Promoting the Accumulation of Tumor-Specific T Cells in Tumor

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. of new immunotherapies. tic factor for survival of patients with different cancer types cancer different with patients of survival for factor tic prognos is an independent CTLs of accumulation Intratumoral the Development protocolof cancer immunotherapy cancer and surveillance immune natural during immunity anticancer of effectiveness the in factor limiting key the as TME the into entry cell T tumor-specific of importance the highlight observations David David L Bartlett University Pittsburgh, of Pittsburgh, Pennsylvania, USA. tumor growth tumor control to system immune the of blockade) ability the demonstrated (checkpoint have molecules checkpoint immune of tors inhibi and immunotherapies cellular involving studies Recent I T tumor tissues, our approach allows detection of intratumoral the analyses of systemictumor-specific and intratumoral immunity is performed over 30–40 d. experimental procedure, including tumor D implantation, to quantify tumor-specific to induce tumor-specific tumors. of the vaccination-induced of mice receiving dendritic cell (D T 1 Nataša Obermajer and chemokine-modulating agents T cells in tumor tissues by dendritic cell vaccines Promoting the accumulation of tumor-specific into tumor lesions tumor into entry CTL effective promote to differ modulation TME and of forms organs, ent lymphoid murine in CTLs tumor-specific USA. the changes in the numbers of tumor-specific CTLs in tumor tumor in CTLs lesions tumor-specific of numbers the in changes the Published online 18 January 2018; Correspondence should be addressed to P.K. ( of immunotherapy do not predict clinical responses clinical predict not do immunotherapy of course the in cells T tumor-specific circulating in increases that ods include the generation of murine type-1-polarized DCs meth type-1-polarized murine of The generation the include ods attraction. CTL enhanced for TMEs reprogram to mice cells in and cancer-bearing present type-1 strategies specific with CTLs with that are tumors in infiltrated of already the effective therapy larly ex vivo ade therapies ade block checkpoint of effectiveness clinical for the is and required material tumor autologous with loaded Department of Surgery,of Department University Pittsburgh, of Pittsburgh, Pennsylvania, USA. NTRO his his detection method can be performed using basic laboratory skills, and facilitates the development and preclinical evaluation his protocol describes how to induce large numbers of cytotoxictumor-specific Here, we a describe protocol to enhance the numbers of tumor- 3 University Cancer Pittsburgh of Institute, Hillman Cancer Center, University Pittsburgh, of Pittsburgh, Pennsylvania, USA. D

1 CTL 9 T UCT during immunotherapy. during he he protocol contains detailed information describing how to generate a specialized D 6–1 assay takes 6 h to set up and 5 h to develop. In contrast to other methods of evaluating immunity tumor-specific in I 2 ON 6–11,27,2 . 1– 6 . To date, immunotherapies have been particu been have .To immunotherapies date, 16–1 1 & Pawel Kalinski 8 1 . By contrast, multiple studies have shown shown have studies multiple contrast, By .

8 . 31–3 We further outline methods to evaluate evaluate to methods outline Wefurther

, Julie Urban CTL CTL 4 CTL doi:10. . s. We also describe methods to modulate the production of chemokines in the s s in the lymphoid organs and tumor tissues of mice receiving different treatments. s s to tumor tissues. We also describe how to evaluate the numbers of tumor-specific 1038/nprot.2017.13 C ) ) vaccines and how to modulate tumor microenvironments ( [email protected] © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 2 1 , Eva Wieckowski – 14,1 5 5 5 Department of Bioengineering, University Bioengineering, of Department Pittsburgh, of Pittsburgh, Pennsylvania, USA. , the induction of of induction the , 0 29,3 0 . These These . C vaccine generation, ( chemokine-modulating 20–2 1 13,1 ). , T 2 -cell -cell responses to weaklynonmanipulated immunogenic cancers. , 6 ------6 4 2

, MuthuswamyRavikumar Immunotransplantation Center,Immunotransplantation University Pittsburgh, of Pittsburgh, Pennsylvania, delivery of CTL-attracting chemokine genes to tumors genes chemokine CTL-attracting of delivery CD8 cancer vaccine cancer DCs can be generated generated be can DCs maturation. and generation DC of Modes CD8 tumor-antigen-specific of cross-priming enhanced components, in TME resulting other and (APCs) cells antigen-presenting lesions. Several strategies have been shown to activate endogenous tumor in CTLs of accumulation the promote to Newstrategies ligands or interleukin (IL)-18 (refs. (refs. (IL)-18 interleukin or ligands cancer cells and induction of type-1 interferons (IFNs) in DCs (IFNs) interferons type-1 of induction and cells cancer enhanced CTL accumulation CTL enhanced enous type I enous type IFNs exog involving adjuvants CKM combinatorial to tissues tumor gens and adjuvants to DCs (reviewed in Kreutz Sipuleucel-T (Provenge) receiving Sipuleucel-T cancer prostate hormone-refractory with patients in seen was infiltration CTL intratumoral promote to vaccines DC of ability The agents. as therapeutic used be can DCs gen-loaded e individuals, cancer-bearing in DCs therapies. cell Dendritic vivo (TAAs) antigens tumor-associated against immunity T-cell of induction and activation DC drive can and tumor a of presence the detects pathway (STING) genes interferon of stimulator The 37,3

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T cells T cells, augmented local chemokine production, and and production, chemokine local augmented cells, T 8 . Alternative strategies involve targeting exogenous anti exogenous targeting involve strategies . Alternative T in in vivo cells ( 4 8 . 4 0 in with Toll-likecombination receptor (TLR) involves of early innate immune recognition CTL natureprotocols s) s) in the spleens and lymph nodes x vivo ex 4 To bypass dysfunction of endogenous endogenous of dysfunction To bypass 7 C , the first FDA-approved APC-based APC-based FDA-approved first the , 1 T vaccine with augmented ability , Roshni Ravindranathan T M he he presented 34,3 E s) s) to ensure effective homing from bone marrow or blood blood or marrow bone from 4 5 Department of Immunology,of Department . Productive cross-priming of of cross-priming Productive . C KM) KM) approaches, and x vivo x 16,18,41–4 T M | VOL.13 NO.2VOL.13 E EL and show how -generated and anti and -generated Mouse and human human and Mouse et al. I S 6 T Deceased. CTL pot-based pot-based he he combined protocol 4 3

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. cells into TME. CKM, chemokine modulation. 1 effector immune cells should be planned at least 4–5 d after CKM regimen, as a shorter period may not suffice for proper migration of DC-induced effector immune responses responses by DCs (Step 9C, (apoptotic bodies, heat shock proteins, and urate crystals) urate and proteins, shock heat bodies, (apoptotic cells damaged or dying by released molecules pattern molecular ( DCs (Step 9), and monitoring of the therapeutic and immunomodulatory effects (Steps 10–24). Start with Flt3L-B16 injection of C57BL/6J mice on Figureday 1 (TNF)- factor necrosis tumor or IL-4 with colony-stimulating combination in or alone (GM-CSF), factor granulocyte/monocyte using precursors 336 RNA) inactivated hemagglutinin, toxin, filamentous cholera peptidoglycan, (LPS), lipopolysaccharide as such components microbial (e.g., ligands other tissues other DC and nodes, of lymph marrow, spleen, bone in DCs and numbers progenitors the increase to used be can development, DC 3 kinase (Flt3L), a ligand tyrosine fms-like with factor promoting of experimental mice to evaluate tumor progression/survival (Steps 14–16) and immune monitoring ( of the TME to attract the vaccination-induced T cells. Repeat the cycles of DC therapy in combination with CKM regimen at weeklyintratumoral intervals. Usedose separateof type-1sets polarized DCs, combination of IFN- with tumor-loaded type-1 polarized DCs at day 4 of tumor growth (Step 15). 3 d after DC vaccination, follow up with CKM regimen (Steps 15 to anddetermine 16; e.g.,the baseline intensity of the tumor-related bioluminescence signal in each mouse (Step 12). For optimal results, start vaccinating11) for thethe micemonitoring of the therapeutic and immunomodulatory effect can be initiated at any time point. A day before DC vaccination, mice are imaged Fig. 2 administering DCs to experimental mice. Basic QC should involve confirmation of the mature DC phenotype (Step 9A, expression of theco-stimulatory monitoring molecules, of the therapeutic and immunomodulatory effects are time-wise independent parts of the protocol. QC (Step 9) toshould cryopreservation be performed of beforemature type-1 polarized DCs (Step 6). The thawing of DCs (Step 8) can be performed at any time point; therefore, the QC of DCs and from from thymic or splenic progenitor cells nity α Box protocol Time line(d) ad IL-1 and , DC DC maturation is needed for effective induction of T-cell immu

[16 h] [1h–48 h] [7–10] [2] [1] 6 | 1 VOL.13 NO.2VOL.13 0 a ), followed by CD11c . Factors that can induce maturation of DCs include TLR TLR include DCs of maturation induce can that Factors . ) and the ability of DCs to produce IL-12 upon CD40L stimulation (Step 9B, 6 1

| , cytokines (including type I interferons, IFN- interferons, I type (including cytokines , Overview of the protocol, with associated timing. The three main parts of the protocol are generation of tumor-loaded DCs (Steps 1–7), QC of ( tumor-specific immunity In vivo ( Phenotype analysisoftheDCs ( In vivo type-1 immunecellinduction( In vitro ( Analysis ofIL-12production Step 9E,Box5 Step 9A Experimental mice(naive) QC ( Step 9D,Boxes4and5 Step 9B Bacillus β 58,5 Step 9 evaluationofsystemic evaluationofDCmigration evaluationofeffector , rsalni E (PGE E2 prostaglandin ), ; ; in vivo Fig. 2a Fig. 2b 9 | . ; 2018 Fig. 2 ; Calmette–Guérin (BCG), Calmette–Guérin or double-stranded Fig. 2e ) ) (Step 9E and ) Fig. 2

| + α

cell isolation and induction of type-1 polarized tumor-loaded DC maturation on day 0 ( ) natureprotocols ; 49–5 Fig. 2d Step 9C c 5 ), and measurement of their lymph node migratory capacity (Step 9D and ) . Mouse DCs can also be generated generated be also can DCs . Mouse ; Boxes 4 Fig. 2c Thawing ofDCs( Thawing ofDCs( Thawing ofDCs( © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. ) 56,5 and 7 2 . Pretreatment of mice ), damage-associated damage-associated ),

5 , Step 8 Step 8 Step 8 Fig. 2 ) ) ) Time line(d) e ). After successful QC, the injection of tumor cells into the experimental mice (Steps 10 and (4 h) (0) (–1) (–2) (–11) α , Ampligen and COX2 blockers, or chemokine-expressing vaccinia virus) to enhance the ability γ , TNF- , 6 2 , or or , ( (Flt3L-B16 cellinjection) Immature DCs Donor mice Thawing ofDCs( Thawing ofDCs( Thawing ofDCs( ( mouse DCsandcryopreservation Collection ofmaturetype-1polarized mouse DCs( Generation oftype-1polarized Preparation ofUVB Box 1 Steps 3–7 -

) cancer patients DC-vaccinated in outcomes clinical positive the of predictive is produce only very low amounts of IL-12p70 (refs. (refs. IL-12p70 of amounts low very only produce producing DCs producing IFN- with LPS of for early clinical trials involved TNF- involved trials clinical early for immune responses immune for IL-12, of and cell production type-1 high activation necessary DCs with high levels for molecules necessary of co-stimulatory T- generate to ligand TLR one than more of mixtures or (poly-IC), phosphorothioate-guanine (CpG), polyinosinic:polycytidylic acid NKT, or (NK), killer (T,natural cells immune other by provided signals (refs. (refs. tion of Th1/CTL/NK-cell-dominated type-1 immunity type-1 Th1/CTL/NK-cell-dominated of tion induc the for critical is IL-12p70 produce to vaccines DC of ity prompted a search for alternative DC maturation factors. The abil ) Fig. 2 Boxes 1and3,Steps1–3 Step 8 Step 8 Step 8 γ Tu cells( b 64,6 ). Additional tests may involve ) ) ) γ δ 5 T T cells) Box 2 . oee, bevtos ht PGE that observations However, ). ) 75–7 66,70,71,78–8 Box ) γ 6 7 to induce highly immunogenic, high-IL-12- immunogenic, highly induce to Time line(d) 13,61,81–8 3 . Among others, we proposed the combination 6 . A maturation cocktail used to generate DCs to .used generate cocktail A maturation and Steps 17–24). Analysis of tumor-specific type- Box +25–35 +17 +14 +13 +10 +7 +6 +3 0 –1 3 0 , . LPS may be replaced with cytosine- with replaced be may LPS . 3 Fig. 2 . Boxes 2 progression ( progression ( progression ( ( Steps 15and16 with tumorcells( tumor-infiltrating specific ( for survival( Monitoring oftheanimals Step 14 Monitoring ofthetumor type-1 immunecells progression ( ( Monitoring oftumor Monitoring oftumor ( Inoculation ofmice Monitoring oftumor Steps 15and16 (tumor-bearing) Experimental mice Steps 15and16 d in vitro ), and ability to induce type-1 Induction of ( ( Step 14 Step 14 ) and Steps 12and13 Steps 12and13 Steps 12and13 Step 18 α induction of type-1 immune

Step 12 3 ) ) ) Step 11 , IL-1 , and Step 1). Proceed ) ) ; Fig. 3c,d ) ) β ) ) ) , IL-6, and PGE and IL-6, , ( Ex vivo ( In vivo Steps 17–24 Boxes 5and6 ) 2 -matured DCs DCs -matured cytotoxicityassay immunomonitoring 13,66–6 ; 70–7 Fig. 4 )

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. eGFP) transgenic mice (as described in DC generation section generation DC in described (as mice transgenic eGFP) 12 production in ID8A-loaded DC cultures ( cultures DC ID8A-loaded in production 12 co-stimulatory and maturation markers (CD40, CD80, and CD86) and lymph node-driving chemokine receptor CCR7 on DCs. Note the expression of IA/E expression the Note DCs. on CCR7 receptor chemokine node-driving lymph and CD86) and CD80, (CD40, markers maturation and co-stimulatory of Pittsburgh. FSC, forward scatter; SSC, side scatter. side SSC, scatter; forward FSC, of Pittsburgh. University of the (IACUC) Committee Use and Care Animal Institutional the by approved were experiments Animal UK. of Birmingham, P. Lane, University mouse/average ratio loaded:unloaded in naive mice)) × 100. CD40L-transfected J558 (J558-CD40L) (Balb/c mouse strain) cells strain) mouse (Balb/c (J558-CD40L) J558 CD40L-transfected × 100. mice)) naive in loaded:unloaded ratio mouse/average in vivo in ( shown. h are 48 and 15 at mice of three nodes lymph popliteal and of footpads BLI Representative points. time separate at (photons/s/area) points data individual as shown are Results photons/s/area. as recorded intensity the and circles) red by marked is of interest (region gated was signal luminescence The nodes. lymph popliteal draining the from signal the detect to order in re-imaged, and tape masking black with covered were mice imaged of the footpads the footpads), the in signal (luminescence image first the obtaining after twice: imaged was mouse Each h. 48 and , 40, , 24 15 1, after IVIS by imaged × 10 0.5 per of spots (no. unpaired Student’s using performed was evaluation Statistical 9C). (Step cells tumor IFN- produce to ability their for assay ELISpot by analyzed and harvested were splenocytes 7 of activation, *** s.d.). (± unpaired Student’s using performed was evaluation Statistical 2 Figure CFSE and H2K and and for DC-vaccinated mice, 441 (CFSE mice, 441 DC-vaccinated for and data of splenocytes from naive mice were 704,397 (ungated), 506,563 (R1, 71.9% of ungated), 609 (R2, 0.12% of R1), 329 (CFSE 329 of R1), 0.12% (R2, 609 of ungated), 71.9% (R1, 506,563 (ungated), 704,397 were mice naive from of splenocytes data used approach is the use of peptides or recombinant proteins proteins recombinant or peptides of use the is approach used commonly One vaccines. DC of activity therapeutic the for cal versus isotype control (light gray) of 1 × 10 gray) (light control isotype versus and GM-CSF, and loaded with ID8A tumor cells were set up in in up set were cells tumor ID8A with loaded and GM-CSF, and Selection of a relevant antigen source is another aspect criti aspect another is source antigen relevant a of Selection low . Gating strategy and evaluation histograms are presented. A histogram including CFSE including A histogram presented. are histograms evaluation and strategy . Gating and CFSE and b e d b a

, but the absence of CD14 expression (macrophage marker) in mature DCs. CD14, CD40, CD80, CD86, CCR7, IA/E CCR7, CD86, CD80, CD40, CD14, DCs. mature in marker) (macrophage expression of CD14 absence the , but |

Key features of type-1 polarized DCs and their functions. ( functions. their and DCs polarized of type-1 features Key % Of max.

SSC 100 120 K 20 40 60 80 P 30 K 60 K 90 K 0

< 0 0 Photons/s/area 0.001. ( 0.001. high e 10 10 10 10 10 ID8A-loaded DCCD40L IA/E ) ) 4 5 6 7 , respectively. Only 0.05–0.5% of the cells are expected to be CD45.1 be to expected are cells of the 0.05–0.5% Only , respectively. 2 In vivo In 040 20 0 500 K0 b 10 -FITC ID8A-loaded DC 4 c 6 ) ) 1.0 M cells) and means (± s.d.). *** s.d.). (± means and cells) 10 specific cytotoxicity assay. Example of the evaluation of the cytolytic capacity of effector type-1 cells, induced by DCs DCs by induced cells, type-1 of effector capacity cytolytic of the evaluation of the assay. Example cytotoxicity specific In vitro In 6 Footpads FSC Time (h) 10 1.5 M cells =506,563 0 induction of tumor-specific type-1 effector cells by DCs. DCs matured in the presence of IFN- presence the in matured DCs DCs. by cells effector type-1 of tumor-specific induction 10 CD14-FITC No. of 2.0 M R1 2 low 200 0 10 ) and 81 (CFSE 81 ) and © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 4 n 5

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In vitro 60 12) with and without J558-CD40L cells (indicated by by (indicated cells J558-CD40L without and with 12) FL-3 10 10 10 10 10 IL-12 production 0 3 4 6 8 10 IL-12 (pg/ml) 0 b CD40L-induced ) Type-1 polarized DCs produce high levels of IL-12 when re-stimulated with J558-CD40L cells. IL- cells. J558-CD40L with re-stimulated when of IL-12 levels high produce DCs polarized ) Type-1 R1-gated 0 Footpads 10 CCR7-PE 400 2 high 10 P 10

). Percentage of specific killing is defined as (100 – (ratio loaded:unloaded in experimental experimental in loaded:unloaded – (ratio (100 as defined is killing of specific Percentage ).

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0.001. ( 0.001. , in vitro in 10 Steps 1–5) and injected into the footpads of the C57BL/6J mouse ( mouse C57BL/6J of the footpads the into injected and 1–5) Steps t CFSE 0 800 600 tests (two-tailed). Results are shown as individual data points (pg/ml) and means means and (pg/ml) points data individual as shown are Results (two-tailed). tests 10 6 4 10 0 *** cultures with splenocytes ( splenocytes with cultures cells =609 a d 10 ) Flow cytometry overlay histograms showing the surface expression of emblematic of emblematic expression surface the showing histograms overlay cytometry ) Flow 10 H2K No. of ) ) 6 48 h 15 h R2 2 In vivo In - b 10 -FITC 10 4 R2 inR1-gated 8 use of tumor lysates or tumor apoptotic bodies as a source of of source a as antigens DC-presented bodies apoptotic tumor or lysates tumor of use representing defined TAAs defined representing 10 DC migration assay. DCs were generated from firefly luciferase (CAG-luc- luciferase firefly from generated were assay. DCs migration DC Photons/s/area 2 ×10 1 ×10 3 ×10 5 ×10 2 ×10 Counts 6 100 60 80 20 40 c Splenocytes -ID8A 10 0 t 10 tests (two-tailed). Results are shown as individual data points points data individual as shown are Results (two-tailed). tests 0 0 5 4 5 5 5 CFSE 0 0 10 D0ACC8-P CD40-APC CD86-APC CD80-APC

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. intralymphatic administration can be also to used to DCs deliver administration intralymphatic CTLs tumor-specific activate effectively to ability their for important administration. DC of Routes induce induce polyclonal immune responses and can a antigens provide of vaccines range whole-tumor-based 338 of administration of the APC-based Sipuleucel-T (Provenge) Sipuleucel-T APC-based the of administration of ated tumor (UVB tumor ated (UVB gamma and beta UV antigens, tumor-rejection that tumor do against types not common have well-characterized tumor antigens tumor with transfected total mRNA or DNA be from tumors to induce unique CTLs against also can DCs antigens. tumor unique cific TAAs, are patient-spe multiple a of source convenient including netd DCs injected intraperitoneally or intravenously than better nodes lymph the to migrate to shown been have intradermally administered DCs protocol Box 2 Box 1  loading isindicated in 5. Resuspend the tumor cellsat1×10 gamma irradiator). 4. Resuspend the pelletin10mlof culture medium and irradiate the cellswith200Gy(the specifictime depends onthe type of the room temperature (the high speedisrequired topelletthe apoptotic bodies). 3. Placethe plateunder the UVB 4. Resuspend the splenocytes in magnetic-activated cell sorting (MACS) buffer for subsequent CD11c follow the procedure outlined in should beused. fully killedwithUVBand gamma irradiation). Staining willdetermine ifthe cellsshould bere-irradiated oranew aliquot of tumor cells V and propidium iodide (PI)staining should beperformed toconfirmthe irradiation waseffective(i.e., the tumor cellswere success 2. Resuspend tumor cellsat1×10 2. After10–12d(the CD11c  isolated from one spleenis30–40×10 1. Inject1×10  for 5–10min,until the cellsdetach. Harvest the tumor cellsand centrifuge at400 flask. Discard the 1mMEDTA inPBSand add a new 10ml of 1mMEDTA inPBS.Putthe cellculture plateinto the incubator at37°C 1. Remove the culture medium from the tumor cellculture plate, washthe cellswith10mlof 1mMEDTA inPBSby tilting the culture P P on the number of vaccine cycles, number of mice treated, and number of injections percycle. The expected number of CD11c culture; alsodetermine the number of mice needed for injection of Flt3-expressing B16cells. The number of CD11c  provides information on the strain and number of mice required for the generation of DCs for specific QC and experimental testing. 3. Make acutinto the leftflank of the mouse toenter the peritoneal cavityand collectthe spleen for CD11c dislocation. 6. Prepare anextra aliquot of cells(take a100–200 5. Once eluted from the column, resuspend the CD11c microbeads, following the manufacturer’s instructions. Splenocytes from two spleens can be combined and loaded onto a single column. gamma irradiation (UVB

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| CR CR CR CR VOL.13 NO.2VOL.13 93,9 I I I I T T T T ure 4 I I I I . Although the intravenous route is the standard mode mode standard the is route intravenous the . Although CAL CAL CAL CAL | | Generation ofimmature DCs Preparation ofapoptotictumorcellsforDCloadingusingUVBand

95–9 STEP STEP STEP STEP 9 2 | 6 7 2018 . Flt3L-expressing B16cellss.c. into C57BL/6Jmice. γ . Ultrasound-guided intranodal injection or or injection intranodal Ultrasound-guided . Flt3L-expressing B16cellsshould bepassaged atleastone totwotimes before injection into mice. It iscritical thatthe tumor cellsprepared inthisstepbeapoptotic before their usefor vaccine production. Annexin Ensure the thatcancer cellline thatwillbeinjectedinto the experimental mice isusedfor DCloading. Determine the number of DCsneeded for vaccination and the number of CD11c -Tu)cells |

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. formly express highly immunogenic model antigens, such as as Dranoff in such (reviewed (OVA) antigens, ovalbumin model immunogenic highly uni express that lines formly cell tumor modified genetically and OT-II), or OT-I (e.g., receptors T-cell defined geneti with mice from manipulated cells cally T of use the involve tissues tumor in cells T tumor-specific of numbers the evaluating of methods Common alternative techniques with Comparisons tumors. sectable unre advanced against effectiveness their enhance to therapies or T-cell blockade, adoptive checkpoint vaccines, with be combined can or resection, tumor following therapies self-standing used as be can lesions tumor the into CTLs the of entry the mote pro to ligands TLR with combination in IFNs I type involving adjuvants combinatorial of application The blockers). COX2 or IDO checkpoints, immune (e.g., TME the in factors inhibitory character of immune responses (reviewed in Ngiow Ngiow in (reviewed and responses immune magnitude of the both character affect substantially to tumor known is manipulated cells genetically such of immunogenicity high (refs. (refs. their entry into tumors. We anticipate that they may be com be may pathways bined the tumor with of survival blockade they that anticipate We tumors. into entry their and antigens tumor-relevant different recognizing CTLs induce therapies. viral or DCs, of injection tumoral as systemic or local application of combinatorial adjuvants, intra forms or of systemically of locally applied TME,modulation such different and vaccines DC using immunization systemic involve approaches The immunotherapy. of course the in numbers ing chang tumor- their evaluate to of and lesions tumor numbers within CTLs the specific enhance to designed is protocol This the protocolApplications current of activity. antitumor local their and tumor lesions into influx CTL both promoting role, dual a have tocol pro this in described blocker COX-2 with therapies viral or DC combining approaches combinatorial The immunity. antitumor (ref. virus therapies virus antigenically heterogenous. antigenically are and antigens strong express rarely which immunotherapy tumors, human the of to relevant fully be not may models such Box 3 6. Count the cellsusing ahemocytometer before useinspecific experiments. 70- 5. Add 30mlof PBSand centrifuge the cellsat400 ? rotate the conical centrifuge tubetomix.Incubate the cellsatroom temperature for 4min. of ammonium–chloride–potassium (ACK) lysisbuffertoremove red bloodcells. Add anadditional 3mlof ACK lysisbufferontopand 4. Centrifuge the cellsat400 ? piston from a3-mlsyringe whilecontinuously washing the strainer withPBS. it through a22-gauge needle into the spleen;repeat three tofivetimes tothoroughly flush the cells. Mash the spleenusing arubber 3. Transfer the spleen onto a 100- 2. Make acutinto the leftflank of the mouse toenter the peritoneal cavityand collectthe spleen. 1. Euthanizethe mouse perIACUC protocol, followed bycervical dislocation. P The presented DC-based approaches can be used to efficiently to efficiently used be can approaches DC-based presented The

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TROU TROU µ 11 m strainer. 110–11 3 ) inhibitors also predispose the TME toward effective effective toward TME the predispose also inhibitors ) B B LES LES | Preparation ofthesplenocyte suspension 2 11 ) and indoleamine 2,3-dioxigenase 1 (IDO1) (IDO1) 1 2,3-dioxigenase indoleamine and ) H H 5 OOT OOT , TNF superfamily ligands superfamily TNF , I I N N G G g and room temperature (20–23°C)for 3–5min,discard the supernatant, and resuspend the cellsin1ml µ m strainer placed on top of a 50-ml conical centrifuge tube. Fill a 10-ml syringe with PBS and inject © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 11 7 ). As the artificially artificially the As ). 11 6 , and blockers of of blockers and , g 11 and room temperature for 3min,then resuspend in20mlof PBSand transfer toa 4 , oncolytic et al. et 11 8 ), ------

analyses of CD8 of analyses by single-cell and analysis, potentially or ELISpot flow cytometry cancer relevant cells. cells can Cytokine-producing be detected by sive T cells) upon stimulation of tumor-isolated lymphocytes with (IFN- cells cytokine-producing of detection the on relies method Our methods the protocol current of other Limitations and with comparison strains. mouse and models tumor multiple to TCR-antigen interactions, and its immediate general applicability high-affinity on focus narrow the to related bias the epitopes, alleviating immunogenic relevant the of identification prior for its ability to evaluate responses to multiple TAAs without the need are technique the cell. current of cancer Thus, any the key advantages recognize to able are that cells T and immunotherapy-induced arising spontaneously of enumeration for allows protocol epitopes are known to be weaker than antiviral responses or or responses antiviral than weaker be to known are epitopes tumor to However, responses T-cell tetramer-posi as analyzed. be can cells of tive subsets and markers various for stained ally addition be can cells is that by tetramers detection of advantage assay and detection by tetramers are very similar (1 in 3–10 3–10 in analysis) (1 ELISpot in similar positive are cells very are tetramer-positive tetramers by detection and assay cells. antigen-specific of proportion a of apoptosis in result professional APCs) to detect tumor-specific responses, which may be rechallenged must (TALs) lymphocytes tumor-associated and/or (TILs) phocytes lym tumor-infiltrated However, isolated cells. cancer recognize lesions tumor infiltrating of capable nating the need for surrogate antigens or genetically manipu genetically or antigens surrogate for need the nating elimi by readout, translatable direct more ‘self’ a provides overexpressed antigens) or (mutated antigens weak multiple to tumor-related responses T-cell polyclonal evaluate to ability The Advantages the protocolcurrent of the design of tetramers or defined defined or tetramers of design the of antigen specificity, threshold selected an reflected in arbitrarily cancer cells. It the need eliminates for known and cancer antigens TAAs by different expressed recognizing T of cells evaluation the allowing and (TCRs) receptors T-cell artificial with cells T lated and target cell killing cell target and In terms of the ability to detect very low cell numbers, ELISpot ELISpot low numbers, very cell to detect the ability In of terms cells that are Our antigen-specific of approach allows detection γ and potentially other mediators of effector and suppres and effector of mediators other potentially and ●

T-cell degranulation (such as CD107 assay) as CD107 (such T-cell degranulation T IMI 12 in in vitro natureprotocols 0 . N G with the original cancer cells (non cells cancer original the with 1h α / in vivo in β TCR combinations. Our Our combinations. TCR

| VOL.13 NO.2VOL.13 and that specifically specifically that and protocol | 2018 12 1 . The The . |

339 11 ------9

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. per group) were left untreated (control), treated with matured type-1 effector T cell-inducing DC (DC, 3 × 10 DC generation starts with administration of Flt3L to C57BL/6J C57BL/6J to Flt3L of progression. administration tumor with starts on generation DC vaccine DC of effectiveness the of ing monitor and QC by points, followed and time generation, DC with separate starting at performed be can parts these of Each ( cells tumor with in inoculated mice DCs of and monitoring of the therapeutic and immunomodulatory effect combinatorial approaches targeting immunosuppressive T DC vaccination and chemokine modulation. with i.p. injection of IFN- of 3 × 10 chemokine-expressing oncolytic virus induction of mature type-1 polarized DCs on day 0, including including 0, day on DCs polarized type-1 mature of induction The three main parts of the protocol are generation of DCs DCs of generation are protocol ( the of parts main three The the protocolof Overview available. epitopes tumor potential the of range narrow relatively a only of detection for allows tetramers ( s.c., followed by CKM regimen for TME modulation. COX-2 inhibitors should be administered frequently (every 2–3 d) during combinatorial therapy. significant, group) were left untreated (control) or treated with i.p. injection of IFN- mice on day day on mice responses to model antigens, such as OVA, as such antigens, model to responses 340 Figure 3 Science Center at San Antonio. Animal experiments were approved by the IACUC of the University of Pittsburgh. CKM, chemokine modulator.in vitro versus DC s.c. per group) were left untreated (control), treated with matured type-1 effector T cell-inducing DC (DC, 3 × 10 cocktail consisting of IL-1 (e.g., antibodies blocking PD1, PDL1, CTLA4). ( to promote tumor infiltration of DC-induced tumor-specific type-1 effector T cells. IL-12 (produced by DCs), a combination of IFN DCs must produce IL-12 upon stimulation with CD40L ( type-1 effector CTLs. Besides expressing relevant co-stimulatory molecules and chemokine receptor CCR7 (to effectively migrate into lymph nodes), mature c Fig. protocol c –

e No. of surviving mice

) Anticipated prolongation of survival following induction of tumor-infiltrating specific type-1 immune responses. ( 0 1 2 3 4 5 6 7 | VOL.13 NO.2VOL.13 1 02 30 20 10 0 malignant transformation of C57BL/6 mouse ovarian surface epithelial cells (MOSECs), was a generous gift from T.J. Curiel, University of Texas Health , Steps 1–7), control the of quality DCs (QC: MC38

5 | DC injected i.p. on days 8 and 15, and followed up for survival. ( Combinatorial strategy to increase the numbers of tumor-specific TILs. ( P

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aspirin). i.t., intratumoral. ID8A (luciferase-expressing), a cell line derived from spontaneous analysis by by analysis 316 13 CKM i.t. 1 , Step 9), 04 60 40 20 injection Days DC α COX2 inhibitori.t. s.c. DC and Ampligen and aspirin on days 8, 15, and 22, and followed for survival (not P -

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. ground as that of the experimental mice to avoid allogeneic allogeneic avoid to mice we CD11c protocol, use In this responses. immune experimental the of that as back ground genetic same the of mice from generated be should DCs design Experimental instead. used be may models tumor MHC-compatible relevant the with combination in strains mouse other mice, C57BL/6 the using developed been has protocol current the Although application. adjuvant after CKM d 4–5 performed typically is cells immune type-1 specific tumor- of Enumeration monitoring. immune and monitoring progression/survival tumor for used are mice experimental of sets Separate mice. tumor-bearing the of survival the prolong to cycles of DC therapy and CKM regimen should be repeated weekly isolated CD11c isolated ( mice donor tumor-bearing B16 Flt3L-expressing from isolated the the generation. DC cells. tumor matching with injected mice experimental and use the DCs generated to TILs induce into tumor-specific the Box Box Box 5 Box 4 mice) todetermine the frequencies of migrated DCs.  mice, starting atthe foot and pulling ittoward the body. 3. At 1,15,24,40,and 48hafterDCinjection ( 2. Inject50 specific experiments. 5. Resuspend the CFSE-labeledcellsin2 mlof culture medium and keep at37°Cfor no longer than 30–60minbefore use in 3 minat4°Ctowashthe cells. 4. Centrifuge the cellsat400 3. Add 10mlof coldculture medium and incubate cellsfor an additional 10minin the refrigerator. diluted CFSEreagent tothe cells. 10. Perform the flowcytometry. Analyze bygating onCD45.1 4% (wt/vol)paraformaldehyde. blocking buffer. After20–30minof incubation at4°C,washthe cellswith200 9. of blocking buffertocount and analyze byflowcytometry. 8. Wash the cellswith10mlof serum-free RPMImedium, centrifuge at400 7. Passage remaining pieces through an18-gauge needle and transfer/filter the cellsinto a15-mlconical tube. min. 15 for 50 add well, the in pieces small into node lymph the cut scalpel, a Using 6. 5. Placelymphnode in50 along the center of the backof the leg.Squeezethe knee joint area and the popliteallymphnode should popoutfrom the cut. 4. Holding the carcass firmlybythe foot and tail,stretch outthe legwiththe back facing up. Using ascalpel, make ashallowcut  2. Add 1 1. Resuspend the cells(from Step9E(iii);orfrom step1of P 1. ThawDCs, asdescribed inStep8,and adjustthe celldensity to1×10 P DC migration by flow cytometry

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n vitro in Flow analysis CR CR 1 ) to generate DCs. During the maturation, we expose the the expose we maturation, the During DCs. generate to ) I I T T I I CAL CAL µ l of CFSEreagent and incubate the cellsat37°Cfor 10min. | | generation Extraction ofcellsfrom thelymphnodesofmicetoinvestigate LabelingofthecellswithCFSE

µ STEP STEP l of CFSE-labeledDCs(

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cells to UVB Special attention should bepaid toavoiding disruption of the underlying muscle tissue. To perform staining with20×dilutedCFSE, firstdissolve1 14,8 2 µ of bone marrow-derived DC with with DC marrow-derived bone of l of serum-free RPMIina96-wellV-bottomplate. g γ for 3minat4°C,then resuspend the cells in10mlof culture medium, and centrifuge againat400 Tu cells (e.g., MC38 or ID8A cells) © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. Box 5 ) orDCsfrom Cd45.1mice (5×10 Fig. 2

splenocytes splenocytes d ● ), euthanizemice perIACUC protocol and remove the skinfrom the legsof the

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N ● G developed maturation cocktail maturation developed method in f oh CD8 both of sion B16 Flt3L or recombinant cells results in Flt3L-expressing expan either of Administration instead. done be can days consecutive 10 of injection daily Alternatively, Flt3L. recombinant of cost the and injections II Vaccine generation. Vaccine and/or ligands TLR cytokines to exposed and animals Flt3L-treated binant recom the or cells Flt3L-B16 with injected mice of spleens the the right flank of C57BL/6 mice C57BL/6 of flank right the in tissue subcutaneous into cells B16 Flt3L-expressing Weinject of of B16-Flt3L cells to enhance the efficiency and reduce the costs in vivo nity, DCs must be mature and produce high levels of IL-12 IL-12 of levels high produce (refs. and mature be must DCs nity, ducing capacity) of the mature DCs. To achieve translational translational DC vaccines achieve to clinical our Toanalogy DCs. mature the of capacity) ducing IL-12-pro (i.e., activity and functional the phenotype affect will

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6 ) cellpermlinculture medium. 58,122,12 g 13,61,70–77,81–8 5 for 5minatroom temperature, and resuspend in200 induction of intrasplenic DC accumulation after injection after DC injection accumulation intrasplenic of induction µ ) inPBSinto the footpad of the mouse. 8 N l of CFSEreagent in1mlof PBSand add 50 µ 2 was replaced with the mouse variant of our previously previously our of variant mouse the with replaced was µ l of collagenase to the well, and incubate at 37 °C °C 37 at incubate and well, the to collagenase of l G l of blocking bufferand resuspend in100–200 3 30–45min .CD11c ex vivo ex µ g of Flt3L subcutaneously to mice for 9–11 9–11 for mice to subcutaneously Flt3L of g α to generate type-1 polarized DCs. polarized type-1 generate to

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. type-1 polarized DCs. polarized type-1 1 rmIL- rmGM-CSF, cytokines murine recombinant the contains 342 DCs with UVB with DCs ity to migrate to draining lymph nodes ( nodes lymph draining to migrate to ity abil their is DCs of characteristic functional important Another flow-cytometry analysis for expression of markers such as IA-E as such markers of expression for analysis flow-cytometry sistency in and phenotypic functional characteristics. We perform con for vaccines DC of batch each test we mice, tumor-bearing DCs. tumor-loaded of QC vaccine. of batch single a of doses ~500 for suffice will QC testing, after which, DCs, to million 150 polarized DCs produce IL-12p70 upon stimulation with CD40L ( CCR7 and molecules co-stimulatory expressing have been matured ( ( H2K tor CCR7 (Giermasz CD86, and CD83) and the lymph node-homing chemokine recep of key co-stimulatory and maturation markers (e.g., CD40, CD80, DCs can secrete IL-12p70 while showing high levels expression of 16–48 to 4 h for optimal DC stimulatory capacity. The 4 h–maturedfrom reduced to be needed DCs mouse of period maturation the totic tumor cells (UVB cells tumor totic cross-present tumor antigens tumor cross-present and IL-12p70 of levels high produce that DCs polarized type-1 after injection. (BLI) imaging bioluminescence by traced be (CAG-luc-eGFP) can mice transgenic luciferase firefly from generated DCs of tion T cells T tumor-specific of numbers high induce cocktail type-1-inducing of type-1 immunity, tumor-loaded DCs matured in the presence of process maturation the completed have that Fig. 2 Box Box protocol β Box 6 unloaded CFSE 2. Labelthe tumor-loaded splenocytes withCFSEand the unloaded splenocytes with20×dilutedCFSEasdescribed inBox 5.Mixthe described inStep3. loaded platebackand forth toensure evendistribution of cells. After4hof incubation at37°C,harvesttumor-loaded splenocytes as for preparation of UVB  immunized mice (3–5dafterStep14)and untreated (control) tumor-bearing mice and atleast2naive (non-tumor bearing) mice. ( 4. Perform the flowanalysis. Make ahistogram of livecells expressing lowand high levels of CFSEto determine the specifickilling 3. Harvest spleens 16hafterinjection and isolatethe splenocytes asdescribed in 5. (Optional) Stainthe cellsfor CD45.1asdescribed instep9of CFSE 1. Isolate splenocytes from aB6CD45.1mouse asdescribed in P We recommend 5×10 per mland plate2mlperwellintwo12-wellculture plates. Immediately add 100 ● We observed that, compared with our human protocol In a single process using ten donor mice, we can generate up up generate can we mice, donor ten using process single a In We use a UVB a We use

, rmIFN Fig. 2 rocedure

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STEP In vivo G In vivo low γ | γ -Tu cells during their maturation to generate generate to maturation their during cells -Tu 2–3h 2018 high irradiation-based technique to prepare apop prepare to technique irradiation-based - and loaded CFSE The number of injected cells is not critical, although higher cell numbers facilitate their detection by flow cytometry. in vitro γ cultures of syngeneic splenocytes ( splenocytes syngeneic of cultures cells. Only0.05–0.5%of the cellsare expected tobeCD45.1 et et al. , rmTNF migration of DCs fromDCs B6 miceCD45.1 of or migration 6 | γ

cellspermouse. γ -Tu cells)tothe wellsof one culture plateonly. Leavethe cellsinthe other plateunloaded. Gently rock the natureprotocols -Tu cells) for antigen loading antigen for cells) -Tu immunomonitoring: 1 with Th1-inducing cocktail. Apart from 4 ; Before vaccine administration in in administration vaccine Before Fig. 2a 15,9 α , and to poly-IC convert DCs into 1 . high , b © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. γ ). -Tu cells is reduced in DCs DCs in reduced is -Tucells -labeled splenocytes ata1:1ratio inPBSto2.5×10 Box 12 3 7 ). . Thus, we load load we Thus, . Fig. 2 Fig. In vivo In 13,66,124,12 a 15,91,99,12 ), type-1 type-1 ), Fig. 2 Fig. migra in vivo- 12 c Box ). b 5 Box - - - - - 8 6 , ,

3 . Resuspend the splenocytes inculture medium at5×10 4 Mouse tumor models. immune response assaytype-1 ( cer vaccine development has been recently reviewed recently been has development vaccine cer prehensively be evaluated by the marker. CD45.1 Furthermore, the for the functionalstaining by activity of detected DCs be can mostcan comcells the as labeling, ( time over mice same the in monitored be can migration that is node-infiltrating cells. The advantage of using CAG-luc-eGFP DCs summarized by Mac Keon Mac by summarized activity, resistance tosuppressive apoptosis, antigenicity, chemokineof production),manipulation as(e.g., recently mecha questions nistic specific address to feasibility and costs their regarding colorectal cancer MC38 and cells) offer multiple ID8A advantages, especially cancer ovarian used have (we models transplantable immune editing, and immunosuppressive events. At the same time, chronic inflammation, development of immune tolerance, tumor is particularly relevant to the physiological situation, as they involve genetically manipulated mice or carcinogen-induced in models tumor tumor modelsinducible using immunotherapy of assessment are allowed to form metastases, reflecting the human disease human the reflecting and metastases, form to organ allowed are relevant the into implanted are lines cell tumor the is critical for effective immunotherapy. To promote the migra the Topromote immunotherapy. effective for critical is tissues. tumor into T cells tumor-specific of infiltration the Promoting immunotherapies. described mice, geneic C57BL/6J to which are susceptible the both similarly syn in intraperitoneal) and (subcutaneous implantation outcomes tumor MC38 of sites therapeutic alternative two involves and protocol our Therefore, progression these tumor microenvironments, affect immune can different to rise inoculation give tumor can of sites different As epi cancer. human ovarian of thelial characteristics the mirroring stages, late the in The mice ID8A tumor-bearing develop ascites massive malignant carboxyfluorescein succinimidyl ester (CFSE)-labeled DCs from DCs (CFSE)-labeled ester succinimidyl carboxyfluorescein C57BL/6J mice C57BL/6J Fig. 2 Fig. . Make ahistogram of liveCD45.1 specific cytotoxicity assay d ). Generation of CD45.1 of Generation ). Homing of effector type-1 immune cells into the TME TME the into cells immune type-1 effector of Homing

. µ Box l perwellof the prepared UVB 13 3 0 . can be detected by flow cytometry of lymph lymph of cytometry flow by detected be can 7 cellspermland inject200 The evolving role of animal models in can et al. et in vivo Fig. 2 13

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. tumor-bearing mice (1 × 10 the induction of tumor-specific TILs in MC38-tumor-bearing mice following DC vaccination in combination with CCL5-expressing vaccinia virus therapy. MC38- points (number of spots in each well) and bars (means ± s.d.). Statistical evaluation was done with ANOVA. ** of spots in MC38-challenged wells. Histograms represent pooled data of the triplicates from all the mice in the group. Results are shown as individual data marked MC38-challenged, whereas the lymphocytes in the top and bottom margin wells of each sample were left unchallenged. ( lymphocytes (splenocytes—bottom three rows and TILs—top four rows) from 12 mice (three mice per treatment group). Tumor cells were addedfurther to ELISpotthe analysis.wells Top and middle interfaces are enriched in myeloid cells and tumor cells. ( gradient centrifugation for IFN- data points (number of spots in each well) and bars (means ± s.d.). Statistical evaluation was done with ANOVA. ** number of spots in ID8A-challenged wells. Histograms represent pooled data of the triplicates from all the mice in the group. Results are shown as individual mice (4 × 10 showing the induction of tumor-specific TALs in ID8A-tumor-bearing mice following DC vaccination in combination with CKM treatment. ID8A-tumor-bearing et al. et ance of immunosuppressive and effector cells bal (reviewed local in the Devaud correct to order in TME, the alter and manipulate rial CKM adjuvants CKM rial combinato using chemokines effector-attracting of induction Alternatively, may the vaccination be the combined with selective ( booster 3 by d intratumoral later a second followed is vaccine subcutaneous first the In injection. DC of doses vidual TME of ( strategies two modulation developed we tumors, to CTLs of tion CCL5-expressing San Antonio. Animal experiments were approved by the IACUC of the University of Pittsburgh. ANOVA, analysis of variance; transformationPFU, plaque-forming ofunit; vv-CCL5,C57BL/6 mouse ovarian surface epithelial cells (MOSECs), was a generous gift from T.J. Curiel, University of DC Texastherapy with HealthCKM regimenScience resultsCenter in at an increase in tumor specificity. ID8A (luciferase-expressing), a cell line derived from spontaneous Figure 4 in triplicates, as shown in ID8A tumor cells. ( were harvested on day 13 and lymphocytes were analyzed for IFN- α combination of 3 × 10 8) only, treated with intratumoral injection of CCL5-expressing vaccinia virus (1 × 10 triplicates, as shown in MC38 tumor cells. ( were harvested on day 14 and lymphocytes were analyzed for IFN- ands (TLR-Ls). TLR-Ls trigger broad inflammatory responses that c a and Ampligen and COX-2 inhibitor celecoxib on day 8, treated with 3 × 10 No. of spots (5 × 105 cells) 13 1,000 200 400 600 800 5

). A potent way to manipulate TME is the use of TLR lig TLR of use the is TME manipulate to way ). potent A | 0

Monitoring of the immunotherapy-associated changes in the numbers of tumor-specific T cells in TME versus the spleen. ( Control Tissue –TIL 6 , 3 per group) were left untreated (control), treated with type-1 polarized DCs (DC, 3 × 10 Vaccinia i. 3 Fig. e c ) Results are shown as individual data points (number of spots in each well) and bars (means ± s.d.) of TALs from each mouse evaluated

) Results are shown as individual data points (number of spots in each well) and bars (means ± s.d.) of TILs from each mouse, evaluated in DC 5 myeloid cells Tumor and TILs/TALS cells Fat/tumor DCs s.c., day 5 and i.p. injection of IFN- a Figure 4 vv-CCL5 1 virus. . u bsc rtcl obns w indi two combines protocol basic Our ). 6 Figure 4 . Multiple chemokines can be targeted to targeted be can chemokines Multiple . 6

s.c., 3–4 per group) were left untreated (control), treated with type-1 polarized DCs (DC, 3 × 10 DC + vv-CCL5 Control MC38-challenged γ b ELISpot assay (Step 18). Following Percoll gradient centrifugation, the bottom interface of lymphocytes must be collected for . ( b d . ( ) To determine tumor specific responses, the average value of spots from control wells were subtracted from the number f ) To determine tumor-specific responses, the average values of spots from control wells were subtracted from the Control MC38- MC38- Control b

Control 0 Tissue –TIL Treatment groupandmousenumber DC vv-CCL5

α DC + vv-CCL5 and Ampligen and COX-2 inhibitor celecoxib on day 8. Peritoneal washes/ascites and spleens Fig. 3b vv-CCL5 γ γ production by ELISpot. The cells were either left unstimulated (control) or challenged with production by ELISpot. The cells were either left unstimulated (control) or challenged with *** , c ). 5 - - - - 1321321 DCs s.c., day 5 and additional dose of 3 × 10 200 400 600

Control 0 DC IC), and COX2 inhibitors (such as celecoxib or aspirin) ( aspirin) or celecoxib as (such inhibitors COX2 and IC), the combination interferons, of type-1 TLR ligands (such as poly- Ls are poly-IC (TLR3 agonist) and CpG (TLR9 agonist) (TLR9 CpG and agonist) (TLR3 poly-IC are Ls adaptive immune reaction immune adaptive the of activation the promote and immunity innate rapid elicit the effector cell-attracting chemokines and T and chemokines cell-attracting effector the both of composed response systemic nonselective substantial a Although systemic application of the individual TLR-Ls can induce selectively induces desirable chemokines desirable induces selectively and tissues tumor on activity their of focus the allows pathway the COX2and blockers of cytokines with ants, combination their 7 Splenocytes PFU) on day 11, or with the combination of both. Tumors and spleens DC vv-CCL5 DC + vv-CCL5 DC +vv-CCL5 ** *** b ) Representative image of ELISpot plate readout analyzing 32132 f

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) ELISpot data DC + IFN + polylC Splenocytes α DC + polylC DC + DC i.p. malignant | α 2018 + polylC DC + DC i.p. ** Fig. 137,13 |

343 3 ). 8 - .

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. cells REAGENTS M Another approach to selectively target tumor lesions 344 optimal therapeutic outcomes therapeutic optimal T tumor-specific enhance cells efficacy of intratumoral to achieve to approaches combinatorial in or modalities individual as used agents immunotherapeutic additional of efficacy DC vaccines, has been shown to reprogram the TME and increase or vectors viral of administration intratumoral using tumors, to Detection and quantification of tumor-specific immune immune temic immunization. tumor-specific of responses in the tumor quantification tissues compared with evaluation of sys and Detection • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • to stimulate multiple TLRs multiple stimulate to genetically manipulated viruses duction of effector cell–attracting chemokines by administration of Ampligen (polyI:polyC Poly-IC (Sigma-Aldrich, cat. no. P9582-5MG) Recombinant mouseIL-7 (research Miltenyi, grade; cat. no. 130-094-636) Recombinant mouseIL-2 (research Miltenyi, grade; cat. no.130-094-055) Recombinant mouseIFN- Recombinant mouse GM-CSF (research grade; Miltenyi, cat. no. 130-094-043) Recombinant mouseIFN- cat. no. 130-094-085) Recombinant mouseTNF- Recombinant mouseIL-1 RPMI-1640 medium (Gibco, Invitrogen, cat. no. 61870-010) Laboratories, cat. no. PK6100) ELISpot kit(VectorAEC substrate Laboratories, cat. no. SK-4200) (Vector ABC reagent (Vector Laboratories, cat. no. PK-6100) AN18 anti-IFN no.3321-6-250) cat. anti-IFN- R4-6A2 OVA SIINFEKL(Genescript, peptide cat. no. rp10611) luciferin from light. ! 2 pyruvateSodium (Gibco; Invitrogen, cat. no. 11360-039) Non-essential (NEAAs; aminoacids Gibco; Invitrogen, cat. no. 11140-035) Penicillin–streptomycin (10,000 U/ml; Gibco; Invitrogen, cat. no. 15140-114) l Trypan blue (0.4%(wt/vol); Corning Cellgro, cat. no. MT-25-900-CI) (Sigma-Aldrich,Aspirin cat. no. A5376-100G) Celecoxib (Biovision, cat. no. 1574100) gen issaferpoly-IC. andmorewith in specific comparison ! Ethanol (diluted 70%(vol/vol); PittPharmco, cat. no. 111000200CS) Luciferin (GoldBiotechnology, cat. no. Luck-1g) chemical fumehood. Avoid skin, contact with eyes, andclothing, gloves ina itwith andhandle cat. no. C34554) Carboxyfluorescein succinimidyl ester (CFSE; Life Technologies, EDTA (500mM; Life Technologies, cat. no. 15575-020) CulturePBS (Cellgro Cell PBS(1×); Corning, cat. no. MT-21-040-CV) Percoll (Sigma-Aldrich, cat. no. P1644) cat. no. 130-096-730) Tumor Kit D, Dissociation (enzymes R, and A; Miltenyi Biotec, no.C0130-100MG) cat. Sigma-Aldrich, stock; (wt/vol) (10% Collagenase Mouse CD11c microbeads (Miltenyi Biotec, cat. no. 130-052-001) ACK lysis buffer (Life Technologies, cat. no. A10492-01) FBS (Gemini Foundation B, cat. no. 900-208) chemical fumehood. Avoid skin, contact with eyes, andclothing, gloves ina itwith andhandle skin, swallowed, andif anditishazardous to theaquatic environment. protocol ATER

-Glutamine ( β The presented DC vaccination and CKM approaches may be be may approaches CKM and vaccination DC presented The CAUT CAUT -mercaptoethanol (2

| 13 VOL.13 NO.2VOL.13 9 I I . Directing cytokines (e.g., IFN- (e.g., cytokines Directing . I ON ON ALS Ethanol isflammable, anditmayeye causeskinand irritation. β -mercaptoethanol istoxic uponinhalation, uponcontact with l γ -glu; Gibco; Invitrogen, cat. no. 25030-024) -coating (Mabtech, antibody cat. no. 3321-3-1000)  γ |

2018 biotinylated detecting antibody (Mabtech, (Mabtech, antibody detecting biotinylated CR I T 12 β I The ME; Sigma-Aldrich, cat. no. M3148) U; Hemispherx Biopharma) CAL β | α γ α

(research Miltenyi, grade; cat. no. 130-094-053) (research Miltenyi, grade; cat. no.130-094-048) natureprotocols (research Miltenyi, grade; cat. no.130-093-131) (research Miltenyi, grade; Protect thereagent from light. in vivo

14 1 and predominantly infect cancer cancer infect predominantly and 136,14 145,14 tumor-specific cytotoxicity assay 0 6 © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. . Oncolytic viruses are known . α and IL-12) specifically specifically IL-12) and 

CR  I

CR I 76,142–14 CAL 13 I T I 9 Protect the

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- - Antibodies for flowAntibodies cytometry ( mice tumor-bearing in response immune tumor-specific temic sys the of magnitude the determine to method straightforward using i.v. injection of a 1:1 mixture of nonloaded CFSE nonloaded of mixture 1:1 a i.v.using of injection • • • • • • • • • • • • • • • • • • • • • • • • • • Cells and antigen-loaded CFSE antigen-loaded and defined peptide-loaded target cells. target peptide-loaded defined relevant antigens, such as whole cancer cells ( by followed centrifugation, gradient Percoll subsequent and digestion tissue using ascites) malignant and tissues primary solid (e.g., tissues cancerous from cells immune of isolation the involves protocol numbers of tumor-specific cells in splenocytes in cells tumor-specific of numbers sensitive highly a ex vivo developed we issue, this address To tissues. tumor within cells T tumor-specific of numbers the in increase the promote to therapies cancer new of effectiveness the of ation Box expressing EL-4-B5 cells are available from Kerafast (cat. no. EVU301); froma gift P. Lane, University Birmingham. of Alternatively, CD40L- H or inhaled. Avoid skin andeyes, contact gloves. with itwith andhandle with skin, swallowed upon isflammable andisharmful strate contact or if Sciences, cat. no. 15714-S) Paraformaldehyde, solution 32%(wt/vol) (PFA; Electron Microscopy fume hood. skin,contact with eyes, andclothing, gloves inachemical itwith andhandle flammable, anditmay causesevereeye and skindamage.burns Avoid Flt3L-expressing B16melanoma cells it. working with chemical andwear fumehood protective gloves you andamaskwhen are  fromgift M. Kronenberg, LaJolla Institute for andImmunology Allergy  with skin and eyes and handle it with gloves. itwith skinandeyes andhandle with swallowed or inhaled. skin, if upon contact with of Avoid contact IgG IgG2 IgG-PE (Armenian hamster IgG, 0.2mg/ml; BioLegend, cat. no. 400908) IA/E CCR7-PE (IgG CD14-FITC (IgG CD86-APC (IgG CD40-APC (IgG no.104714) cat. BioLegend, mg/ml; 0.2 IgG, hamster (Armenian CD80-APC CD45.1-APC (IgG IgG CD40L-transfected J558 (J558-CD40L) (Balb/c mouse strain) cells mousestrain) (Balb/c CD40L-transfected J558(J558-CD40L) section. design (Invitrogen, cat. no. intheExperimental 14-8001-80)asdescribed  authentic mycoplasma. andare notinfected with dH BSA (Sigma-Aldrich, cat. no. A9647) IL-12 ELISAKit (R&D, cat. no. DY419) KH ELISA substrate (FisherELISA substrate Scientific, cat. no. ENN301) ! Na NaCl (Sigma-Aldrich, cat. no. S7653) 10× PBS(Cellgro PBS(1×); Corning, cat. no. MT-46-013-CM) Tween 20(Fisher Scientific, cat. no. BP337-500) H2K IgG

CAUT 2

CR CR CR SO 2 2 2 1 2a 1 HPO O (Life Technologies, cat. no. 10977) PO -APC (IgG -FITC (IgG b b a 6 I I I -FITC (IgG -FITC (IgG -FITC (IgG -APC (IgG 4 T T T IFN- and and (Sigma-Aldrich, cat. no. 339741) I I I I 4 ON CAL CAL CAL (Sigma-Aldrich, cat. no. P5655) 4 ·2H ELISpot Substrate ELISpot reagent isflammable andisharmful B16-Flt3Lcells replaced can be recombinant with Flt3L cell regularly The linesshouldbe checked to ensure are they Protect from theantibodies light. Fig. 2 Fig. γ ex vivo ex 2 ELISpot-based protocol us to allowing ELISpot-based compare the O (Sigma-Aldrich, cat. no. 71643) 2a 1 1 , BDBiosciences, cat. no. 555751) 2a 2a 2a , 0.2mg/ml; BioLegend, cat. no.120106) 2a , 0.5mg/ml; BDBiosciences, cat. no. 553953) 2a 2a 1 , 0.5mg/ml; BDBiosciences, cat. no. 553739) , 0.2mg/ml; BioLegend, cat. no. 400219) , 0.2mg/ml; BioLegend, cat. no. 105012) , 0.2mg/ml; BioLegend, cat. no. 124612) 2a , 0.5mg/ml; BDBiosciences, cat. no. 554688) , 0.5mg/ml; BioLegend, cat. no.114406) , 0.5mg/ml; BioLegend, cat. no. 116505) e , 0.2mg/ml; BioLegend, cat. no. 110714) ). However, this strategy does not allow evalu allow not does ).However, strategy this exposure of the isolated immune cells to the the to cells immune isolated the of exposure !

CAUT high splenocytes I ON 14 PFA isahazardous reagent; usea 7 (C57BL/6Jmousestrain), a !

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5 Fig. ( Sulfuric acid is highly ishighly Sulfuricacid ! Boxes

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. protocol are indicated in  Mice immune responses andtumor rejection. as themice injected, be into which thecells will to avoid any allogeneic • • • • • • • • • • • • • • • • • • • • • • • • • • •  EQUIPMENT C57BL/6J (The JacksonC57BL/6J (The Laboratory, cat. no. 000664) FVB-Tg(CAG-luc,-GFP)L2G85Chco/J—backcrossed on theC57BL/6 line Institute (protocol 14063339). Care andUse Committee (IACUC) attheUniversity Cancer Pittsburgh of animalstudies reportedThe here were approved by theInstitutional Animal committee. animalethics thelocal relevant guidelines andregulations of institutional andgovernmental animalprotocols andcomply the with procedures involving shouldfollow animalexperiments approved nos. 305195, 305155, 305109) 305109) 305155, 305195, nos. cat. Biosciences, (BD gauge 27 gauge, 22 gauge, 18 Needles, care.Handle them with Science Center atSan Antonio cells,epithelial from wasagift T.J. Curiel, University Texas of Health in vitro ID8A (luciferase-expressing), acell linederived from spontaneous mycoplasma.and are notinfected with cell regularlyThe linesshouldbe checked to ensure are they authentic  substituted for theCD40L-expressing cells, inStep 9B(iv). asdescribed (MEGACD40L; Enzo Life Sciences, cat. no. ALX-522-120-C010) canbe 0.5-ml Syringes (BDBiosciences,0.5-ml Syringes cat. no. 305620) care. with Scissors (Fisher Scientific, cat. no. 08-951-20) Forceps (Fisher Scientific, cat. no. 08-880) platesELISpot (Millipore, cat. no. MAHAS4510) no.T2795-1000EA) cat. Sigma-Aldrich, ml; (2 tubes centrifugation Eppendorf Conical (15ml; tubes centrifuge Denville, cat. no. c1018-p) Conical (50ml; tubes centrifuge Denville, cat. no. C1060-p) (70 strainers Cell (100 strainers Cell Corning, cat. no. 07-200-35) High-binding 96-wellplates for ELISA(Costar 96-wellEIA/RIAplates; cat. no. CLS3894) Sterile cell culture plates (96well, V-bottom; Corning Costar, no. t1096) cat. Denville, flat-bottom; well, (96 plates culture cell Sterile Sterile cell culture flasks(75mm Sterile cell culture plates (12well; Denville, cat. no. t1012) 12 ×75-mmPolystyrene (Falcon, tubes cat. no. 14-959-11B) Alcohol prep (Fisher pads Scientific, cat. no. 22-363-750) 50-ml Pipettes (sterile, Denville, wrapped; individually cat. no. p7135) 10-ml Pipettes (sterile, Denville, wrapped; individually cat. no. p7134) 5-ml Pipettes (sterile, Denville, wrapped; individually cat. no. p7133) 2-ml Pipettes (sterile, Denville, wrapped; individually cat. no. p7132) 14063339). (protocol Institute Cancer Pittsburgh of University the at IACUC the by approved were here reported studies animal The committee. ethics animal local the of regulations and guidelines relevant the with comply and protocols animal governmental and institutional approved follow should experiments mycoplasma.with regularly checked to ensure are they authentic andare notinfected ! no.(cat. 008450) Laboratories Jackson Wurzburg,the of from or Hospital B6.SJL- University Cancer Pittsburgh Institute of (protocol 14063339). tee. animalstudies reported The here were approved by the IACUC atthe commit animalethics thelocal the relevant guidelines andregulations of approved institutional andgovernmental animalprotocols andcomply with expressing) colorectalMC38 (C57BL/6Jmousestrain) cancer cells (luciferase- authentic mycoplasma. andare notinfected with for however, we have never them. used cat. no. 002014) no. 002014) cat.

CR CR CAUT

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14 generations I I T CAL CAL I malignant transformation of C57BL/6 mouse ovarian surface C57BL/6mouseovarian of transformation malignant I Ptprc ON CAL Donor and experimental mice used in separate steps of the Donormice andexperimental inseparate used steps of background thesamegenetic Cancer cell of linesshouldbe proceduresAll involving shouldfollow animalexperiments 14 The cell regularly The linesshouldbe checked to ensure are they a 9 Pepc (available from Kerafast, cat. no. ENH204) ! µ

b CAUT µ m; Falcon, cat. no. 08-771-2) /BoyJ (C57BL/6 CD45.1; The Jackson Laboratory, Laboratory, Jackson The CD45.1; (C57BL/6 /BoyJ m; Falcon, cat. no. 08-771-19) 15 0 —can be obtained from obtained —can be A. Beilhack, University Table I ON All procedures involving animal animal involving procedures All 1  ! andlisted below.

2 CAUT

; Falcon, cat. no. 13-680-65) CR  I T

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different vendors are used. We prepare all cytokines as 100× or 1,000× 1,000× used. are or vendors Wedifferent 100× as cytokines all prepare from cytokines when made be must companies. specific Adjustments from cytokines specific to Wechallenging. referring maybe information provide activity companyfrom company, to of greatly measurement that suggesting available. not is Valuesinformation this differ can cytokines, some for activity. However, functional of basis the on comparison for form correct the DC maturation medium maturation DC in indicated vary, as concentrations months.The 6 is time storage recommended mum 1.051 g/ml) PBS from 13.5 g NaCl, 0.125 g of Na of g 0.125 NaCl, g 13.5 from PBS g/ml) 1.051 KH • • • • • • • • • • • • • • • • • • • • tion medium before use. • • • • Cell cultureCell medium °C. 37 at it keep and use before SIP prepare Freshly (SIP). solution Percoll isotonic standard (vol/vol) 34% and (vol/vol), 45% (vol/vol), 60% prepare to medium culture with diluted further and 10:1 PBS acidic with mixed be must Percoll months. 3 is time storage recommended maximum The aliquots. 4-ml in °C 4 at store and before to adding thecultures. toadding themedium. Allow theprepared medium to equilibrate to 37°C arecytokines stored at isolated CD11c depends onneeded thenumber of medium before maturation.maturation uptheDC setting volume The at aliquots storethe and solutions stock concentrated Cytokines store at4°C. maximum The recommended storage is1month. time (14.3 mM)2 (vol/vol) (1mM)sodiumpyruvate, 1%(vol/vol) NEAAs, 0.1%(vol/vol) 1%(vol/vol)1640 medium with (100U/ml)penicillin–streptomycin, 1% solution Percoll isotonic Standard at years 2 to up for stored be can serum The avoided. be should cycles Freeze–thaw again. frozen and should tubes 50-ml aliquots in serum prepared be then and °C, 56 at min 30 for heat-inactivated and at Supplement asindicated in cytokines culture medium with isolated from DCs medium isrequiredration one spleen. of for maturation FBS REAGENT SETUP UVB irradiator. agamma with is hazardous. Appropriate measures taken safety shouldbe working while Pipetman (P10-P100; Gilson,Pipetman cat. no. F123615G) (Eppendorf,Centrifuge no. model 5810R) cat. no. 51000001) Temperature-controlled freezing container Scientific, Thermo (MrFrosty; In vivo ELISA reader (Perkin Elmer, no. model Victor 2) readerELISpot (ImmunoSpot, Cellular Technology) usingaUVsource.while hazardous. Appropriate personal protective equipment worn shouldbe Multichannel pipette (20–300 3121.000.020, 3121.000.054, 3121.000.062) Pipettes (0.5–10 Scientific) (Drummond Pipet-Aid (P100-P1000; Gilson,Pipetman cat. no. F123602G) QuadroMACS separator (Miltenyi Biotec, cat. no. 130-090-976) MACS MultiStand (Miltenyi Biotec, cat. no. 130-042-303) FACS (Accuri instrument or Fortessa; BDBiosciences) Microscope (Carl Zeiss, no. model Axiovert 200) Scientific, cat. no. 02-671-10) Countess cell counter (Invitrogen) or Neubauer hemocytometer (Fisher Pasteur pipettes (Denville, cat. no. p0458-9) Sterile beakers (Pyrex, cat. no. 02-540M) Scientific, (Thermo cat.Cryovials no. 12-565-164N) 1,000 200, (10, tips Pipette (BDBiosciences,10-ml Syringe cat. no. 309604) (BDBiosciences,3-ml Syringe cat. no. 309585) Gamma irradiator (NordionGamma irradiator International) Kendro, no. model Heracell 150) incubatorCell (temperature

− 2

20 °C. Individual bottles should be slowly thawed (at 4 °C overnight) overnight) °C 4 (at thawed slowly be should bottles Individual °C. 20 We purchase the serum in frozen 500-ml bottles and store them directly We and store directly them bottles in the frozen serum 500-ml purchase γ light (Spectroline, light no. model EB-160C) 4 bioluminescence imager (IVIS; Perkin Elmer) , and 200 ml dH ml 200 ,and

Many cytokine concentrations are indicated in IU/ml, which IU/ml,which in indicated are concentrations Manycytokine Table β ME, 1%(vol/vol) (2mM) µ 2 l, 20–200 .

To prepare cell culture medium, supplement RPMI-

−

20 °C and should be completely20 °Candshouldbe thawed atRT before

2 O. Sterilize acidic PBS by 0.22- by PBS acidic O.Sterilize To DCs, polarized prepare generate effector type-1 natureprotocols µ µ 

= l, 100–1,000 l; MidSci, cat. nos. nos. AV10R-H, cat. MidSci, l; AV200, AV1000) µ

37°C, CO CR l; Scientific, Thermo cat. no. 4661140) I T

I Prepare 10× acidic (pH: 4.6, 4.6, (pH: acidic 10× Prepare CAL l -glu, and10%(vol/vol) FBSand Freshly prepare matura theDC 2 µ level l; Eppendorf, cat. nos. !

CAUT !

CAUT |

2 = VOL.13 NO.2VOL.13

+ HPO

5%(vol/vol); Thermo/ cells—~10 ml of matu cells—~10 mlof I ON

− − I ON 4 Gamma irradiation Gammairradiation

20 °C. The maxi °C. 20 The 20 °C. 20 ·2H protocol µ UV light is UVlight m filtration, filtration, m Table 2 O, 2.1 g O, 2.1 | 2018 2 . The

- 345 is is - - © 2018 Nature America, Inc., part of Springer Nature. All rights reserved. DCs. When using 0.3×10 CFSE reagent tor solutions before use. models). ligen in PBS for injecting 50 COX-2 inhibitors mouse (for i.p. tumor models) and 2 × 10 through a0.22- 500mMEDTAby 1mlof adding to 500mlPBSandsterile-filter thesolution EDTA inPBS recommended storage is1month. time lyophilized of to CFSE. thevial Store CFSEreagent at 200 injecting working solutionsaspirin) inPBS immediately before (for administration (12.5% (vol/vol) DMSO, celecoxib) and2.5mg/ml(1.25%(vol/vol) DMSO, maximumThe recommended storage is6months. time We make 500 inDMSO andstore aspirin 200 mg/mlstock aliquots at solution of 10 CKM adjuvants for modulation theof TME storage is1month. time (20% (vol/vol)). Store at4°C. media both maximum The recommended RPMI-1640 medium (40%(vol/vol)), FBS(40%(vol/vol)), andDMSO 50%(vol/vol)1640 medium with FBS. Freezing medium 2(FM2)contains Cell-freezing medium a T 346 MACS buffer is3months.time One donor mouse isexpected toyield ~30–40×10 a and 18; sion and survival (Steps 12, 13, Monitoring of the tumor progres 17–24; 17–24; Ex vivo In vivo Fig. 2 tumor-specific immunity (Step 9E; In vivo E 9B; 9B; Analysis of IL-12 production (Step (Step 9A; Phenotype analysis of the DCs (Step (Step 9D; In vivo (Step 9C; type-1 immune cell induction In vitro protocol 4 xperimental purpose b IU/ml IFN-

le le | VOL.13 NO.2VOL.13 Fig. Fig. 2 1 1 e immunomonitoring (Steps cytotoxicity assay ( evaluation of systemic evaluation of DC migration  ) evaluation of effector Fig. Fig. | Fig. Fig. 3c

CR Donor and experimental mice used in separate steps of the protocol. Fig. Fig. 2 b Fig. Fig. 2 Box µ

) I For MACS buffer BSAand 2mMEDTA (0.5%(wt/vol) in For tumor cells, detachment of prepare 1mMEDTA inPBS l per mouse).l per Prepare CFSEreagent by inthedark 18 adding 4 T α µ I ) and 0.25 mg/ml Ampligen in PBS for injecting 200 m filter. Store at4°C.The maximum recommended storage CAL 4

, c We prepare celecoxib a4mg/mlstock solution of anda 6 ; ; d a | type-1polarizedDCsperinjection, one donor mouse allowsfor generation of DCsfor ~35–40vaccinations. ) 2018 ) ) Fig. Fig. 2 Freshly prepare the CMK adjuvant solutions before use.

For freezing medium 1(FM1), supplement RPMI- | d µ

 natureprotocols ) l intratumorally per mouse (for s.c. MC38 tumor

CR Box I T I 6 CAL - ) 6 CD11c 5 Freshly prepare theCOX-2 inhibi IU/ml IFN- © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. loaded DCs) C57BL/6J mouse (UVB loaded DCs) C57BL/6J mouse (UVB loaded DCs) C57BL/6J mouse (OVA- loaded DCs) C57BL/6J mouse (UVB loaded DCs) C57BL/6J mouse (UVB DCs) mouse (UVB CAG-luc-GFP or B6 Cd45.1 loaded DCs) C57BL/6J mouse (UVB loaded DCs) C57BL/6J mouse (UVB

We prepare a solution 5of × +

cells. ~1×10 a D C vaccine generation

α − and 1.0 mg/ml Amp

20 °C. maximum The 6 γ UVB -Tu-loaded γ -Tu cellsare needed for loading of 30–40×10 µ l of DMSO l of

µ − l i.p. per γ γ γ γ γ γ

20 °C. µ Donor mice -Tu- -Tu- -Tu- -Tu- -Tu- -Tu- g/ml

- - splenocytes) B6 B6 Cd45.1 or C57BL/6J mouse (OVA-loaded CFSE CFSE mouse (UVB B6 Cd45.1 or C57BL/6J C57BL/6J C57BL/6J mouse Luciferin Freshly prepare thedigestion buffer before use.  by onereagents adding drop washbuffer. each of A andBto 10mlof ABC reagent mended storage is3months. time Tweenof 20. Store atroom temperature (20–23 °C). maximum The recom Digestion bufferDigestion ommended storage is6months. time PBS. 35mlof Store solution32% (wt/vol) with at4°C. maximum The rec Paraformaldehyde solution 4%(wt/vol) maximumThe recommended storage is6 months. time cold PBSandfreeze 33.3mlof at with in thedark D, 50 constitution information). Per digestion medium, 2.5mlof 100 KitDissociation atroom temperature manufacturer’s (see for re instructions dH 9liters of in PBS)by 10×PBSwith diluting 1liter of Wash buffer 1 month. a 0.22- PBSandsterile-filter thesolution BSAto through 50mlof 1.5gof adding Blocking buffer maximumThe recommended storage is3months. time EDTA andsterile-filter thesolution through a0.22- 500mM BSAand2mlof 2.5gof PBS), PBSwith supplement 500mlof Induction of type-1

+ CR

splenocytes) effector cells I µ T µ l of enzyme R, enzyme and12.5 l of I m filter. Store at4°C.The maximum recommended storagetime is CAL

γ Thaw luciferinatroom temperature. Reconstitute luciferin 1gof -Tu loaded Freshly prepare ABC reagent before use.

We prepare washbuffer (0.05%(vol/vol) 10liters of Tween 20 Prepare avidin–peroxidase complex (Vector Laboratories) 6

CD11c

Prepare blocking buffer BSAinPBS)by (3.0%(wt/vol) Thaw D, digestion enzymes R, and A from theTumor

cells, which should yield ~15×10

out out DC vaccination) C57BL/6J mice 3–5 3–5 C57BL/6J mice injection of DCs) C57BL/6J mice 3–5 C57BL/6J mice i.p. i.p. vaccine/cycle; 1–2 cycles) 3–5 C57BL/6J mice (1 s.c. vaccine naive C57BL/6J mice 1 i.p. vaccine/cycle; 1–2 cycles) 7–10 C57BL/6J mice (1 s.c. vaccine 1 i.p. vaccine/cycle; 3 cycles) 8–10 C57BL/6J mice (1 s.c. vaccine µ recommended number of mice per per ofmice number recommended l of enzyme enzyme A are required.l of E xperimental mice (and (and mice xperimental experimental group) experimental

Dilute 5 ml of paraformaldehydeDilute 5ml of 6 type-1polarizedantigen-loaded

− µ

80 °Cin1-mlaliquots. m filter. Store at4°C.

+ +

2 control (with 2 control (no 2 O and adding 5ml O andadding 

CR µ l of enzyme enzyme l of I T I

CAL +

+ +

1 -

- © 2018 Nature America, Inc., part of Springer Nature. All rights reserved. example, if mice need toreceive three cyclesof vaccination and twovaccine doses percycle, then freeze the DCsinatleast to freeze the DCsinatleast asmany cryovials asthe number of planned vaccine doses plusanadditional vial for QC.For frozen percryovial depends onthe number of planned vaccine doses and the number of mice receiving the vaccine. Prepare 6| of DCmaturation medium, add 100 the DCcultures for the density and general appearance of the cells, using aninvertedmicroscope. Immediately afteraddition 1 mlof cellsuspension toeachwellof a12-wellculture plate. Add 1mlof DCmaturation medium toeachwell.Examine 1| 3| be extended to4.5hinthe casethatmany plates must beharvested. viability isbelow50–70%,werecommend discarding the batchof DCsand repeating Steps1–5. the UVB 2| tumor antigens bymaturing DCs.  cells tothe maturing DCs. loading (see 4| 7 (2×3 C 5| C Generation of type-1polarizedmouseD PROCE  at 37°Cand 5%(vol/vol)CO  of the DCsinbatches sothatthe platesare not kept at4°Cfor longer than30min,asthismight affectDCfunction. ? again and collectthe cellsinthe 15-mltube. Repeatuntil allcellsare collected. cold PBSand check for remaining adherent cellsusing amicroscope. Ifsome cellsremained attached, vigorously pipette at 4°Cisimportant toreduce the adherence tothe plastic and recover high numbers of viable DCs. polarized mature DCsare more adherent tothe plastic, displaying more elongated morphology. Incubation of the plates T a IFN- IL-1 TNF- Poly-IC C IFN- GM-CSF ollection ollection of mature type-1 polarized mouse D ryopreservation and thawing of type-1 polarized mature D

TROU

ytokine b

CR CR CR le le Keep the harvestedtype-1 polarizedmature DCsat4°Cwhilepreparing for cryopreservation. The number of DCs Centrifuge the cellsat400 Detach DCsbyvigorous pipetting and collectthem ina15-mlconical centrifuge tube. Refilleachwellwith1ml of Transfer the platestoa refrigerator for 5–10min.Depending onthe number of plates, youmay perform harvesting Gently rock the platebackand forth toensure evendistribution of cells. Placethe culture platesinthe cellincubator Resuspend the CD11c β α γ α I I I T T T 2 2 D I I I B URE γ | CAL CAL CAL

LES -Tu-loaded DCs(orunloaded DCs)into one tube. Assess viability of the cellsbyTrypan blueexclusion. Ifthe +

DC DC growth factors and DC maturation factors.

1)cryovials. Freeze atleast1×10

H STEP STEP STEP Box OOT 2 Add UVB Ensure thatthe same cancer cellline thatwillbe injected into the experimental mice isusedfor DC Incontrast tothe classic cytokine-based maturation cocktail(supplemented withPGE I N for preparation of UVB G

+ 0.1% 0.1% (wt/vol) BSA in PBS 0.1% 0.1% (wt/vol) BSA in PBS γ

cells(see -Tu cellswithin10minafteraddition of DCmaturation medium toensure optimal take upof 2 20 20 mM acetic acid for 4h.The target maturation time is4hbefore harvesting, butthe maturation period can g for 5minat4°C.Resuspend cellpelletsinalltubeswith1-mlof coldPBSand combine RPMI-1640 © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. Diluent dH dH µ l of 1×10 2 2 Box O O γ C 1 Tu cells) s for CD11c ● 6 DCspercryovial.

6 T UVB IMI C s s . To generate control unloaded type-1polarizedDCs, do not add UVB ● N γ S

G -Tu cellspermlwell(40:1CD11c

+ tock concentration T

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. strong predictor of their overall functional status. tumor-specific type-1immunity represents the most definitive test ofDCfunction, the analysis ofIL-12 production isavery DC migration, and option Efor production, option Cfor Place the cells on ice for 10 min and add the same volume of FM2. Place the cells in a temperature-controlled freezing freezing temperature-controlled a in cells the Place FM2. of volume same the add and min 10 for ice on cells the Place for settingupthe ( (B) ( 9| Quality control of the generated D  at store and Frosty) Nalgene,Mr (e.g., container 7| 348 8| 400 after thawing. Spindown the DCs(5min,400 protocol C A (iii) (iii) (iii) (vi) (iv) (iv) (vi) (iv) )

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g | In vitro A P VOL.13 NO.2VOL.13 To check the quality of the DCs, follow option Afor phenotype analysis of the DCs, option Bfor analysis of IL-12 To thawthe cells, placethe vials inawaterbathat37°Cand transfer the cellstocoldPBS(20ml)immediately 400 at DCs mature polarized type-1 the Centrifuge , 4°C).Resuspend the cellsin1mlof culture medium and count the cells.

henotype analysis of theD nalysis of I and 5%(vol/vol)CO for 5minat4°C. 10 1 of CCR7-PE,and 2.5 1 1 1  cells. (MEGACD40L, 1 times. 1 month. (37 °C,5%(vol/vol)CO with DCs. Add 100 Wash the wells byadding 200 Prepare the ELISpot plate. To do this, add 100 On day 4of culture, add IL-2(25U/ml)and IL-7 (0.5ng/ml) tothe DC–splenocyte co-cultures. Prepare the splenocyte suspension ( Add 0.25mlof tumor-loaded DCsincellculture medium toeachwellof a48-wellplate(4.0×10 Adjust the celldensity of the type-1polarizedmature DCsto1×10 Perform IL-12ELISA following the manufacturer’s instructions ( Prepare 5×10 Add 100 Adjust the celldensity of the type-1polarizedmature DCsto1×10 Perform flowanalysis following the instructions of the flowcytometer manufacturer ( Discard the supernatant and add 100 Discard the supernatant, add 200 Add 200 Prepare fivestaining mixes byadding the following antibodies to100 Adjust the celldensity of the type-1polarizedmature DCsto1×10 and potency. and 1 × 10 × 1 ELISpot plateinPBS,wrap the platewithParafilm, and incubate overnight at4°C. buffer and resuspend in100 a 96-wellplateand mix.After20–30minincubation at4°Cinthe dark, washthe cellswith100–200 Start cultures of J558-CD40L cells in cell culture medium at 2 × 10 × 2 at medium culture cell in cells J558-CD40L of cultures Start µ µ µ µ

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cellspermlof J558-CD40Lcellsinculture medium and add 100 µ S natureprotocols Supernatants may bestored at pot plate, and~5hfor developing the g/ml; EnzoLifeSciences, cat.no. ALX-522–120-C010) (ref. in vitro µ 6 µ 2 l of culture medium tothe remaining 3wellsfor controls. Placethe culture plateinanincubator µ cells per ml for at least 3–5 d after thawing before their use, to ensure their high viability viability high their ensure to use, their before thawing after d 3–5 least at for ml per cells for 7–10d. l of IgG-PE, 2.5 l of CD80-APC(mix1); 2 µ in vivo ) and collectthe media from the cellsafter24h.Alternatively, solubleCD40Lwithenhancer evaluation of effectortype-1immune cellinduction, option Dfor l of CD86-APC(mix3); µ µ © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. C l of IgG l of CD40-APC(mix2); s ● µ C ● l of 4%(wt/vol)PFA. µ

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. (D) imaging ( imaging of instead analysis cytometric flow by migration DC of evaluation perform must you mice, C57BL/6J from or mice, Cd45.1 (ref. 008450) no. cat. Laboratory, Jackson The Tg(CAG-luc,-GFP)L2G85Chco/J; O ( 

(xvii) (xiii) (viii) (xvi) (xiv) E (xii) (vii) V (iii) (xv) (iii) (iv) (xi) (ix)

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T mal handling and care toimage the mice. 1-cc syringe and a27-gauge or30-gauge needle) before imaging. Followthe instructions outlined inStep12for ani with a1-ccsyringe and a 27-gauge or30-gauge needle into the flank of naive mice. C57BL/6J mouse. ABC reagent according to the manufacturer’s instructions. PBS/0.5% (wt/vol)BSA and incubate atroom temperature for 2h.10minbefore the incubation isfinished, prepare (PBS/0.05% (vol/vol)Tween 20).Afterthe washes, add 100 the cellsusing ahemocytometer. the cellsat400 at 400 at 1.0 PBS to35–40mland centrifuge at400 medium to3wellsof control DCsplenocytes. Incubate the cultures at37°C,5%(vol/vol)CO  plate is72hat4°C.  37° C;however, such incubation islessefficient. ing the splenocytes inthe next steptoensure maximal efficiency. Alternatively, the plate may be coated for 2hat load at 0.5 0.5 at load to tubes the of one in splenocytes to OVA peptide Add tubes. centrifugation conical 15-ml two into divide both tubes at 37 °C for 1.5–2.0 h. Fill the tubes with PBS to 12–15 ml and centrifuge at 400 at centrifuge and ml 12–15 to PBS with tubes the Fill h. 1.5–2.0 for °C 37 at tubes both Cd45.1 mouse as described in in described as mouse Cd45.1 room temperature. room allow the platetodry. Readthe plateusing anELISpot reader ( visible, butbefore background appears, rinse the platethoroughly indH unloaded CFSE 100 room temperature for 5minand resuspend the irradiated tumor cellsat4×10 Label loaded splenocytes withCFSEand unloaded splenocytes with 20×dilutedCFSEasdescribed in Resuspend the cellsinPBSat3×10 Resuspend the unloaded DC cells(from Step8)inculture medium at1×10 Image mice byIVIS after1,12,24,and 48h( Inject 50 Adjust the celldensity of UVB Add 100 Wash the platethree times bysoaking 3–5minin wash buffer, followed byanadditional twowashes inPBS.While Wash the platefivetimes asinStep9C(xiii),add 100 Mix the ABCreagent and letitstand atroom temperature for 30min. Add 100 Resuspend the tumor cellsat1×10 Harvest the tumor cells(usethe tumor cellline thatwasusedfor loading of DCs)inculture with1mMEDTA inPBSas Discard the culture medium inthe anti-IFN- Harvest the DC-stimulated splenocyte co-cultures ondays 7–10ina15-mlconical centrifuge tube. Centrifuge the cells Block the wellsbyadding 200 Develop theELISpotplate. washing the plate, prepare AECsubstrate according tothe manufacturer’s instructions. described instep1of Antigen loading of target cells target of loading Antigen for 1h. I CAL

PAUSE CR Box Box evaluation of effectortype-1 immunecellinduction µ evaluation of D I To conduct this analysis, generate the DCs from firefly luciferase (CAG-luc-eGFP) transgenic mice (FVB- mice transgenic (CAG-luc-eGFP) luciferase firefly from DCs the generate analysis, this To conduct l of 1×10 T g µ C I atroom temperature for 3–5min,discard the supernatant, and resuspend the cellsin5mlof PBS.Centrifuge 4 g per10 CAL s, 3–4hfor preparation of target cells, and3–4hfor flow analysis of harvested splenocytes µ µ ).

µ PO l of AECreagent perwell.Spotsappearafter5–20min,depending onthe assay. Once spotsare clearly l of irradiated tumor cells(4×10 l of DCs(5×10 µ

I STEP g per 10 per g NT low g The maximum recommended storage time for ananti-IFN- 5 6 and room temperature for 3min,then resuspend the cellsin0.5mlof cellculture medium and count - and loaded CFSE splenocytes insixwellsof the plate. cells. Incubate ina50-mlconical centrifugation tubeat37°Cfor 1.5–2.0h.Fillthe tubewith The IFN C 6 Box migration cells. Add nothing or a control peptide to the splenocytes in the other tube. Incubate Incubate tube. other the in splenocytes the to peptide control a or nothing Add cells. To do this, firstwashthe platefivetimes bysoaking 3–5minin200–300 γ 5 ELISpot platemust bepre-coated withIFN 2 © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. ) inPBSwitha1-ccsyringe and a27-gauge or30-gauge needle into the footpad of the and centrifuge at400 γ . 3–5 d after vaccination with OVA-loaded DCs, isolate splenocytes from a B6 B6 a from splenocytes isolate DCs, OVA-loaded with vaccination after d 3–5 . µ -Tu-loaded DCsto1×10 Box Box l of culture medium for 30minat37°C. ● high 3 6

6 cellspermlinculture medium and irradiate with20Gy. Centrifuge at400 T . Resuspend splenocytes at 2 × 10 × 2 at splenocytes Resuspend . cellspermlinPBSand inject3–4×10 -labeled splenocytes ata1:1ratio inPBS to2.5×10 IMI g for 5minatroom temperature. N G 4 γ 10–20minfor D cells)to3wellswithDC-stimulated splenocytes. Add 100 antibody precoated and blocked plate(from Step9C(vii))and plate Fig. 2

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C Fig. 2 T injectionand~15minfor imagingperfive mice IMI µ N c g/ml R4-6A2biotinylated secondary antibody in G γ ). 8–10hfor preparation and injectionof capture antibody atleast1dbefore harvest 2 7

O. Remove the plastic lid from the plateand cells per ml in culture medium and and medium culture in ml per cells 15 γ 7 antibody precoated and blocked ELISpot 0 permland add OVA peptide tocells 5 ). If you generate the DCs from B6 B6 from DCs the generate you If ). OVA-loaded DCs/0.1mlof PBSs.c. 5 natureprotocols cellsperml. µ l of 30mg/ml i.p.witha 7 cellspermland inject 2 for 24–48h.

g | VOL.13 NO.2VOL.13 for 5 min at at min 5 for Box µ l of washbuffer protocol µ l of culture 5 . Mix | 2018

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349 - - © 2018 Nature America, Inc., part of Springer Nature. All rights reserved. (5 ×10 described instep1of 10| s.c. injection (localized/solid tumor). 11|  exposure time. allows more light toreach the charge-coupled device) increase sensitivity. Images should take no longer than5minof of view sizeaffectsthe sensitivity. Note thatlonger exposure time, higher binning, and alowerF/Stop(awider aperture setup’) toautomatically setthe imaging parameters. Adjust the field of view depending on number of mice imaged. The field 13| We image mice every5–7d. ? tumors). s.c. MC38 (for caliper a with size tumor assessing by or tumors) i.p. (for circumference or weight mouse in increase by monitored concomitantly be should progression tumor therefore, model), s.c. MC38 the in ulceration of because or model (B) ( 350  imaging (firefly luciferase signal peaksafter10min and then declines). imaging. Wait 8minafterthe luciferin injection before transferring the mice toa5%(vol/vol)isoflurane chamberbefore 12| Inoculation Inoculation of mice with tumor cells Monitoring Monitoring of tumor progression protocol A (vii)

TROU (vi)

) Intraperitoneal injection (v) CR CR (i) (i)

Culture the relevant tumor cellsfor atleasttwopassages afterthawing before harvesting. Harvest the tumor cellsas Injectthe tumor cells. Followoption Atoperform i.p.injection (disseminated peritoneal tumor model) oroption Bfor Use the control panel tomanually setthe imaging parameters orusethe software’s Imaging Wizard (under ‘sequence Injectmice i.p.withluciferin (100 | S VOL.13 NO.2VOL.13 I I ubcutaneous injection

T T  penetrating the abdominal wall(~4–5mm),attempt toaspirate (pulling backslightly onthe needle) toensure that pointed down. Enter the abdominal cavitywiththe needle bevel-side upand slightly angled (15–20degrees). After injections), manually restrain the mice withdorsal recumbence (abdomen side up)and the cranial end of the animal develop, otherwise withdraw the needle slightly. the needle ~2–3mmbelowthe skinsurface sothatthe tipisvisibleunder the skin.When injecting, ablebshould right side pointed up.When penetrating the skin,hold the needle bevel-side upand angled 15–25degrees. Advance abdominal organs, such asthe bladder orcolon,havenot beenpenetrated.  6–8-week-old B6mice for colorectal orovarian cancer models, respectively. on CFSE detected byflowcytometry. We aim for 5×10  the procedure slightly abovethe initial siteof injection. aspirating. Ifswelling occursorifthere isresistance tothe injection, remove the needle from the tailand repeat the tail,whilekeeping the needle parallel tothe vein.Injectthe material inaslow, smooth motion, without with the bevelfacing upward atthe middle orslightly distalpartof the tail.Insert the needle atleast3mminto thumb and forefinger and cleanitwith70%(vol/vol)ethanol. Keep the tailunder pressure and placethe needle making sure not tooverheat the animal. Afterplacing the animal inarestraint device, hold the tailbetweenthe 200 in  (unvaccinated) mice. Inject 0.5×10 Use a1-ccsyringe and a27-gauge needle toinject 1×10 (Optional) Stainthe cellsfor CD45.1asdescribed instep9of Harvest spleens 16hafterward and isolatesplenocytes asdescribed in Flow analysis. 6 I B I permlfor MC38Luc or20×10 CAL CAL

LES

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l i.v. into DC-vaccinated mice (3–5daftervaccination withOVA-loaded DCs)and into atleasttwonaive STEP T T T T STEP OOT I I I I low CAL CAL CAL CAL | 2018 and CFSE Optimal I Luciferase signal can decrease at later time points (owing to ascites accumulation in the ID8A ID8A the in accumulation ascites to (owing points time later at decrease can signal Luciferase e N

. STEP STEP STEP STEP Determine the specifickilling bythe comparative analysis of liveCFSE G | 6

Box MC38Luc cellsin100 natureprotocols Fors.c. injections, shave the right flank of the mice and then manually restrain the mice withthe To make the injection into lowerleftabdominal quadrant (the preferred injection sitefor i.p. The number of injectedcellsisnot critical, although more cellsare better, asthe cellswillbe Forthe i.v. injections, keep the mouse warmunder aheat lamp(orother heating device), high in vivo 2

. Wash the cellstwice with10–20mlof PBSand resuspend inPBSfor subsequent injection cells. imaging time should bedetermined bytaking images overincremental periods of time. © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. ●

T IMI 6 ● µ permlfor ID8Acells).

l of 30mg/ml using a1-cc syringe and a27-gauge or30-gauge needle) before T N IMI G µ ~15 min per five mice N l PBSs.c. into the lowerright flank. G 5–10 min per five mice 6 cellspermouse.

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved.  for injection into five mice  diameter, or ulceration occurs (for s.c. tumors). (anorexic, emaciated, lethargic), changes in behavior occur (immobile and/or non-responsive), tumor size exceedswhen one 2 cm of in the following end points is met: weight exceeds 140% of initial body weight, mice appear to be in distress  treatments. 10B(i)), ondays 3–7afterinjecting tumor cellsasarepeat injection twotofour times, witha1-weekinterval between ascites (inthe caseof the i.p.tumor model; option B). 19| before day 30(MC38model) orday 40(ID8Amodel). 18| however, such incubation islessefficient. with Parafilm toprecoat the plate. Incubate overnight at4°C.Alternatively, the plate may becoated for 2hat37° C; 17| the type-1polarizedmature DCsoroption Bfor CKMadjuvant-mediated modulation. 15| 14| untreated and treated sets. ( plate Ex vivo (B) ( Induction of tumor-infiltrating specific type-1 immune cells cells permlinPBSand inject 3–4×10 ? i.p. every2–3dstarting asearlyday 4aftertumor inoculation. 16| A A

(iii) TROU (iv)

(ii) ) Harvesting ) D (v) CR CR CR (i) (i) (i) Monitor animals in accordance with institutional guidelines. Kill mice by CO Isolate the TILs/TALs from the tumors (inthe caseof the s.c. tumor model; option A)orperitoneal washes/cancer Add 100 Perform TMEmodulation 3daftereachs.c. DCvaccination. Followoption Atomodulate the TMEbyi.p.injection of ThawDCsasdescribed inStep 8.Referto Inject0.2mlof 500 C

I I I KM adjuvant injection

C  T T T continuously washwithPBStoafinal volume of 10–20ml. 100- add 500  digestion buffertocover the tumor pieces. scalpels to of PBS. IFN- mature DCsin0.05mlintratumorally (for the s.c. model). conical centrifuge tubetomix.Incubate the cellsat room temperature for 4min. 1 mlACK lysisbuffertoremove the red bloodcells. Add anadditional 3mlof ACK lysisbufferontop and rotate the Add 30mlof PBSand centrifuge the cellsat400 Centrifuge the cellsat400 Incubate tumors at37°Cfor 20–45minonarotator. Afterthe tissuedigestion, transfer the tumors toapre-wetted Add 2mlof PBStoaPetri dishtocoverthe bottom.Transfer the harvestedtumor tissueand mince itwithtwo Make acuttoenter the peritoneal cavityand collectthe tumor tissueinto 15-ml conical tubesfilledwith 2-ml Inject 1×10 Inject 3–4×10 PBS and transfer the mixture toa70- injection immunomonitoring I I I B

CAL CAL CAL

LES CR CR α µ I I We recommend imaging and weighing mice 1dbefore therapy initiation and grouping mice randomly into

T H and 50 m strainer placedontopof a50-mlconical centrifuge tubeand mash withthe rubberpistonof the syringe and STEP STEP T µ I OOT I CAL l of 15 CAL µ T l of digestion buffer, and then mash withthe rubberpistonof the syringe. I Donot allowmice todie due tohigh tumor burden. Typically, the untreated mice must beeuthanized Ifas.c. tumor model isinvestigated, DCsshould beinjecteds.c. into the oppositeflank. I

< Initiate Step17before the mice from Step18approach the relevant time point. L N

s from tumors STEP 4 0.5-mm pieces. Transfer the tumor pieces toanew 15-mlconical centrifugation tubeand add enough G IUof IFN- µ µ g Ampligen in0.05mlintratumorally (for the s.c. model). 5 type-1polarizedmature DCsin0.2mlof PBSi.p.(for the i.p.model) or3–4×10 g/ml AN18anti-IFN Ifthe tumor pieces are verysmall, transfer them directly toa2-mlEppendorf centrifugation tube, µ g/ml celecoxib (in12.5%(vol/vol)DMSO) or0.2mlof 2.5mg/ml (1.25%(vol/vol)DMSO) aspirin ●

α T IMI and 50 g © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. for 3–5minatroom temperature, discard the supernatant, and resuspend the cellsin N G 5 8–10hfor settingupan DCsper0.1mlof PBSs.c. into the leftflank of tumor-bearing mice (asinStep µ g Ampligen ( γ coating antibody inPBStoeachwellof anELISpot plateand wrap the plate µ m strainer. T able

1 todetermine the number of vials needed. Dilutethe cellsto3×10 g Fig. 3 for 3minatroom temperature, then resuspend them in20mlof b ) in0.2mlof PBSi.p.(for the i.p.model) or1×10 ●

T EL IMI I S N pot plateand~6hfor developing the G 20–30 min for vaccine preparation and ~10 min 2 inhalation, followed by cervical dislocation natureprotocols

| VOL.13 NO.2VOL.13 5 type-1polarized protocol EL I 4 | S 2018 IUof pot

6 351

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. 23| media. Blockthe plateby adding 200 22| culture medium. as described instep1of six wellsof the ELISpot plate. Harvest the tumor cellsinculture with1mMEDTA inPBS and centrifuge atroom temperature 20 Gy. Add 100 CO medium tomake three control wellsof splenocytes and TIL/TALs ( 21| wells. ? ? 24| 20| (B) Harvesting 352  resuspend the samplesin0.7mlof the cellculture medium. (viii) protocol (vii) (vii)

(iii) TROU TROU (vi) (iv) (vi)

(ii) 2 (v) CR (i)

for 24–48h. Discard the culture medium from the plateand dispense 100 Wash the ELISpot platefrom Step17three times byadding 200 Isolate splenocytes asdescribed in Developthe ELISpot plate asinStep9C(xiii). Adjust the cellconcentration to1×10 | VOL.13 NO.2VOL.13 I

T PBS, and transfer the mixture toa70- conical centrifuge tubetomix.Incubate the cellsatroom temperature for 4min. 1-ml of ACK lysisbuffertoremove the red bloodcells. Add anadditional 3mlof ACK lysisbufferontopand rotate the  cell culture medium. 15 mlwithPBS,and centrifuge at600 ? effective centrifugation. transfer them to15-mlconical tube. technique tolayer3mlof 34%(vol/vol)SIPontopof the 45%(vol/vol)SIPlayer. shallow angle down the side of the centrifuge tube. Take care not tomixwiththe bottomlayer. Repeatwithasimilar 50-ml conical tube. conical 50-ml a to fluid peritoneal the Transfer sample. collected the discard aspirated, is blood or material fecal If ruptured. been has intestine the that indicate may material fecal or Blood angle. the changing and millimeters few a for backward it pulling by needle the readjust to try case, such In penetrated. been not have colon, or bladder the as such organs, abdominal the that Ensure possible. as fluid peritoneal much as aspirate then and cavity i.p. the into PBS inject quadrant, abdominal left lower the into mm) (~4–5 wall abdominal the penetrating After PBS. of ml 3–5 ? Collect the bottominterface ( Centrifuge the tubesat2,400 Layer 3mlof 45%(vol/vol)SIPslowlyontopof the 60%(vol/vol)SIPlayerbyexpelling the 45%(vol/vol)SIPata Centrifuge the cellsat400 Count the cellsusing ahemocytometer. Add 30mlof PBSand centrifuge the cellsat400 Centrifuge the cellsat400 Centrifuge the cellsat400 Count the cellsusing ahemocytometer. Use a 10-ml syringe and an 18-gauge needle to obtain the peritoneal washes/cancer ascites. Fill the syringe with with syringe the Fill ascites. washes/cancer peritoneal the obtain to needle 18-gauge an and syringe 10-ml a Use transfer them toa15-mlconical tube. I B B

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G G 4 ) tumor cellstothree wellswithsplenocytes and TIL/TALs ( 2

. Resuspend the tumor cells at8×10 g g g © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. for 3minatroom temperature, resuspend the cellsin3mlof 60%(vol/vol)SIP, and for 3–5minatroom temperature, discard the supernatant, and resuspend the cellsin for 3–5minatroom temperature, resuspend the cellsin3mlof 60%(vol/vol)SIP, and g Fig. 4 for 30minatroom temperature without applying the brake. µ l of culture medium for 30minat37°C. Box a 6 ) (~2–3ml)into anew 15-mlconical centrifugation tube, fillthe tubeto permlwithcellculture medium. Ifthe cellnumber is 3 µ g 4–5dafterTMEmodulation (Step15)and resuspend to5×10

for 10minatroom temperature. Resuspend the cellpelletin0.5mlof the m strainer. g for 3minatroom temperature, then resuspend them in20-mlof µ Fig. 4 l (5×10 5 µ cellspermlof culture medium and irradiate with l of RPMI-1640and waiting 3–5minbefore removing b ). Incubate the cultures at 37°C,5%(vol/vol) 5 ) splenocytes and 1×10 Fig. 4 b ). Add 100

1 ×10 5 TILs/TALs into 6 µ perml, l of culture 6 permlin

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. ● Steps Steps 1–8, generation, collection, cryopreservation, and thawing of type-1 polarized DCs from donor mice, day 0: 10 h Box Box Box Box Box Box Generation of tumor-loaded D This protocol covers a minimum of 30 d. A workflow schema is provided in T Troubleshooting advice canbefound in ? Box step 3 of Step Step 13 Step Step 16 Box step 4 of 19A(vi) Step Step Step 4 19B(viii) Step and 24 Steps 23 a S

TROU

tep b T le le IMI 6 5 4 3 2 1 3 3 , , preparation of UVB , , flow analysis of the spleens for , labeling of the cells with CFSE (several time points): 30–45 min , extraction of cells from the lymph nodes of mice, days 1–3: 2 h , preparation of the splenocyte suspension, day 0: 1 h , , Flt3L-B16 cell injection into donor mice, day 3 3 N B | LES G

Troubleshooting table. H exposure time Images take Low Low splenocyte count Precipitation of celecoxib viability Low splenocyte count/ Low Low TIL count/viability Low Low yield of DCs No layers visible no distinct dots visible High background and/or P OOT roblem I N G

5 5 min of γ -Tu cells, day C s © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. used, adherence and DC recovery may vary If plates from other manufacturers are depending on the plate manufacturer. Adherence to plastic of cells differs cells cells growing in the mice There are no luciferase-expressing tumor The cell strainer is clogged temperature The suspension was left too long at room compromises compromises cell viability Prolonged exposure to ACK buffer effective effective cell isolation incubation time that is too short can limit compromises cell viability, whereas an Prolonged exposure to digestion buffer of CD11c Insufficient washing after maturation Mixing of layers while adding SIP Brake was applied during centrifugation Tumor cells are overgrown Too few lymphocytes were plated P in in vivo ossible reason T

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changing the plate manufacturer intensity and number of washing steps) when Adapt the DC-harvesting technique (washing and control frequently using a microscope cells off takes practice. Increase the washing steps Application of the correct pressure for washing the the presence of luciferin in a cell culture plate tumor cells express luciferase by imaging them in Verify that the luciferin reagent is working and that bigger Image mice after 2–3 d, allowing the tumors to grow wash wash extensively with PBS Make sure to mash the splenocytes thoroughly and higher DMSO content tion or consider making the final suspension with a Make sure to prepare celecoxib prior to administra after after incubation in ACK buffer centrifugation time. Dilute cells with culture medium Decrease incubation time in ACK buffer; decrease isolated isolated cells the digestion buffer to increase the viability of prolonged, add FBS or BSA (1–2% (vol/vol)) to In the case that the incubation time must be Adjust the incubation time in the digestion buffer to to avoid disturbing the bottom layer Add SIP with a lower concentration, using caution centrifugation and that the acceleration is slow Make sure that the brake is not applied during mice of the same group Consider combining lymphocytes from two to three or decreasing the incubation time Consider irradiating tumor cells with a higher dose S olution 1 natureprotocols

| VOL.13 NO.2VOL.13

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© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. therapy of genetically manipulated tumors withDCsloaded withstrong antigens alternative cytokines, and thus are abletodetect Tcellswithdifferent functions. modified toinclude additional elements and readouts, for example, ELISpot assaysthat detect Tcellsthatproduce for selectiveattraction of specificsubsets of immune cells. Inthis regard, the currently presented protocol canbe and chemokine-expressing induced byadministration of aCKMregimen, e.g., IL-12-producing DCs;combination of IFN- antitumor effectiveness of immune responses inthe cancer microenvironment, TMEmodulation iscritical and canbe intratumoral production of CCL5,CXCL9, and CXCL10 and localinfiltration of CD8 or aCKMregimen alone ( aspirin) onprolongation of mice survival(Steps15and 16).Typically, mice treated withs.c. injection of DCsonly-( toneal administration of DCswithintraperitoneal injection of CKM(IFN- intraperitoneal tumor–bearing mice. The data from thistherapeutic model are incontrast toprevention models AUT A versus 35dfor control; significant prolongation of mouse survival(median survival administration of DCs(ondays 8and 15)significantly prolongs survival of the mice (median survival DCs (3×10 in animal survivalare shown in Typical results of immunotherapy-induced changes (following DC therapy and combinatorial approaches with TME modulation) ANT Step 24, developing of ELISpot plate to detect intratumoral type-1 immune cells from experimental mice, days 16/17: 6 h Steps 18–23, isolation of splenocytes and TILs/TALs from experimental mice and setup of the Step 17, ELISpotpreparation of assay,ELISpot plate to detect intratumoral days type-1 immune 13–15: cells from 8–10experimental mice,h day 12: 10 min Monitoring of the therapeutic and effects immunomodulatory DCs (and CKM), weekly from days 0 and 3 : 20–30 min for vaccine preparation and ~10 min for injection into five mice Steps 14–16, induction of tumor-infiltrating-specific type-1 immune responses in experimental mice with type-1 polarized Steps 12 and 13, monitoring of tumor progression (BLI) of experimental mice, weekly from day Steps 10 and 11, inoculation of experimental mice with tumor cells, day loaded DCs, 3–4 h for preparation of target cells, and 3–4 h for flow analysis of harvested splenocytes Step 9E, Step 9D, of the ELISpot plate, and ~5 h for developing the ELISpot plate Step 9C, ~6 h for ELISA Step 9B, ELISA to evaluate the capacity of DCs from donor mice to produce IL-12, day 2: 20–30 min for culture set-up and Step 9A, phenotype analysis of the DCs, day 2: 40–50 min for staining and ~20 min for flow analysis donor mice, day 1: 6 h Step 9, phenotype analysis, Q 354 N.O. and P.K. wrote the manuscript. R.M., R.R., E.W., P.K., and D.L.B. participated in the design of the protocol; N.O. and J.U. selected and evaluated the experimental data; N.O., J.U., Regional Therapy Cancer Center (D.L.B.). 2015 CRI Clinical Strategy Team Grant (to P.K.), and by the David C. Koch comments. This work was supported by NIH grant CA132714 (to P.K.), by a with monitoring the mice and BLI, and P. Bailey for critical review and editorial M. Kronenberg for Flt3-L-expressing B16 cells. We thank K. Lemon for assistance ID8A ovarian cancer cell line, P. Lane for CD40L-expressing J558 cells, and unexpectedly on 14 December 2015). We thank T.J. Curiel for providing the P CXCR3) isassociated withhigh CTLinfiltration incancers production of CCL5/RANTES (ligand for CCR5)and CXCL9/MIG, CXCL10/IP10, and CXCL11/ITAC (three known ligands for protocol c

k The current protocol isdesigned toenhance the infiltration of tumor-specific CTLs into the TME( = of D no H

0.0021; | I OR VOL.13 NO.2VOL.13 C w I

le CONTR PATE C dgm in in vivo in vitro s in in vivo 5 D IB ents cellsondays 5and 12),usedasasingle treatment, willnot significantly prolong the survival of MC38 n UT RESULTS

= evaluation of effector type-1 immune cell induction, day 2: 8–10 h for preparation and injection of OVA- evaluation of DC migration, day 2: 10–20 min for DC injection and ~15 min for imaging per five mice I | evaluation of effector type-1 immune cell induction, day 2: 1–2 h for setup of the IVS culture, ~2 h for setup

We dedicate this article to Eva Wieckowski (who died ONS 2018

10mice pergroup) (

N.O., J.U., and R.R. participated in data generation; |

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10mice pergroup)) ( in in vitro e Figure 3c ) do not demonstrate anadvantage insurvival.However, the combinatorial approach shows © 2018 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. viruses. However, additional combinatorial approaches are likely tobeeffective evaluation of type-1 immune cell induction, and J558-CD40L restimulation of DCs from Fig. 3 , d . It may beexpected thatthe s.c. injections of tumor-loaded type-1polarized c ). Furthermore, weevaluated the effectof substituting additional intraperi Fig. 3

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