Innate-Like lymphocyte ILL Unconventional lymphocytes. B-1 cells, Marginal zone (MZ) B cells, Mucosal-associated invariant T (MAIT) cells, Natural killer T (NKT) cells, γδT cells.

1. Innate-like Lymphocytes
(ILLs)
Ahmed Adel Abdallah
Advanced Immunity
Islamic University of Gaza
08 Oct. 2021

2. Introduction
• Innate-like lymphocytes (ILLs) are a group of recently discovered cells that do not fit into either
side of the historical innate-adaptive classification.
• They exhibit phenotypic and functional properties of both the innate and adaptive immune
system, and are referred to as innate-like T and B cells or “Inbetweeners”.
• As Innate lymphoid cells (ILCs) lack RAG-dependent antigen receptors expressed by B and T cells.
innate-like lymphocytes (ILLs) use RAG-1 and RAG-2 mediated recombination to generate their
TCRs.
• They preferentially utilize specific TCR genes, resulting in the expression of semi-invariant
receptors that limit the range of antigens these cells recognize.
• Their antigen receptors encoded by very few common gene rearrangements result in a very
limited diversity; in addition to they only present in certain locations within the body.

3. Introduction
• ILLs are localized to areas of high antigenic exposure in the epithelial layer of the gut or in
specialized niches within organs such as the marginal zone in the spleen.
• Innate-like lymphocytes exist in a naturally activated state. This phenotype facilitates rapid
activation upon antigen recognition and innate-like lymphocytes may perform their effector
functions within hours after antigenic challenge.
• Several types of ILLs have been identified, including B-1 cells, Marginal zone (MZ) B cells,
Mucosal-associated invariant T (MAIT) cells, Natural killer T (NKT) cells, γδT cells, etc.

5. 1. γδ T cells
• γδ T cells are a subset of T cells which express a TCR γ chain in combination with a TCR δ
chain.
• In humans, peripheral γδ T cells most frequently utilize the Vγ9 and Vδ2 chains, and
represent 1–5% of T cells in healthy adult, but can reach up to 50% of T cells in a matter
of days following an infection.
• γδ T cells are enriched in the human intestine, mostly as intra-epithelial lymphocytes.
• In contrast to αβ T cells which are MHC-restricted, γδ T cells can recognize antigens in
both MHC-dependent and MHC-independent ways.

6. γδ T cells
• γδ T cells are activated by both microbial- and host-derived compounds.
• They recognize the microbial compound (E)-4-hydroxyl-3-methyl-but-2-enyl
pyrophosphate (HMB-PP)[4], an essential metabolite in isoprenoid biosynthesis,
generated by the majority of gram negative bacteria and some gram positive bacteria.
• They also recognize host-derived phosphoantigens such as isopentenyl pyrophosphate.
• Recent work has focused on the mechanism behind phosphoantigen sensing and
presentation, and several studies have identified butyrophilins
as responsible for presenting HMB-PP.

7. γδ T cells
• Once activated, γδ T cells have a range of functions, such as killing infected or stressed
target cells, priming CD4 and CD8 T cells, providing B cell help, inducing DC maturation,
and promoting survival of neutrophil and monocytes.
• Recent studies have demonstrated the role that γδ T cells play in limiting transepithelial
pathogen invasion.
• Edelblum et al,. observed that higher numbers of S. typhimurium are seen in the gut of
TCR δ defective mice, and that migration of γδ IELs was critical to their
function.

8. γδ T cells
• During microbial dysbiosis, resident Vδ1+ T cells are indispensable for triggering early
protective inflammatory responses, which are essential for wound healing.
• In such cancer or stressed cells, isopentenyl pyrophosphate (IPP) gradually accumulates
and boosts the activation of Vγ9Vδ2 T cells, which only take up a small fraction of
lymphocytes in peripheral blood (PB), but harbor an antineoplastic effect.
• When barrier damage results in microfloral translocation, γδ T cells are indispensable for
triggering early acute inflammatory responses against the penetration of microbiota
across the impaired intestinal mucosa.

9. γδ T cells
• For example, TCRγδ-deficient mice are more susceptible to harmful microbial cues and
show early spread of pathogens to other organs.
• a compelling feature seen in γδ T cells is their ability to secrete interleukin-17 (IL-17) in
early pathogen invasion, which seems to benefit the host initially.
• It is probable that γδ T cells recognize pathogen-associated molecular patterns through a
series of toll-like receptors (TLRs), indicating the direct effect of bacterial products to
drive the expansion of γδ T cells.

10. γδ T cells
• On one hand, γδ T cells promote Th1-committed αβ T cell responses via interferon γ
(IFN-γ) production and enhance the secretion of inflammatory cytokines including tumor
necrosis factor α (TNF-α).
• On the other hand, γδ T cells, employed as antigen-presenting cells, potentiate the
secretion of IL-22 by CD4+ cells and release of calprotectin in an inducible T-cell co-
stimulator ligand (ICOSL)/TNF-α- dependent manner.
• γδ T cells are continuously activated by sustained exposure to bacterial metabolites,
thereby leading to the exhaustion of protective γδ T cell subtypes and activation of
chronic inflammation.

12. 2. B1-Cells
• B1 cells, which are distinct from conventional B cells, are a rare B lymphocyte
subpopulation that plays a pivotal role in innate immunity.
• B1 cells were discovered in mice over 30 years ago as a rare B lymphocyte subpopulation
with unique cell surface antigens that spontaneously secretes immunoglobulin M (IgM).
• This subpopulation is distinguished from conventional B cells, so-called B2 cells, by their
surface phenotype, function, localization, and ontogenesis.
• B1 cells play important roles in innate immunity by producing natural antibodies,
whereas B2 cells elicit adaptive humoral immunity.
• These natural antibodies have weak affinities for antigens, recognize molecules
expressed by pathogens and self-antigens, and can be produced in the absence of
exogenous stimulation.

13. B1-Cells
• In contrast to conventional B cells (B2 cells), B1 cells were identified at a relatively low
frequency in the spleen, but they were abundant in the peritoneal cavity.
• Importantly, B1 cells differ functionally from B2 cells in their spontaneous secretion of
natural IgM that is a more germ-line-like immunoglobulin than that in B2 cells because of
their minimal N-region addition, broad reactivity, restricted repertoire, and auto
reactivity.
• Such natural IgM secreted from B1 cells plays an important role in the early defense of
bacterial and viral infections.
• B1 cells are divided into two subsets according to CD5 expression; CD5+ B1a and CD5−
B1b cells.

14. B1-Cells
• It has been estimated that 80–90% of resting serum IgM, and 50% of serum IgA is
derived from B-1a cells.
• The main function of B-1a cells in the innate immune system is spontaneous secretion of
natural Abs, thereby maintaining resting immunoglobulin levels in the body without any
stimulus, while B-1b cells are critical in development of IgM memory cells.
• IgM antibodies comprise both natural IgM antibodies and adaptive IgM antibodies.
• NAbs are primarily derived from B1 cells that spontaneously secrete T-cell–independent
antibodies, whereas adaptive IgMs are secreted by B2 cells in a T-cell–dependent
manner.
• Natural IgM is produced by a small subset of B1 (CD5+) do not require affinity maturation
to provide early protection.

15. B1-Cells
• It has been shown that in animals treated with endotoxin, peritoneal B-1a cells migrate
to the spleen where they secrete higher amounts of Abs.
• B-1a cell-derived natural Abs are able to recognize phosphorylcholine (PC), an invariant
constituent of Gram positive microbial membranes such as the S. pneumonia bacterial
cell wall, as well as membranes of other bacterial pathogens.
• In addition, natural Abs produced by B-1a cells recognize phosphatidylcholine, a key
constituent of senescent red blood cell membranes.
• Interaction of such natural Abs with an infectious agent can act either by direct
inhibition, complement activation, or opsonization leading to phagocytosis and/or Ab
dependent cell-mediated cytotoxicity.

17. 3. Natural killer T (NKT) cells
• Natural Killer T (NKT) cells belong to a subset of T cells that can influence the status of
the innate and adaptive immune systems.
• NKT cells are defined as “CD1d-restricted and TCR-αβ positive T cells”.
• The levels of NKT cells are low in humans, comprising about 0.1% of human T cells in
peripheral blood.
• The greater part of the NKT cells, called invariant NKT cells (iNKT cells) which have a
specific TCR α-chain rearrangement, associated with limited diverse Vβ chains.

18. Natural killer T (NKT) cells
• Unlike peptide-MHC restricted T cells, which emerge from the thymus “naïve,” iNKT cells
leave the thymus fully matured and able to perform their effector functions without
priming.
• iNKT cells respond rapidly and without the need for priming, they are some of the first
cells within an immune response to be activated and therefore act as a “bridge” between
the innate and adaptive immune systems.

19. Natural killer T (NKT) cells
• Invariant natural killer T (iNKT) cells are stimulated by CD1D-expressing antigen-
presenting cells (APCs) that are loaded with α-galactosylceramide (α-GalCer).
• iNKT cells receive stimulatory signals from APCs (through IL-12) and, in return, the iNKT
cells stimulate APCs by secretion of IFN-γ.
• After stimulation, iNKT cells can exert effector functions (for example, cytotoxicity
through FAS–FAS-ligand and perforin/ granzyme pathways), also immunoregulatory
functions (for example, secretion of the cytokines IL-4, TGF-β and IFN-γ) can be exerted.
• The early burst of cytokine secretion by iNKT cells might also be crucial for the
differentiation of CD4+ T cells to T helper 1 (TH1) or TH2 cells.
• B cells and natural killer (NK) cells might also become activated by iNKT cells.

22. 4. Mucosal-associated invariant T (MAIT) cells
• MAIT cells express semi-invariant T cell that recognize microbial vitamin B2 (riboflavin)
derivatives presented by the MHC class Ib molecule, MR1.
• In humans, their T cell receptor is composed of a Vα7.2-Jα33/20/12 chain, combined
with a restricted set of Vβ chains.
• MAIT cells are predominantly located at barrier sites and, upon activation, rapidly
produce either Th1- or Th17-associated cytokines.
• MAIT cells are the most abundant innate-like T cell population in humans. MAIT cells are
very abundant in the liver (up to 40% of resident T cells) and in mucosal tissues, such as
the lung and gut.

23. Mucosal-associated invariant T (MAIT) cells
• Because the riboflavin synthesis pathway is broadly conserved among many species of
bacteria and fungi, MAIT cells respond to a wide array of microbes, including known
commensals.
• Upon recognition of MR1 ligands, MAIT cells have the capacity to rapidly eliminate
bacterially infected cells through the production of inflammatory cytokines (IFNγ, TNFα,
and IL-17) and cytotoxic effector molecules (perforin and granzyme B).
• Thus, MAIT cells may play a crucial role in antimicrobial defense.

24. Mucosal-associated invariant T (MAIT) cells
• The major histocompatibility complex (MHC) class-I related molecule MR1 is a
monomorphic and evolutionary conserved antigen (Ag)-presenting molecule that shares
the overall architecture of MHC-I and CD1 proteins.
• However, in contrast to MHC-I and the CD1 family that present peptides and lipids,
respectively, MR1 specifically presents small organic molecules.
• MR1 can binds riboflavin (vitamin B2) biosynthesis precursor derivatives, such as
5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) produced by most,
but not all, bacteria and yeasts
• During bacterial infections, derivatives of the riboflavin biosynthesis pathway are
captured by MR1 and presented on the surface of APCs, subsequently leading to
the activation of MAIT cells via the MAIT TCR-MR1.
• Thus, MAIT cells are considered to play a protective role in antibacterial
immunity.

25. Mucosal-associated invariant T (MAIT) cells
• In contrast, during viral infection (e.g. dengue, hepatitis C and influenza viruses), infected
cells release IL-18, IL-12 and IFNα, which can activate MAIT cells in a MR1-independent
manner.
• Upon activation, MAIT cells rapidly produce inflammatory cytokines in Th1/Th17-like
responses and may also express granzyme B and perforin, killing the infected cells.

26. Mucosal-associated invariant T (MAIT) cells
• Internalization of bacteria by an antigen-presenting cell, either through infection or
actively by phagocytosis.
• Lysis of the bacteria, within endocytic compartments, releases 5-A-RU, which is
converted to 5-OE-RU or 5-OP-RU and binds and stabilizes MR1.
• The stable MR1 translocates to the cell surface, where it is presented along with other
co-stimulatory molecules, e.g., CD80 or CD86.
• Bacterial components trigger pathogen recognition receptors (PRR), such as TLR8.
• PRR triggering drives cytokine expression, such as IL-12, and the activation of the
inflammosome, resulting in the release of active-IL-18

27. Mucosal-associated invariant T (MAIT) cells
• mucosal-associated invariant T (MAIT) cells can be activated in a MHC class I-related
molecule (MR1)-dependent manner, through their T cell receptor (TCR) recognizing MR1-
presenting bacterial ligands presented along with other co-stimulatory molecules, e.g.,
CD28.
• Activation of MAIT cells can also be done through an MR1-independent manner by
inflammatory cytokines such as IL-12 and IL-18, produced by infected cells and sensed by
MAIT cells through cytokine receptors.
• Activated MAIT cells express pro-inflammatory cytokines, e.g., IFNγ, TNFα, and IL-17.
• These cytokines can directly act anti-bacterially, or recruit and stimulate other immune
cells, e.g., neutrophils by IL-17.

28. Mucosal-associated invariant T (MAIT) cells
• Activation of MAIT cells upregulates perforin and granzyme B expression, which kill
infected cells.
• Activated MAIT cells can also promote monocyte recruitment and activation.
• Theoretically, the degranulation of cytotoxic granules into infected cells, via recognition
of MR1, could induce cell death and, thus, the potential clearance of infected cells.

31. References.
• Bennett, M. S., Round, J. L., & Leung, D. T. (2015). Innate-like
lymphocytes in intestinal infections. Current opinion in infectious
diseases, 28(5), 457.
• Aziz, M., Holodick, N. E., Rothstein, T. L., & Wang, P. (2015). The role
of B-1 cells in inflammation. Immunologic research, 63(1), 153-166.
• Wolf, B. J., Choi, J. E., & Exley, M. A. (2018). Novel approaches to
exploiting invariant NKT cells in cancer immunotherapy. Frontiers in
immunology, 9, 384.
• Toubal, A., Nel, I., Lotersztajn, S., & Lehuen, A. (2019). Mucosal-
associated invariant T cells and disease. Nature Reviews
Immunology, 19(10), 643-657.