individuals with obesity are at increased risk of several health conditions including type 2 diabetes , dyslipidaemia , liver steatosis , hypertension , cardiovascular disease , osteoarthritis , as well as various forms of cancer ( faulds and dahlman - wright , 2012 , mauro and marelli - berg , 2012 , norata et  al . , 2015 ) .
a strong link between obesity and the establishment of chronic systemic inflammation has recently emerged ( bjrndal et  al . , 2011 ) and
is often related to increased expression of  adipokines ( i.e. , leptin , chemerin , and resistin ) , myokines , and pro - inflammatory mediators ( i.e. , tumor necrosis factor [ tnf]- , interleukin [ il]-1 , and il-6 ) , and decreased expression of the anti - inflammatory cytokine , adiponectin ( norata et  al . , 2015 ) .
in obesity , activated immune cells ( including t  cells , macrophages , and dendritic cells [ dc ] ) infiltrate adipose tissue , promoting in turn the production of pro - inflammatory cytokines , such as tnf- , il-6 , resistin , and monocyte chemotactic protein-1 ( mcp-1/ccl2 ) ( mathis and shoelson , 2011 , norata et  al . , 2015 ) .
this process is thought to underlay the activation of both the innate and adaptive arms of the immune system , which results in the chronic inflammatory response observed in obese subjects ( larbi et  al . , 2008 , shaw et  al . , 2010 ) . while t  cells , b cells , macrophages , neutrophils , and mast cells are increased in number in the visceral adipose tissue ( at ) of obese individuals , specific subsets of t  cells ( helper t  cell type [ th2 ] , regulatory t  cells , and invariant natural killer t [ inkt ] cells ) , as well as eosinophils are reduced ( cildir et  al . , 2013 ) .
obesity - related inflammation , caused by excessive and inappropriate activation of the immune system , is pivotal to disease progression and the development of complications , such as atherosclerosis and type 2 diabetes ( norata et  al . , 2015 ) .
t lymphocytes play a major role in the development of these inflammatory processes both via direct cytotoxic activities and secretion of cytokines that influence the severity and outcome of the inflammatory reaction ( hamann et  al . , 2000 ) . based on the observation
that t  cells in the adipose tissue display specific t  cell receptor ( tcr ) rearrangements , it has been proposed that obesity might  be associated with an autoimmune response ( gerriets and rathmell , 2012 ) .
the mechanisms of enhanced t  cell activation and tissue infiltration during obesity , however , remain elusive .
altered antigen presentation by dc has been proposed to underlie the activation of adaptive immunity observed in dyslipidaemia .
although at - derived dc in obesity display an immature phenotype with lower expression of activation markers , such as  cd40 , cd80 , and cd86 ( chen et  al . , 2014 ) , they promote the differentiation of pro - inflammatory interferon ( ifn) cd4 t  cells ( chen et  al . , 2014 ) .
a recent report has implicated dietary long- and medium - chain fatty acids ( fa ) in t  cell differentiation toward th1 and th17 subsets in autoimmune responses of the cns ( haghikia et  al . , 2015 ) ; however , whether fa can directly affect t  cell activation and differentiation toward specific cell subsets during metabolic overload remains to be established . in this study
, we investigated the phenotypic , functional , and migratory features of memory cd4 t  cells in experimental models of saturated fa - induced metabolic stress , as well as in human cd4 t  cells obtained from lean , overweight , and obese individuals in a large cohort of free - living people ( progressione della lesione intimale carotidea [ plic ] study : n  = 2,606 ) .
mechanistic studies show that direct exposure of cd4 t  cells to saturated fa ; i.e. , palmitate , which is enriched in high - fat diet ( hfd ) , induces cd4 t  cells to acquire a specific cd44-ccr7-cd62l - cxcr3-lfa1 pro - inflammatory functional phenotype via enhanced engagement of a pi3k p110-akt - dependent pathway upon priming , but independent of the modality of antigen presentation by dc .
given that lymphocyte infiltration of non - lymphoid tissues is a prominent feature of chronic inflammation , we first assessed whether hfd - induced metabolic stress directly promotes the generation of t  cell populations with specific migratory features .
to this aim , memory cd4 t  cells were generated in rag2-deficient tcr transgenic marilyn female mice , which harbor monoclonal cd4 t  cells specific for the male antigen h - y ( lantz et  al . , 2000 ) , by intraperitoneal ( i.p . )
cd4 t  cells were then isolated from the lymph nodes of donors fed 8-week chow ( cd ) or hfd and adoptively transferred in hfd- or cd - fed ( i.e. fed chow or high - fat diet for 8  weeks , respectively ) c57bl/6 male recipients that had been i.p .
injected with zymosan and ifn 48  hr earlier to induce localized inflammation ( figures s1a  s1c ) ( fu et  al . , 2014 ) . in  vivo - primed cd4 t  cells isolated from hfd donors exited from the blood circulation of cd recipients with a kinetic 24  hr faster than their cd counterpart ( figure  1a ) .
cd4 t  cells that were primed in hfd donors also trafficked preferentially to the non - lymphoid , inflamed sites ( peritoneal cavity ) of both hfd and cd recipients ( figure  1b ) .
in contrast , primed cd4 t  cells from cd donors preferentially localized to the mesenteric lymph nodes of both cd and hfd recipients ( figure  1c ) .
no difference in cd4 t  cell localization to the spleen was observed when comparing equal numbers of adoptively transferred cd and hfd donors ( figure  1d ) .
these results indicate that memory t  cell populations induced in cd- or hfd - fed animals are characterized by different trafficking patterns . to establish the pathophysiological relevance of these differential trafficking patterns , we performed hy - mismatched skin grafts , whereby male skin was implanted on the upper right back of cd- or hfd - fed marilyn female recipients .
we found that rejection of hy - mismatched skin was significantly accelerated in hfd as compared to cd female recipients ( figure  1e ) ( molinero et  al . , 2016 ) .
activation of cd4 t  cells and their localization to the graft are known to be instrumental to rejection in this system ( chen et  al . , 2003 ) .
indeed , we observed that hy - mismatched skin graft rejection was completely abrogated in rag2ko mice ( that are deficient for mature t and b cells ) independent of the diet they received ( figure  s1d ) .
reconstitution of hfd rag2ko mice with cd4 t  cells isolated from marilyn mice resulted in a rapid rejection of the transplanted male - derived skin ( figure  s1d ) , showing the crucial role these cells play in such system .
these findings support the conclusion that biased differentiation of cd4 t  cells toward a pro - inflammatory effector memory phenotype takes place during hfd - induced metabolic stress . based on the distinct homing preferences of memory t  cells generated in hfd- or cd - fed animals and the stronger immune response elicited in hfd animals
, we asked whether a biased differentiation of activated cd4 t  cells toward a pro - inflammatory phenotype  and associated homing receptors  takes place upon priming following hfd - induced metabolic stress . using the same immunization procedure
as that used for the experiments presented in figures 1a1d , we found that hfd - fed hosts displayed an increase in the size of the cd44-ccr7-cd62l - cxcr3-lfa1 effector memory - like cd4 t  cell population and no difference in the cd44-ccr7-cd62l central memory cd4 t  cells as compared to cd4 t  cells primed in cd - fed mice ( figures 2a2d and s2a  s2d ) .
in contrast , the size of naive t  cells was reduced in hfd ( figures 2a , 2b , s2a , and s2b ) .
these data suggest a preferential cd4 t  cell differentiation toward an effector memory phenotype under hfd regimen in concomitance with a reciprocal reduction of the naive cell population without affecting the size of the central memory pool . no difference was observed in the expression of the activation markers cd25 and cd44 ( figures 2e , 2f , s2e , and s2f ) by memory cd4 t  cells activated in cd or hfd hosts , indicative of similar priming efficiency in either metabolic background .
in agreement with the observed phenotypes , a significantly larger fraction of in  vivo - primed cd4 t  cells isolated from the lymph nodes of hfd mice migrated in response to the pro - inflammatory chemokine cxcl10 ( a cxcr3 ligand ) in ex  vivo chemotaxis assays , while most in  vivo - primed cd4 t  cells isolated from the lymph nodes of cd mice migrated to the homeostatic lymphoid tissue chemokines ccl19 and ccl21 ( ligands of ccr7 ) ( figure  2 g ) . to investigate the cellular sources of cxcl10 in  vivo
injection of ifn. at 48  hr later , intracellular staining of cxcl10 in a number of immune cell populations retrieved from the peritoneal cavity was performed .
we chose this system because of its relevance to the peritoneal recruitment system shown in figures 1a1d .
data indicate that resident ( peritoneal ) and to a less extent infiltrating ( monocyte - derived ) macrophages are main sources of cxcl10 in this model ( figures s2 g and s2h ) .
furthermore , we observed an increase in the ifn and il4 populations upon priming following hfd ( figures 2h and 2i ) .
our findings are in line with previous work showing that increased expression of cxcr3 in inflammatory conditions , such as obesity and atherosclerosis , drives accumulation of inflammatory t  cells in fat sites .
indeed , cxcr3- or cxcl10-deficient mice are protected from obesity- and atherosclerosis - induced inflammation in association with reduced accumulation of effector t  cells and macrophages in at and atherosclerotic plaques ( gupta et  al . , 1997 , rocha et  al . , 2014 ,
a cohort of 1,172 subjects was selected by probability sampling from a large survey of free - living people ( plic study : n  = 2,606 ; average age 66 years ) . the selected cohort was representative of the entire plic cohort in terms of age , gender , and clinical parameters according to the kolmogorov - smirnov test ; individuals were stratified in three groups ( i.e. lean , overweight , and obese ) , based on bmi , and a number of biochemical and cellular markers of their peripheral blood were analyzed ( table s1 ) .
in particular , this analysis revealed increased total lymphocyte counts in the blood of overweight and obese as compared to lean individuals ( figure  3a ; table s1 ) . from this cohort ,
a detailed immunophenotype was performed in a group of n  = 187 subjects . again
, these individuals were selected by probability sampling and were representative of the entire cohort according to the kolmogorov - smirnov test .
specific subsets of circulating cd4 t  cells were assessed by 10-parameter/8-color polychromatic flow cytometry with cd3 , cd4 , cd45ro , cd45ra , ccr7 , ccr5 , cxcr3 , and hla - dr as markers .
the main cd4 t  cell subsets present in the blood were identified by flow cytometry based on a  well - defined gating strategy ( figures s3a and s3b ) .
the percentage of total t - helper lymphocytes was equal in the three groups ( figure  3b ) ; however , we found a strong association of the obese condition with reduced naive t  cells and increased effector memory t  cells ( figures 3c and 3d ) .
to gain further insights into the relationship in humans between obesity and the effector memory phenotype of cd4 t  cells , we  focused on the few very obese patients in our cohort ( bmi > 35  kg / m2 , n  = 5 ) and observed that the percentage of effector memory t  cells was raised to 18.2% ( 11.221.3 ) as compared to 13.4% ( 8.818.25 ) of the entire obese population ( figures 3d and 3e ) and to 10.2% ( 8.412.5 ) of the lean population ( see figure  3d ) .
to address the potential link of this phenotype with obesity - related chronic inflammation , we also analyzed the blood levels of c reactive protein ( crp ) in the lean , overweight , and obese subjects .
as expected , crp levels rose progressively with increasing bmi ( figure  s4a ) , corroborating the correlation between obesity and inflammation .
however , no significant correlation was observed between crp levels and effector memory t  cells ( figure  s4b ) . furthermore , no correlation between fasting  glucose levels and effector memory t  cells was observed ( p  =  0.117 ) .
also , effector memory t  cells were not significantly increased in type 2 diabetes subjects ( n  = 13 ) as compared to the rest of the subjects in the cohort ( p  = 0.069 ) .
the body mass fat distribution of each individual was also analyzed by dual - energy x - ray absorptiometry ( figure  s4c ) , and the resulting android / gynoid ratio was correlated with the percent of various t  cell subsets .
the android / gynoid ratio correlated negatively with the naive t  cell subset ( figure  3f ) and positively with the cxcr3 effector memory t  cell subset ( figures 3 g and 3h ) .
no significant correlation between the android / gynoid ratio and hladr or ccr5 effector memory t  cell subsets was observed instead ( figures s4d and s4e ) .
prompted by previous suggestions that altered antigen presentation by dc might affect t  cell activation during metabolic stress ( chen et  al .
, 2014 , shamshiev et  al . , 2007 ) , we assessed the effect of splenic dc purified from hfd or cd animals on t  cell activation and differentiation .
we found that in  vitro co - cultures of dc isolated from the spleen of cd - fed c57bl/6 male mice with cd4 t  cells obtained from the lymph nodes of cd - fed marilyn mice induced a strong proliferative response of marilyn cd4 t  cells , which was reduced when cd4 t  cells were isolated from donors fed hfd ( figures 4a and s5a ) .
in contrast , dc purified from the spleen of c57bl/6 male mice fed hfd were significantly less effective in promoting cd4 t  cell proliferation compared to their  cd - fed
 counterpart , irrespective of the t  cell metabolic status ( i.e. , cd or hfd cd4 t  cell donors [ figures 4a and s5a ] , in line with previous reports ; shamshiev et  al . , 2007 ) .
these data suggest that the metabolic status of dc plays a role in determining their ability to promote cd4 t  cell proliferation
. however , it did not affect the differentiation of cd4 t  cells to central versus effector memory phenotype .
indeed , hfd cd4 t  cells showed a higher percent of cd44-ccr7-cd62-cxcr3 cells as compared to cd cd4 t  cells ( consistent with figures 2a2c ) , independent of whether they were co - cultured with dc from hfd- or cd - fed donors ( figure  4b ) .
similar results were obtained in  vivo when cd4 t  cells isolated from female marilyn mice were co - injected with dc from hfd or cd c57bl/6 male donors in c57bl/6 female recipients ( i.e. , reduced cd4 t  cell proliferation in the presence of dc from hfd - fed mice and no effect on central memory versus effector memory differentiation ; figures s5b and s5c ) . in line with previous reports ( shamshiev et  al . , 2007 ) ,
dc isolated from hfd - fed mice showed impaired activation as indicated by reduced expression of the activation markers cd80 and cd86 as compared to lean dc ( figure  4c ) .
having ruled out a possible role for altered antigen presentation by dc in biasing cd4 t  cell differentiation to the cxcr3 effector memory phenotype observed during hfd - induced metabolic stress , we sought to establish whether cd4 t  cell intrinsic alterations were responsible for this event by interrogating pathways controlling nutrient responses in t  cells .
pi3k / akt and mammalian target of rapamycin complexes ( mtorc1 and mtorc2 ) are known to be rapidly activated upon t  cell priming and control transcriptional and metabolic programs that sustain cell activation ( finlay et  al . , 2012 , macintyre et  al . , 2011 ) .
we found a substantial increase in the levels of akt activation ( phosphorylation at serine 473 , a target of mtorc2 ) in in  vivo - primed cd4 t  cells isolated from the lymph nodes of hfd- as compared to cd - fed mice ( figure  5a , compare lanes 68 versus 12 ) .
similar results were obtained with cd4 t  cells isolated from the peripheral blood of obese versus lean individuals selected from the plic cohort ( figure  5b ) . in contrast , threonine 308 on akt and the mtorc1 target s6 did not undergo any significant modulation when comparing in  vivo - primed cd4 t  cells isolated from the lymph nodes of hfd or  cd  mice ( figures 5a and s6a ) . in line with recent literature ( finlay et  al . , 2012 , macintyre et  al . , 2011 ) , our data suggest that cd4 t  cell priming following hfd regimen triggers a pi3k / akt pathway via mtorc2 , but does not result in downstream mtorc1 activation .
a recent report has implicated direct signaling induced by dietary fa in t  cell differentiation in the gut , with middle- and long - chain fa supporting th1 and th17 cell differentiation and short - chain fa promoting treg cell development ( haghikia et  al . ,
, using lipidomic analysis , we found that hfd alters the fatty acid and phospholipid profile of t  cells ( figure  s6b ) , resulting in increased membrane fluidity ( figure  5c ) .
this might underlay a reduced threshold for tcr clustering and activation in hfd ( swamy et  al . ,
, imagestream analysis enabled us to observe that in  vitro activation of t  cells isolated from hfd as compared to cd mice results in increased aggregation of cholera enterotoxin ( ctx ) b staining ( figures 5d and s6c ) , indicative of enhanced formation of lipid rafts , a key signaling event downstream of tcr engagement ( viola et  al . , 1999 ) .
the saturated fa palmitate has been shown to directly promote the activation of the inflammasome via nlrp3 and asc ( wen et  al . , 2011 ) and of the toll - like receptor ( tlr ) 4/myd88 pathway ( eguchi et  al . , 2012
we therefore reasoned that the observed effector memory phenotype and enhanced activation of the pi3k / akt pathway during cd4 t  cell  hfd priming  could be due to a direct effect of the saturated fa palmitate , whose levels increase during hfd . mirroring the results shown in figures 2a2d
, we found that palmitate enriched diet ( ped)-fed animals displayed an increase in the size of the cd44- ccr7-cd62l - cxcr3-lfa1 effector memory - like cd4 t  cell population and no difference in the cd44-ccr7-cd62l central memory cd4 t  cells as compared to cd4 t  cells primed in the corresponding palmitate control diet ( pcd ) fed - mice ( figures 5e5h ) .
in contrast , the size of the naive t  cell subset was reduced upon ped ( figures 5e5h ) .
further , cd4 t  cells were cultured overnight with il-7 in the presence or absence of either palmitic or linoleic acid at a concentration which mirrors plasma levels in obese individuals ( 200  m ) , followed by activation with anti - cd3 and anti - cd28 for the indicated time points .
as shown in figure  5i , cd4 t  cell activation in the presence of palmitic acid for 2  hr showed a trend of enhanced akt activation compared to linoleic acid or untreated control without any modulation of s6 . in similar experimental settings , only cd4 t  cell activation in the presence of palmitic , but not stearic , acid for 48  hr led to reduced levels of ccr7 and cd62l mrna ( figure  5j ) . in line with previous reports ( eguchi et  al .
, 2012 , wen et  al . , 2011 ) , these data suggest the existence of specific sensing mechanisms for palmitate . to further link the activation of the pi3k / akt pathway to the observed specific cd4 t  cell differentiation during hfd priming , we activated cd4 t  cells via cd3/cd28 antibody ligation in the presence or absence of the akt activator sc79 .
as expected , such treatment led to marked akt phosphorylation ( figure  s6d ) .
importantly , enhanced akt activation led to reduced levels of ccr7 and cd62l mrna , as well as ccr7 protein ( figures 5k and 5l ) , indicating that increased akt activation during cd4 t  cell priming directly promotes the pathway leading to the differentiation of effector memory - like t  cells .
we addressed the question as to whether enhanced pi3k / akt activation as we observed during hfd priming in cd4 t  cells was instrumental to the biased t  cell differentiation to the observed cxcr3 effector memory phenotype .
the p110 subunit of pi3k is crucial for correct tcr signaling and t  cell activation ( okkenhaug et  al . , 2002 ) .
furthermore , p110 engagement during t  cell priming is instrumental for akt activation and differentiation toward an effector memory phenotype , at least in cd8 t  cells ( finlay et  al .
we therefore took genetic and pharmacologic approaches to selectively target p110 and akt activities in  vivo .
first , allogeneic cd4 t  cells were primed in  vivo in hfd - fed p110 ( expressing kinase - dead p110 ) or wild - type ( wt ) mice .
genetic inhibition of p110 activity completely reversed cd4 effector memory differentiation in hfd - fed animals ( figures 6a6d ) .
this was accompanied by a reduction of both akt activation on serine 473 ( figure  6e ) and migration in response to the chemokine cxcl10 ( figure  6f ) .
similarly , administration of the selective p110 inhibitor ic87114 during priming prevented biased differentiation of allogeneic cd4 t  cells toward an effector memory phenotype upon priming ( figures 6g6j ) . of note ,
administration of ic87114 completely blocked akt activation in hfd cd4 t  cells , but only partially in cd mice ( figure  5a , lanes 911 versus 35 ) . finally , to further causally link pi3k and akt , we also treated cd wt mice with the akt - selective activator sc79 .
similar to what we had observed in  vitro ( figures 5k and 5l ) , this treatment led to increased effector memory differentiation to levels similar to those observed in hfd - fed animals ( figures 6g6j and s6e ) .
given the important role of fa oxidation ( fao ) in the generation of memory t  cells ( osullivan et  al .
, 2014 ) , we assessed whether this metabolic pathway contributes to enhanced effector memory differentiation upon priming following saturated fatty acid - enriched diet ( ped ) .
as shown in figures 5e5h , enrichment of dietary palmitate led to enhanced differentiation of primed cd4 t  cells toward an effector memory phenotype . in  vitro
, naive t  cells increased their rate of oxidative phosphorylation ( ocr : oxygen consumption rate ) only in response to palmitic , but not stearic , acid ( figure  7a ) .
etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase-1 ( cpt-1 ) , an upstream enzyme in the fao cascade .
with etomoxir during priming in ped - fed mice and found that this treatment completely prevented biased differentiation of effector memory cd4 t  cells we observed upon priming following ped ( figures 7b7e ) .
chronic systemic inflammation in obesity is characterized by the secretion of th1 cytokines , as well as aberrant t  cell infiltration of non - lymphoid tissue , including adipose tissue and arterial wall .
obese at contains more th1 lymphocytes and higher levels of ifn than its lean counterpart ( rocha et  al . , 2008 ) .
in this study , we have tested the hypothesis that excess dietary saturated fa directly affects the differentiation and trafficking of cd4 t  cells to non - lymphoid tissues .
we report that differentiation of memory cd4 t  cells primed in hfd - fed mice is biased toward a cd44-ccr7-cd62l - cxcr3-lfa1 effector memory - like phenotype without major changes in the size of the central memory pool .
these t  cells display preferential trafficking to non - lymphoid , inflammatory sites , as well as increased effector function following adoptive transfer .
although metabolically imbalanced dc from obese / dyslipidaemic hosts tend to support th1/th17 responses ( chen et  al . , 2014 ,
, 2012 , reynolds et  al . , 2012 , shamshiev et  al . , 2007 ) ,
our data show that they play no role in determining cd4 t  cell commitment to effector memory - like phenotype nor homing .
the cd4 t  cell developmental bias is instead mediated by direct exposure of t  cells to saturated fa ; i.e. , palmitate , leading to enhanced activation of a pi3k p110-akt - dependent pathway upon priming .
our data suggest that this pathway might involve mtorc2 leading to akt activation , but does not result in the downstream activation of mtorc1 .
the key role of the pi3k p110/akt axis is  further confirmed by our observation that selective inactivation of p110 in hfd can reestablish homeostatic cd4 t  cell responses .
a similar signaling cascade has previously been shown to direct the differentiation of cd8 effector t  cells ( finlay et  al . , 2012 , macintyre et  al . , 2011 ) ,
whereby high levels of akt activation led to the downregulation of cd62l and ccr7 expression and redirected the trafficking of effector cd8 t  cells away from the secondary lymphoid tissues into the sites of inflammation .
specifically , it has been shown that downstream of tcr engagement , akt controls transcriptional programs directing cytotoxic t  cell fate , but is dispensable for t  cell metabolism ( macintyre et  al . , 2011 ) , while pdk1 directly regulates mtor and hypoxia - inducible factor 1 , leading to an integrated control of metabolism and migration of cd8 t  cells ( finlay et  al . , 2012 ) .
in addition to showing that similar signaling events underlay the differentiation of cd4 t  cells , our data expand the reach of these studies from the basic mechanisms of t  cell activation and effector functions control to the biased establishment of unwanted t  cell responses ; i.e. , specific pro - inflammatory effector and migratory phenotypes , in a pathophysiologic context such as obesity .
this is in line with a recent study showing the link between obesity and metabolic diseases via increased th17 t  cell differentiation ( endo et  al . , 2015 ) .
it has also been reported that obesity could cause impairment in the t  cell memory response to secondary influenza infection , thereby affecting vaccine efficacy ( karlsson et  al . , 2010 ) . as we did not observe major differences in the size of the central memory population upon priming following hfd or cd , it is possible that central memory cells are functionally impaired in obesity . in line with the above study
interestingly , our data shown in figure  5a suggest that in physiologic conditions ( cd ) , different upstream kinases might control the activation of akt induced upon t  cell priming , as the p110 inhibitor ic87114 did not completely block akt phosphorylation .
by contrast , in hfd mice , akt phosphorylation was completely abrogated by ic87114 , indicating that the enhancement of akt activation by dietary lipids is particularly dependent on p110. the molecular mechanisms of lipid exchange on the t  cell membrane in physiology and during hfd remain to be fully established , as well as the question as to whether fa act by altering the cell membrane lipid balance or by binding to specific receptors , as recently suggested ( eguchi et  al . , 2012 , haghikia et  al . , 2015 , wen et  al . , 2011 ) .
our data showing increased membrane fluidity , as well as enhanced formation of lipid rafts upon tcr engagement , might provide some clues as to how excess dietary saturated fa impact on t  cell responses .
similarly , our data suggest that specific mechanisms exist for palmitate sensing , which remain to be defined . in this context
, we have observed that t  cell exposure to fa at the time of tcr triggering in  vitro is not sufficient to induce the biased effector memory differentiation we observed in  vivo , suggesting that palmitate sensing and alteration of the t  cell membrane must occur prior to antigen recognition .
hence , the need to maintain naive t  cell viability over prolonged culture with or without fa and in the absence of antigen stimulation has been a major challenge in setting up in  vitro models to study t  cell differentiation following prolonged exposure to fat overload .
we have partially overcome this issue by adding il-7 to the culture of naive t  cells during incubation with or without fa prior to tcr stimulation , and this led to the results we showed in figures 5i and 5j . defining in  vitro systems of immunization following prolonged exposure to certain nutrients would be extremely useful to test pathways involved with t  cell differentiation , such as palmitate sensing .
the observation that etomoxir prevents biased differentiation of effector memory cd4 t  cells upon priming following saturated fatty acid - enriched diet supports the concept that preferential utilization of fao during t  cell activation promotes effector memory cd4 t  cell differentiation following hfd .
ampk - induced fao has been shown to play an important role in the generation of memory t  cells ( osullivan et  al . , 2014 ) .
increased fao is also a feature of hfd ( cole et  al . , 2011 ,
however , reduced ampk activity is also observed in hfd ( lindholm et  al . , 2013 , liu et  al . , 2006 ) , and its reactivation by metformin is known to ameliorate glucose tolerance in obesity / type 2 diabetes .
thus , during hfd , ampk - induced fao is unlikely to play a role in the biased effector memory cd4 t  cell differentiation we observe . whether and how an enhanced pi3k / akt pathway leads to enhanced fao remain open questions .
the biased development of pro - inflammatory effector memory cd4 t  cells occurred in mice within 48  weeks from exposure to hfd , a time that precedes overt obesity and associated complications .
this might imply a causative role of altered t  cell differentiation in the pathology accompanying metabolic stress , and that immune challenges occurring naturally in individuals before the establishment of overt obesity could drive cardiovascular disease later in life .
finally , our observation that selective pi3k p110 and fao inhibition can correct cd4 biased differentiation might provide targets for the therapeutic control of inflammation during metabolic stress .
likewise , our study supports the notion that by modulating metabolism , we can affect immune responses , a relatively novel concept with substantial translational implications . in this regard ,
hints and hopes come from few recent studies , such as the report that cd4 t  cells from systemic lupus erythematosus ( sle ) patients exhibited enhanced glycolysis and mitochondrial metabolism that correlated with their activation status and their excessive ifn production was significantly reduced upon in  vitro treatment with metformin .
the same study also showed that in a number of murine lupus models the combination of metformin and 2-deoxy - glucose led to normalization of cd4 t  cell metabolism and overall reversed disease biomarkers ( mehta and chandel , 2015 , yin et  al . , 2015 ) .
human blood was obtained from healthy donors according to ethics approval obtained from the universit degli studi di milano ( cholesterol and health : education , control and knowledge - studio check [ sefap / pr.0003 ] - reference number fa-04-feb-01 ) .
all in  vivo experiments were conducted under the uk home office regulation ( ppl 70/7443 ) .
the plic study ( a substudy of the check study ) is a large survey of the general population living in the northern area of milan ( n  = 2,606 ; average age 66 years ) ( baragetti et  al .
. , 2007 ) , followed at the center for the study of atherosclerosis , bassini hospital ( cinisello balsamo , milan , italy ) .
for all participants , information on medical histories and therapies was obtained , and bmi ( kg / m ) and waist / hip ratio ( w / h ) were calculated . after an
overnight fast , blood samples were collected from the antecubital vein for determination of lipid profile and glucose levels , as previously described ( ammirati et  al . , 2012 ,
information on total leukocytes and subfraction counts was available for n  = 1,172 subjects , representatives of the entire population in terms of age , gender distribution , and clinical parameters according to the kolmogorov - smirnov test .
this cohort was divided by bmi in accordance to the world health organization definition ( lean for bmi  < 25 kg / m ; overweight for 25 kg / m  bmi  < 30 kg / m ; and obese for bmi  30
polychromatic flow cytometry ( 10-parameter/8-color ) was performed ( as previously described ; ammirati et  al . , 2012 ) to obtain a detailed characterization of a total of n  = 57 subsets of circulating cd4 t  cells from n  = 187 subjects of the plic study , representative of the entire population in terms of age , gender distribution , and clinical parameters according to the kolmogorov - smirnov test .
whole blood from each subject was collected in edta anticoagulated tubes ( bd vacutainer ) .
we previously verified that there was no significant difference in the investigated marker levels in samples stained immediately after collection in comparison to samples stained up to a maximum 24  hr time interval post - sampling and stored at room temperature . in order to reduce cellular loss and analysis sampling bias in the specimen , the no wash
briefly , for each specimen , 50  l of a mixture of eight antibodies ( cd3/cd4/cd45ro / cd45ra / ccr7/ccr5/cxcr3/hla - dr ) was added to 100  l of whole blood , followed by 20  min incubation in the dark at room temperature . in particular , the panel used consisted of the subsequent cellular surface markers : cd3 ( pacific blue - labeled , clone hit3a , bd pharmingen ) , cd4 ( apc - cy7 , sk3 , bd biosciences ) , ccr5/cd195 ( pe , 2d7 , bd pharmingen ) , cxcr3/cd183 ( fitc , 49801 , r&d systems ) , ccr7/cd197 ( pe - cy7 , 3d12 , bd pharmingen ) , hla - dr ( quantum red , hk14 , sigma - aldrich ) , cd45ro ( apc , uchl1 , caltag ) , and cd45ra ( ecd , 2h4 , iotest , beckman coulter ) . after staining , the red blood cells were lysed and fixed with the immune - prep system ( beckman coulter ) .
white cells , diluted in 1  ml total volume , were analyzed on a lsr ii flow cytometer ( bd biosciences ) equipped with four lasers and standard optics .
furthermore , to reduce the non - specific fluorescence background and optimize the fluorescence signal , we used appropriately titrated directly conjugated monoclonal antibodies . to appropriately identify positively stained cells and differentiate them from background autofluorescence for gate inclusion , we used the fluorescence minus one strategy .
fluorescence intensity for each signal measured was standardized using multiple peak rainbow calibration particles ( code rcp-30 - 5a , spherotech ) to allow reproducible and comparable median fluorescence intensity throughout the study period , as previously described ( ammirati et  al . ,
we identified seven principal t  cell subsets and 50 t  cell subpopulations derived by principal subsets by means of the combination of the surface markers .
all data were acquired in fcs format using facsdiva software 5.0 ( bd biosciences ) .
data were processed and analyzed using fcs express v3 research edition ( de novo software ; http://www.denovosoftware.com ) .
cell viability was > 99% , as assessed using the molecular probes patented live / dead viability ( invitrogen ) , according to the manufacturer instructions . in  vitro : murine cd4 t  cells were isolated with commercially available isolation kits ( negative selection ; easysep , invitrogen ) according to manufacturer s instructions from pooled lymph nodes ( inguinal , mesenteric , axillary , brachial , superficial and deep cervical , lumbar , and sacral ) of c57bl/6 mice and activated with plate bound anti - cd3 ( 1  g / ml , ebioscience ) , anti - cd28 ( 5  g / ml , ebioscience ) , and il-2 ( 10  ng / ml , roche ) in the presence or absence of the akt activator sc79 ( 500  nm , tocris ) . in alternative , isolated cd4 t  cells were cultured over night with il-7 ( 1  ng / ml , peprotech ) in the presence or absence of palmitic , linoleic , or stearic acid ( 200  m , sigma ) , followed by activation with plate bound anti - cd3 ( 0.5  g / ml , ebioscience ) and anti - cd28 ( 2.5  g / ml , ebioscience ) . in  vivo : in rag2ko
marilyn c57bl/6 female mice fed cd or hfd ( test diet ; table s2 ) for 8  weeks memory antigen - specific ( hy - specific ) cd4 t  cells were generated by i.p .
immunization with wt c57bl/6 male splenocytes ( 1.5   10 ) for 14  days .
t  cells were then isolated from pooled lymph nodes of these mice ( figures 1a1d , 2a2 g , and s2a  s2f ) and injected i.v . in recipients ( see experimental procedures , in  vivo peritoneal recruitment , below ) or used for chemotaxis or flow cytometry . in p110 or wt c57bl/6 mice fed hfd ( or cd as control ) or ped ( or pcd as control ; test diet ; table s2 ) for 8  weeks , memory cd4 t  cells were generated by intraperitoneal immunization with cba / balbc splenocytes ( 1.5   10 ; allogeneic immunization ) for 7  days .
t  cells were then isolated from mesenteric lymph nodes of these mice and analyzed by facs or western blot ( figures 2h and 2i , 5a , 5e5h , 6a6e , 6g6j , and 7 ) . in this system ,
the  pi3k p110 inhibitor ic87114 ( tocris ) was administered i.p . at a dose of 50 
mg / kg daily during the 7-day immunization ( ying et  al . , 2012 ) .
the akt activator sc79 ( tocris ) was administered i.p . at a dose of 7  mg / kg daily during the 7-day immunization ( moreira et  al . , 2015 ) .
etomoxir ( sigma ) was administered i.p . at a dose of 15  mg / kg daily during the 7-day immunization ( shriver and manchester , 2011 ) . in ot - ii
p110 mice fed cd or hfd for 8  weeks , memory antigen - specific ( ova - specific ) cd4 t  cells were generated by intraperitoneal immunization with ovalbumin peptide ( 10  g , cambridge research biochemicals ) and poly ic ( 1:1 , sigma ) for 7  days .
t  cells were then isolated from mesenteric lymph nodes of these mice ( figure  6f ) and analyzed for chemotaxis . in  vivo activated t  cells from rag2ko marilyn female mice were isolated from pooled lymph nodes of hfd- or cd - fed donors , labeled with cfse ( hfd ; 3.3  m , invitrogen ) or pkh26 ( cd ; 2  m , sigma ) , pooled together , and intravenously injected in hfd or cd recipient male mice ( 5   10 cells / mouse ) that had received an intraperitoneal injection with zymosan ( 1  mg / mouse , sigma ) and ifn ( 600  u / mouse , peprotech ) 48  hr earlier . at 48  hr after injection , blood , peritoneal lavage , peritoneal membrane , mesenteric lymph nodes , and spleen were collected and analyzed by facs for presence of cfse- and pkh26-labeled t  cells .
dead cells were excluded from analysis by staining with fixable aqua dead cell stain ( invitrogen ) .
isolated lymphocytes or dc were stained for surface markers : cd3 , cd4 , cd8 , cd25 , cd44 , ccr7 , cd62l , cxcr3 , lfa1 , v6 , cd40 , cd80 , cd86 , mhcii , cd11c , cd19 , cd14 , and f4/80 with fluorescently conjugated primary antibodies ( 1:200 , ebioscience / biolegend ) at 4c for 30  min ( 37c for ccr7 ) in facs buffer ( pbs  + 2% fcs  + 0.1% sodium azide ) and fixed at 4c for 30  min with 1% pfa in facs buffer . for intracellular staining , isolated lymphocytes were incubated in permeabilization / fixation buffer ( ebioscience ) at 4c for 1  hr .
samples were washed in permeabilization buffer and stained for the cytokines / chemokine ifn , il4 ( 1:200 , ebioscience / biolegend )  and cxcl10 ( 1:200 , stratech ) with fluorescently conjugated primary antibodies at 4c for 30  min in permeabilization buffer .
all samples were then assessed by flow cytometry using a lsrfortessa ( bd biosciences ) and flowjo version 10 software .
dot plots were concatenated to be representative of the independent biological replicates used in each experiment .
chemotaxis assays were performed in 5  m transwell inlays ( corning ) . in  vivo primed lymphocytes
were isolated from mice fed cd or hfd for 8  weeks , suspended in migration medium ( rpmi 2% fcs ) , and seeded in the upper transwell chamber ( 3   10 cells / transwell ) .
chemokines ( peprotech ) were added to the lower chamber , cxcl10 ( 300  ng / ml ) , or ccl19/21 ( 200  ng / ml of each chemokine ) . migrated t  cells were counted with a haemocytometer 3 , 6 , and 24  hr after seeding , then the percent of migrated cells were calculated .
protein lysates were prepared from : ( 1 ) mesenteric lymph nodes isolated from in  vivo primed mice fed cd or hfd for 8  weeks , ( 2 ) cd4 t lymphocytes isolated by magnetic beads ( mylteni ) from lean subjects ( median bmi 22.43 kg / m , minimum 17.67 kg / m , and maximum 24.54 kg / m ) and obese subjects ( median bmi 31.64 kg / m , minimum 30.48 kg / m , and maximum 41.79 kg / m ) , and ( 3 ) cd4 t lymphocytes isolated from pooled lymph nodes and activated as described above for the indicated time points .
samples were lysed in  nonidet p-40 lysis buffer ( 50  mm hepes ph 8.0 , 350  mm nacl , 1% nonidet p-40 , 1  mm edta , 1  mm na3vo4 , 1  mm naf , 20  mm glycerol-2-phosphate , 1  mm pmsf , 1  mm dtt , 10  g / ml aprotinin , 10  g / ml leupeptin , and a protease inhibitor mixture ; roche ) .
equivalent amounts of protein as determined by standard bradford assay ( bio - rad ) were separated by sds / page and transferred to a nylon membrane ( ge healthcare ) .
membranes were blocked for 2  hr at room temperature in 5% milk / tbs - t ( 5% bsa / tbs - t for phosphorylation antibodies ) , incubated overnight at 4c with primary antibodies ( 1:1,000 ) , and subsequently with hrp - conjugated secondary antibody ( amersham bioscience ) ( 1:5,000 ) . antibodies against p - akt ( s473 and t308 ) , akt ,
p - s6 ( s235/236 ) , s6 and -actin were purchased from cell signaling .
density of bands was calculated relative to akt , s6 , or -actin as shown in figures via the use of imagej software .
rna was isolated using commercially available kits ( qiagen ) according to the manufacturer s instructions and assessed for quality and quantity using absorption measurements .
reverse transcription to cdna was performed according to the manufacturer s instruction ( applied biosystems ) .
gene expression analysis was done using sybr green supermix ( bio - rad ) in cfx connect light cycler ( bio - rad ) , according to the manufacturer s instructions .
gene - relative expression was calculated using the ct method and normalized to a reference control ( gapdh ) : f 5-ggctcatgaccacagtcca-3 ; r 5-cacattgggggtaggaacac-3. ccr7 and cd62l primers sequences were the same as published in finlay et  al .
t  cells were isolated from pooled lymph nodes of rag2ko marilyn female mice fed cd or hfd for 8  weeks and were labeled with cfse ( 3.3  m ) in pbs for 10  min at room temperature .
t  cells ( 2   10/well ) were then stimulated with cd11c dc ( 7   10/well ) isolated from the spleen of c57bl/6 male mice fed cd or hfd for 8  weeks .
cd11c dc were treated with mitomycin c to prevent proliferation prior to incubation with t  cells .
t  cells were harvested 3  days later and cfse dilution was assessed by flow cytometry ( figures 4a , 4b , and s5a ) . in  vivo : t  cells were isolated from pooled lymph nodes of rag2ko marilyn female mice , labeled with cfse ( 3.3  m ) in pbs for 10  min at room temperature , and intravenously injected in c57bl/6 recipient female mice ( 5   10 cells / mouse ) .
concurrently , cd11c dc were isolated from the spleen of c57bl/6 male mice fed cd or hfd for 8  weeks and intraperitoneally injected in the c57bl/6 recipient female mice ( 1/4 spleen derived dc / mouse ) .
cells were harvested from the peritoneal lavage , spleen , and draining lymph nodes 5  days later and cfse dilution was assessed by flow cytometry ( figures s5b and s5c ) .
rag2ko and marilyn female mice fed cd or hfd for 8  weeks received a c57bl/6 male - derived skin graft on the upper right back .
some rag2ko mice were i.v . injected with 1   10 cd4 t  cells isolated from marilyn females 9  days post - transplantation .
t cells isolated from pooled lymph nodes of c57bl/6 mice fed cd or hfd for  8  weeks were activated as described above for 4  hr or left inactivated .
cells were stained with ctxb - fitc ( 8  g / ml , sigma ) and cd4-pecy7 ( 1:200 , ebioscience ) at 4c for 30  min in facs buffer ( pbs  + 2%fcs  + 0.1% sodium azide ) and fixed at 4c for 30  min with 1% pfa in facs buffer .
samples were assessed using imagestream flow cytometer ( amnis ) and ideas software . for quantitative image analysis of lipid raft aggregation , bright
the bright detail intensity feature calculates the sum of intensity values from the brightest areas within a cell that are morphologically defined as the peak fluorescence distributions of 3 pixel radius or less . there were 5,000 cd4 t  cells that were analyzed per condition .
as described before , isolated naive cd4 t  cells were cultured over night with il-7 in the presence or absence of palmitic or stearic acid .
2   10 cd4 t  cells were then seeded in a 96-well microplate in xf assay modified dmem and incubated in a non - co2 incubator for 1  hr prior to the assay .
statistical analysis of human data was performed using the spss v.21.0 for windows ( ibm corporation ) program .
a kolmogorov - smirnov test was performed to test whether the studied cohort was representative of the entire plic population in terms of age , gender distribution , and clinical parameters .
a shapiro - wilk test was performed to verify the normal distribution of linear variables ; median and inter - quartile range ( iqr ) was reported and mann - whitney u non - parametric test was performed .
outliers detection ( above and below 1.5   iqr ) was performed by use of grubb s test .
anova test was performed to compare clinical parameters and leukocyte subfraction among subjects divided by bmi and then adjusted for age , gender , and therapies by using analysis of covariances ( ancova ) . for all analysis , statistically relevant differences were considered for p values  < 0.05 . for murine studies , data are expressed as mean   sem .
two - tailed student s t test was used to compare two groups with parametric data distribution . for multiple comparison analysis , 1- , 2- , or 3-way anova was used , unless otherwise specified ( figure  1e ) . in all cases ,
g.d.n . , and f.m.m .- b . performed the experiments : c.m . , j.s . , d. cucchi , d. coe , f.b . ,
 summarylow - grade systemic inflammation associated to obesity leads to cardiovascular complications , caused partly by infiltration of adipose and vascular tissue by effector t  cells . 
 the signals leading to t  cell differentiation and tissue infiltration during obesity are poorly understood . 
 we tested whether saturated fatty acid - induced metabolic stress affects differentiation and trafficking patterns of cd4 + t  cells . 
 memory cd4 + t  cells primed in high - fat diet - fed donors preferentially migrated to non - lymphoid , inflammatory sites , independent of the metabolic status of the hosts . 
 this was due to biased cd4 + t  cell differentiation into cd44hi - ccr7lo - cd62llo - cxcr3 + -lfa1 + effector memory - like t  cells upon priming in high - fat  diet - fed animals . 
 similar phenotype was observed in obese subjects in a cohort of free - living people . 
 this developmental bias was independent of any crosstalk  between cd4 + t  cells and dendritic cells and was mediated via direct exposure of cd4 + t  cells to palmitate , leading to increased activation of a pi3k p110-akt - dependent pathway upon priming . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions