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\begin{document}

\title{Targeting T-cell immunometabolism during transplantation}



\author[1]{SHADAB KAZMI}%
\author[1]{Mohammad Khan}%
\author[1]{Talal Shamma}%
\author[1]{Abdullah Altuhami }%
\author[1]{Abdallah Assiri}%
\author[1]{Dieter Broering}%
\affil[1]{King Faisal Specialist Hospital and Research Center}%


\vspace{-1em}



  
  \date{May 07, 2024}


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\selectlanguage{english}
\begin{abstract}
The balance between the immune system and its metabolism is becoming an
effective therapeutic alternative in various inflammatory diseases,
including organ transplantation. The interaction between the immune and
metabolic pathways play a critical role in dictating disease pathology
and progression, and the differences in the bioenergetic demands between
immune cells enable them to differentiate into effector and regulatory
cells. Recent studies have suggested that changes in intracellular
metabolic programs control T cell activation, proliferation and
differentiation into T effector (Teffs) or T regulatory cells (Tregs),
and metabolic differences between Tregs and Teffs can shift the balance
toward a more specific immune tolerance in organ rejection.
Pharmacological targeting of T cells metabolism affects the balance
between effector and regulatory function of T cells.This therapeutic
modulation are of great interest in cancer, autoimmunity, and organ
transplantation. In this review, we discuss major metabolic pathways
that influence the activation, proliferation and differentiation of
Tregs, and also special emphasis on liver kinase B1 (LKB1) pathway that
provide stability of Tregs. We also highlight how Tregs metabolic
regulome and LKB1 signaling pathway relationship contribute to rescue
organ transplants from associated injuries and chronic rejection.~%
\end{abstract}%



\sloppy


\textbf{BACKGROUND:}

The metabolic heterogeneity of immune cells provide an opportunity for
different immune cells to differentiate into effector or regulatory
cells. Targeting the metabolism of immune cells with metabolic
inhibitors is a new approach in immunosuppression. Therefore, selective
inhibition of basic metabolic pathways, which are necessary for all
healthy cell survival, can still show selectivity toward immune cells.
It all depends on the degree of reliance on immune cells on those
pathways. The delicately balanced interplay between protective immunity
and inflammatory conditions is critical for the
activation/differentiation of various immune cells. On encounter with
antigen and receiving appropriate co-stimulatory signals, na\selectlanguage{ngerman}ïve T cells
activate downstream metabolic reprogramming for rapid cell growth and
proliferation to fuel their specific effector functions.
Immunometabolism provides a greater room for a therapeutic window in
transplantation settings and autoimmune disease treatment. Furthermore,
LKB1 is an important upstream kinase that strengthen the stability of
Tregs, and their signaling can be co-regulated to enhance the stability
of Tregs. Notably, LKB1 is a metabolic regulator that connects cellular
metabolism to immune cell functions. Recent work has demonstrated that
modulating Treg function by targeting metabolic pathways and LKB1
signaling in models of transplantation and inflammatory diseases do not
cause any visible toxicity, which advocates this strategy as a new
therapeutic intervention for treating inflammatory diseases.

\textbf{~}

\textbf{IMMUNOMETABOLISM:~~~~~~~~}

Immunometabolism has emerged as one of the exciting areas in the field
of translational research. More recently, the role of cellular
metabolism in the development and function of immune cells in healthy
and diseased individuals has been described in diseases such as cancer,
autoimmunity, and organ transplantation. Metabolic reprogramming of
distinct T cell subsets is the center for appropriate T cell
differentiation and function {[}1-3{]}. In this line, T cells use many
different metabolic pathways to generate ATP for their survival and
boost numerous molecular biosynthetic precursors production for their
proper proliferation and differentiation {[}4{]}. These metabolic
pathways entail diverse end products, but they are interconnected on
shared fuel ground. For example, fatty acid synthesis pathway is
dependent on the availability of intermediate products from the
glycolysis and tricarboxylic acid (TCA) cycle metabolism. Thus,
metabolic regulome of immune cells is a key factor in the control of
immune responses.

\textbf{~}

\textbf{CELL ENERGY:} Cells mostly use glucose as the source of energy;
however, few cells use fatty acids or amino acids as the source of
energy depending on their energetic demands {[}5-7{]}. Typically, the
cell allows the uptake of these energy-rich molecules through glycolysis
and enter the TCA cycle, and the electron transport chain (ETC) to
produce ATP {[}8, 9{]}. Glycolysis involves the uptake of extracellular
glucose into cells through the glucose transporter 1 (GLUT1) and
subsequent conversion into pyruvate along with numerous other products
inside the cytosol {[}10{]}. This glucose metabolism is a relatively
inefficient pathway in terms of cellular ATP production; however, it
provides a way to maintain redox balance inside the cell (to reduce
NAD\textsuperscript{+} into NADH), which is subsequently used by various
enzymes as a cofactor and enables the diversion of metabolic
intermediates to biosynthetic growth pathways to support anabolic growth
{[}11, 12{]}. The TCA cycle occurs in the mitochondrial matrix and it
uses pyruvate as a substrate {[}13{]}, which is converted into acetyl
coenzyme A (CoA) to generate energy-rich electron carriers NADH and
FADH2. These electron carriers transport electrons through oxidative
phosphorylation (OXPHOS) to the inner mitochondrial membrane into the
ETC to generate a hydrogen gradient required for the synthesis of ATP
{[}14{]}. Besides, the fatty acid oxidation (FAO) pathway, which is a
key catabolic pathway for energy production, involves the mitochondrial
conversion of fatty acids into several products that are subsequently
used by the cell to generate energy using fatty acids instead of glucose
{[}7{]}. Activation of the fatty acid is the first step of FAO to occur
in the cytosol that generates acetyl-CoA, which then enters the TCA to
generate electron carriers, and then enter the ETC to generate energy
{[}15{]}. ~In the context of immunity, a high rate of glucose metabolism
has been found in lipopolysaccharide (LPS)-activated macrophages and
dendritic cells (DCs), activated natural killer (NK) cells, ~activated
effector T cells, and activated B cells {[}16-20{]}. Indeed, this
upregulation of glycolysis in immune cells can be considered a hallmark
metabolic change for immune cells that are undergoing rapid activation,
explicitly in response to stimulation of pattern recognition receptor
(PRR), cytokine receptors, or antigen receptors. Aerobic glycolysis has
been associated with inflammatory and rapidly proliferating immune
cells; however, dependency on FAO has been observed in many
non-inflammatory immune cells and show raised cellular lifespans,
including M2 macrophages, Tregs, and memory T cells {[}7, 21-23{]}. In
contrast to FAO, fatty acid synthesis regulates the generation and
activation of pro-inflammatory immune cells. Therefore, inflammatory
signal leads to fatty acid synthesis of inflammatory immune cells,
whereas tolerogenic stimuli drive FAO of non-immune cells. Moreover,
activated T cells also use few amino acids, especially glutamine
metabolites to generate \selectlanguage{greek}α-\selectlanguage{english}ketoglutarate as a substrate of the TCA cycle
to generate energy, and most importantly, through the mTOR pathway for
sensing amino acid levels inside the cell and for nucleotide synthesis
{[}24-26{]}.

\textbf{T CELL ENERGY:} The metabolic pathways described above play a
significant role in determining T cell functional outcomes. In the
primary lymphoid organs, undifferentiated na\selectlanguage{ngerman}ïve T cells rely on
mitochondrial OXPHOS for energy, as well as on extrinsic cell signals
such as IL-7 to maintain energy for immune surveillance before
activation {[}3{]}. Upon activation in the secondary lymphoid organs, a
substantial change in the metabolism of naïve T cells allows them to
differentiate into various Teff cells. Teffs such as activated
CD4\textsuperscript{+} and CD8\textsuperscript{+} T cells depend on
aerobic glycolysis for their bioenergetic demands due to the need for a
rapid immune response {[}27{]}. In contrast to Teffs, Tregs use lipid
metabolism instead of glycolysis {[}28{]} and mainly rely on OXPHOS
generating long-lasting energy. Memory T cells (Tmem) also depend on FAO
and OXPHOS for energy and have a high mitochondrial mass to generate
energy rapidly in secondary exposure to the same antigen to execute
rapid response. Notably, FAO is essential to maintain mitochondrial
spare respiratory capacity (SRC) in Tmem cells. SRC is a functional
parameter of mitochondria that measure maximum capacity of a cell to
consume oxygen during stress {[}29{]}. Therefore, Treg and Tmem mainly
rely on OXPHOS and FAO, for their energy demand, and express low levels
of Glut1 and high rate of lipid oxidation that also further strengthen
their dependency on FAO {[}28, 30{]}. As stated earlier, Teffs (Th1,
Th2, and Th17) cells need glycolysis and to the lower extent rely on
OXPHOS for their energy requirements and express high levels of Glut1
that underpins their high glycolytic nature {[}31{]}. Subsequent
research studies demonstrated that Tregs have elevated expression of FAO
related genes such as AMP-activated protein kinase (AMPK) compared to
Th17 cells {[}32{]}. Previous research suggests that long chain FAO
impart Tregs and Tmem cells survival. This conception is mainly due to
inhibition of carnitine palmitoyltransferase 1A (CPT1A) with the drug
etomoxir. CPT1A is the rate-limiting enzyme for long chain FAO and
facilitate transport of long chain fatty acids into mitochondria for its
\selectlanguage{greek}β-\selectlanguage{english}oxidation. In turn, further findings show that Treg cells and Tmem
cells utilize various pathways to transport fatty acids and use short-
or medium-chain fatty acids whose translocation is not dependent on
CPT1A {[}33,34{]}. Recent research shed new light that, engagement of
the inhibitory programmed cell death 1 (PD1) receptor on T cells with
its ligand PD-L1 resulted in enhanced expression of CPT1A and elevated
FAO. Hence, this engagement of PD1 prevents effector cell development
{[}35{]}. Notably, PD1 is a crucial regulator of immune homeostasis, and
its engagement increases the longevity of T cells in an oxidative
environment. In marked contrast, cytotoxic T lymphocyte antigen 4
(CTLA4) inhibits glycolysis without augmenting CPT1A and FAO. Under
normal conditions, Teffs cells downregulate FAO during their activation
process and enhance fatty acid synthesis for their growth {[}27{]}. This
downregulation of FAO by Teffs is attributed to Teffs functional
inhibition and enhanced immune tolerance by FAO. Moreover, the
glycolytic-lipogenic pathway and glutamine metabolism are used to fuel
mitochondrial OXPHOS through the TCA cycle as an alternative input for
the TCA cycle to support ATP production. It is mainly associated with
Teffs differentiation through the mTOR pathway {[}36{]}, which is the
central player of CD4 T cells differentiation {[}37{]}. These different
metabolic programs are tightly regulated to facilitate the energy
production required for each distinct T cell subset. Glycolysis, OXPHOS,
and glutaminolysis, are intertwined and properly managed to fulfill the
bioenergetics demand of immune cells for proper proliferation and
differentiation \textbf{(Figure 1)}. Collectively, these studies
indicate key roles of metabolic programming in determining T-cell fate
and function along with other signals such as the strength of TCR
stimulation, inflammatory cytokines and transcriptional factors {[}38,
39{]}.

\textbf{REGULATORY T CELLS AND EFFECTOR T CELLS METABOLISM:} Tregs are a
subset of CD4\textsuperscript{+} T cells that play a vital role in
maintaining immunological homeostasis, preventing autoimmunity, and
graft rejection due to their potent immunosuppressive and reparative
activities {[}40-44{]}. To accomplish ~these multiple roles in control
of the disproportionate inflammatory response and tissue injury Treg
cells ~express various effector molecules for their activation and
stability {[}45{]}. Tregs have two main subsets, thymus-derived natural
Treg (nTreg) and peripherally (induced) derived Treg (iTreg) cells.
iTregs are generated at peripheral sites from na\selectlanguage{ngerman}ïve T cells after TCR
stimulation in the presence of TGF\selectlanguage{english}-\selectlanguage{greek}β, \selectlanguage{english}or they can arise from
conventional effector T cells {[}46{]}.

In transplantation, donor-specific tolerance is a crucial parameter for
a high survival rate, which could be achieved by increasing Tregs or by
suppressing donor reactive T cells. Therefore, the differences in the
metabolism between effector and regulatory T cells make
immuonometabolism a promising therapeutic intervention that could allow
for a more specific immune tolerance in the field of transplantation
{[}47{]}. Numerous preclinical models of autoimmune diseases and
transplantation have shown that Treg cells maintain peripheral tolerance
after tissue injury and exposure to intracellular antigens or
alloantigen {[}48{]}. Various T cells adjust their metabolic programming
to meet the energetic demands necessary for their cellular functions. As
discussed in the earlier section, for respiration, a na\selectlanguage{ngerman}ïve T cell mainly
depends on OXPHOS, and later on, upon antigen encounter, T cell
activates aerobic glycolysis necessary for effector cytokine production
{[}47, 49{]}. Activated T cells use aerobic glycolysis as a fast
generating energetic mechanism to satisfy the immediate energetic
demands for proper proliferation and differentiation. This use of
aerobic glycolysis by T cells was first described by Otto Warburg in
cancer cells and hence is known as the Warburg effect {[}50{]}. The two
main subsets of T cells (CD4\textsuperscript{+} and
CD8\textsuperscript{+} T cells) show many similarities in their
activation. Both activated subsets enhance their dependency on
glycolysis and increase Glut1 expression for glucose uptake; however,
they have different metabolic profiles. CD8\textsuperscript{+} T cells
rely on glycolysis less than CD4\textsuperscript{+} T cells due to
diminished glycolytic enzyme expression {[}51, 52{]}. Alternatively,
CD4\textsuperscript{+} T cells display a marked increase in
mitochondrial mass as compared to CD8\textsuperscript{+} T cells, while
CD8\textsuperscript{+} T cells show greater dependency on OXPHOS for
cytokine production {[}53{]}. Activated CD4\textsuperscript{+} T cells
differentiate into Teffs (Th1, Th2, or Th17 cells) by triggering
different metabolic pathways downstream of the TCR and by the
availability of essential metabolites.

Tregs have different metabolic profiles compared to
CD4\textsuperscript{+} T effector cells. Tregs utilize FAO for their
differentiation, unlike Teffs cells {[}54, 55{]}. Furthermore, Tregs
depend on OXPHOS for proliferation and steady, long-lasting suppressive
functions as seen in Tmems {[}23, 28{]}. In Tregs, FOXP3 expression use
fatty acids, upregulate ETC, and ATP generation through OXPHOS.
Moreover, FOXP3 initiates increased fatty acid \selectlanguage{greek}β-\selectlanguage{english}oxidation, which
results in the selective protection of FOXP3\textsuperscript{+} cells
from fatty acid-induced cell death {[}56{]}. This phenomenon is crucial
to provide a key target for modulating Treg function and selection in
clinical settings. Recent findings have identified an inverse relation
for the metabolic phenotype in mouse Tregs and human Tregs. In murine
models, iTregs displayed low glycolytic rate, whereas,~ human iTregs,
preferentially use glycolysis for their development and function because
of enolase-1 suppresses the transcription of the exon 2-containing FOXP3
splicing variant unless engaged in glycolysis {[}32, 57{]}. This
splicing variant is important for Treg mediated suppression.

The metabolic profiling of T cell subsets also reveals their dependency
on glycolysis or OXPHOS. The enzyme pyruvate dehydrogenase (PDH) has
been found as a central node in the programming of T cells to use either
glycolytic or oxidative metabolism for their differentiation. Indeed,
pyruvate metabolism plays a crucial role in Teffs and Tregs conversion.
The enzymatic activity of PDH is inhibited by PDH kinases (PDHKs). PDHKs
are highly expressed on Th17 cells, but not on Th1 cells and at low
levels in Tregs under the influence of hypoxia inducible factor 1- \selectlanguage{greek}α
\selectlanguage{english}(HIF1-\selectlanguage{greek}α\selectlanguage{english}) {[}2, 58-60{]}. Inhibiting PDHK1 with specific inhibitors such
as dichloroacetate (Table 1), which promotes oxidative phosphorylation
selectively suppresses Th17 cells~therefore favors Tregs generation
{[}61{]}.~This suppression causes the production of reactive oxygen
species (ROS), and treating with ROS scavengers such as N-acetyl
cysteine (NAC) restore Th17 cell generation {[}2{]}.

Lipids, especially short-chain fatty acids, are a preferential source of
acetyl groups for histone acetylation and epigenetic reprogramming.~ FAO
reprogram cellular metabolism and this oxidation is also the primary
source of lipid-derived acetyl-CoA {[}62{]}. A recent finding shows that
post-translational modifications such as acetylation, ubiquitination,
and phosphorylation control FOXP3 expression, therefore, FAO supports
the immunosuppressive function of Treg by regulating histone acetylation
in the FOXP3 locus {[}63, 64{]} which stabilize the FOXP3 expression
{[}65, 66{]} (\textbf{Figure 1}).

In addition to glucose and fatty acids, cells also utilize glutamine and
leucine amino acids, which play a vital role in Teff's differentiation.
CD4\textsuperscript{+} T cells deprived of glutamine differentiate into
Tregs in- vitro condition {[}67{]}; however, iTregs show less dependency
on amino acids for their energy requirements {[}68{]}. Glutamine
metabolism involves the influx of glutamine in the TCA-cycle in the form
of \selectlanguage{greek}α-\selectlanguage{english}ketoglutarate. This intermediate encourages Th1 cell
differentiation by enhancing the expression of the inflammatory
transcription factor T-bet {[}28, 68{]}. The deletion of
neutral-amino-acid transporter genes (Slc7a5 and Slc1a5 (also known as
ASCT2) reduce glutamine uptake, glucose metabolism, and overall Teffs
differentiation, however, it does not affect the generation of iTregs
{[}69, 70{]}. These metabolic variances show that Tregs have less
dependency on amino acid metabolism for producing energy compared to
Teffs.

Similarly, the metabolic by-products of tryptophan, such as kynurenine,
triggers iTreg proliferation by its binding to the aryl hydrocarbon
receptor {[}71, 72{]}. However, iTregs in the absence of tryptophan can
activate the amino-acid-starvation sensor GCN2 (general control
nonderepressible-2) kinase that inhibits Th17 cell differentiation
{[}73, 74{]} and supports Treg stability. It has been reported that
Tregs upregulate amino-acid-consuming enzymes including ARG1 (arginase
1), HDC (histidine decarboxylase), TDH (threonine dehydrogenase), and
IL4I1 (interleukin-4 induced 1) in skin graft as compared to fresh skin
to induce tolerance. These findings suggest that Tregs can modulate the
concentration of essential amino acids and their catabolic products in
the intrinsic cell milieu through the activation of amino acid
starvation sensors {[}75{]}. In this way, Tregs trigger suppression
through amino acid starvation and limit the pathology during the normal
immune response.

Thus far, we have discussed activation and differentiation of T cells
that lead to metabolic programming. Likewise, in the hostile envoirnment
such as in the case of tumor, cellular metabolism reshape in ways that
are central to T cells exhausation and at the same time Tregs
sustainability. The tumor microenvironment poses metabolic hurdles such
as hypoxia, low glucose, and high lactate concentration, meanwhile tumor
cells require immune tolerance to evade host immunity. The upregulation
of lactate~impairs effector, and cytotoxic T cells function through
LDH-mediated NAD depletion. However, Tregs resist this environment
through FOXP3 expression {[}76{]}, which acts as an intrinsic metabolic
regulator that suppresses mTOR- and Myc- signaling pathways that
activate- glycolysis while enhancing OXPHOS and NAD+/NADH generation and
levels of FAO. These metabolic adaptations in Tregs allow them to
sustain in severe inflammatory microenvironments without affecting their
function, survival, and suppression, which is vital to maintain
peripheral immune tolerance {[}56, 76, 77{]}.~ Additionally,
tumor-infiltrating Tregs frequently display substantially upregulated
expressions of co-inhibitory receptors, such as T cell immunoreceptor
with immunoglobulin and ITIM domains (TIGIT), lymphocyte activation gene
3 (LAG3), neuropilin 1 (NRP1), PD1, and CTLA4, to sustain stable FOXP3
activity, FOXO1 nuclear localization and higher levels of suppressive
function {[}78{]}.

Contrary to the tumor microenvironment, in other inflammatory
conditions, such as in transplantation settings or autoimmunity, Tregs
lose their suppressive function and convert into effector cells that
produce pro-inflammatory cytokines (IFN\selectlanguage{greek}γ, \selectlanguage{english}IL-17, IL-4) and have
increased expression of associated master regulator transcription
factors (such as T-bet, IRF4, and ROR\selectlanguage{greek}γ\selectlanguage{english}t, or IRF4 and GATA3). This
functional impairment of Tregs causes loss in FOXP3 expression as well
as hyperactivation of the inflammatory PI3K pathway {[}77{]}.

\textbf{IMMUNOMETABOLIC REGULATION OF TREGS: ~}Multiple \textbf{}
signaling networks, \textbf{} for instance, PI3K/Akt, mTOR-HIF-1\selectlanguage{greek}α \selectlanguage{english}axis,
and LKB1--AMPK pathways coordinate after immune cells are stimulated to
mediate metabolic reprogramming. Here we focus on LKB1 signaling, in
which LKB1 protein that restrains the activation and pro-inflammatory
function of Teffs. LKB1 is a bioenergetic sensor that is expressed by
the serine-threonine kinase 11 (STK11) gene and regulates cell polarity
and function {[}79- 82{]}. It is an important upstream kinase that
phosphorylates AMPK\selectlanguage{greek}α \selectlanguage{english}(AMPK is heterotrimer having catalytic subunit
(AMPK\selectlanguage{greek}α\selectlanguage{english}) and regulatory subunit (AMPK\selectlanguage{greek}β \selectlanguage{english}and AMPK\selectlanguage{greek}γ\selectlanguage{english}) ) that contributes to T
cell differentiation and function and for maintaining functional fitness
of Tregs in conditions of energy stress.~ {[}83{]}. Maclver et al. have
reported that LKB1-AMPK signaling negatively regulates T cell effector
function through the regulation of mTOR activity {[}84{]}. Of note,
later studies have reported that the LKB1 signaling pathway promotes
OXPHOS and FAO to maintain Treg survival and function. Furthermore,
Nanhai et al. \emph{} reported that LKB1 is crucial for maintaining
cellular metabolism and energy homeostasis in Tregs and this metabolic
phenotype is independent of AMPK and mTOR signaling pathways {[}85,
86{]}. To proof this, Nanhai et al. \emph{} and others ~have deleted
~the LKB1 gene in Treg and catalytic subunit deletion of AMPK in Tregs.
They found that LKB1 deletion resulted in loss of Tregs number and
function and leads to impaired cellular metabolism in Tregs and
uncontrolled immune activation. Contrary to this, the catalytic subunit
deletion of AMPK in Tregs does not cause any abnormalities in the murine
model. These findings are similar to those mice having mutations or
deletion of FOXP3, where FOXP3 deletion resulted in loss of Tregs
number. These studies further advocate the importance of LKB1 in Treg
cell metabolism~ {[}80, 85-87{]}.

LKB1 stabilizes FOXP3 expression in Tregs that maintain immunological
self-tolerance and homeostasis {[}80{]}. Di Wu \emph{et al.} have
reported that LKB1 governs Treg survival and its lineage identity
{[}80{]}. T cell's specific deletion of LKB1 causes a halt in Treg
regulatory function that leads to impaired immune responses {[}80,
87{]}. Moreover, LKB1 deficient Tregs have been characterized by
defective mitochondria, compromised OXPHOS, depleted cellular ATP, and
altered cellular metabolism pathways {[}80{]}. Treg-specific deletion of
LKB1 leads to the development of fatal autoimmune inflammation {[}80,
88{]}, and causes disrupted Treg survival and reduced mitochondrial
mass, its membrane potential, and increased the generation of ROS {[}85,
86, 89{]}. Mechanistically, LKB1 deficiency causes the diminished
release of intracellular ATP and induces aberrant expression of immune
regulatory molecules such as PD-1, and TNF receptor GITR, and OX40
{[}85, 90-93{]}. Yang K et al. in his classic work has been reported
that LKB1 function in Tregs does not depend on AMPK signaling or the
mTORC1--HIF-1\selectlanguage{greek}α \selectlanguage{english}axis as described earlier, however, it depends on
LKB1-\selectlanguage{greek}β-\selectlanguage{english}catenin signaling to regulate PD-1 and TNF receptor proteins such
as GITR and OX40 expression on Treg cell~ {[}9, 21, 85{]}. \selectlanguage{greek}β-\selectlanguage{english}catenin is
a key mediator of Wnt signaling and suppresses the aberrant expression
of PD-1 and GITR in Treg cells. LKB1 deficient Treg cells show the
degradation of \selectlanguage{greek}β-\selectlanguage{english}catenin. Briefly, these findings indicate the role of
LKB1-\selectlanguage{greek}β-\selectlanguage{english}catenin signaling in the control of Th2 response by modulating
PD1 and other Treg signature molecules {[}85, 94{]}.

It has been reported in many clinical studies that LKB1 is mutated in
20\%--30\% of NSCLC (Non-small-cell lung carcinoma) patients, which
causes enhanced sensitivity to metabolic inhibitors or stress-induced
mitochondrial dysfunction {[}95{]}. Further, Yang \emph{et al}. have
reported that Tregs need the LKB1 gene to manage their metabolic and
immunological homeostasis function, and deficiency of LKB1 resulted in
the apoptotic and functional exhaustion of Tregs {[}85{]}. Xiuhua Su
\emph{et al}. have demonstrated that Tregs from acute graft- versus-
host disease (aGVHD) patients show an exhausted phenotype, which is
characterized by the unstable FOXP3 expression, diminished suppressive
functions, defective migration capacity, increased apoptosis, and
downregulation of LKB1 expression {[}96{]}. In addition to maintaining
suppressive activity, LKB1 maintain FOXP3 stability in Tregs by
demethylation of conserved non-coding sequences (CNS2) at the FOXP3
locus {[}75{]} through the activation of signal transducer and activator
of transcription 4 (STAT-4), and partially through suppressing nuclear
factor-\selectlanguage{greek}κ\selectlanguage{english}B (NF-\selectlanguage{greek}κ\selectlanguage{english}B) signaling {[}97{]}. Meanwhile, LKB1 promotes Treg
suppressor function by increasing the expression of various
immunosuppressive genes by enhancing transforming growth factor-\selectlanguage{greek}β
\selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english}) signaling. The deletion of TGF-\selectlanguage{greek}β \selectlanguage{english}leads to autoimmune
glomerulonephritis and impaired Treg activity {[}98{]}. Thus, TGF- \selectlanguage{greek}β
\selectlanguage{english}pathway is vital for nTreg and iTreg development {[}94, 95{]}.

Treg cells exert their suppressive function in several different ways.
One mechanism is through the LKB1 pathway that activates the mevalonate
pathway, which is crucial for Treg functional fitness and stability
{[}85{]}. This pathway also activates its metabolite geranyl
pyrophosphate (GGPP), which phosphorylates STAT-5 via IL-2 signaling and
subsequently support Treg function and lineage stability. Activation of
mevalonate genes by LKB1 is required for Treg proliferation and, thereby
suppressing the interferon-gamma (IFNy) and interleukin-17A (IL-17A)
expression, which is independent of the AMPK signaling {[}85{]}.
Furthermore, LKB1 induced the mevalonate pathway was also found to
maintain intracellular cholesterol homeostasis {[}87, 91{]}. LKB1
signaling is key for Tregs to maintain their metabolic and immunological
balance to curb apoptotic and functional exhaustion, thereby reinforcing
homeostatic control of Tregs. Altogether, these preclinical
investigations highlighted that LKB1 is a primary regulator of lipid
metabolism in Tregs, which play a regulatory role in modulating Tregs
suppressive activity and maintaining the phase of immunotolerance
(\textbf{Figure 2}).

\textbf{IMMUNOMETABOISM AS A THERAPEUTIC TARGET IN TRANSPLANTATION:}
Organ transplantation is the last therapeutic approach used to treat
end-stage organ failure. Current therapeutic approaches for organ
rejection target overall immune suppression, which is associated with
severe side effects, cancer, and mortality. However, targeting a more
specific immune suppression has become more favorable in clinical
settings to treat transplant rejection. The long term survival of
transplanted graft is limited by surgical trauma and ischemaic
reperfusion injury (IRI). These unavoidable events trigger innate immune
cell activation that eventually promote sterile inflammation. This
prolong inflammation causes immunometabolic rewiring especially after
ischemia and IRI to fulfill oxgen demand. Ischemia or Hypoxia reduces
OXPHOS and hence, induce tissue reliance on aerobic glycolysis {[}99{]}.
This event enhance the production of pro-inflammatory cytokine that
subsequently leads to T cell activation. In fact,the metabolic
requirements of Teffs and Tregs and the conclusive role of Teffs/Tregs
ratio have been crucial parameters to decide the fate of the
transplanted organ. These metabolic requirements could be used as a key
tool in specific therapeutic approaches to contain graft associated
microvascular injuries and induce specific immune tolerance. In
alloimmune inflammation post-transplantation, donor immune cells rely
less on lipid metabolism for their energy requirements, whereas the
recipient cell depends on aerobic glycolysis {[}74, 98{]}. Specifically,
CD4\textsuperscript{+}T cell activation in solid organ transplantation
more closely resembles the classical metabolic reprogramming that is
seen during normal T cell activation {[}1{]}.

It has been reported that inhibiting glycolysis by metabolic inhibitors
such as 2DG (2-Deoxy-D-glucose) and metformin (inhibits complex I of
ETC) (Table 1) can considerably decrease {[}74{]} the glycolysis of an
activated Teff and their cytokine production {[}101-103{]}. These two
drugs and 6-Diazo-5-oxo-L-norleucine (DON) (Table 1), a~glutamine
analogue, has been reported to abolish the immune response. In
transplant settings, combined therapy such as 2DG, metformin, and DON
could suppress the proliferation of pathogenic Teff cells and promote
the generation of antigen-specific Tregs; however, combination therapy
does not cause the global inhibition of immune responses; instead, it
selectively inhibits effector responses while promoting Treg responses
based on differential metabolism of immune cells resulted in a specific
immune suppression {[}103{]}. This combinational therapy targets
explicitly allogeneic Teffs that cause graft rejection and keeps other
immune cells and healthy tissues relatively unharmed. This combined
therapy reduces skin and heart allografts rejection through the
inhibition of allogeneic Teffs, and by stimulating Treg proliferation
and activation {[}103-105{]}. Furthermore, metformin (AMPK agonist) and
Soraphen A (acetyl-CoA carboxylase inhibitors) (Table 1) enhance pTreg
differentiation by inhibiting allogeneic Teffs in autoimmune mouse
models of allergic asthma and experimental allergic encephalomyelitis
respectively (\textbf{Figure 3}) {[}60, 106-109{]}.

Various strategies have been implemented to increase the numbers and
potency of Tregs in vivo to reduce the severity of graft rejection
{[}110{]}. Besides targeting metabolic pathways, some studies have also
highlighted that the augmentation of the LKB1 pathway is immensely
beneficial in reducing graft rejection, and strategies that could
activate the LKB1 pathway might be employed as a future treatment in
transplantation settings {[}96{]}. Some strategies such as
overexpressing LKB1 in lentivirus vectors and then use these vectors to
create genetically reprogrammed Tregs and then the adoptive transfer of
these modified Tregs would be an attractive strategy to prevent or treat
Graft vs Host Disease (GVHD) {[}111{]}. It has been established that
LKB1 downregulation in Tregs in a GVHD pathological condition and this
is significant for targeting LKB1-related pathways to treat GVHD. As
demonstrated in that, JQ1 (BET bromodomain inhibitor) (Table 1) enhanced
the expression of LKB1, ATG5, and LC3-II genes and resulted in the
phosphorylation of AMPK, ULK1, and ATG14 in allografts. This
phosphorylation process has been reported to prolong heart allograft
survival and inhibits the release of inflammatory cytokines {[}112{]},
which further supports the role of the LKB1 gene in graft survival.
Metabolic manipulation to Tregs and its expansion protocol holds
tremendous promise in maintaining Treg-based tolerance during
inflammatory disorders. It has been reported that rapamycin (Table 1)
causes enhanced cell expansion and Treg stability {[}113{]}. In
addition, recent progress in chimeric antigen receptors (CARs)
technology affects the metabolic properties of Tregs {[}114-116{]} and
enhance the specificity and functionality of Tregs {[}35, 117, 118{]}.
It is an indirect strategy to modulate metabolic pathways in a
cell-type-specific manner. Thus, future studies should emphasize to use
a combination of antimetabolites with tolerance-inducing regimens such
as co-stimulatory blockade or LKB1 pathway augmentation or modified Treg
adoptive transfer to provide specific and effective long-term graft
acceptance.

\textbf{CONCLUSION:}

The role of immune signaling networks in immunometabolism is emerging as
a promising area of research, which could provide a valuable tool to
redesign therapeutic options to contain allograft rejection and the
progression of chronic fibrosis. Here, we have discussed the critical
roles of T cell's energy generation, roles in immunomodulation, and the
impact of immunomodulation on transplantation. Altogether, the power of
these metabolic signals contributes to the differentiation of Teffs;
therefore, our understanding of the metabolic disparity among T-cell
populations would open up new avenues to design therapeutic strategies
to prevent inflammatory-related diseases.

~

~

\textbf{LIST OF ABBREVIATIONS:}

~

Teffs: T effector cells; Tregs: T regulatory cells; ~Tmem: T memory
cells; APCs: Antigen-presenting cells; LKB1: Liver kinase B1; TCA:
Tricarboxylic acid cycle; ETC: Electron transport chain; GLUT1: Glucose
transporter 1; ATP: ~Adenosine triphosphate; NAD: Nicotinamide adenine
dinucleotide; CoA: Coenzyme A; OXPHOS: Oxidative phosphorylation; LPS:
Lipopolysaccharide; DC: Dendritic cell; NK: Natural killer; PRRs:
Pattern Recognition receptor; FAO: Fatty acid oxidation; IL:
Interleukin; CD: Cluster of differentiation; Th: T helper cells; CPT1A:
Carnitine Palmitoyltransferase 1A; PD1: Programmed cell death 1; nTreg:
natural Treg; iTreg: induced Treg; pTreg: peripheral Treg; PDH: pyruvate
dehydrogenase; PDHKs: PDH kinases; NAC: N-acetyl cysteine; GCN2: general
control nonderepressible-2; ARG1: arginase 1; HDC: histidine
decarboxylase; TDH: threonine dehydrogenase; IL4I1: interleukin-4
induced 1; LDH: lactate dehydrogenase; mTOR: mammalian target of
rapamycin; TIGIT: T cell immunoreceptor with immunoglobulin and ITIM
domains; LAG3: lymphocyte activation gene 3; NRP1: neuropilin 1; CTLA4:
cytotoxic T lymphocyte antigen 4; AMPK: AMP-activated protein kinase;
ROS: reactive oxygen species; GITR: Glucocorticoid induced TNF receptor;
HIF1- \selectlanguage{greek}α: \selectlanguage{english}Hypoxia inducible factor 1- \selectlanguage{greek}α; \selectlanguage{english}mTORC1: mammalian target of
rapamycin complex 1; IRI: Ischemaic reperfusion injury; aGVHD: acute
graft- versus- host disease; CNS2: conserved non-coding sequences;
STAT4: signal transducer and activator of transcription 4; NF-\selectlanguage{greek}κ\selectlanguage{english}B:
nuclear factor-\selectlanguage{greek}κ\selectlanguage{english}B; TGF-\selectlanguage{greek}β: \selectlanguage{english}transforming growth factor-\selectlanguage{greek}β; \selectlanguage{english}GGPP: geranyl
pyrophosphate; 2DG: 2-Deoxy-D-glucose; DON: 6-Diazo-5-oxo-L-norleucine;
ATG5: Autophagy related 5; LC3-II: light chain 3-II; ULK1: unc-51-like
kinase 1; ATG14: Autophagy Related 14; BET: Bromodomain and
Extra-Terminal motif; PPP: Pentose phosphate pathway; SRC: spare
respiratory capacity.

~

\textbf{Consent for publication:} As per journal rules, I disclose that
the work has not been published or submitted for publication elsewhere,
either completely or in part, or another form or language

\textbf{Availability of data and material:} Not applicable

\subsection*{ACKNOWLEDGEMENTS}\label{acknowledgements}

\subsection*{The author thanks all research staff of the organ transplant
research center at King Faisal Special Hospital and Research
Centre.}\label{the-author-thanks-all-research-staff-of-the-organ-transplant-research-center-at-king-faisal-special-hospital-and-research-centre.}

\subsection*{Funding: Not Available}\label{funding-not-available}

Contributions: \textbf{} SK planned, and wrote the manuscript, MAK has
been involved in writing, and revising it critically for publication
standards. TS and AA contributed to various editing work. AS and DB
provided the theme of the proposed research plan.

Corresponding author: \textbf{} Correspondence to~Dr. Shadab Kazmi

\textbf{ETHICS DECLARATION}

Ethics approval and consent to participate: \textbf{} Not applicable.

Consent for publication: \textbf{} Not applicable.

\subsection*{Competing interests: The author declares that they have no
competing
interests.}\label{competing-interests-the-author-declares-that-they-have-no-competing-interests.}



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\par\null

\textbf{Figure legends and Tables}

\textbf{Fig.1} \textbf{Factors that influences metabolic phenotype of
inflammatory and anti-inflammatory immune cells towards
anti-inflammatory phenotype}: The various immune cell subtype~ reliance
on distinct metabolic pathways to promote their~ survival, lineage
subtype and function in the presence of several factors. mTOR-HIF1-\selectlanguage{greek}α
\selectlanguage{english}signaling axis promote inflammatory macrophages (M1) and effector T
cells (Th1,Th2,Th17)~ proliferation and function through utilizing
glycolysis, fatty acid synthesis and amino acid metabolism as a main
energy source. Contray to this, Tregs, Tmem and M2 macrophages, which
show a more tolerant phenotype, use the TCA cycle and fatty acid
oxidation~ for their energy source under the influence of LKB1
signaling. Figure created with Biorender.com

~

\textbf{Fig.2}~ \textbf{LKB1 and AMPK Signaling allows metabolic
programming in T cells}: Concerning with low nutrient and energy status,
the energy stress pathway kinases LKB1 and AMPK are triggered through
TCR and CD28 co-stimulatory signals, with AMPK activity mainly triggered
by most understood Ca2+-CAMMK2 (calcium calmodulin kinase kinase 2)
pathway. Bioenergetic fluctuations in cells such as deprivation of
glucose or glutamine or elevation of AMP/ADP-to-ATP ratio, can also
activate LKB1--AMPK signaling. The LKB1 cellular localization and
post-translational modifications play an important role in its activity.
The role of many upstream regulators of LKB1 signaling in T cells
metabolic programming is still undiscovered. The activation of LKB1
promote mitochondrial fitness and increased mevalonate metabolism in
Treg cells. The downstream kinase AMPK is the best-known kinase of LKB1.
LKB1 is a critical metabolic regulator that regulates energy homeostasis
in Tregs. AMPK also promotes mitochondrial fitness by increasing
mitochondrial mass. Figure created with Biorender.com

~

\textbf{Fig.3} \textbf{Strategies to modulate metabolism to regulate T
cell function in diseases}: The figure provides a summary of strategies
to pharmacologically target (green) metabolic pathways crucial for T
cells responses (black). This figure depicts the utilization of three
key metabolic pathways namely glycolysis, FAO and glutaminolysis for the
treatment of experimental autoimmune encephalomyelitis (EAE), multiple
sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus
(SLE), and transplantation. Figure created with Biorender.com

~

\textbf{Table 1.~ Pharmacological inhibitors of metabolic pathways and
their effects on Treg and in diseases}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-1/Fig-1}
\caption{{~
{\label{428906}}%
}}
\end{center}
\end{figure}

\textbf{Figure 1.} \textbf{Factors that influences metabolic phenotype
of inflammatory and anti-inflammatory immune cells towards
anti-inflammatory phenotype}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-2/Fig-2}

\end{center}
\end{figure}

\textbf{Figure 2.}~ \textbf{LKB1 and AMPK Signaling allows metabolic
programming in T cells}

\par\null

\textbf{}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-3/Fig-3}

\end{center}
\end{figure}

\textbf{}

\textbf{Figure 3.} \textbf{Strategies to modulate metabolism to regulate
T cell function in diseases}

\par\null\par\null

\textbf{~}

\textbf{~}

\textbf{~}

\textbf{Table 1.~ Pharmacological inhibitors of metabolic pathways and
their effects on Treg and in diseases}

\textbf{~}

\textbf{~}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
Metabolic Pathway & Drug & Target & Effect on Tregs & Diseases & Model
animal & Reference\tabularnewline
Glucose metabolism & 2-DG ~ ~ ~ Dichloroacetate & Glycolysis ~ ~ ~ PDHK
& Induce Treg differentiation ~ ~ Induce Treg generation & Skin and
heart transplantation ~ ~ EAE & Mouse ~ ~ ~ Mouse & {[}101{]} ~ ~ ~
{[}61{]}\tabularnewline
Lipid metabolism & Soraphen A ~ ~ ~ ~ & acetyl-CoA carboxylase
inhibitors ~ ~ & Induce pTreg differentiation ~ ~ ~ & EAE ~ ~ & Mouse &
{[}108{]}\tabularnewline
Glutamine metabolism & DON & Glutamine uptake & Induce Treg generation &
Skin and heart transplantation ~ & Mouse & {[}103{]}\tabularnewline
Metabolic regulators & Rapamycin ~ ~ ~ Metformin & mTORC1 ~ ~ ~ AMPK &
Enhance Treg cell expansion and stability ~ Induce Treg differentiation
& Graft Rejection ~ ~ ~ Skin and heart transplantation ~ & Human ~ ~
Mouse & {[}113{]} ~ ~ {[}106{]}\tabularnewline
Epigenetic inhibtor & JQ1 & BET bromodomain inhibitor & Enhanced
expression of LKB1 and provide stability to Treg & Heart transplantation
~ & Mouse & {[}112{]}\tabularnewline
\bottomrule
\end{longtable}

\textbf{~}

\selectlanguage{english}
\FloatBarrier
\end{document}

