Toll-like receptor agonist therapy can profoundly augment the antitumor activity of adoptively transferred CD8 + T cells without host preconditioning

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1 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 DOI /s RESEARCH ARTICLE Open Access Toll-like receptor gonist therpy cn profoundly ugment the ntitumor ctivity of doptively trnsferred CD8 + T cells without host preconditioning Michelle H. Nelson 1*, Jco S. Bowers 1, Stefnie R. Biley 1, Mrshll A. Diven 1, Croline W. Fugle 1, Andrew D. M. Kiser 2, Cludi Wrzesinski 2, Bei Liu 1, Nichols P. Restifo 2 nd Chrystl M. Pulos 1* Astrct Bckground: Lymphodepletion enhnces doptive T cell trnsfer (ACT) therpy y ctivting the innte immune system vi microes relesed from the rdition-injured gut. Microil components, such s LPS, re key meditors of totl ody irrdition (TBI) enhncement, ut our ility to strtegiclly use these toll-like receptor (TLR) gonists to olster the potency of T cell-sed therpies for cncer remins elusive. Herein, we used TLR4 gonist LPS s tool to ddress how nd when to use TLR gonists to effectively improve cncer immunotherpy. Methods: To determine the mechnisms of how innte immune ctivtion vi lymphodepletion potentited ntitumor T cell immunity, we utilized the pmel-1 melnom mouse model. B16F10-ering mice were preconditioned with 5Gy TBI nd given triprtite ACT therpy (consisting of trnsferred pmel-1 CD8 + T cells, vccintion with fowlpox encoding gp100, nd IL-2) long with TLR4 gonist LPS. The timing of LPS dministrtion nd the requirement of individul components of the triprtite therpy were evluted sed on tumor growth nd the phenotype of recovered splenocytes y flow cytometry. We lso evluted the role of non-toxic nd cliniclly used TLR4 nd TLR9 gonists monophosphoryl lipid A (MPL) nd CpG Oligodeoxynucleotide (CpG ODN), respectively for ACT therpy. Results: Here we report tht while exogenous dministrtion of LPS ws le to enhnce doptively trnsferred CD8 + T cells tumor destruction, LPS tretment lone did not replce individul components of the triprtite ACT regimen, or ovite TBI. Moreover, we found tht sequentilly dministering LPS during or one dy prior to ACT therpy compromised tumor regression. In contrst, dministering LPS fter ACT potentited the ntitumor effectiveness of the regimen, therey supporting the expnsion of trnsferred tumor-specific CD8 + T cells over host CD4 + Tcells.Welso found tht non-toxic TLR gonists MPL nd CpG potentited the ntitumor ctivity of infused CD8 + T cells. Finlly, TBI ws no longer needed to regress tumors in mice who were depleted of host CD4 + T cells, given triprtite ACT regimen nd then treted with low dose LPS. Conclusions: Collectively, our results identify how nd when to dminister TLR gonists to ugment T cell-sed immunotherpy in the sence or presence of host preconditioning for tretment of dvnced mlignncies. Our findings hve clinicl implictions for the design of next genertion immune-sed therpies for ptients with cncer. Keywords: Totl ody Irrdition, Adoptive immunotherpy, CD8 + T lymphocytes, Innte immunity * Correspondence: nelsonmh@musc.edu; pulos@musc.edu 1 Microiology nd Immunology, Hollings Cncer Center, Medicl University of South Crolin, Chrleston, SC 29425, USA Full list of uthor informtion is ville t the end of the rticle 2016 Nelson et l. Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 2 of 14 Bckground The humn digestive trct contins 100 times more single-celled orgnisms thn the sum totl of ll cells within the ody [1]. The microiome hs evolved in symiotic wys to ply key role in sustining humn helth, including the sorption of nutrients, mintennce of mucosl integrity nd the regultion of intestinl homeostsis [2 5]. Homeosttic disruption cn induce microil trnsloction, triggering switch in the commensl host-microe reltionship from mutulistic to pthogenic [6, 7]. This phenomenon excertes grftversus-host disese, inflmmtory owel disese nd HIV/AIDS infection [8 10]. Surprisingly, the deleterious effect of this phenomenon vi chemo- nd/or rdiotherpy is eneficil for T cell-sed tretments for cncer; including doptive T cell trnsfer (ACT) therpy [11 14]. Lymphodepletion with chemotherpy nd/or rdition preprtive regimens dministered prior to doptive immunotherpy medites ojective clinicl responses in ~50 % of ptients with metsttic melnom nd hs recently shown promise in metsttic cervicl cncer [15 17]. Likewise, ACT tretment with nti-cd19 chimeric ntigen receptor (CAR)-engineered T cells medites roust tumor immunity in preconditioned ptients with hemtologicl mlignncies [18 22]. Additionlly, tumor muttion-specific T cells re now eing exploited for ACT therpy [23]. How lymphodepletion ugments ACT in these vrious clinicl trils hs een elucidted in cliniclly relevnt mouse models of melnom. A non-myeloltive lymphodepleting preprtive regimen with 5Gy TBI prior to n ACT regimen cn induce destruction of B16F10 melnom in mice y removing cytokine sinks, depleting suppressive T reg cells, trnsiently lting myeloid derived suppressor cells (MDSCs) nd ctivting the innte immune system [11]. Interestingly, innte immune ctivtion vi microil LPS nd other microil gonists, lierted from the rdition-injured gut, ws responsile for triggering tolllike receptor 4 (TLR4) signling nd is mechnism underlying the enhnced ACT effectiveness in mice [11, 24 27]. Removl of signling components, through the use of mice deficient in TLR4, reduced the eneficil effect of TBI [11]. Likewise, cncer ptients who crry TLR4 loss-of-function llele relpsed more quickly fter chemotherpy thn those crrying the norml TLR4 llele [28]. Additionl investigtion reveled tht incresing the intensity of irrdition from 5 to 9Gy TBI, which requires hemtopoietic stem cell (HSC) support, further enhnced tretment outcome [29]. Collectively, these findings illuminte n importnt role for innte immunity in ugmenting T cell-sed tumor immunity. Microes relesed from the rdition-injured gut contin plethor of TLR lignds esides LPS. Any of these lignds my contriute to ctivting the innte immune system. However, herein, we used TLR4 gonist LPS s tool to ddress how nd when to use djuvnts to improve cncer immunotherpy. We sked if TLR4 gonist LPS could replce nd/or enhnce vccines, trnsferred CD8 + T cells nd/or host lymphodepletion in n ggressive model of melnom. We found tht ctivting the innte immune system in non-irrdited mice with exogenously delivered LPS could not replicte the effectiveness of TBI when used in conjunction with ACT therpy. Yet, exogenous LPS could ugment the ntitumor ctivity of infused CD8 + T cells in mice receiving non-myeloltive, low level of irrdition (5Gy TBI). We lso found tht dministering LPS fter, ut not efore or simultneous with, ACT tretment ws optiml for potentiting the ntitumor ctivity of infused CD8 + T cells. Additionl investigtion reveled tht codministrtion of LPS plus ntiody depletion of host CD4 + T cells triggered roust tumor erdiction. Surprisingly, this comintion eliminted the previous requirement for TBI. LPS is not the only TLR gonist cple of enhncing ACT. We found tht the use of MPL or CpG in similr conditions lso ugmented ACT. Collectively, these findings provide insight into how to use other TLR gonists with ACT regimens nd suggests lterntive regents to lymphodepletion tht might sfely tret ptients sensitive to chemotherpy/ rdiotherpy. Results Lymphodepletion ugments trnsferred ntitumor CD8 + T cells y ctivting DCs nd lting host lymphocytes Lymphodepletion with 5Gy TBI enhnces triprtite ACT tretment consisting of PFI (P = infusion of 1e 6 pmel-1 CD8 + T cells, F = vccintion with fowlpox encoding hgp100 nd I = high dose IL-2) in mice with B16F10 melnom to greter extent thn in lymphoreplete mice (Fig. 1). We evluted how lymphodepletion impcts the ctivtion of the innte immune system nd the degree of pmel-1 CD8 + T cell engrftment vs. host cell depletion. The improved outcomes in 5Gy TBI + PFI treted mice compred to non-irrdited + PFI treted mice correlted with higher reltive frequency of donor pmel-1 CD8 + T cells in the tumor (Fig. 1). Moreover, we found tht irrdited mice hd n incresed numer of ctivted DCs expressing the co-stimultory molecules CD86, OX40L, ICOSL nd 41BBL; likely promoting the prolifertion of infused CD8 + lymphocytes (Fig. 1c). Given tht 5Gy TBI decreses the cellulr density in mice over time, we evluted the solute numer of immune cells following TBI. We found tht incresing the intensity of lymphodepletion from 0 to 5Gy TBI ws ssocited with greter reduction in the solute numer of splenic host CD4 + nd CD8 + T cells

3 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 3 of 14 c d Fig. 1 Lymphodepletion ugments the ntitumor ctivity of trnsferred CD8 + T cells vi depleting host suppressor T cells nd ctivting APCs. TBI ugmented ntitumor responses in mice. C57BL6 mice ering sucutneous B16F10 tumors estlished for 8 dys received non-myeloltive 5Gy TBI or were not irrdited. One dy lter, mice received n ACT tretment regimen consisting of the doptive trnsfer of 1e 6 cultured tumorrective pmel-1 CD8 + T cells, fowlpox hgp100 vccintion nd hil-2 or were left untreted (NT). Dt (men +/- SEM, n = 5 mice per group) re representtive of 5 independent experiments. PFI vs. 5Gy PFI, **P <.01, ANOVA. TBI incresed the engrftment of infused pmel-1 CD8 + T cells over host CD8 + or host CD4 + T cells reltive to non-irrdited mice. Rtios of trnsferred pmel-1 CD8 + T cells (Vβ13 + Ly5.1 + ) reltive to returning host CD8 + nd CD4 + T cells re shifted towrd pmel-1 CD8 + T cells in irrdited mice. B6 mice were given 5Gy TBI or not followed y the trnsfer of 1e 6 pmel-1 CD8 + T cells (Ly5.1 + ), fowlpox hgp100 vccintion nd hil-2. Splenocytes otined 1 week fter trnsfer were simultneously nlyzed for infused pmel-1 CD8 + nd reconstituting host cells. c TBI up-regultes costimultory molecules on host CD11 + CD11c hi dendritic cells. Splenic DCs were isolted t 24 h from mice treted with 5Gy TBI or without. The expression of costimultory molecules (CD86, ICOSL, OX40L nd 41BBL) on CD11c hi -gted DCs were nlyzed y flow cytometry nd displyed in histogrm form. d TBI depletes endogenous CD4 + nd CD8 + T cells nd trnsiently promotes ctivtion of CD11c hi dendritic cells. Splenocytes were isolted from 0 nd 5Gy irrdited mice 2 dys fter TBI. Asolute numers of CD4 +,CD8 + nd ctivted CD11 + CD11c hi CD86 hi DCs in the spleens of TBI nd non-irrdited C57BL6 mice were enumerted. Dt shown re representtive of 2 independent experiments. **P <.01, unpired t-test (Fig. 1d; Dy 3 CD4 + nd CD8 + cells: 0Gy TBI-10.6 nd 6.45e 6 ; 5Gy TBI-0.35 nd 0.56e 6, respectively). Consistent with previous work [11, 30], the solute numer of ctivted dendritic cells in the spleen trnsiently incresed s the intensity of irrdition ws incresed from 0 to 5Gy TBI (Fig. 1d; Dy 1 CD11 + CD11c hi CD86 hi cells: 0Gy TBI-0.5e 4 ; 5Gy TBI-13.5e 4 ). Collectively, these dt reveled tht the ddition of TBI correlted with greter depletion of endogenous lymphocytes nd ctivtion of the innte immune system. The ntitumor ctivity of trnsferred CD8 + T cells is compromised in MyD88 deficient mice post-irrdition We reported tht TLR4 signling ws criticl for roust ntitumor response, s TLR4 knockout mice hd reduced ntitumor enefits following TBI [11]. As other

4 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 4 of 14 innte microes nd chnges in gut microes in generl, likely triggered y TBI, were responsile for ctivting the innte immune system, we sought to elucidte if glol ltion of MyD88 ( universl dpter protein used y lmost ll TLRs (except TLR3) to ctivte trnscription fctor NF-κB [31]) signling would lso impir tretment outcome y ACT therpy in lymphodepleted nimls. To ddress this question, we exmined the consequences of triprtite ACT therpy (PFI) in WT versus MyD88 deficient nimls with or without 5Gy TBI. Tumor destruction ws significntly impired in irrdited MyD88 -/- mice (P < 0.02; lck dimond vs. lck circle) compred to irrdited WT mice (Fig. 2). Conversely, we oserved no difference etween the effectiveness of ACT tumor tretment in non-irrdited WT or MyD88 -/- mice (Fig. 2). Additionlly, we discovered tht TBI dmges the integrity of the gut y pthologicl score (Fig. 2) nd permitted microil trnsltionl in irrdited mice, s detected y LPS in the serum of irrdited mice 6 dys fter TBI (Fig. 2c). Our preliminry work shows tht LPS cn e detected confidently t dys 5 to 7, ut not efore this time point (not shown). Thus, our dt identified n essentil role for MyD88 in the induction nd stility of ntitumor CD8 + T cells in irrdited mice. We surmised tht microil TLR gonist (such s LPS nd eyond) enhnced ACT tumor tretment vi MyD88-dependent signling in irrdited nimls only. c Administrtion of LPS fter ACT enhnces ntitumor immunity only in irrdited nimls Becuse microil LPS ws detected in the ser of irrdited nimls (Fig. 2c), we used TLR4 gonist LPS s tool to ddress how nd when to use TLR gonists to potentilly improve doptive T cell trnsfer cncer immunotherpy. We posited tht dministering ultrpure LPS to non-irrdited mice would ypss the previous need for TBI to ugment ACT therpy. We first determined the highest dose of LPS tht could e tolerted in non-irrdited mice given ACT therpy. To this end, incresing doses of ultrpure LPS, rnging from 0.1 to 10 μg, were dministered to non-irrdited nimls one dy fter n ACT nd PFI therpy nd their tolernce ws monitored y their overll ppernce nd survivl. In contrst to our hypothesis, we found tht even the highest tolerle dose of LPS (5 μg) dministered to non-irrdited mice could not enhnce the tretment or their survivl (Fig. 3). Treting mice with 10 μg of LPS ws toxic to the mice with limited survivl 2 dys fter its dministrtion (not shown). Collectively, these dt showed tht merely incresing the dose of LPS to the highest tolerle dose ws not sufficient to improve ACT in non-irrdited nimls compred with ACT in irrdited mice. Fig. 2 TLR-MyD88 signling triggered y TBI is criticl for ugmenting the ntitumor ctivity of trnsferred CD8 + T cells. Tumor ering mice were given triprtite ACT therpy consisting of n infusion of 1e 6 trnsgenic pmel-1 CD8 + T cells with TCR tht recognizes the gp100 peptide on B16 tumors, virl vccintion encoding gp100 peptide nd IL-2 cytokine support in WT versus MyD88 deficient nimls with or without 5Gy TBI. The effectiveness of tretment ws decresed in irrdited mice geneticlly deficient in MyD88. WT nd MyD88 / tumor-ering mice were irrdited nd then received ACT tretment or were left untreted. Dt (men ± SEM; 5 mice per group) re representtive of 2 independent experiments. 5Gy PFI > WT vs. 5Gy PFI > MyD88 -/-,**P <.01,ANOVA. TBI compromises the colon. Colon of mice were nlyzed t 3 dys post-tbi nd scored y pthologist unwre of the tretment groups. Dt shown (3 mice per group) re representtive of 5 independent experiments. *P <.05, unpired ttest.c TBI promotes trnsloction of gut-derived LPS. Serum from non-irrdited nd 5Gy irrdited mice were collected nd nlyzed for the presence of LPS using LAL ssy 6 dys fter TBI. Dt shown (3 mice per group) re representtive of 5 independent experiments. *P <.05, unpired t-test We next sought to determine wht dose of LPS might sfely nd most effectively enhnce ACT tretment in nimls given 5Gy TBI. Thus, LPS doses rnging from 0.1 to 25 μg were dministered to irrdited nimls one dy fter tretment. In contrst to our findings in nonirrdited nimls, 1 μg of LPS could significntly

5 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 5 of 14 potentite CD8 + T cell-medited tumor erdiction in irrdited nimls. Likewise, doses of LPS exceeding 1 μg of LPS improved ACT tretment (Fig. 3) nd doses lower thn 1 μg of LPS did not ugment ACT tretment. Interestingly, LPS doses tht were toxic in nonirrdited mice were well tolerted in irrdited hosts given ACT tretment. Thus, 25 μg of LPS, dose highly toxic to ll non-irrdited mice, ws well tolerted in irrdited nimls (not shown). The reduced toxicity oserved in irrdited, ut not in non-irrdited, mice given LPS might e due to the fct tht TBI ultimtely reduces (trnsiently) the solute numer of innte immune cells triggered y LPS [32]. Collectively, our dt reveled tht LPS gretly improves tretment outcome, ut only in conjunction with TBI. Fig. 3 Administrtion of LPS enhnces ntitumor immunity in irrdited ut not lymphoreplete mice. LPS does not ugment ntitumor responses in non-irrdited mice. Mice ering sucutneous B16F10 tumors were estlished for 8 dys. Mice received n ACT tretment comprised of the doptive trnsfer of 5e 5 cultured pmel-1 T cells, fowlpox hgp100 vccintion nd hil-2 or were left untreted. The next dy, mice received ultr-pure LPS rnging from 0.5 to 5 μg or left untreted. Dt shown (men ± SEM, 10 mice per group) re representtive of 2 independent experiments. PFI vs. PFI + 0.5, 1 or 5 LPS, NS, ANOVA. LPS ugments the ntitumor ctivity of pmel-1 CD8 + T cells in irrdited mice. Mice ering sucutneous B16F10 tumors estlished for 8 dys received 5Gy TBI. One dy fter TBI, mice received n ACT tretment comprised of the doptive trnsfer of 5e 5 cultured pmel-1 T cells, fowlpox hgp100 vccintion nd hil-2 or were left untreted. The next dy, mice received LPS rnging from 0.5 to 5 μg or left untreted. Dt shown (men ± SEM, 5 10 mice per group) re representtive of 2 independent experiments. 5 Gy PFI (white circle) vs. 5Gy PFI LPS (white squre), NS. ANOVA. 5Gy PFI LPS (white squre) vs. 5 Gy PFI + 1 or 5 LPS (grey or lck squre), ***P <.001, ANOVA LPS induces CD25 on ntitumor CD8 + T cells nd supports their persistence How exogenous LPS impcts the phenotypic signture nd prolifertive cpcity of infused pmel-1 CD8 + T cells in mice given TBI remins incompletely elucidted. Thus, we investigted how LPS influenced the expression of CD62L, CD44 nd CD25 on trnsferred T cells in non-irrdited nd irrdited mice 5 dys postinfusion. Interestingly, LPS doules the expression of CD25, receptor for IL-2, on ll trnsferred cells, regrdless of therpy, ut irrdition clerly enhnces CD25 expression (86 %) on infused donor pmel-1 T cells (non-irrdited, 31 %; Fig. 4). These dt imply tht infused tumor-specific CD8 + T cells from irrdited mice given LPS might hve n dvntge in consuming the homeosttic cytokine IL-2 in vivo. In contrst, there were no differences in the expression of CD62L (Fig. 4) nd CD44 (not shown) on the trnsferred T cells in irrdited or non-irrdited mice given LPS. To investigte how LPS impcts the in vivo prolifertive cpcity of the infused pmel-1 CD8 + T cells, we treted the mice with BrdU nd determined its percent incorportion into infused pmel-1 cells 3 dys post-trnsfer. We found tht the trnsferred cells from irrdited mice given LPS incorported significntly more BrdU thn in mice receiving TBI lone (Fig. 4). Moreover, LPS ws less effectivetdrivingtheprolifertionofinfusedcd8 + T cells in non-irrdited nimls. These dt might imply tht lting suppressive lymphocytes with TBI while concomitntly intensifying innte ctivtion through dministrtion of higher concentrtion of LPS to the host unmsked the prolifertive cpcity of the trnsferred pmel-1 CD8 + T cells. However, dditionl experiments will e needed to confirm this postultion. Also, it will e insightful to know whether the expression of receptors for IL-7, IL-15 nd IL-21 on donor T cells were up-regulted following TBI. The solute numer of pmel-1 CD8 + T cells ws lso considerly elevted in the spleen nd lood of irrdited mice receiving LPS (compred with irrdited mice not receiving LPS) 35 dys fter tretment (Fig. 4c). Collectively, our dt indicte tht dministering LPS to irrdited nimls

6 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 6 of 14 c Fig. 4 LPS enhnces CD25 expression on trnsferred CD8 + T cells nd improves their long-term persistence in irrdited nimls. LPS enhnces the expression of CD25, ut not CD62L, on doptively trnsferred pmel-1 CD8 + T cells (Ly5.1 + ) when nlyzed on dy 7 from irrdited B6 mice given fowlpox hgp100 nd hil-2 s demonstrted y flow cytometry. LPS enhnces the initil prolifertion of doptively trnsferred cells s indicted vi BrdU incorportion. On dy 3 post-act, mice were given 1 mg of BrdU nd scrificed 2 h lter. Trnsferred cells were nlyzed for BrdU uptke(5 mice per group).c LPS incresed the solute numer of trnsferred pmel-1 T cells in the spleen nd lood of irrdited hosts. Asolute numers of trnsferred pmel-1 cells (CD8 + Ly5.1 + ) in the spleens nd lood were enumerted from Thy1.1 mice. Dt shown (men ± SEM, 5 mice per group) re representtive of 2 independent experiments. *P <.05, **P <.01, unpired t-test drives the prolifertive cpcity nd increses the persistence of trnsferred cells. Infused CD8 + T cells, vccine nd IL-2 re required to erdicte melnom in irrdited mice treted with LPS Becuse LPS olstered the persistence of infused CD8 + T cells nd medited curtive responses in irrdited mice, we hypothesized tht LPS dministrtion might drive tumor regression without the need for vccintion or IL-2. To ddress this ide, we replced infused tumor-specific CD8 + T cells, vccintion vi fowlpox expressing hpg100 or high dose IL-2 with LPS in irrdited mice. Although dministrtion of LPS reproducily medited tumor regression in irrdited mice receiving the triprtite ACT tretment, LPS did not replce individul components of the regimen (Fig. 5). Indeed, replcing infused cells or vccintion with LPS in the triprtite regimen impired the tretment chieved in irrdited mice given the triprtite regimen (Fig. 5 nd c, respectively). In contrst, replcing IL-2 with LPS ws comprle to tretment seen in irrdited mice only given the triprtite regimen (in other words: 5Gy PFI (Fig. 5; white circle) = 5Gy PF + LPS (Fig. 5d; grey dimond)), implying tht LPS might improve the cpcity of trnsferred cells to cquire endogenous IL-2. This ide is plusile given our finding tht LPS incresed CD25 expression on infused CD8 + T cells (Fig. 4). Nonetheless, replcing IL-2 with LPS ws less effective thn tretment driven y LPS in irrdited mice receiving the entire triprtite regimen (5Gy, PFI + LPS; lck circle)-which ws the most efficcious tretment. Collectively, we found tht LPS dministrtion enhnced ACT therpy in irrdited mice ut did not replce individul components of the regimen. LPS dministrtion, reltive to ACT therpy, impcts tretment outcome TLR gonists re used cliniclly lone or in comintion with tumor vccines [33 37]. Although TLR gonists plus vccine comintions hve shown success in triggering T cell immune responses in ptients, they hve not consistently medited tumor regression [38]. Interestingly, when testing TLR gonist comintions with different therpeutic strtegies, investigtors hve not determined the optiml timing for dministering TLR gonists reltive to vccines, checkpoint modultors nd/or T cell-sed therpies [39]. Although we found tht dministering LPS fter ACT therpy potentites the effectiveness of the triprtite therpy in nimls (for exmple Fig. 3), it remined

7 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 7 of 14 c d Fig. 5 Vccintion, olus IL-2 nd infusion of tumor-rective lymphocytes re required to potentte ACT tumor tretment in irrdited mice given LPS. LPS ugmented the ntitumor ctivity of CD8 + T cells in irrdited mice given the triprtite tretment. Removl of pmel-1 T cells; c fowlpox hgp100 vccine or d olus IL-2 impired the enhnced ntitumor response medited in irrdited mice given triprtite therpy nd LPS. One dy fter TBI, mice received n ACT tretment comprised of the doptive trnsfer of 5e 5 cultured pmel-1 T cells, fowlpox hgp100 vccintion nd hil-2 or were left untreted. The next dy, mice received 2 μg of LPS or left untreted. Dt shown (men ± SEM, 5 10 mice per group) re representtive of 2 independent experiments. 5Gy (white tringle) vs. 5Gy PFI (white circle, *P <.05), 5Gy FI+ LPS (grey tringle, NS), 5Gy PI + LPS (grey squre, *P <.05)or 5Gy PF + LPS (grey dimond, *P <.05), ANOVA. 5Gy PFI + LPS (lck circle) vs. 5Gy PFI (white circle, *P <.05), 5Gy FI + LPS (grey tringle, ***P <.001), 5Gy PI + LPS (grey squre, **P <.01) or 5Gy PF + LPS (grey dimond, *P <.05), ANOVA unknown whether this time-point ws idel to deliver TLR gonists reltive to T cell infusion, vccintion nd IL-2. To ddress this question, s depicted in Fig. 6, we dministered LPS either one dy prior, during or one dy fter the triprtite ACT regimen nd monitored tumor growth in irrdited nimls. Consistent with previous experiments, dministering LPS fter ACT potentited CD8 + T cell-medited tumor regression (Fig. 6). In strk contrst, treting mice with LPS one dy efore ACT tretment sttisticlly impired tumor growth nd mice rpidly died (Fig. 6c). Administering LPS t the sme time s ACT did not impct tretment outcome compred to ACT tretment lone (Fig. 6d). We next sought to explore how the timing of LPS dministrtion reltive to the ACT regimen impcts the engrftment of infused pmel-1 CD8 + T cells versus host CD4 + T cells in vivo. We found tht delivering LPS fter the ACT regimen incresed the solute numer of donor CD8 + T cells (Fig. 6e) to host CD4 + T cells (Fig. 6f) in the spleen of irrdited mice 7 dys fter the tretment regimen, which likely explins why this pproch effectively regresses B16F10 tumor. Conversely, giving LPS efore the regimen supported host CD4 + T cells over infused pmel-1 CD8 + T cells (Fig. 6e nd f), which likely impired the ntitumor ctivity of CD8 + T cells. If LPS ws given to mice fter ACT, it incresed the rtio of infused CD8 + T cells to host CD4 + T cells in the tumor of mice (Fig. 6g). In contrst, high rtio of host CD4 + T cells to infused CD8 + T cells ws oserved in the tumor of mice given LPS prior to the ACT

8 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 8 of 14 c d e f g h i Fig. 6 The timing y which LPS is dministered reltive to ACT therpy differentilly impcts tretment outcome nd regultes the innte nd dptive immune system. Schemtic showing the time t which LPS is dministered to tumor ering mice reltive to the triprtite ACT tretment regimen. One dy fter TBI, mice received n ACT tretment comprised of the doptive trnsfer of 5e 5 cultured pmel-1 T cells, fowlpox hgp100 vccintion nd hil-2 or were left untreted. Either on dy prior (), during (c) or one dy fter (d) ACT, mice received 2 μg of LPS or were left untreted. Dt shown (men ± SEM, 5 mice/group) re representtive of 4 independent experiments. 5Gy (lck dimond) vs. 5Gy PFI (white dimond, **P <.01), 5Gy PFI pre-lps (red circle, *P <.05), 5Gy LPS during PFI (lue circle, **P <.01) or 5Gy PFI post-lps (green circle, **P <.01),ANOVA. 5Gy PFI (white dimond) vs. 5Gy PFI pre-lps (red circle, *P <.05), 5Gy LPS during PFI (lue circle, NS) or 5Gy PFI post-lps (green circle, **P <.01), ANOVA. (e g) Splenocytes were hrvested from irrdited mice on dy 7 post-t cell infusion nd solute cell counts were determined y flow cytometry. *P <.05, **P <.01, unpired t-test. (h &i) CD11c hi dendritic cells were sorted y flow cytometry from single-cell B16F10 tumor suspensions prepred 6 dys fter ACT tretment from mice given LPS t different time points (s shown in scheme A). Tumor-infiltrting DCs were co-cultured for 4 dys with CFSE-leled, negtively-isolted pmel CD8 + T cells t 10:1 T cells: APC rtio. DCs were exposed to ntigen during in vivo fowlpox vccintion. Tumor ntigen ws not dded to the co-culture. Representtive flow cytometry plots (h) nddotplots(i) of CFSE dilution of pmel-1 cells. ****P <.0001, **P <.01, ANOVA

9 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 9 of 14 triprtite regimen. Note, our preliminry dt show tht there re more host CD25 high FOXP3+ CD4 + T cells nd less donor pmel-1 CD8 + T cells in irrdited mice given LPS efore PFI versus in mice given LPS fter PFI (Additionl file 1 A nd B). These dt indicte tht the timing in which TLR gonists re used reltive to ACT cn drmticlly impct tretment outcome, likely y ltering the rtio of tumor-specific CD8 + T cells to host immune cells. Dendritic cells residing within tumors of mice treted with LPS fter triprtite ACT regimen stimulte pmel-1 CD8 + T cell prolifertion Becuse LPS impired the engrftment nd ntitumor ctivity of infused pmel-1 CD8 + T cells, we ssumed tht LPS premturely ctivted host DCs nd thus compromised their ility to tke up, process nd present ntigen delivered vi vccintion. To test this ide, we sought to oserve the ility of tumor-residing DCs (from tretment groups in Fig. 6, c, d) to stimulte the division of pmel-1 CD8 + T cells. Single cell suspensions of estlished melnom from mice treted with LPS either efore, during or fter ACT therpy ws sorted y flow cytometry for dendritic cells. Sorted DCs were then co-cultured with CFSE-leled, pmel-1 TCR trnsgenic splenocytes t 10:1 rtio of T cells: DCs. DCs isolted from tumors of mice treted with LPS prior to or during ACT regimen filed to roustly induce pme l-1 CD8 + T cell division (Fig. 6h). In contrst, DCs isolted from B16 tumors of mice treted with LPS fter the triprtite ACT regimen stimulted strong in vitro CD8 + Tcell prolifertion within 4 dys of co-culture (Fig. 6h). These findings show tht DCs from mice given LPS fter ACT promoted ex vivo prolifertion of pmel-1 CD8 + T cells were significnt nd reproducile (Fig. 6i). Collectively, our dt suggest tht LPS potentites the ility of DCs to drive pmel-1 CD8 + T cell responses to tumors in vivo when dministered one dy fter the triprtite regimen. Next, we sought to test our hypothesis tht LPS eneficilly increses co-stimultory molecules only if given fter PFI. We found tht giving LPS to mice fter ACT only slightly incresed the expression of co-stimultory molecules CD80 nd CD86 on conventionl DCs s well s on monocytes from the spleens of mice (3 dys post ACT). Moreover, minor increse in these molecules ws induced on APCs if LPS ws given efore ACT (Additionl file 1 C nd D). We did not see n increse in co-stimultory molecules 41BBL, OX40L or ICOSL on conventionl DCs or monocytes y dministering LPS to irrdited mice (either efore or fter PFI). Perhps we did not see n increse in these prticulr molecules ecuse TBI itself induces them. As shown in Fig. 1c, TBI induces these molecules, ut they re lower on the APCs from non-irrdited cohorts. Collectively, our dt imply tht LPS slightly enhnces DC ctivtion, which might contriute to improving ACT therpy. Administrtion of MPL or CpG enhnces ntitumor immunity in irrdited mice Owing to its inherent toxicity, it is importnt to find n lternte gonist to LPS for tumor immunotherpy in the clinic. Moreover, some ptients hve TLR4 polymorphisms, rendering their innte immune system resistnt to microil LPS y chemotherpy or TBI [28]. Thus, we sought to determine whether TLR2/TLR4 monophospholipid A (MPL- detoxified version of LPS) could lso ugment ACT tretment in irrdited hosts. Similr to ultrpure LPS, we found tht MPL ws effective in mediting tumor regression y the trnsferred cells (Fig. 7). Importntly, we lso found tht nother cteril-derived gonist CpG-DNA (TLR9 gonist; Fig. 7) ugmented PFI tretment in irrdited mice. These dt re importnt, s these gonists hve een sfely used in the clinic. Tumor erdiction vi ACT therpy cn e chieved without host preconditioning We next posited tht lymphodepletion with 5Gy TBI or chemotherpeutics, which cn hve toxic side effects in ptients, could e ypssed in nimls with estlished melnom if they were trnsiently depleted of host CD4 + T cells, given triprtite ACT therpy nd then treted with LPS. As shown in Fig. 8, WT mice were first ntiody depleted of CD4 + T cells prior to nd during n ACT regimen nd then treted with LPS one dy fter the cells were infused. Indeed, we found tht the dministrtion of LPS enhnced tumor destruction only in non-irrdited mice tht were depleted of host CD4 + T cells (PFI + nti-cd4 + LPS > PFI + LPS or PFI + nti- CD4; Fig. 8). Long-term cures (>70 dys) were oserved in these mice. Conversely, the depletion of CD4 + T cells lone or the ctivtion of the innte immune system with LPS lone induces wek ntitumor CD8 + T cell immune responses in non-irrdited nimls with melnom (Fig. 8). We were curious if depleting CD4 + T cells would impct the expnsion of trnsferred CD8 + T cells. Although, the rtio of donor pmel-1 CD8 + T cells to host CD4 + T cells significntly increses when nti-cd4 ntiody is given (Additionl file 2), the numer of CD8 + T cells did not chnge. It is possile tht trnsiently removing host CD4 + T cells permits infused CD8 + T cells to etter interfce nd e ctivted y APCs when mice re given LPS. Collectively, we speculte tht depletion of host CD4 + T cells (which cn function nd cytokine sink or suppressors) plus LPS tretment olster tht ntitumor properties (function nd persistence) of infused pmel-1 CD8 + T cells in non-irrdited mice. Given the

10 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 10 of 14 Fig. 7 Administrtion of MPL or CpG enhnces ntitumor immunity in irrdited mice. Mice ering sucutneous B16F10 tumors estlished for 8 dys received 5Gy TBI. One dy fter TBI, mice received n ACT tretment comprised of the doptive trnsfer of 5e 5 cultured pmel-1 T cells, fowlpox hgp100 vccintion nd hil-2 or were left untreted. The next dy, mice received either () 5μg MPL (i.v.) or () 10μg of CpG (i.t.), dily for 4 dys, or left untreted. Dt shown (men ± SEM, 5 10 mice per group) re representtive of 2 independent experiments. For MPL tretment: 5Gy PFI vs. NT (*P < 0.05) or 5Gy MPL post-pfi (*P < 0.05), ANOVA. For CpG tretment: 5Gy PFI vs. NT (**P < 0.01) or 5Gy CpG post-pfi (*P <0.05),ANOVA incresed interest to determine nd use novel neontigens to trigger effective T cell responses in ptients with cncer [23], our dt offers importnt insights into how nd when to use TLR gonists to effectively ugment T cell-sed immunotherpies. Collectively, we identified the criticl determinnts for mediting successful ACT with TLR signling nd used this informtion to potentite the ntitumor ctivity of trnsferred CD8 + T cells with vccintion ut without host preconditioning. Discussion Lymphodepletion enhnces doptive immunotherpy vi severl reported mechnisms [40]. In ddition to the removl of myeloid-derived suppressor cells, cytokine sinks nd T reg cells, trnsloction of gut microflor y TBI impcts the outcome of doptive immunotherpy [11]. This intriguing finding tht cteri cn promote tumor regression is reminiscent of Coley s work with ptients treted with repeted inocultions of erysipels pulished in 1893 [41]. Yet, how to properly use cteri or TLR gonists to most optimlly enhnce vccines, checkpoint modultors nd cellulr therpy remins incompletely explored. Herein, we used TLR4 gonist LPS s tool to ddress how nd when to use TLR gonists to effectively improve cncer immunotherpy. We sked whether LPS could replce host lymphodepletion, vccines or trnsferred CD8 + T cells in n ggressive model of melnom. In ptients with dvnced metsttic melnom, non-myeloltive regimen prior to infusion of tumorinfiltrting lymphocytes nd olus IL-2 resulted in n ojective response rte of 50 % [42]. While the tolerted doses of lymphodepletion re well estlished, these systemic pproches re not devoid of toxicities. In mice, we report here tht lymphodepletion to 5Gy TBI correlted with greter innte immune ctivtion. Heightened innte ctivtion ws ssocited with greter impirment of the gstrointestinl trct, s evidenced y destruction of the colon nd greter microil LPS trnsloction [11]. However, we wished to find n effective nd sfe wy to ctivte the innte immune system without compromising the GI trct. Thus, we dministered LPS to non-irrdited mice receiving ACT. LPS could not ugment ACT-medited tumor regression in non-irrdited mice. However, low dose LPS could improve ACT tretment in mice given 5Gy TBI. Additionl investigtion reveled tht LPS incresed the expression of CD25 on pmel-1 CD8 + T cells infused into lymphodepleted mice. We re prticulrly intrigued y the fct tht LPS roustly incresed CD25 on donor pmel-1 CD8 + T cells in irrdited mice. Our new finding complements work from our collegue Mrk Ruinstein. His l recently pulished tht the induction of CD25 on CD8 + T cells vi IL-12 ugments the engrftment potentil nd ntitumor ctivity of doptively trnsferred CD8 + T cells without host lymphodepletion [43]. Mechnisticlly, CD25 (i.e. the IL-2Rα) medites IL-2 signling nd enhnces immunotherpy. Bsed this new finding, we surmise tht it is possile tht LPS induced IL-12 y APCs in vivo. Thus, LPS-induced IL-12 possily up-regulted CD25 on donor CD8 + T cells nd in turn incresing the

11 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 11 of 14 Fig. 8 Long-term curtive responses cn e medited in mice without lymphodepletion y depleting host CD4 + T cells nd ctivting APCs with TLR4 gonists LPS. () Scheme for treting non-irrdited mice with CD4 depleting ntiody, triprtite ACT tretment nd LPS. () Tumor erdiction vi ACT cn e chieved without host preconditioning vi ntiody depleting CD4 lymphocytes tht ct s T reg nd cytokine sinks nd ctivting innte immunity vi TLR signling. One dy efore ACT, mice were ntiody depleted of host CD4 + T cells nd susequently dministered every other dy for totl of 5 doses. The ACT tretment regimen ws comprised of the doptive trnsfer of 5e 5 cultured pmel-1 T cells, fowlpox hgp100 vccintion nd hil-2 or were left untreted. One dy fter ACT, mice received 2 μg of LPS or were left untreted. Dt shown (men ± SEM, 5 mice per group) re representtive of 4 independent experiments. NT (lck dimond) vs. PFI, PFI + LPS, or PFI + nti-cd4, *P <.05. PFI + LPS + nti-cd4 (lck circle) vs. ll groups P < 0.001, ANOVA cpcity of infused CD8 + T cells to consume homeosttic ville IL-2 in the host. We re now conducting follow up studies in our l to ddress this hypothesis. Collectively, our dt reveled tht LPS lone is not enough to ugment ACT tretment in non-irrdited nimls consistent with findings tht TBI provides multiple enefits to mximize ACT therpy, including TBImedited removl of immune cells y suppressive donor T cells. Moreover, eyond LPS, it is now well pprecited tht vriety of microil components induced y TBI/chemotherpy (or induced y other types of immunotherpies), cn drmticlly lter T cell-medited tumor regression. Thus, it is likely tht multiple TLRs, NODs, etc. on vrious innte immune cells re triggered, therey enhncing T cell-sed immunotherpies. Importntly, we found tht LPS doses toxic to nonirrdited mice were well tolerted in mice given low dose irrdition (5Gy TBI), possily due to the fct tht the numer of APCs re reduced following irrdition [32] nd therefore re unle to respond to LPS therpy. Furthermore, cliniclly ville MPL nd CpG-DNA ws sfe in 5Gy TBI mice nd medited CD8 + T cellmedited destruction of tumors. Besides the difference in CpG sequence specificity, one importnt distinction etween mice nd humns is the expression pttern of TLR9 on immune cells. In humns, TLR9 is expressed only on pdc nd B cells, wheres mice dditionlly express TLR9 on cells from the myeloid-linege, resulting in more dynmic response. All other effects of TLR9 lignds on humn immune cells seem to e indirect nd depend on fctors produced y pdcs nd B cells [44 46]. These differences my lter the trnsltility of CpG therpy (found in our work, Fig. 7) etween mouse nd humn. None the less, our dt suggest tht cliniclly ville MPL, CpG, vccines, or perhps recominnt humn OX40 lignd or CD40 lignd could e used to sfely ctivte the innte system [39, 47 49], therey enhncing the infused tumor-rective lymphocytes [50, 51]. Indeed, the enggement of the innte immune system s trigger of the dptive immune system represents powerful pproch to enhnce doptive immunotherpy. Moreover, it is likely tht these therpies lter the contents of the microiome, which mny hve recently pulished, hve long-term nd often positive consequences of minstrem checkpoint modultors [52, 53]. We found tht dministering LPS fter triprtite ACT therpy could improve tumor regression in irrdited (ut not in non-irrdited) nimls. However, dditionl investigtion reveled tht we could ovite the requirement for host preconditioning nd drive curtive responses in non-irrdited nimls if we ntiody lted host CD4 + T cells trnsiently, long with ACT tretment followed y TLR gonist therpy. In retrospect, these findings re not surprising, s LPS hs een shown to preferentilly support the genertion of regultory T cells, known to suppress effector CD8 + T cells. Although some helper CD4 + T cells re known to olster the cytotoxicity of CD8 + T cells, such s Th1 nd Th17 cells, other non-specific CD4 + T cells impir the engrftment of infused CD8 + T cells y competing for cytokines induced y lymphopeni. Given tht lting CD4 + T cells enhnces the ntitumor therpy of pmel-1 CD8 + T cells, it would e interesting to execute these studies in Foxp3 DTR mice, eliminting FoxP3+ T regultory cells. Ongoing studies in our lortory re now focused on

12 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 12 of 14 mechnisticlly understnding how this potent therpy is modulting the immune system to cure lrge estlished melnom. We re lso interested in understnding how nd wht type of host CD4 + T cells re reconstituting in these mice nd if they re influencing the genertion nd memory iology of tumor-specific CD8 + T cells in vivo. Finlly, it is compelling to us tht LPS could ct either to enhnce or hinder therpy depending on when it ws delivered to the host. Though the LPS signl is ment to enhnce the donor CD8 + T cells, prticulrly in the cse of dministering LPS efore T cell infusion, it ppers tht this eneficil signl olsters the genertion of host immune cells over tumor specific T cells (Is this phenomenon restricted to ACT therpy or does it impir vccines, chemotherpy nd/or checkpoint modultors s well?). Moreover, we lso found tht giving LPS during or prior to the triprtite regimen does not s roustly enhnce the host DCs ility to drive the prolifertion of the tumor-specific CD8 + T cells. In contrst, if LPS ws given fter the triprtite regimen, the host DCs effectively medited the prolifertion of pmel-1 CD8 + T cells in vitro. Bsed on these findings, our dt rgue tht it is criticl to consider when distinct therpies re given to ptient, prticulrly given tht mny investigtors, including ourselves, elieve tht the pth forwrd to improved tretment outcome in ptients will e through comintoril therpeutic pproches [54 57]. Conclusion Collectively, our results indicte tht, though often overlooked in the design of cncer immunotherpy sed clinicl trils, tht the temporl rrngement of multiple-tretment delivery is prmount considertion in designing next genertion T cell-sed immunotherpies for cncer nd infectious diseses. Specificlly, our dt identify how nd when to dminister TLR gonists to ugment T cell-sed immunotherpy in the sence or presence of host preconditioning. Methods Mice To investigte the ility of TLR gonists to enhnce ACT, we used the pmel-1 model to trget B16F10 melnom. All mice were red nd housed t NIH or MUSC fcilities. Femle pmel-1 TCR trnsgenic mice were crossed with C57BL/6-Ly5.1 Tg mice (C57BL/6- pmel-1-ly5.1 mice; Jckson Lortory). C57BL/6 nd MyD88 -/- (Tconic) were used s recipients in ACT experiments. Experiments were conducted with the pprovl of the Ntionl Cncer Institue nd Medicl University of South Crolin Animl Use nd Cre Committee. The poorly immunogenic murine gp100 + B16F10 melnom tumor ws used. In vitro ctivtion of pmel-1 CD8 + T cell genertion Pmel-1 splenocytes were cultured in the presence of 1 μm hgp (KVPRNQDWL) with culture medi contining 30 IU/ml of recominnt humn IL-2 (rhil- 2; NCI preclinicl repository), s descried elsewhere [11]. Cells were trnsferred 7 10 dys fter the strt of the culture. Regimen with doptive trnsfer, vccintion, cytokines nd TLR gonists Recipient 6 10 week old mice were injected sucutneously with 5e 5 of the poorly immunogenic B16F10 tumor cells. Eight dys lter, mice received 0.5-1e 6 in vitro ctivted pmel-1 CD8 + T cells. 5Gy TBI ws given to mice the morning of ACT. CD4 + T cells were depleted y i.p. dministrtion of 0.1 mg/mouse of nti- CD4 ntiodies (BD Biosciences) -1, 1, 3, 5 nd 7 dys reltive to cell trnsfer. Mice were vccinted with 2e 7 PFU of recominnt fowlpox virus expressing humn gp100 (Therion Biologics). rhil-2 ws dministered t 36 μg/dose 2X/dy for five doses. Ultr-pure LPS (Invivogen; μg, i.v.) ws dministered either 1 dy pre, during or 1 dy post PFI tretment. In some experiments, MPL (5 μg, i.v.) ws given on dy 1 following ACT. Alterntively, CpG (5 -TCCAT- GACGTTCCTGATGCT-3 ) ws given i.t. t 10 μg/ dy for 4 consecutive dys. Experiments conducted in rndomized fshion nd tumor mesurements recorded over time y individuls unwre of tretment groups. Enumertion nd phenotype of cells At the indicted times, trnsferred pmel-1 cells were enumerted y multiplying the percent of Ly5.1/CD8 + T cells in the spleen y the solute spleen count. Dt were cquired on BD LSRII (BD Biosciences) nd nlyzed using FlowJo softwre (Tree Str, Ashlnd, OR). Ex vivo prolifertion ssy Untouched pmel-1 cells were isolted from splenocytes of n untreted niml, CFSE-leled, nd co-cultured t 10:1 rtio with sorted CD11 + CD11c hi CD86 hi dendritic cells from tumor-ering mice (dy 6 post-act) given LPS t different time points. CFSE dilution ws ssyed on dys 0, 2 nd 4 post ctivtion. Mucosl rrier score nd detection of serum LPS Colons were removed from mice nd plced in 10 % formlin for 48 h, nd then emedded in methylcrylte. 4 5 mm sections were tken long the ppillry-opticl xis. Sections were evluted y pthologist unwre of the identity of the groups using the scores s follows: norml rchitecture = 0, some signs of edem = 1, mild cell infiltrtion nd reduction of crypts nd golets = 2, severe cell infiltrtion nd profound reduction of crypts

13 Nelson et l. Journl for ImmunoTherpy of Cncer (2016) 4:6 Pge 13 of 14 nd golets = 3, severe cell infiltrtion nd visully undetectle crypt nd golets = 4. An LAL ssy (QCL- 1000; Cmrex) ws used to nlyze serum LPS on dy 6. BrdU incorportion Three dys fter tretment, mice were injected i.p. with 1 mg of BrdU (Sigm-Aldrich, St. Louis, MO). After two hours, the spleens from tretment groups were hrvested, homogenized, nd stined for pmel-1. Cells were permeilized using Cytofix/Cytoperm (Phrmingen), treted with DNseI (Sigm-Aldrich) for 1 h t 37 C, then stined with FITC-conjugted nti-brdu (BD Biosciences), nd nlyzed using FACS. Additionl files Additionl file 1: One dy fter TBI, mice received n ACT tretment comprised of the doptive trnsfer of 5e 5 cultured pmel-1 T cells, fowlpox hgp100 vccintion nd hil-2 or were left untreted. Either on dy during or one dy fter ACT, mice received 2 μg of LPS or were left untreted. Flow cytometry dt shown re from splenocytes. (A) Percentge of CD25 high FOXP3 + CD4 + T cells on dy 5 following PFI. (B) Rtio of donor pmel-1 to host CD4 + T cells on dy 5. ***P <.001**P <.01, *P <.05, ANOVA. APCs were gted on either conventionl DCs (C) or monocytes (D) on dy 2 following PFI. (PDF 255 k) Additionl file 2: One dy efore ACT, mice were ntiody depleted of host CD4 + T cells (0.1 mg/tretment) nd susequently dministered every other dy for totl of 5 doses or left untreted. The ACT tretment regimen ws comprised of the doptive trnsfer of 5e 5 cultured pmel-1 T cells, fowlpox hgp100 vccintion nd hil-2 or were left untreted. One dy fter ACT, mice received 2 μg of LPS. The rtio of donor Vβ13tohostCD4cellsreshownfrom splenocytes on dy 5 (men ± SEM, 5 mice per group). ****P <.0001, unpired t-test. (PDF 70 k) Arevitions ACT: Adoptive cell trnsfer; CpG: CpG oligodeoxynucleotides; HSC: Hemtopoietic stem cell; LPS: Lipopolyscchride; MPL: Monophosphoryl lipid A; PFI: P = infusion of 1e 6 pmel-1 CD8 + T cells, F = vccintion with fowlpox encoding hgp100 nd I = high dose IL-2; TBI: Totl ody irrdition; TLR: Toll-like receptor; WT: Wild-type. Competing interest The uthors declre tht they hve no competing interests. Authors contriution MHN prticipted in the concept nd design of the study, developed methodology, cquired dt, interpreted the dt, nd drfted, edited nd revised the mnuscript. JSB contriuted to the development of methodology, nd cquired nd interpreted dt. SRB ssisted in nlyzing nd interpreting ex vivo dt. MAD ssisted in cquiring, nlyzing in vivo dt. CWF ssisted in cquiring, nlyzing nd interpreting follow-up dt. ADK ssisted in cquiring, nlyzing nd interpreting oth in vivo nd in vitro dt. CW ssisted in cquiring, nlyzing nd interpreting oth in vivo nd in vitro dt. BL ssisted nlyzing nd interpreting APC dt. NPR ssisted with the study s design nd interprettion, writing the mnuscript nd providing dministrtive support. CMP ws involved in the design nd conception of the study, developed the methodology, cquired nd interpreted the dt, ssisted in writing nd revising the mnuscript, provided dministrtive support nd supervised the work. All uthors criticlly reviewed nd pproved the mnuscript. Acknowledgements The Intrmurl nd Extrmurl Reserch Progrm of the NIH nd ACS supported this reserch. Specificlly, this work ws supported, in prt, y NIH grnt 5R01CA175061, KL2 South Crolin Clinicl nd Trnsltionl Reserch grnt UL1 TR000062, ACS-IRG grnt , Lefkow Memoril Thorcic Cncer Reserch Awrd, Jene B. Kempner Foundtion grnt, ACS Postdoctorl fellowship ( PF LIB) grnt support, nd Cell Evlution nd Therpy Shred Resource, Hollings Cncer Center, MUSC (P30 CA The uthors hve no conflicting finncil interests. We thnk Angel Mexs nd Mry Jo Turk for thoughtful discussion. We thnk Megn Meek, Logn Huff, Dve Jones nd Pul Speiss for excellent technicl service. We thnk the ls of Zihi Li, Jennifer Wu, Beichu Guo, Shikhr Mehrotr nd Benny Yng for providing mice for follow-up experiments. Author detils 1 Microiology nd Immunology, Hollings Cncer Center, Medicl University of South Crolin, Chrleston, SC 29425, USA. 2 Center for Cncer Reserch, Ntionl Cncer Institute (NCI), Ntionl Institutes of Helth (NIH), Bethesd, MD 20892, USA. Received: 12 Octoer 2015 Accepted: 19 Jnury 2016 References 1. Bckhed F, Ley RE, Sonnenurg JL, Peterson DA, Gordon JI. Host-cteril mutulism in the humn intestine. Science. 2005;307(5717): Gurner F, Mlgeld JR. Gut flor in helth nd disese. Lncet. 2003;361(9356): Hooper LV, Wong MH, Thelin A, Hnsson L, Flk PG, Gordon JI. Moleculr nlysis of commensl host-microil reltionships in the intestine. Science. 2001;291(5505): McFll-Ngi MJ, Ruy EG. Symiont recognition nd susequent morphogenesis s erly events in n niml-cteril mutulism. 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