp-1+, XBP-1+, IRF4+

Plasmablast

Plasmablasts are located in the peripheral immune organs where they undergo rapid proliferation to produce a large pool of terminally differentiated plasma cells. They can be distinguished from plasma cells by low production antibodies, ability to proliferation, and a shorter lifespan.


Mouse Markers:

CD19+

CD20+

CD27hi

CD38hi

CD45R (B220)+

IgM+/-

IgD+/-

IgA+/-

IgG+/-

CD138-

Transitional B cell

Transitional B cells are immature B cell progenitors derived from fetal hematopoietic progenitors which are capable of differentiating to B1 B cells.


Mouse Markers:

CD19+

CD20+

CD21/35+/-

CD23+/-

CD24+

CD45R (B220)+

IgM+

IgDlow

CD93+

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

B1-a/b B cell

B1 cells are derived from fetal progenitors and are localized mainly in the peritoneal cavity and gut-associated lymphoid tissues. B1-a and B1-b cells can be distinguished by the expression of CD5. The B1 BCR is less diverse than that of B2 cells as it is rearranged from a limited number of Ig gene segments and lacks nucleotide editing. B1 cells predominantly secrete IgM and undergo very little somatic hypermutation.


Mouse Markers:

CD19+

CD20+

CD23-

CD43+

CD45R (B220)+

IgM+

IgDlow

CD5+/-

CD1d+

CD11b+

Breg (Regulatory B cell)

Regulatory B cells represent a small fraction of B cells with immunosuppressive capacities. These cells can be phenotypically challenging to identify, as they bear many common markers found on other B cell types.


Mouse Markers:

CD19+

CD20+

CD21/35hi/mid

CD23+/-

CD45R (B220)+

IgM+

IgD+/-

CD5+

CD1dhi

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

HSC (Hematopoietic Stem Cell)

HSCs are pluripotent cells which give rise to all blood cell populations of lymphoid, myeloid, and erythroid lineages and persist throughout the lifespan. They have the potential to self-renew to sustain the stem cell pool or differentiate into other multi-, oligo-, and unipotent progenitors which give rise to terminally differentiated cells (1-3, 5, 6).

 

Human Markers:

Lin- ( CD3-, CD19-, CD56-, CD10-, CD14-, CD66b-, CD335-, CD11c- )

CD45RA-

CD34+

CD38-

CD90+

CD135 (Flt3)+

MPP (Multipotent Progenitor)

MPPs are multipotent progenitors derived from HSCs. They can give rise to other multipotent progenitor (LMPPs), or oligopotent and unipotent progenitors (i.e., CMPs, GMPs, or MDPs), which give rise to terminally differentiated cells of lymphoid, myeloid and erythroid lineages (1-3, 5, 6).


Human Markers:

Lin- ( CD3-, CD19-, CD56-, CD10-, CD14-, CD66b-, CD335- , CD11c- )

CD34+

CD38-

CD90-

CD45RA-

CD135 (Flt3)+

LMPP (Lymphoid-Primed Multipotent Progenitor)

LMPPs are multipotent progenitors derived from MPPs. They generate all cells in the lymphoid lineage and pDCs.

 

Human Markers:

Lin- ( CD3-, CD19-, CD56-, CD14-, CD66b-, CD335- , CD11c- )

CD34+

CD38-

CD90-

CD45RA+

CD10-

CD135 (Flt3)+

MLP (Multi Lymphoid Progenitor)

MLPs are multipotent progenitors derived from LMPPs. They generate all cells in the lymphoid lineage and pDCs.

 

Human Markers:

Lin- ( CD3-, CD19-, CD56-, CD14-, CD66b-, CD335- , CD11c- )

CD34+

CD38-

CD45RA+

CD10+

CD7+

CD90-

CD135 (Flt3)+

Pro-B cell

Pro B cells are differentiated from MLPs and are one of the earliest B cell progenitors identified in the bone marrow. Upregulation of RAG1/RAG2 recombinases induces the rearrangement of the V, D, and J segments of the µ-heavy chain. Meanwhile, TdT (Terminal Deoxynucleotidyl Transferase) adds N- and P- nucleotides to the end of the V, D, and J segments to diversify the specificity of the newly forming BCR. Pro-B cells which successfully rearrange the heavy chain capable of pairing with the surrogate light chains (VpreB and λ5) to form a pre-BCR are able to differentiate further.


Human Markers:

CD34+

CD38+

CD10+

CD127 (IL-7Rα)+

CD19+

CD20+

CD24+

IgM-

IgD-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, SPI1 (PU.1)+

Pre-B cell

After the second wave of RAG1 and RAG2 recombinase upregulation, Pre-B cells undergo V and J segment rearrangement and diversification to form λ and κ light chains. Upon successful pairing of a light chain with the µ-heavy chain, the BCR associates with Ig-α and Ig-β molecules to form a fully functioning receptor.


Human Markers:

CD34-

CD38+

CD10+

CD127 (IL-7Rα)+

CD19+

CD20+

CD24+

Surface IgM-

Cytoplasmic μ heavy chain+

IgD-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

Immature B cell

Immature B cells expressing the newly formed IgM/BCR undergo a negative selection process in which B cells expressing BCRs with a high affinity to self-antigens are eliminated or undergo additional receptor editing to change the antigen specificity. Immature B cells egress the bone marrow to continue their differentiation in blood and other lymphoid organs.


Human Markers:

CD10+

CD127 (IL-7Rα)+

CD19+

CD20+

CD24hi

IgM+

IgD-

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

Transitional B cell

Transitional B cells differentiate from immature B cells which successfully pass negative selection. Transitional B cell maturation occurs primarily in the spleen. Upon encountering antigen, Transitional B cells differentiate to follicular, marginal zone, germinal center, memory, and plasma B cells.


Human Markers:

CD10lo

CD127 (IL-7Rα)+

CD19+

CD20+

CD21+

CD24hi

CD38hi

IgM+

IgDlo

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

Naïve Mature B cell

Mature B-lymphocytes develop from transitional B cell precursors in the spleen. They can be distinguished from their progenitors by expression of both IgM and IgD. Mature B cells can be further differentiated to germinal center and marginal zone B cells.


Human Markers:

CD10+

CD127 (IL-7Rα)+

CD19+

CD20+

CD24+

IgM+

IgD+

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

FO B cell (Follicular B cell)

Follicular B cells can be found within structures called follicles (which contain germinal centers) in secondary and tertiary lymphoid organs like the spleen, Peyer's patches, and lymph nodes. Unlike marginal zone B cells, follicular B cells circulate throughout the body. They are capable of becoming either memory B cells or plasma cells. In most instances, these cells require T cells for full activation.


Human Markers:

CD10-

CD127 (IL-7Rα)+

CD19+

CD20+

CD21+

CD22+

CD23+

CD38lo

IgMlo

IgD+

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

GC B cell (Germinal Center B cell)

Upon antigen encounter and interaction with T follicular cells, B cells cluster into germinal centers within follicles. B cell maturation in GCs is associated with somatic hypermutation of antibody V regions allowing to generate the antibodies with high avidity BCRs. Antigen selected GC B cells leave the germinal center to differentiate further to memory or plasma B cells.


Human Markers:

CD19+

CD20+

CD23+

CD38hi

IgM+

IgD+/-

IgA+

IgG+

 

Transcription Factors: Pax-5+ , XBP-1+ , IRF4+, Bcl-6+

MZ B cell (Marginal Zone B cell)

Marginal Zone (MZ) B cells are non-circulating B cells which can be identified in the marginal zone of lymphoid organs such as the spleen. They typically have a lower threshold of activation compared to follicular B cells and can develop into plasma cells with the help of T cells.


Human Markers:

CD19+

CD20+

CD21hi

CD23+/-

IgM+

IgDlo

 

Transcription Factors: E2A+, Pax-5+ , EBF1+, Oct2+

Memory B cell

Memory B cells are found predominantly in the marginal zone of the spleen, the sub-capsular sinus of the lymph nodes, and under the intestinal epithelium in Peyer's patches and crypt epithelium of the tonsils. Memory B cells develop after infection with a host and retain memory of the antigen allowing for a more rapid immune response upon reintroduction of the host.


Human Markers:

CD19+

CD20+

CD23lo

CD27+

CD38-

IgM+

IgD-

IgA+

IgG+

CXCR4+

 

Transcription Factors: Pax-5+, POU2AF1 (OBF1)+, SPI-B+

Plasma B cell

Plasma B cells are terminally differentiated B cells which circulate throughout the body and produce large volumes of antibodies. They can be identified by high levels of expression of CD27 and CD138.


Human Markers:

CD19lo

CD20-

CD27hi

CD38hi

IgM-

IgD-

CD138+

CXCR4+

 

Transcription Factors: Blimp-1+, XBP-1+ , IRF4+

Plasmablast

Plasmablasts are located in the peripheral immune organs where they undergo rapid proliferation to produce a large pool of terminally differentiated plasma cells. They can be distinguished from plasma cells by low production antibodies, ability to proliferation, and a shorter life-span.


Human Markers:

CD19+

CD20+

CD27hi

CD38hi

IgM+ /-

IgD+ /-

IgA+ /-

IgG+ /-

CD138-

CLP (Common Lymphoid Progenitor)

CLPs are multipotent progenitors derived from Lymphoid Multipotent Progenitors (LMPPs). They give rise to all cells from the lymphoid lineage including T cells, B cells, and NK cells.


Mouse Markers:

Lin- (Ter119-, CD3-, B220- or CD19-, CD11b-, Gr-1- ,CD11c- , NK1.1- )

CD117 (c-kit)int/low

CD135 (Flt3)+

CD127 (IL-7Rα)+

ILC1 (Group 1 Innate Lymphoid Cells)

Like T cells, ILCs are derived from CLPs; however, they do not respond in an antigen-specific manner. Group 1 ILCs include Natural Killer (NK) cells and produce type 1 cytokines including IFN-γ and TNF-α. They play a critical role in protecting the host against pathogens and tumor cells.


NK Cell Mouse Markers

NK1.1+

NKp46+   

CD11b+   

CD49b+

CD122 (IL-2Rβ)+

CD244 (2B4)+

CD314 (NKG2D)+


NK Cell Transcription factors: T-bet, Id2, Nfil3


ILC1 Mouse Markers:

Lin- (CD3-, CD11b-, B220-, Gr1-, Ter119-)

CD4-

CD8-

CD127 (IL-7Rα)+

IL-12Rβ+


ILC1 Transcription factors: T-bet


Learn more about NK and ILC cell development and function on our Natural Killer Cells and Innate Lymphoid Cells webpages.

ILC2 (Group 2 Innate Lymphoid Cells)

Like T cells, ILCs are derived from CLPs; however, they do not respond in an antigen-specific manner. Group 2 ILCs produce cytokines normally associated with Th2 cells including IL-5 and IL-13. They play a key role in response to helminth infection, allergic lung inflammation, and are involved in wound healing.


ILC2 Mouse Markers:

Lin- (CD3, CD11b-, B220-, Gr1-, Ter119-)

CD4-

CD8-

CD127 (IL-7Rα)+

CD90 (Thy1)+

CD117+

Ly6A (Sca-1)+

CD25+

ICOS+

IL-17Rβ+


ILC2 Transcription factors: RORα, GATA-3


Learn more about Innate Lymphoid cell development and function on our Innate Lymphoid Cells webpage.

ILC3 (Group 3 Innate Lymphoid Cells)

Like T cells, ILCs are derived from MLPs; however, they do not respond in an antigen-specific manner. Group 3 ILCs were originally discovered as cells that express NKp46, but do not resemble normal NK cells. Instead, they express RORγt and produce IL-17 and IL-22. ILC3s primarily reside in mucosal tissues where they help maintain intestinal homeostasis. Lymphoid tissue inducer cells (LTi) are considered subset of group 3 ILCs that are involved in lymphoid organ formation during embryogenesis. The exact relationship between LTis and other ILC3s is still being investigated.

 

ILC3 Mouse Markers:

Lin- (CD3, CD11b-, B220-, Gr1-, Ter119-)

CD4-

CD8-

NKp46+

CD127 (IL-7Rα)+

CD90 (Thy1)+

CD121a (IL-1R)+

IL-23R+


ILC3 Transcription factors: RORγt


Learn more about Innate Lymphoid cell development and function on our Innate Lymphoid Cells webpage.

ETP/DN1 (Early T Cell Progenitor/Double Negative Thymocyte 1)

ETP/DN1s are one of the first T cell progenitor populations that have been identified in the thymus. These progenitors are named double negative as they do not express the CD4 or CD8 co-receptors found on mature T cells. Additionally, they do not express the CD3/TCR complex.


Mouse Markers:

TCRαβ-

TCRγδ-

CD3-

CD4-

CD8α-

CD8β-

CD25-

CD44+

DN2 (Double Negative Thymocyte 2)

DN2 thymocytes are differentiated from DN1 progenitors. These progenitors were named double negative as they do not express the CD4 or CD8 co-receptors found on mature T cells. At this stage, upregulation of RAG1/RAG2 recombinases induces VDJ rearrangement of the β-chain and γδ- loci of the TCR. Meanwhile, TdTs (Terminal Deoxynucleotidyl Transferases) add N- and P- nucleotides to the end of the V, D and J segments to diversify the specificity of the newly forming T cell receptor. Determination of the αβ/γδ lineage fate depends on TCR signal strength. This model of lineage determination predicts that the γδ TCR transduces a stronger signal directing cells to the γδ lineage, while a pre-TCR transduces a weaker signal directing progenitors to the αβ lineage (25, 28).


Mouse Markers:

TCRαβ-

TCRγδ-

CD3-

CD4-

CD8α-

CD8β-

CD25+

CD44+

DN3 (Double Negative Thymocyte 3)

DN3 thymocytes are differentiated from DN2 progenitor cells. These progenitors were named double negative as they do not express the CD4 or CD8 co-receptors found on mature T cells. DN3 T cells which have successfully rearranged a TCRβ-chain that is capable of pairing to a surrogate TCRα-chain pass the β-selection checkpoint and undergo clonal division before transitioning to the DN4 stage.


Mouse Markers:

TCRαβ-

TCRγδ-

CD3-

CD4-

CD8α-

CD8β-

CD25+

CD44-

DN4 (Double Negative Thymocyte 4)

DN4 thymocytes are differentiated from DN3 progenitor cells. These progenitors were named double negative as they do not express the CD4 or CD8 co-receptors found on mature T cells. DN4 cells undergo rearrangement of the TCR α-locus to form a functional αβ-TCR.


Mouse Markers:

TCRαβ-

TCRγδ-

CD3-

CD4-

CD8α-

CD8β-

CD25-

CD44-

ISP (Intermediate Single Positive Thymocyte)

ISP thymocytes form an intermediate stage between DN4s and DPs. During this stage, the TCRα-chain undergoes rearrangement to form the αβ-TCR before transitioning to the DP stage.


Mouse Markers:

TCRαβ-

TCRγδ-

CD3-

CD8+

DP (Double Positive Thymocyte)

DP thymocytes differentiate from the ISP stage and can be distinguished by the expression of both CD4 and CD8 co-receptors. The fate of DP cells depends on the interaction of their αβ-TCR with MHCI/II molecules expressed on cortical epithelial cells. Upon TCR interaction with self pMHC molecules, there are 3 possible outcomes:

  1. No Signal: DP thymocytes that receive no signal die of neglect.
  2. Weak signal: DP cells pass positive selection and transition to a CD4+CD8low stage before differentiating to single positive CD4 and CD8 T cells (28).
  3. High Affinity: To avoid potential autoimmunity, DP thymocytes expressing high avidity TCRs differentiate to FOXP3+ Treg cells, CD8αα IELs, or are subjected to clonal deletion through the process of negative selection (28, 27).

 

Alternatively, DP thymocytes that express a rearranged Va14i-TCR interact with glycolipids presented on CD1d molecules and differentiate into NKT cells (23).


Mouse Markers:

TCRαβlow/-

CD3low/-

CD4+

CD5+/-

CD8α+

CD8β+

CD69+/-

CD8αα IELs (CD8αα Intraepithelial Lymphocytes)

CD8αα IELs differentiate as an alternative pathway to clonal deletion of autoreactive T cells. Autoreactive DP cells which do not receive a costimulation signal through B7/CD28 escape clonal deletion during the process of negative selection in the thymic medulla. These cells downregulate both CD4 and CD8 co-receptors to become TCRαβ+ DN cells. From here, they migrate to gut tissues where they differentiate to CD8αα IELs and function to regulate intestinal homeostasis and maintain epithelial barrier function (27).


Mouse Markers:

TCRαβ+

CD3+

CD8α+

CD8b-

Transcription factors: Runx3

γδ-T cells

γδ-T cells diverge from αβ-T cells at the DN2 progenitor stage. γδ-T cells represent a small subset of T cells found in tissues of both lymphoid and non-lymphoid origin. Unlike the αβ-TCR, the γδ-TCR recognizes lipid antigens and doesn’t require antigen presentation by MHC molecules.


Mouse Markers:

CD3+

TCRαβ-

TCRγδ+

 

Transcription factors: SOX13

CD4+CD8low

CD4+CD8low cells differentiate from DP thymocytes through positive selection. CD4+CD8low thymocytes pass lineage commitment and differentiate into CD8+ and CD4+ T cells. According to the kinetic signaling model, the lineage fate decision depends on the persistence of TCR signaling in CD4+CD8low cells. Persistence of TCR signaling in CD4+CD8low intermediate thymocytes block IL-7 signaling and induces differentiation into CD4+ T cells. Cessation or disruption of TCR signaling permits IL-7 signaling resulting in co-receptor reversal and differentiation to CD8+ cells (28).


Mouse Markers:

TCRαβlow

CD3low

TCRγδ-

CD4+

CD8αlow

CD8βlow

CD8 Single Positive Thymocyte

CD8+ T cells or cytotoxic T cells differentiate from CD4+CD8low progenitors and recognize antigens presented on MHC class I molecules. Mature CD8+ T cells which are ready to egress the thymus can be identified by downregulation of CD24 and overexpression of S1PR.


Mouse Markers:

TCRαβ+

CD3+

CD4-

CD8α+

CD8β+

CD24+/-

S1PR+/-

 

Transcription factors: Runx3

CD4 Single Positive Thymocyte

CD4+ T cells or T helper cells differentiate from the CD4+CD8low progenitors and recognize antigens presented on MHC class II molecules. Mature CD4+ T cells which are ready to egress the thymus can be identified by downregulation of CD24 and overexpression of S1PR. In the periphery, mature CD4+ T cells differentiate into different T helper subsets upon encountering an antigen.


Mouse Markers:

TCRαβ+

CD3+

CD4+

CD8α-

CD8β-

CD24+/-

S1PR+/-


Transcription factors: TH-POKGATA-3


Learn more about T helper subsets on our T helper types webpage.

Treg

T regulatory cells are specialized T cells responsible for protecting the organism from autoimmune responses by regulating CD4+ and CD8+ T cell cytotoxic activity. One of the transcription factors important for Treg cell differentiation is FOXP3. Mice with the “Scurfy” mutation of the FOXP3 gene leading to ablation of FOXP3 expression exhibit multi-system autoimmune disorders.


Mouse Markers:

TCRαβ+

CD3+

CD4+

CD5+

CD25+

Helios+

CD134 (OX40)+

CD137 (4-1BB)+

CD152 (CTLA)

CD279 (PD-1)+

CD304 (Neuropilin)+

CD357 (GITR)+

 

Transcription factors: FOXP3Helios


Learn more on our T regulatory cell webpage.

iNKT (Invariant Natural Killer T cells)

DP thymocytes that express a rearranged Va14i-TCR interact with glycolipids presented on CD1d molecules and differentiate into NKT cells (23).


Mouse Markers:

CD1d tetramer+

Vα14+

CD3+

CD4+/-

CD24-

CD44+

 

Transcription factors: PLZF


Learn more about different stages of iNKT development and function on our Natural Killer T Cells webpage

MLP (Multi Lymphoid Progenitor)

MLPs are multipotent progenitors derived from LMPPs. They generate all cells in the lymphoid lineage and pDCs.


Human Markers:

Lin- ( CD3-, CD19-, CD56-, CD14-, CD66b-, CD335- , CD11c- )

CD34+

CD38-

CD45RA+

CD10+

CD7+

CD90-

CD135 (Flt3)+

ILC1 (Group 1 Innate Lymphoid Cells)

Like T cells, ILCs are derived from MLPs; however, they do not respond in an antigen-specific manner. Group 1 ILCs include Natural Killer (NK cells) and produce type 1 cytokines including IFN-γ and TNF-α. They play a critical role in protecting the host against pathogens and tumor cells.


NK Cell Human Markers

CD56+

CD3-

CD314 (NKG2D)+


NK Cell Transcription factors: T-bet , Id2, Nfil3


ILC1 Human Markers:

IL-1R+

CD56+

CD127(IL-7Rα)-

NKp30+

NKp44+

NKp46+


ILC1 Transcription factors: T-bet


Learn more about NK and ILC cell development and function on our Natural Killer Cells and Innate Lymphoid Cells webpages.

ILC2 (Group 2 Innate Lymphoid Cells)

Like T cells, ILCs are derived from MLPs; however, they do not respond in an antigen-specific manner. Group 2 ILCs produce cytokines normally associated with Th2 cells including IL-5 and IL-13. They play a key role in response to helminth infection, allergic lung inflammation, and are involved in wound healing.


ILC2 Human Markers:

CD45hi

IL-7Rα+

CD161+

CRTH2+


ILC2 Transcription factors: RORα, GATA-3


Learn more about Innate Lymphoid cell development and function on our Innate Lymphoid Cells webpage.

ILC3 (Group 3 Innate Lymphoid Cells)

Like T cells, ILCs are derived from MLPs; however, they do not respond in an antigen-specific manner. Group 3 ILCs were originally discovered as cells that express NKp46 but do not resemble normal NK cells. Instead, they express RORγt and produce IL-17 and IL-22. ILC3s primarily reside in mucosal tissues where they help maintain intestinal homeostasis. Lymphoid tissue inducer cells (LTi) are considered subset of group 3 ILCs that are involved in lymphoid organ formation during embryogenesis though the exact relationship between LTis and other ILC3s is still being investigated.

 

ILC3 Human Markers:

CD117 (c-Kit)+

CD127 (IL-7Rα)+

NKp44+/-

NKp46+/-

IL-1R+

IL-23R+


ILC3 Transcription factors: RORγt


Learn more about Innate Lymphoid cell development and function on our Innate Lymphoid Cells webpage.

ETP/DN1 (Early T Cell Progenitor/Double Negative Thymocyte 1)

ETP/DN1s are one of the first T cell progenitor populations that have been identified in the thymus. These progenitors are named double negative as they do not express the CD4 or CD8 co-receptors found on mature T cells. Additionally, they do not express the CD3/TCR complex.


Human Markers:

TCRαβ-

TCRγδ-

CD1α-

CD3-

CD4-

CD8α-

CD8β-

CD7-

CD10-

CD34+

DN2 (Double Negative Thymocyte 2)

DN2 thymocytes are differentiated from DN1 progenitors. These progenitors were named double negative as they do not express the CD4 or CD8 co-receptors found on mature T cells. At this stage, upregulation of RAG1/RAG2 recombinases induces VDJ rearrangement of the β-chain and γδ- loci of the TCR. Meanwhile, TdTs (Terminal Deoxynucleotidyl Transferases) add N- and P- nucleotides to the end of the V, D and J segments to diversify the specificity of the newly forming T cell receptor.

In mice, the αβ/γδ lineage fate depends on TCR signal strength. The signal strength model predicts that the γδ TCR transduces a stronger signal directing cells to the γδ lineage, while a pre-TCR transduces a weaker signal directing progenitors to the αβ lineage (25, 29,30).


Human Markers:

TCRαβ-

TCRγδ-

CD1α-

CD3-

CD4-

CD8α-

CD8β-

CD7+

CD10-

CD34+

DN3 (Double Negative Thymocyte 3)

DN3 thymocytes are differentiated from DN2 progenitor cells. These progenitors were named double negative as they do not express the CD4 or CD8 co-receptors found on mature T cells. DN3 T cells which have successfully rearranged a TCRβ-chain that is capable of pairing to a surrogate TCRα-chain pass the β-selection checkpoint and undergo clonal division before transitioning to the ISP stage. Alternatively, DN3 thymocytes which have a rearranged γδ-TCR continue their differentiation towards the γδ-T cells.


Human Markers:

TCRαβ-

TCRγδ-

CD1α+

CD3-

CD4-

CD8α-

CD8β-

CD7+

CD34+

ISP (Intermediate Single Positive Thymocyte)

ISP thymocytes form an intermediate stage between DN3s and DPs. During this stage, the TCRα-chain undergoes continued rearrangement to form the αβ-TCR before transitioning to the DP stage.


Human Markers:

TCRαβ-

TCRγδ-

CD1α+

CD3-

CD4+

CD8α-

CD8β-

DP (Double Positive Thymocyte)

DP thymocytes differentiate from the ISP stage and can be distinguished by the expression of both CD4 and CD8 co-receptors. The fate of DP cells depends on the interaction of their αβ-TCR with MHCI/II molecules expressed on cortical epithelial cells. Upon TCR interaction with self pMHC molecules, there are 3 possible outcomes:

  1. No Signal: DP thymocytes that receive no signal die of neglect.
  2. Weak signal: DP cells pass positive selection and transition to single positive CD4 and CD8 T cells (28).
  3. High Affinity: To avoid potential autoimmunity, DP thymocytes expressing high avidity TCRs differentiate to FOXP3+ Treg cells or are subjected to clonal deletion through the process of negative selection.

 

Alternatively, DP thymocytes that express a rearranged Va24i-TCR interact with glycolipids presented on CD1d molecules and differentiate into NK-T cells (23).


Human Markers:

CD3lo/-

TCRαβlo/-

CD4+

CD8α+

CD8β+

γδ-T cells

γδ-T cells diverge from αβ-T cells at the DN2 progenitor stage. γδ-T cells represent a small subset of T cells found in tissues of both lymphoid and non-lymphoid origin. Unlike the αβ-TCR, the γδ-TCR recognizes lipid antigens and doesn’t require antigen presentation by MHC molecules.


Human Markers:

CD3+

TCRαβ-

TCRγδ+


Transcription Factors: Id3

CD8 Single Positive Thymocyte

CD8+ T cells or cytotoxic T cells differentiate from DP progenitors and recognize antigens presented on MHC class I molecules.


Human Markers:

CD3+

TCRαβ+

CD4-

CD8α+

CD8β+


Transcription Factors: Runx3STAT5

CD4 Single Positive Thymocyte

CD4+ T cells or T helper cells differentiate from DP progenitors and recognize antigens presented on MHC class II molecules. In the periphery, mature CD4+ T cells differentiate into different T helper subsets upon encountering an antigen.


Human Markers:

CD3+

TCRαβ+

CD4+

CD8α-

CD8β-


Transcription Factors: TH-POKGATA-3


Learn more about T helper subsets on our T helper types webpage.

Treg

T regulatory cells are specialized T cells responsible for protecting the organism from autoimmune responses by regulating CD4+ and CD8+ T cell cytotoxic activity. One of the transcription factors important for Treg cell differentiation is FOXP3. Mice with the “Scurfy” mutation of the FOXP3 gene leading to ablation of FOXP3 expression exhibit multi-system autoimmune disorders. The phenotype of Scurfy mice closely resembles human IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked) syndrome.


Human Markers:

CD3+

TCRαβ+

CD4+

CD25+

CD45RA+

CD127lo


Transcription Factors: FOXP3


Learn more on our T regulatory cell webpage.

iNKT (Invariant Natural Killer T cells)

DP thymocytes expressing the rearranged Va24i-TCR interact with glycolipids presented on CD1d molecules and differentiate into NK-T cells.


Human Markers:

CD1d tetramer+

Vα24+

CD3+

CD56+

CD28+

CD45RO+


Transcription Factors: PLZF


Learn more about different stages of iNKT development and function on our Natural Killer T Cells webpage.