Table of contents :

• cell types of the adaptive immune system : lymphopoiesis

Even the ontogeny of the adaptive immune system recapitulates its phylogeny. All the maturation stages are Ag-independent : the bone marrow produces 10⁶ cells / day. Homo sapiens body contains ~ 2 ^. 10¹² lymphocytes.
• pluripotent lymphoid stem cell (PLSC) / multipotent lymphoid progenitor (MLP) (CD34⁺) in bone marrow
‡
+ SCF
• bipotent lymphoid progenitor (CD34⁺)

BCL11A / EVI9 functions as a myeloid or B cell proto-oncogene in mice and humans, respectively. Bcl11a is essential for postnatal development and normal lymphopoiesis. Bcl11a mutant embryos lack B cells and have alterations in several types of T cells. Phenotypic and expression studies show that Bcl11a functions upstream of the transcription factors Early B cell factor (EBF) and Pax5 / BSAP in the B cell pathway. Transplantation studies show that these defects in Bcl11a mutant mice are intrinsic to fetal liver precursor cells. Mice transplanted with Bcl11a-deficient cells died from T cell leukemia derived from the host. Thus, Bcl11a may also function as a non-autonomous T cell tumor suppressor gene. The early B cell–specific mb-1 promoter is hypermethylated at CpG dinucleotides in HSCs but becomes progressively unmethylated as B cell development proceeded. EBF, a transcription factor required for B lymphopoiesis, potentiated activation of mb-1 by the transcription factor Pax5 (which alone cannot activate endogenous mb-1 transcription in a plasmacytoma cell line). EBF and the basic helix-loop-helix transcription factor E47 each contributed to epigenetic modifications of the mb-1 promoter, including CpG demethylation and nucleosomal remodeling. EBF function is enhanced by interaction with the transcription factor Runx1^ref.
‡
+ IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 (=> up-regulation of antiapoptotic Bcl-2 and MCL-1^ref (which selectively inhibits the proapoptotic protein BIM), is essential both early in lymphoid development and later on in the maintenance of mature lymphocytes), IFN-g, EBF (TF for CD79a / Iga) :
• early B progenitor (CD10 / CALLA⁺19⁺34⁺38⁺45 / B220 / LCA⁺79a / Iga⁺79b / Igb⁺, MHC II⁺, TdT⁺). In bony fishes B cells develop since day 4 in pacreas and since week 3 in pronephros (head kidney), which remains the main source organ.

‡
+ ? (inhibited by IFN-a => TYK2 => DAXX), ZAP70
• early pro-B cell (CD10 / CALLA⁺19⁺24 / HSA⁺34⁺38⁺40⁺43⁺45 / B220 / LCA⁺79a / Iga⁺79b / Igb⁺, MHC II⁺ IgM^- BP1⁺) : D_HJ_H rearrangement.


‡
+ Pax-5 (TF for CD19 and transcriptional repressor for Notch1), cyclin D3^ref, BLNK / SLP65, ZAP70
• late pro-B (a.k.a. pre-BI or stage B/C) cell (CD10 / CALLA⁺19⁺20⁺25^-38⁺40⁺43⁺45 / B220 / LCA^int): V_HD_HJ_H rearrangement. To survive and proliferate, pro-B cells must be able to interact with intramarrow stromal cells and to react to soluble and membrane factors that these cells produce. Moreover, to differentiate into pre-B cells, pro-B cells must initiate and successfully complete the V(D)J recombination program at the IgH locus, and to express on cell membrane a m heavy chain (mmH) capable of pairing with the invariant (i.e. non rearranged) surrogate light (SL / yLC) chains (SLC) (a heterodimer formed by CD179a / VpreB and CD179b / l5 / l-like 1). Such surrogate sIgM joins to CD79a / Iga and CD79b / Igb to form the pre-B cell receptor (pre-BcR / preBcR / pBcR) complex, allowing B cells to proliferate in a ligand-independent manner : the nonimmunoglobulin portion of l5 mediates cell-autonomous pre-BcR signalling via a crosslinking that involves a linking factor produced by the pre-B cell itself or non-covalent interactions between 2 l5 non-immunoglobulin regions. Based on the observations that the surrogate L chains also assemble on the surface of mm^- pro-B cells, it was proposed that membrane protein complexes analogous to pre-BcR and BcR may regulate some of the survival and differentiation processes in early pro-B cells. More recently, Iga and Igb have also been on the surface of mm^- pro-B cells in protein complexes that do not contain surrogate L chains, but 4 other proteins, one of which has been identified as calnexin. m gene recombination is not affected in pro-B cells lacking Iga, Igb, or both molecules, and the size and phenotype of the pro-B cell population is also not affected in vivo.

Until recently, it was generally assumed that all H chain proteins were capable of bringing about these changes; however dysfunctional H chains neither mediate heavy chain allelic exclusion nor drive clonal maturation, due to the inability to pair with SLC and form a pBcR. Significantly, failure to pair with SLC is also indicative of the inability of the H chain protein to pair with conventional L chain. Analysis of the Ig rearrangements from early B cells has revealed that as many as 50% of all H chain proteins are dysfunctional. Since V_H genes have been found that can encode for either a functional or a dysfunctional H chain the inability to pair with SLC must arise as a result of differences within the CDR3 sequence : the CDR3, formed by DNA recombination between V_H, D, and J_H genes, is the only sequence that varies between H chains using the same V_H gene.
‡
+ p110^d subunit of PI3K, CD21 / CR2 / C3dR / EBVR, Syk, ZAP70, myeloid-cell leukemia 1 (MCL1)^ref (a component of the signalling pathway downstream of IL-7, which binds BIM and prevents BIM-mediated activation of the pro-apoptotic proteins BAK1 and BAX), FOXP1 (which binds to the Erag enhancer of RAG1 and RAG2 and is involved in controlling VDJ recombination of the gene encoding IgH in a B cell lineage–specific way)^ref :
• large pre-B cell (CD7⁺9⁺10 / CALLA^+?19⁺20⁺21 / CR2 / C3dR / EBVR⁺24⁺38⁺40⁺43^-45 / B220 / LCA⁺79a / Iga⁺79b / Igb⁺179a / VpreB⁺179b / l5⁺, mmH⁺, MHC II⁺).

‡
• small pre-B cell or early immature cell (CD7⁺9⁺10 / CALLA^+?19⁺20⁺21 / CR2 / C3dR / EBVR^-24⁺38⁺40⁺43^-79a / Iga⁺79b / Igb⁺) : V_LJ_L rearrangement. 76% of antibodies from early immature B cells are self-reactive and polyreactive.

‡
• immature B cell

(CD19⁺20⁺21 / CR2 / C3dR / EBVR^-23^-24 / HSA^hi40⁺79a / Iga⁺79b / Igb⁺ IgM⁺IgD^lo/-, E₂R⁺). 43.1% of antibodies from immature B cells are self-reactive and a few are polyreactive.
‡
+ Syk
• newly formed or new emigrant or transitional (TR) type 1 (T1) B cell

(CD1^lo19⁺20⁺21 / CR2 / C3dR / EBVR^lo/-22^lo23^lo/-24 / HSA^hi40⁺45 / B220 / LCA^lo79a / Iga⁺79b / Igb⁺ IgM^hi/+IgD^lo/-)
Located in bone marrow, peripheral blood, B-cell zones of spleen PALS. 20% of antibodies from T1 B cells are self-reactive, with the potential to contribute to autoimmune diseases.
‡
+ Rac1, Rac2^ref
• transitional (TR) type 2 (T2) B cell

(CD1^hi19⁺20⁺21 / CR2 / C3dR / EBVR^hi22⁺23^hi24^int40⁺45 / B220 / LCA^lo79a / Iga⁺79b / Igb⁺, MHC II⁺, IgM^hiIgD^hi)
Located in B-cell zones of spleen PALS
‡
+ BOB.1 (which downregulates CD22), BLNK / SLP65
• resting (naive) mature B or B0 cell (CD13^-15^-19⁺20⁺21 / CR2 / C3dR / EBVR⁺22⁺23⁺24⁺25⁺32 / FcgRII⁺38⁺39⁺40⁺45 / B220 / LCA^hi72⁺75⁺79a / Iga⁺79b / Igb⁺, MHC II⁺IgM⁺IgD^-).

Approximately 65% of B cells generated in human bone marrow are potentially harmful autoreactive B cells. Most of these cells are clonally deleted in the bone marrow (negative selection by BcR editing as in the thymus for T lymphocytes), while those autoreactive B cells that escape to the periphery are anergized or undergo clonal deletion by AICD before becoming mature B cells. Escape of self-reactive B cells from tolerance permits production of pathogenic auto-antibodies; recent studies suggest that extended B lymphocyte survival is a cause of autoimmune disease in mice and humans. Spontaneous death of resting B cells is regulated by nuclear localization of PKCd that contributes to phosphorylation of histone H2B at Ser¹⁴ (S14-H2B) : treatment of B cells with BAFF prevents nuclear accumulation of PKCd^ref. Those occasionally found in the thymus were once called T_h5 cells in mice. Bone marrow produces 10⁹ B cells/day. Mature B lymphocytes represent 10% of bone marrow lymphocytes, 10-15% of peripheral blood lymphocytes (0.3 ^. 10⁹ B cells/L = 1.5 ^. 10⁹ B cells), and 50% of spleen lymphocytes. Once terminated the Ag-independent development in primary lymphoid organs, mature B cells undergo Ag-dependent maturation in secondary lymphoid organs.
‡
+ interaction between BcR and self-Ag => differentiation, expression and maintenance
• B-1 cell (CD9⁺11b / a_M integrin⁺14^lo15^-19⁺20⁺21 / CR2 / C3dR / EBVR^lo23^lo/-40⁺43⁺45 / B220 / LCA^lo72⁺w125 / IL-5Ra⁺ IgM^hisIgD^lo) : CXCL13 / BLC-mediated homing from fetal liver and omentum to peritoneal and pleural cavities up to adolescence : not recirculating, they are absent from lymph nodes, spleen and peripheral blood. They produce IL-10 and IL-10R (autocrine signalling). They enter S phase in response to phorbol ester alone. B-1 B cells are not usually associated with autoimmune diseases (being able to undergo class switch recombination and somatic hypermutation) : anyway typical B-1 antibodies do not seem to undergo extensive hypermutation, perhaps because of a lack of interaction with T cells. They represent the cell of origin for B cells chronic lymphocytic leukemia (B-CLL).  On Ag encounter they might give rise to plasma cells that produce a V_H selected nonimmune repertoire of "natural" serum IgM (natural antibodies (NAb)) and substantial amounts of IgA in the gut and mesenteric lymph nodes. They are considered to be innate lymphocytes as among the known BcR specificities typical of B-1 cells are autoantigens (=> natural autoantibodies (nAAbs)), such as plasma membrane phospholipids (eg V_H11-V_k9 or V_H12-V_k4 Ig genes - expressed by 5-15% of the peritoneal B-1 cells in normal mice - react with phosphatidylcholine (PtC)), and conserved epitopes present on common pathogens, such as polysaccharide moieties : this set of specificic VDJ rearrangements encoded in the germline with short or absent N sequences is evolutionarily retained and provides at low affinity a degree of serological protection against a range of microorganisms prior to the immunization that accompanies microbial pathogenesis. There is mounting indirect evidence that self-Ags drive the development of naive B cells, but Hardy's murine model, dealing with a monoreactive autoantibody directed against a membrane-bound Ag, is, to date, the only direct proof of such a positive selection of B cells. However, as mentioned above, nAAbs are mostly polyreactive, and mainly react with conserved soluble self-Ags such as ssDNA, IgG, thyroglobulin, or cytokines. Precisely because of the polyreactivity profile of these nAAbs, demonstrating the role of a given autoantigen by its elimination is difficult. B-1 B cells are the first B cells to express fully mature levels of CXCR5, thereby promoting the development of follicular dendritic cells (FDC) in spleen^ref.
• B-1S cell (precursor in adult bone marrow => in spleen) CD11b / a_M integrin^-
• B-1P cell (precursor in fetal liver => in peritoneal cavity) CD11b / a_M integrin⁺
• B-1a B cells (CD5 / Ly-1⁺11b / a_M integrin^-23^-24 / HSA^lo43⁺45 / B220 / LCA^int IgM^hisIgD^lo). Their number is reversibly increased in mice deficient for serum "natural" IgM (negative feedback ?).
• B-1b B cells (CD5 / Ly-1^-11b / a_M integrin⁺23^-24 / HSA^lo43⁺45 / B220 / LCA^int IgM^hisIgD^lo) : CD5 / Ly-1 binds membrane lipids and modulates BcR signalling through SHP-1 / PTPN6 recruitment, causing an altered responsiveness to BcR ligation compared with their B-2 cohorts (marked diminished ability for intracellular Ca²⁺ mobilization, aberrant proliferation, and increased apoptosis after BcR cross-linking). B-1b cells produce IgM natural antibodies against a1-3G7a1b1-4GlcNAc (aGal). These can be tolerized by non-myeloablative induction of mixed chimerism using Gal⁺ donor marrow. The role of CR1/2 was assessed in this model for induction of tolerance of B-1b cells. Mixed hematopoietic chimerism was induced in a1-3-galactosyltransferase (GalT^-/-) and GalT^-/-Cr2^-/- mice with aGal⁺ BALB/c marrow donors. Anti-Gal Ab and anti-Gal Ab-producing B cells became undetectable in GalT^-/- chimeras, whereas they persisted in chimeric GalT^-/-Cr^-/- mice. To determine whether CR1/2 expression on stromal cells and/or hematopoietic cells was critical for B-1 cell tolerance, GalT^-/- radiation chimeras were generated in which CR1/CR2 was expressed on either stromal cells, hematopoietic cells, neither, or both. After induction of mixed chimerism from aGal⁺ allogeneic bone marrow (BM) donors, anti-Gal-producing B cells were rendered tolerant in reconstituted recipients expressing only stromal CR1/CR2. Follicular dendritic cells may pick up immune complexes via CR1/CR2 receptors and induce tolerization of B-1b cells^ref
... or ...

without interaction between BcR and self-Ag

• B-2 cell (conventional B cell) (CD1^lo5 / Ly-1^-19⁺21⁺23⁺24 / HSA⁺72⁺sIgM⁺sIgD^-TLR1⁺TLR6⁺ TLR7⁺TLR9⁺TLR10⁺RP105) :

enter S phase in response to anti-Ig stimulation; located in :
• bone marrow
• peripheral blood : 16 to 28% (of these, 4-12.7% have sIgG, 1-4.3 have IgA, 6.7-13% have IgM, and 5.2-8.2 have IgD).
• spleen :
+ p110^d subunit of PI3K
• marginal zone (MZ) B cells(CD1D^hi9⁺11a / a_L integrin⁺18 / b₂ integrin⁺21^hi22^hi23^lo/-24 / HSA^int29 / b₁ integrin⁺49d / a₄ integrin⁺ IgM^hiIgD^lo/-S1P₁⁺S1P₃⁺) : lower threshold for activation, mainly involved in responses against TI-Ags ; express Gab1 and Id2. MZ B cell localization to the marginal zone depends on responsiveness to the blood lysophospholipid S1P, with S1P₁ signaling overcoming the recruiting activity of CXCL13. In mice lacking the chemokine CXCL13, S1P₁-deficient marginal zone B cells reacquired a marginal zone distribution. Exposure to LPS or antigen caused marginal zone B cells to downregulate S1P₁ and S1P₃ and to migrate into the splenic white pulp^ref. CXCL13-independent homing; non-recirculating thanks to a₄b₁integrin---CD106 / VCAM1 and a_Lb₂ integrin---CD54 / ICAM-1 interactions (both ligands are upregulated by LT-b); upon stimulation with bacterial products they undergo CXCL13-dependent migration to the B-cell follicles at the T-B junction of the spleen where they divide and generate large clones of IgM-secreting plasma cells within 3 days. They are considered to be innate lymphocytes. Their number is reversibly increased in mice deficient for "natural" serum IgM (natural antibodies (NAb)) (negative feedback ?).
• follicular (FO) B cells (CD1D^int9^-21^int22^hi23^hi IgM^int/loIgD^hi) (CXCL13-dependent homing from adult bone marrow into the follicles; recirculating)
• lymph nodes (recirculating)
They enter S phase in response to anti-Ig stimulation. Tonsillar B cells exhibit constitutively high levels of TLR1, TLR2, TLR7, TLR9 and TLR10, which are not developmentally regulated during the B-cell differentiation process. Ongoing microbial infections, such as chronic tonsillitis, do not appear to affect the TLR profile in B cells^ref.
... or ...

+ Notch1 (vs. mNumb)

• common T-cell and NK progenitor (C-TNKP) (CD3deg⁺19^-34⁺117 / c-kit / SCFR⁺)
‡
• NK progenitor or pro-T1 (CD2^-3deg⁺56⁺161⁺)

‡
+ IL-2, IL-15
• natural killer (NK) cell / large granular lymphocyte (LGL) / null cell (CD2⁺3^-4^-8^-(rarely 8ab⁺)14^-15^-16a / FcgRIIIA⁺19^-46⁺55 / DAF⁺56 / NKH1 / NCAM / Leu-19⁺57 / HNK1 / Leu-7^{+ (absent in cord blood NK cells !)}85⁺94⁺178 / Apo1L / FasL⁺226 / DNAX accessory molecule 1 (DNAM1)⁺244 / 2B4⁺, MHC II^-, TcR^-, asyalo-GM₁⁺) : small eosinophilic granules in the cytoplasm; 5÷15% of blood lymphocytes (more abundant in TAP^-/- individuals), localized mainly in spleen but not in thymus and lymph nodes. CD94.1-expressing blood cells in the urochordate Botryllus schlosseri might be ancestral NK cells : they mediate cytotoxic lesion at the point of contact between colonies.


• NK gene complex (NKC) is on human chromosome 12p13
• leukocyte receptor cluster (LRC) on chromosome 19q13.4 (evolved by gene duplication, together with FcaR gene; 15 haplotypes => > 100 genotypes) : diversity at the KIR locus has been generated by an evolutionary history of expansion and contraction, presumably due to combinations of duplication and unequal crossing over within the locus. Up to 12 KIR genes appear to be expressed, and KIR haplotypes consist of various combinations of KIR genes. The number of putatively expressed KIR genes present on a single haplotype ranges from 7 to 11, depending primarily on the presence or absence of activating KIR loci. Estimates of the extent of KIR genotype diversity within the population suggest that far < 0.24% of unrelated individuals can expect to have identical genotypes. The most common Caucasian haplotype, the "A" haplotype (frequency of ~ 47-59%), contains only 1 activating KIR gene (KIR2DS4) and 6 inhibitory KIR loci (KIR3DL3, -2DL3, -2DL1, -2DL4, -3DL1, and -3DL2). The remaining "B" haplotypes are very diverse and contain 2 to 5 activating KIR loci (including KIR2DS1, -2DS2, -2DS3, and-2DS5).
NK cell receptors don't cover the whole set of class I HLA alleles and are not expressed on 100% of NK cells.
Diversity in KIR gene content, KIR specificity for particular HLA allotypes, and the unlinked physical location of the KIR loci relative to the HLA loci give rise to the possibility that, for some KIR loci, any given individual may encode receptor only, ligand only, both receptor and ligand, or neither one. NK cell receptors (NKR) are all type II transmembrane proteins.
• inhibitory receptors (also expressed on memory CD8⁺ >> CD4⁺ ab and gd T cells) ...
• ... have cytoplasmic ITIM(s) that, upon binding to MHC I, become phosphorylated by c-Src and allow recruitment of SHP1 / PTPN6 and/or SHP2, LCK and talin :
• killer cell Ig-like (or inhibitory) receptors (KIR) / CD158 family : they undergo Zn²⁺- (and Co²⁺- ?) induced multimerization.
• KIR2DLs (long intracytoplasmic tail with 2 ITIMs)
• KIR2DL1 / CD158a / p58.1 (D1 and D2 Ig domains) --- group 2 HLA-C allotypes (HLA-C2)
• KIR2DL2 / CD158b / p58.2 (D1 and D2 Ig domains) --- group 1 HLA-C molecules (HLA-C1)
• KIR2DL3 / CD158b / p58.3 (D1 and D2 Ig domains) --- group 1 HLA-C molecules (HLA-C1)
• KIR2DL4 (D0 and D4 Ig domains) --- HLA-G
Anyway it has activating function !!!
• KIR3DLs (3 Ig domains (D0, D1 and D2), long intracytoplasmic tail with 2 ITIMs)
• KIR3DL1 / NKB1 / NKR p70 --- HLA-B serotypes that have the Bw4 >> Bw6 epitope (determined by amino acids positions 79-83 in a₁ domain of the molecule), e.g. HLA-B27 : HLA Bw4 molecules with Ile⁸⁰ (Bw4-80I) may be better ligands than those containing Thr⁸⁰.
• KIR3DL2 / NKR p140 --- HLA-A3 and HLA-A11
• killer cell lectin-like receptors (KLR) (belonging to C-type (Ca²⁺-dependent) lectin SF).
• CD94 / KLRD1
• KLRC1-derived NKG2A isoform (both ITIMs are required for mediating the maximal inhibitory signal, but opposite to KIR, the membrane distal ITIM is of primary importance rather than the membrane-proximal ITIM : this probably reflects the opposite orientation of the ITIMs in type II vs type I proteins).
• KLRC1-derived NKG2B isoform
The receptor may occur either as a CD94 homodimer or as a disulfide bound CD94 heterodimer with NKG2A or NKG2B) --- HLA-E charged with HLA-A, HLA-B, HLA-C, or HLA-G leader peptides (so cells that don't express MHC Ia have no ligand on their surfaces : such charging is TAP-independent as leader peptides are already available in RER lumen) or with a peptide from the chaperonin HSP60, up-regulated in stressed cels (in this latter case the complex cannot be recognized by CD94/NKG2A, allowing killing of stressed cells). Expression of both CD94 and NKG2A is induced by IL-12. CD94/NKG2A is continuously recycled between the cell surface and cytoplasm, and that this recycling is independent of ligation and the transmission of inhibitory signals. Because CD94/NKG2A receptors are in the constant presence of ligand expressed by normal cells, the detachment of recycling from ligation/inhibitory processes likely facilitates the maintenance of a pool of CD94/NKG2A receptors on the cell surface available for interaction with HLA-E
• Ig-like transcripts (ILTs) / leukocyte inhibitory receptors (LIRs) / monocyte-macrophage inhibitory receptors (MIRs) family
• CD85 / LIR-1 / MIR7 / ILT-2 --- HLA class Ia and HLA-G1
• LIR-2 / MIR10 /  ILT-4 --- HLA class Ia and HLA-G1
• ... have a different unknown STP :
• tetraspanin family
• CD81 / TAPA-1 --- protein E2 on HCV envelope
• CD161 / KLRB1 / NKR-P1A / NKR-P1B, NKR-P1D, and NKRP1F^ref (CD161b/d/f) (contains an extracellular domain with several motifs characteristic of C-type lectins, a transmembrane domain, and a cytoplasmic domain. The KLRB1 protein is classified as a type II membrane protein because it has an external C terminus.) --- C-type lectin superfamily 2, member D (CLEC2D) / osteoclast inhibitory lectin (Ocil) / Clr-b / CLAX / LLT-1, a member of a previously cloned group of C-type lectin-related (Clr) proteins linked to the NKR-P1 receptors in the mouse NK gene complex (NKC). Expression of Ocil/Clr-b on mouse tumor cell lines inhibits NK cell-mediated killing. Inhibition is blocked with a new mAb (4A6) specific for Ocil/Clr-b. Ocil/Clr-b is displayed at high levels on nearly all hematopoietic cells, with the exception of erythrocytes, in a pattern that is similar to that of class I MHC molecules. Remarkably, Ocil/Clr-b is frequently down-regulated on mouse tumor cell lines, indicating a role for this receptor-ligand system in a new form of "missing self-recognition" of tumor cells^ref.
• tyrosine phosphorylation of the cytoplasmic tail of SH2D1B / EAT-2 and ELF3 / ERT adaptors, which associate with the SLAM-related receptor CD244 / 2B4, represses natural cytotoxicity and IFN-g secretion^ref
• activating receptors are linked to intracellular adapters :
• adapter FceRI g chain or CD247 / z chain (ITAM-containing) => ZAP70 and Syk => PI(3)K STP
• CD16a / FcgRIIIA --- IgG (mediates ADCC)
• NKp30 / 1C7 / B144 / LST1 / D6S49E --- ? (also used for DCs killing).

[pp65, the main tegument protein of HHV-5 / HCMV, inhibits NK cell cytotoxicity by a direct and specific interaction with the activating receptor NKp30, leading to dissociation of the linked CD3z from NKp30^ref]
• NKp46 --- HA_{Influenza virus}, ?
• Killer cell Ig-like (or inhibitory) receptors (KIR) / CD158 family : they undergo Zn²⁺- (and Co²⁺- ?) induced multimerization.
• p50^KIR2DSs (2 Ig domains (D1 and D2), short intracytoplasmic tail with no ITIM) --- HLA-C, non-MHC molecules (foreign or microbial antigens expressed on infected cells, normal cell surface proteins that are aberrantly expressed, or complexes of pathogen-derived peptides bound to MHC class I molecules), ?
• KIR2DS1 / CD158a / p50.1 ---
• KIR2DS2 / CD158b / p50.2 --- group 1 HLA-C molecules (HLA-C1)
• KIR2DS3 ---
• KIR2DS4 / PAX / p50.3 ---
• KIR2DS5 ---
• KIR3DS1 (3 Ig domains (D0, D1 and D2), short intracytoplasmic tail) --- at least some of the HLA-Bw4 allotypes in HIV-1⁺ individuals.
• adapter p14^{DNAX-activating protein of 12 kDa (DAP12) / KARAP} homodimer (recruited by electrostatic interaction between transmembrane domains; ITAM-containing) => ZAP70 and Syk => PI(3)K STP. This STP is required to trigger NKG2D-dependent cytokine release by NK cells.
• killer cell lectin-like receptors (KLR) (belonging to C-type (Ca²⁺-dependent) lectin SF).
• CD94 / KLRD1
• NKG2C / KLRC2
• NKG2E / KLRC3
• NKG2I / KLRE1 (40% homology with CD94 and NKG2D, but lacks any signalling motifs or positively charged residues in its putative transmembrane region, which are characteristics of the NKG2 family, and shows little similarity to other family members in its putative ligand-binding domain, indcating that it is likely to interact with unique ligands; homodimer or heterodimer ? The earlier report indicated that it can dimerize with an unidentified partner containing an ITIM motif and form an inhiitory receptor complex^ref, while a later report suggests it is an activating NK-cell receptor involved in initiating pathways that lead to NK-cell-mediated allograft rejection^ref, just like CD94)
The receptor may occur either as a CD94 homodimer or as a disulfide-bound heterodimer --- HLA-E charged with HLA-A, HLA-B, HLA-C, or HLA-G leader peptides (so cells that don't express MHC I molecules have no HLA-E on their surfaces ! Such charging is TAP-independent as leader peptides are already in RER lumen)
• NKp44 --- HA_{Influenza virus}, ?
• adapter DNAX-activating protein of 10 kDa (DAP10) / phosphoinositide-3-kinase adaptor protein (PIK3AP) (no ITAM motif => PI(3)K and AKT STP !)
• killer cell lectin-like receptors (KLR) (belonging to C-type (Ca²⁺-dependent) lectin SF)
• KLRK1 / NKG2D / NKG2F / KLRC4-L (homodimer) ---
• MICA and MICB (K_d = 5 ^. 10^-7 M)
• ULBPs
The binding of NKG2D to its various ligands is generally of higher affinity than many immunoreceptor-ligand interactions, with a K_dranging from 10^-6 M to 4 ^. 10^-9 M. Despite the marked differences in their amino-acid sequences, the different ligands interact with NKG2D similarly (diagonally across the a-helical surface of each of these ligands, similar to the mode of binding of a TcR to an MHC molecule), and the receptor does not seem to undergo marked conformational changes (induced fit) to accommodate different ligands. Notably, most of the receptor amino-acid residues that dominate binding to the different ligands are the same, and several of the contact residues on the ligands are conserved, expecially those that are thought to contribute most of the binding energy. Another interesting feature of the structures is that different residues in the 2 NKG2D monomers that make up the homodimer dominate binding to the asymmetric a₁ and a₂ domains of the ligands. The NKG2D signal is strong enough to overcome inhibitory signalling by MHC-specific receptors in some cases.
The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor^ref
• adapter SAP / SH2D1A
• CD244 / 2B4 --- CD48 / Blast-1 / OX-45 / Hu Lym3
• adapter ? => ERK STP
• CRACC --- ?
• DNAX accessory molecule 1 (DNAM1) / CD226 --- poliovirus receptor (PVR) and the d and a isoforms of CD115 / poliovirus receptor-related 2 (PRR2) / herpesvirus entry mediator B (HVEB) / nectin-2 (at adherens junctions of myelo-monocytic and megakaryocytic hematopoietic, neuronal, endothelial and epithelial cells)
The susceptibility of the target cell to killing by NK cells depends on the balance of the ligands for activating and inhibitory receptors. This is reminiscent of the balance between proapoptotic and antiapoptotic members of the Bcl-2 family proteins that control intrinsic apoptosis and presumably reflects the fact that "fuzzy" decision-making mechanisms are more resistant to errors than discrete, all-or-none processes. Peroxiredoxin (PRDX1) is required in both NK cells and non-immune cells for optimal NK function.
See also activation of NK cells.
• KLRH1
Comprising the third largest lymphocyte population, NK cells recognize and kill cellular targets and produce pro-inflammatory cytokines. These potentially self-destructive effector functions can be controlled by inhibitory receptors for the polymorphic MHC class I molecules that are ubiquitously expressed on target cells. However, inhibitory receptors are not uniformly expressed on NK cells, and are germline-encoded by a set of polymorphic genes that segregate independently from MHC genes. Therefore, how NK-cell self-tolerance arises in vivo is poorly understood. NK cells acquire functional competence through 'licensing' by self-MHC molecules. Licensing involves a positive role for MHC-specific inhibitory receptors and requires the cytoplasmic inhibitory motif originally identified in effector responses. This process results in 2 types of self-tolerant NK cells—licensed or unlicensed—and may provide new insights for exploiting NK cells in immunotherapy. This self-tolerance mechanism may be more broadly applicable within the vertebrate immune system because related germline-encoded inhibitory receptors are widely expressed on other immune cells^ref.
Redirected killing is an experimental system for determining the capacity of a NK-cell receptor to induce cytotoxicity. NK cells coated with antibody specific for a candidate receptor are assessed for their ability to kill target cells that express an Fc receptor to which the antibody binds.
NK cell development is thought to occur in the bone marrow. GATA-3 and CD127 (IL-7R) are molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127⁺ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and IL-7. The CD127⁺ NK cells had a distinct phenotype (CD11b^loCD16^-CD69^hiLy49^lo) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56^hiCD16^- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56⁺CD16⁺ NK cells. Bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets^ref.
Human NK cells can be divided into two phenotypically distinct functional subsets based on their cell surface expression of CD56 (CD56^bright and CD56^dim). As mouse NK cells do not express CD56, comparable mouse NK cell subsets have proven difficult to identify. Mouse NK cells can be subdivided by the expression of CD27. The CD27^hi and CD27^lo mouse NK cell subsets show some intriguing similarities to but also some distinct differences from the human CD56 NK cell subsets in terms of their function and phenotype^ref
Bibliography : Carrington, Mary and Norman, Paul : The KIR gene cluster, Bethesda (MD):National Library of Medicine (US), NCBI; 2003.
Web resources : NKcells.info by Volker Huppert

or ...

+ ? to become a :

• primary prothymocyte / haematopoietic precursor / bone-marrow derived progenitor / pro-T2 (CD7⁺34^- or CD7⁺34⁺) : needs ..
• Ikaros (which associates with nucleosome-remodelling histone (NuRD) complex, a multisubunit complex containing nucleosome remodeling and HDAC activities, including methyl-CpG binding domain protein 3 (MBD3), metastasis-associated gene family, member 2 (MTA2), p66a, ...)
• PU.1 (=> up-regulation of M-CSFR, G-CSF-R and IL-7Ra : whereas the transcription factor PU.1 regulates IL-7R expression in mouse pro–B cells via a GGAA motif, GA binding protein (GABP) ab₂ binds to this site and is essential in the regulation of IL-7R expression in T cells, where PU.1 is not expressed^ref)
• Notch1
• Pax3
• GATA3
• IL-15Ra
... to undergo T-cell commitment (see also T-cell development). CCR7 is crucial for migration of early thymic immigrants from the corticomedullary region to the subcapsular region (more important in the adult structured thymus) and migration of positively selected thymocytes from the cortex to the medulla (more important in newborns than in adult mice)^{ref1, ref2}. The only progenitors in blood with efficient T lineage potential are Lin^-SCA1^hic-kit^hi (LSK cells). The blood LSK population, like its counterpart in the bone marrow, contains HSCs and nonrenewing, multipotent progenitors, including early lymphoid progenitors and CD62L⁺ cells previously described as efficient T lineage progenitors. CLPs can not be identified in the circulation, suggesting they are not physiological T lineage precursors. Blood LSK cells are the principal circulating progenitors with T lineage potential^ref.
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+ ?
• secondary prothymocyte / early T lineage progenitor (ETP)  (CD7⁺, cCD3deg) : 10⁶ primary prothymocytes enter the thymus daily through ...
• the capsule and the septa (in embryo)
• blood vessels (after thymus became vascularized) at corticomedullary junction
... then placing in cortical subcapsular portion. Developing thymocytes within the thymic microenvironment are subject to an osmotic stress that is effectively countered by NFAT5-dependent responses. ETPs can also develop via a common lymphoid progenitors (CLP)-independent pathway.
See also in vitro techniques to study the development of thymocytes.
‡
+ IL-1b, IL-2, IL-6, IL-7, + Notch1 on T cells (vs. mNumb) --- Notch ligands (on TECs), ...
IL-7 induces the proliferation of recent thymic emigrants (RTE) in neonates. The survival and proliferative effects of IL-7 on human RTE can be distinguished, on the basis of dose as well as duration of IL-7 administration. A dose of 0.1 ng/ml of IL-7 is sufficient to promote viability whereas cell cycle entry is observed only at doses > 1 ng/ml. Moreover, a short 1h exposure to high dose IL-7 (10 ng/ml) induces long-term survival but continuous IL-7 exposure is necessary for optimal cell cycle entry and proliferation. Distinct signaling intermediates are activated under conditions of IL-7-induced survival and proliferation: STAT5 tyrosine phosphorylation does not correlate with proliferation whereas upregulation of the glucose transporter Glut-1 as well as increased glucose uptake are markers of IL-7-induced cell cycle entry. Glut-1 is directly regulated by PI3K and indeed, inhibiting PI3K activity abrogates IL-7-induced proliferation^ref
• thymic lymphoid double-negative (DN1) progenitor (CD1a^-4^-8^-25^-34⁺44⁺117 / c-kit / SCFR⁺CCR7⁺) : it is located in the deep cortex and needs CD117 / c-kit / SCFR/ CD132 / g_c, preTcRa/ CD132 / g_c, BCL11B, and IL-7Ra to maturate.

‡
• tertiary prothymocyte / pro-T DN2 cell (CD1a⁺4^-5⁺7⁺8^-19^-25⁺34⁺44⁺CCR7⁺, cCD3deg). It is located in the midcortex.

‡
+ BMP2, BMP4, myeloid-cell leukemia 1 (MCL1)^ref (a component of the signalling pathway downstream of IL-7, which binds BIM and prevents BIM-mediated activation of the pro-apoptotic proteins BAK1 and BAX)
• early pre-T DN3 cell (CD1a⁺2 / LFA-2⁺19^-25⁺44^-CCR7⁺) : it is located in the outer third of the cortex and needs ...

‡
+ RAG1, RAG2, Ku80, DNA-PK_CS / XRCC7, TcRbd, cCD3egx, p56^LCK, Jak3, Csk, 45 / B220 / LCA, ZAP70, Syk, Fyn, CD117 / c-kit / SCFR, IL-2Rg, IL-7Ra, TCF1a, GATA3^ref, Rho, LAT, SLP76, Vav1, c-Myb^ref, survivin^{ref1, ref2}, and Runx2
induction of preTCR signaling resulted in rapid elevation of c-Myc protein levels, only required for preTCR induced proliferation but dispensable for developmental progression from the DN to the DP stage^ref.
... to undergo b-selection / pre-TcR checkpoint : b-chain rearrangements occur and successfully rearranged b-chain associates with preTcRa and CD3 g-, d-, e, and z-chains to form the preTcR complex. Signaling from the complex leads to survival, differentiation from DN3 to DN4, proliferation, and allelic exclusion : it channels cells into the ab-lineage because it reduces the number of gd T cells with productive TcRb rearrangements. The absence of signaling due to the lack of functionally rearranged b-chain leads to cell death and a developmental block at the DN3 stage. There exists an obvious similarity of proximal signaling events between the constitutively signaling pre-TcR and the ligated TcRab : in a TcRa^-/- background, the pre-TcR promotes a biased positive selection of CD8⁺T cells. The pre-TCR complex can deliver its signal autonomously through oligomerization of the pre-TCR a-chain mediated by charged residues in the extracellular domain^ref
• late pre-T DN4 or triple-negative (TN) cell (CD1a⁺3^-4^-8^-19^-25^-44^-)

‡
It is controversial whether cells commit to the T-cell lineage prethymically or intrathymically^{ref1, ref2}. Aided by modern cell-sorting techniques, many studies in the mouse have recently readdressed these issues. Adult murine BM has been demonstrated to contain precursors with a restricted T-/B-lymphoid potential, so-called common lymphoid progenitors (CLPs)^{ref1, ref2}. However, the earliest thymic immigrants were shown to differ from CLPs in several aspects^ref. In peripheral blood, T-lineage potential appeared to be restricted to Lin^–Sca-1⁺c-Kit⁺ (LSK) progenitor populations, rather than to CLPs^ref. Recently, 2 detailed analyses of the earliest subpopulations in the murine thymus showed variable lineage potential of different subsets^{ref1, ref2}. Together, these studies point toward a model in which a range of BM-derived progenitors colonize the thymus, probably including multipotent progenitors and more lineage-restricted precursor cells^ref. In both humans and mice, the most immature cells in the thymus do not express CD4 and CD8 and therefore are called double negative (DN). In humans, the DN stage can be further subdivided by staining for CD34 and CD1^{ref1, ref2}. Studies in the murine thymus have shown that the DN1 subset (the earliest DN subset in the mouse) contains multilineage progenitors^{ref1, ref2, ref3, ref4, ref5}. CD34 levels decline as the cells mature^{ref1, ref2}, concomitantly with expression of CD1^ref. This is usually detected by staining for CD1a, but CD1b, CD1c, CD1d, and CD1e are also expressed. The CD34⁺CD1a^–cells start to rearrange their TCRD genes, but mostly have their TCRG and TCRB loci still in germ line position, whereas expression of CD1a is accompanied by rearrangements of TCRD (V to DJ), TCRG, and TCRB (D to J) loci^ref. Gene expression, lineage potential, and self-renewal capacity of the 2 most immature subsets in the human thymus, namely CD34⁺CD1a^- and CD34⁺CD1a⁺ thymocytes, were examined. DNA microarrays revealed the presence of several myeloid and erythroid transcripts in CD34⁺CD1a^– thymocytes but not in CD34⁺CD1a⁺ thymocytes. Lineage potential of both subpopulations was assessed using in vitro colony assays, bone marrow stroma cultures, and in vivo transplantation into NOD/SCID mice.
• CD34⁺CD1a^– cells (0.4% of all thymocytes^ref) contained progenitors with
• lymphoid (both T and B), myeloid, and erythroid lineage potential
• B-cell precursor potential of CD34⁺CD1a^– thymocytes has never been reported, despite significant numbers of B cells in the human thymus^{ref1, ref2, ref3}. Furthermore, in the thymus we could detect the presence of all BM B-cell progenitor stages, indicating that B cells develop in the thymus, albeit at much lower frequencies than in the BM^ref.
• the capability of human CD34⁺ thymocytes to develop into NK cells and DCs has been demonstrated by several studies, in both fetal^{ref1, ref2} and adult^{ref1, ref2} thymus.
• myeloid potential of CD34⁺ thymocytes was investigated by a number of studies several years ago, yielding contradictory results. Although one study could generate myeloid colonies from CD34⁺ as well as from CD34^– thymocytes^ref, others could not confirm any myeloid potential in CD34⁺ thymocytes^ref. De Yebenes et al^ref demonstrated that myeloid DC precursors can be generated from CD34⁺ thymocytes in cultures containing M-CSF.
• megakaryocytic potential was suggested by the expression of NF-E2 in CD34⁺CD1a^– thymocytes and was confirmed by culturing thymocytes in suspension in the presence of TPO and SCF. Although CD34⁺CD1a⁺ thymocytes did not survive, CD34⁺CD1a^– thymocytes could be cultured in this condition for at least 14 days and developed into large cells resembling megakaryocytes, although definitive flow cytometric characterization (eg, by staining for CD41) was hindered by high autofluorescence^ref
Remarkably, development of CD34⁺CD1a^– thymocytes toward the T-cell lineage, as shown by TcR gene rearrangements, could be reversed into a myeloid-cell fate. A close lineage relation between myeloid cells and T cells has also been suggested by experiments in the fetal mouse, by Kawamoto et al.^ref. These cells resemble hematopoietic stem cells but, by analogy with murine thymocytes, apparently lack sufficient self-renewal capacity. Both CD34⁺CD1a^– and CD34⁺CD1a⁺ thymocytes failed to repopulate NOD/SCID mice.  In the mouse it has been shown that proliferation of a precursor that enters the thymus is limited to several weeks and that no permanent endogenous stem cell exists^ref. This makes it likely that, similar to the corresponding cells in the mouse, CD34⁺CD1a^- cells have very limited self-renewal. Thus, it is possible that the thymus is seeded by a HSC that rapidly loses self-renewal capacity and reduces non–T-cell potential after entering the thymus and interacting with molecules on the thymic epithelium, for instance, Notch ligands. Another possibility is that only a minute fraction of the cells that seed the thymus retains sufficient self-renewal and that this population is too rare to be detected with currently used assays. In line with this, it may be that a similar situation exists in the mouse, but that very stringent lineage depletion or insufficient cell numbers used in colony assays have excluded detection of, for instance, erythroid potential. An alternative explanation for the fact that CD34⁺CD1a^– thymocytes could not repopulate NOD/SCID mice is that they lack BM homing capability. One of the adhesion molecules necessary for BM homing is CXCR4^{ref1, ref2}, but using flow cytometry no significant differences in CXCR4 expression was detected between CD34⁺ UCB cells, which efficiently repopulate NOD/SCID BM, and CD34⁺ thymocytes; absence of other homing signals in CD34⁺ thymocytes cannot be excluded. Poor homing capability can be considered as an additional argument against the existence of a thymic HSC, because murine HSCs were shown to have high BM-seeding efficiency^ref. Frequencies of myeloid and erythroid potential, as determined by in vitro colony assays, were markedly lower in the CD34⁺CD1a^– thymocytes than in CD34⁺ UCB cells, probably because UCB contains higher numbers of more mature progenitors. This is visible, for instance, in the higher expression of CD13/33 and MPO on CD34⁺ cells from UCB. Also in the OP9 cocultures we found low frequencies of myeloid and B-lineage progenitors in the CD34⁺CD1a^– subset^ref.
• CD34⁺CD1a⁺ cells (0.6% of all thymocytes^ref) yielded only T-cell progenitors (a very limited NK and no DC precursor activity of CD34⁺CD1a⁺ cells^ref), demonstrating their irreversible commitment to the T-cell lineage. In contrast to the immature characteristics of the CD34⁺CD1a^-– cells, the CD34⁺CD1a⁺ thymocytes showed a T-cell–specific gene-expression pattern and did not yield any non–T-lineage cells in in vitro cultures. Together these findings suggest that human CD34⁺CD1a⁺ thymocytes are the equivalents of murine DN3 cells, in which final commitment to the T-cell lineage occurs^ref. This confirms a recently proposed scheme in which the stages of human T-cell development are highly similar to those in the mouse^ref.
These precursor cells exit the thymus before TcR rearrangements and permanently colonize the precursor compartment of lymphoid organs such as the thymus and the gut for the generation of 'euthymic like' CD8aa⁺ intraepithelial lymphocytes^ref.
+ Ets2
• intermediate single-positive (ISP) T-cell / intrathymic T progenitor (ITTP) (CD1a⁺2 / LFA-2⁺3^-4⁺8ab^-19^-25^-34^-44^-TcRab^- or CD1a⁺3^-4^-8ab⁺25^-34^-44^-TcRab^-) : they express IL-4, but attenuate GATA3 expression, recruit DNA methyltransferases (Dnmts) to the Il4-Il13 locus and downregulate IL-4 expression as they mature into T cells. They contain mature TCRB (V to DJ) rearrangements^ref

‡
+ IL-7, IL-15 (which induces local chromatin modifications specific for the variable gd-region gene segment and enhanced accessibility conducive to subsequent targeted gene rearrangement. This cytokine-directed tissue-specific TCR repertoire formation probably reflects distinct TCR repertoire selection criteria for gd and ab T cell lineages adopted for different antigen-recognition strategies^ref)
• gd T lymphocytes (1÷5%) : in the inner cortex. (CD1abcde⁺2 / LFA-2⁺3deg⁺4^-7⁺8ab^+/-10^-15^-19^-45 / B220 / LCARA^-RO⁺NKG2D⁺, z⁺h⁺TcRgd^hi)
• CD2 / LFA-2⁺3deg⁺4^-5⁺7⁺8ab^+/-10^-13^-15^-19^-, TcRgd^hi : they represent 3-4% of peripheral blood lymphocytes and 50% of intraepithelial T lymphocytes (IELs) in skin, intestinal and pulmonary epithelium. They act as a first line of defense against infections and cancers on the basis of their ability to respond directly to soluble protein and non-protein antigens of endogenous origins (e.g. hsp). In many animal species, gd T cells are the first lymphocyte lineage to be generated during fetal development : although theoretically ~ 10²⁰ different TcR could be realized, an enormous selective pressure is exerted on the development of gd T cells throughout life to produce populations of cells that express TcR encoded by specific gene segments. The human gd T cell repertoire has the greatest diversity during the prenatal stage of development : after birth and up to 6-10 years age there is a progression to oligoclonality (human Vg gene nomenclature is that of Lefranc). Intrinsic molecular constraints - rather than antigen selection - limit the generation of functional receptors in these populations :
• Vg3 gd T cells can be generated by fetal liver hematopoietic stem cells (FL HSC), but not by adult long-term reconstituting HSC (LT-HSC).
• Vg4 gd T cells can be generated by fetal liver hematopoietic stem cells (FL HSC), but not by adult long-term reconstituting HSC (LT-HSC).
• VgX-Vd1gd T cells use CD31 / PECAM1a for transendothelial migration (TEM). They are mainly located in bowels (intestinal IELs (iIELs / i-IELs)) and represent 25-60% of all gut T lymphocytes, being partially activated CD8aa⁺44^lo/int45 / B220 / LCARB^hi103 / a_E integrin⁺134 / OX40^lo178 / Apo1L / FasL^loLy-6C^lo : they express high levels of FasL gene whose surface expression is controlled at posttranscriptional level. Their TcR binds to
• MICA and MICB expressed on enterocytes (binding poorly occurs also through the costimulatory receptor NKG2D - as for NK cells - which anyway doesn't deliver any costimulatory signal 2)
• lipohexapeptides (during Lyme arthritis)
• HLA-A2
• HLA-A24
• CD1C
• Most have naive phenotype and no canonical sequence motif. Vd1-expressing T lymphocytes are often clonally expanded in human transplant patients that are infected with HHV-5 / HCMV, a virus that is known to cause the upregulation of MIC expression by infected cells. Clones derived from transplanted patients, including Vd1⁺, Vd3⁺ and Vd5⁺ cells, responded strongly to CMV-infected cells^ref [13], killed the infected targets and limited CMV propagation in vitro. This reactivity was specific for CMV among herpesviridae but the same clones also responded to intestinal epithelial tumor cells, again suggesting the involvement of an (unidentified) autologous ligand.
• Vg9-Vd2gd T cells (> 97% all human gd T cells in peripheral blood !) use CD161 / NKRP1a for transendothelial migration (TEM). They have unique reactivity toward nonpeptidic Ags generated by the 1-deoxy-D-xylulose 5-phosphate (also referred to as Rohmer metabolic route) (many eubacteria, algae, plants, and Apicomplexa) and mevalonate (eukaryotes, archaebacteria, and certain eubacteria) pathways of isoprenoid biosynthesis. Because the former pathway is absent from human cells, it proves an ideal target for focusing efficiently the antimicrobial selectivity of human gd T lymphocytes. Also compounds synthesized by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isopentenyl pyrophosphate synthesis^ref
• organic phospholigands / monoalkyl phosphates / natural phosphoantigens from
• Mycobacterium spp.^ref : 4 different phosphoantigens termed TUBag1 to TUBag4 with a common 3-formyl-1-butyl-pyrophosphate moiety and isopentenyl-pyrophosphate have been isolated and identified from mycobacteria. IPP does not induce down-modulation of the TcR·CD3 complex, which likely results in the highly sustained signaling and release of high levels of TNF-a^ref. The mechanisms by which mycobacteria induce gd T cell reactivity are not fully defined. Previous studies have shown cell contact dependency, suggesting a requirement for antigen presentation, and a current study using a human Vg9Vd2⁺ clone capable of responding to alkylphosphate, alkylamine and aminobisphosphonate, as well as purified protein derivative (PPD) from Mycobacterium tuberculosis, shows that only human cells satisfy the contact requirement, for all of these antigens^ref [11].
• mycobacterial mycolylarabinogalactan peptidoglycan, a cell-wall component of Mycobacterium bovis, recently reported to induce bovine gd T cells to IFN-g production in the presence of IL-2 and accessory cells^ref [10].
• Escherichia coli : phosphoantigens exert bioactivities on gd T cells with similar potencies to the mycobacterial phosphoantigens at 5-15 nM concentration^ref
• Plasmodium falciparum
• Legionella pneumophila : in samples obtained 4 to 6 days after the onset of the disease, the mean percentage (± the standard deviation) of Vg9Vd2⁺ T cells among CD3⁺ cells was 1.0% ± 0.5%, compared to 5.0% ± 3.9% in healthy control subjects (P < 0.001). Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset, mean peak levels were as high as 15%. During the next 6 months, values slowly declined, although without reaching the normal range. Percentages of gd⁺ T cells expressing TNF-a or IFN-g in response to phorbol myristate acetate were assayed in vitro. At 14 to 16 days after the onset of disease, the expression of both cytokines was increased (P < 0.01), whereas at 5 to 7 weeks, the expression of tumor necrosis factor alpha was decreased (P < 0.05), possibly reflecting modulation of an inflammatory response. In conclusion, Pontiac fever was found to be associated with a pronounced and long-lasting expansion of Vg9Vd2 T cells, implying that the subset may also be pathophysiologically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, the expansion was preceded by a depletion of circulatory Vg9Vd2 T cells. Possibly, Vg9Vd2 T cells are initially recruited to a site of infection before they expand in response to antigen and occur in high numbers in blood.^ref
• Francisella tularensis (but not after tularemia vaccination)^{ref1, ref2}
• pyrophosphomonoesters :
• isopentenyl pyrophosphate (IPP)^ref
• 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP)^ref
• 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP)
• hydroxydimethyl allyl-pyrophosphate
• prenyl phosphates
• aminobisphosphonates (zoledronate and pamidronate) (during tuberculoid leprosy), which block the mevalonate pathway in target cells, leading to accumulation of natural phosphoantigens that in turn activate Vg9Vd2⁺ cells. Activation of primary gd T cells by pamidronate strictly depends on the presence of monocyte-lineage cells, unlike that by pyrophosphomonoesters. Thus, although pamidronate induced cell clustering, proliferation, and IFN-g production of gd T cells in the culture of PBMC, it failed to induce any of these activities in the culture of purified primary gd T cells. By adding back the purified monocytes, however, both cell clustering and IFN-g production of  T cells by pamidronate could be restored. The pamidronate-pulsed, but not untreated, myelomonocytic line, THP-1, was capable of activating the purified gd T cells to produce IFN-g, which was associated with the down-regulation of gd TCR. Furthermore, pamidronate-pulsed THP-1 cells were significantly more susceptible to gd T cell-mediated cytotoxicity than untreated THP-1. Also, TCR-defective Jurkat T cells transfected with gd TCR genes produced a significant level of IL-2 in response to the pamidronate-pulsed THP-1 cells. These results have suggested strongly that human  T cells are functionally activated via  TCR by aminobisphosphonate Ag presented on the surface of monocyte lineage cells rather than directly by its free form^ref
• nonphosphorylated alkylamines
• nucleotide conjugates
• bromohydrin pyrophosphate (BrHPP)/phosphostim, a potent synthetic agonist for which the mechanism of action is similar to natural phosphoantigens. Although of very short half-life, injection of BrHPP leads to strong activation of Vg9Vd2, inducing production of a high level of T_h1 cytokines. Combination of BrHPP with low-dose rhIL-2 induces specific amplification of effector-memory peripheral Vg9Vd2 in blood in a dose-dependant manner. This transient response returns to baseline within 10–15 days. Successive infusions of BrHPP and rhIL-2 induce less vigorous expansions, suggesting a progressive exhaustion of the response. As no toxicity is detected with or without IL-2, this scheme represents a promising immunotherapeutic strategy for induction of systemic T_h1 cytokines and massive expansion of  T cell subset with antitumor and anti-infectious propert