a synthetic cd8a:myd88 coreceptor enhances t-cell ... · ciated macrophage (6) and th2 cytokine...

Microenvironment and Immunology

A Synthetic CD8a:MyD88 Coreceptor EnhancesCD8þ T-cell Responses to Weakly Immunogenicand Lowly Expressed Tumor AntigensSabina Kaczanowska1, Ann Mary Joseph1, Jitao Guo1, Alexander K Tsai1,Jackline Joy Lasola1, Kenisha Younger1, Yuji Zhang1,2, Cruz Velasco Gonzales3,and Eduardo Davila1,4

Abstract

T cell–based immunotherapies are a promising approach forpatients with advanced cancers. However, various obstacleslimit T-cell efficacy, including suboptimal T-cell receptor(TCR) activation and an immunosuppressive tumor environ-ment. Here, we developed a fusion protein by linking CD8aand MyD88 (CD8a:MyD88) to enhance CD8þ T-cellresponses to weakly immunogenic and poorly expressedtumor antigens. CD8a:MyD88–engineered T cells exhibitedincreased proliferation and expression of effector and costi-mulatory molecules in a tumor antigen–dependent manner.These effects were accompanied by elevated activation of TCR

and Toll-like receptor signaling-related proteins. CD8a:MyD88–expressing T cells improved antitumor responses inmice. Enhanced antitumor activity was associated with aunique tumor cytokine/chemokine signature, improved T-cellinfiltration, reduced markers of T-cell exhaustion, elevatedlevels of proteins associated with antigen presentation, andfewer macrophages with an immunosuppressive phenotype intumors. Given these observations, CD8a:MyD88 represents aunique and versatile approach to help overcome immunosup-pression and enhance T-cell responses to tumor antigens.Cancer Res; 77(24); 7049–58. �2017 AACR.

IntroductionT cell–based immunotherapies are one of the most promising

treatments for patients with advanced cancers, including melano-ma. Several approaches have been developed to harness the anti-tumor activity of cytotoxic T cells, including vaccine-based thera-pies, adoptive cell transfer of tumor-infiltrating lymphocytes (1, 2)or T cells engineered to express a tumor-reactive T-cell receptor(TCR; refs. 3, 4), and antibody-mediated checkpoint blockade (5).Despite encouraging clinical results demonstrating tumor regres-sion and prolonged survival, durable antitumor responses areobserved only in a subset of patients, highlighting the need toimprove these therapies. Limitations include low tumor antigen-specific T-cell precursor frequencies, suboptimal TCR responsesdue to low TCR affinity (or avidity) toweakly immunogenic tumorantigens, and low antigen expression and/or presentation ontumor cells (1, 2, 5). Furthermore, various suppressivemechanismswithin the tumor microenvironment (TME), such as tumor-asso-

ciated macrophage (6) and Th2 cytokine accumulation hamperantitumor T-cell responses. Moreover, chronic exposure to factorsin the TME induces the expression of receptors that foster T-cellexhaustion, such as Tim-3, Lag-3, and PD-1 (7).

Various studies have demonstrated that activating MyD88signaling in T cells via Toll-like receptor (TLR) engagementenhances cytokine production, cytotoxicity, and proliferation(8–10). Yet, applying TLR costimulation specifically to T cells ischallenging due to their low and transient expression of TLRs,insufficient intratumoral TLR agonist localization for T-cell costi-mulation, and the potential for TLR engagement on tumor cells topromote tumor growth (10).

Here, we present a novel strategy to activate MyD88 signalingwithin CD8þ T cells in a TLR-independent but TCR-dependentfashion to overcome weak tumor antigenicity and an immuno-suppressive TME. This strategy utilizes the CD8a chain, an endog-enous TCR coreceptor that interacts with MHC I. By fusing CD8ato MyD88 (CD8a:MyD88), we developed a novel platform thatenhances various parameters of T-cell function. CD8a:MyD88–expressing T cells respond to suboptimal levels of tumor antigenand display reduced levels of exhaustion markers and increasedproduction of effector molecules. The enhanced antitumor activ-ity of CD8a:MyD88–expressing T cells is associated with theirability to reshape various facets of the TME, including the cytokinemilieu aswell as the phenotype of other immune cells, resulting ina TME that favors T-cell infiltration and antitumor activity.

Materials and MethodsPlasmids, retroviral production, and T-cell transduction

The CD8a:MyD88 coreceptor was designed by fusing themurine CD8a extracellular and transmembrane domain sequences

1University of Maryland, Marlene and Stewart Greenebaum ComprehensiveCancer Center, Baltimore, Maryland. 2Department of Epidemiology and PublicHealth, University of Arkansas for Medical Sciences, Little Rock, Arkansas.3Department of Pediatrics, University of Arkansas for Medical Sciences, LittleRock, Arkansas. 4Department of Microbiology and Immunology, University ofMaryland, Baltimore, Maryland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Eduardo Davila, University of Maryland, 655 WBaltimore St, Baltimore, MD, 21201. Phone: 410-706-5055; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-17-0653

�2017 American Association for Cancer Research.

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to the human MYD88 death domain and intermediate domainsequences (Supplementary Fig. S1A). The CD8aDIC constructlacked any intracellular domains. Genes were cloned into thepMIG-w vector containing a GFP reporter gene. Retroviral vectorsupernatants were produced from Phoenix Ampho and Ecopackaging cell lines we have described previously (11). Forgenerating engineered mouse T cells, pmel or OT-I T cells wereactivated for 48 hours using 1 mg/mL hgp10025-33 or SIINFEKLpeptide, respectively, and 50 U/mL of IL2 prior to transduction.Efficiency was determined by assessing the frequency of GFPþ

cells byflow cytometry. For generatingDMF5-CD8a:MyD88 cells,human peripheral blood mononuclear cells (PBMC) were engi-neered to express DMF5 as described previously (11), expandedwith beads (Invitrogen) coated with HLA-A2/MART-127-35 (BDPharmingen) and anti-CD28 antibody plus 100 U/mL IL2 andthen engineered to express the indicated CD8a vectors. Viablecells were enriched by Ficoll gradient prior to setting up all assaysor injection into mice.

Mice, tumor model, and ex vivo analysisStudies were approved by the UMB Institutional Animal Care

and Use Committee. C57BL/6J and pmel (B6.Cg-Thy1/Cy Tg(TcraTcrb)8Rest/J) mice were purchased from The Jackson Labo-ratory. T cells from IRAK-4 kinase deadmicewere kindly providedby Dr. Stefanie Vogel at the University of Maryland (Baltimore,MD). C57BL/6J mice were injected with 2 � 105 B16-F1 mela-noma cells subcutaneously on the rightflank.Micewere irradiatedwith 550 radians on day 9 after tumor inoculation and intrave-nously injected with engineered pmel T cells on day 10. Mousebody weight and tumor size were monitored every 2 to 3 days.Tumor volume was calculated by the ellipsoid formula: length �width� height� (4/3)p. Specific tissues were harvested oneweekafter T-cell transfer for ex vivo analyses including flow cytometryand cytokine/chemokine Luminex. The following antibodieswere used: CD8a, Lag3, Tim3, I-A/I-E, CD86, CD11b, CD11c,F4/80, CD206, Gr-1, NK1.1 (BioLegend), CD45.2, CD8a, CD19(BD Pharmingen), MHC class I H2 Kb þ Db (Abcam).

T-cell proliferation assays, cytokine measurements, andintracellular staining

Splenocytes from C57BL/6J mice were irradiated (3,000 radi-ans) and pulsed with varying concentrations of hgp10025-33 orSIINFEKL peptide for 2 hours at 37�C. Pmel or OT-I T cells werecocultured with peptide-pulsed splenocytes at a 1:1 ratio, andsupernatant was collected after 24 hours. Alternatively, B16-F1cells (ATTC CRL-6323, obtained within three years of usingthem) were irradiated (20,000 radians) and plated at variouscell numbers together with 1 � 105 transduced T cells for48 hours. Cytokine concentrations were determined by ELISA(eBioscience) or the Milliplex Cytokine/Chemokine Kit (Milli-pore). IFNg and TNFa production by DMF5 T cells was eval-uated at 2:1 and 1:2 T cell to PBMC ratios after 4 days ofstimulation.We assessed T-cell proliferation by adding 1 mCi/wellof tritiated thymidine (methyl-3H, PerkinElmer) and measuredthymidine incorporation 24 hours later. Intracellular levels ofsignaling proteins were evaluated by flow cytometry. Briefly,cells were permeabilized in BD Pharmingen Phosflow PermBuffer III (BD Bioscience) and stained with anti–p-p65 and anti-rabbit IgG F(ab0)2 Fragment-PE (Cell Signaling Technology), oranti-p-ERK1/2-Pacific Blue and anti–p-JNK-PE, or anti–p-p38-Pacific Blue and anti–p-Zap70-PE (BD Biosciences). For flow

cytometry–based proliferation assays, transduced T cells werepulsed with cell proliferation dye eFluor 450 (eBioscience),washed, and cocultured with hgp10025-33-pulsed splenocytesat a 1:1 ratio for 72 hours. In other experiments, T cells werecoincubated with tumor antigen-pulsed splenocytes (mousepmel or OT-I T cells) or autologous PBMCs (human DMF5 Tcells) for 48 or 96 hours with brefeldin A added the last 6 hoursof incubation prior to staining. For evaluating human T-cellproliferation, transduced T cells were cocultured with Malme-3Mmelanoma cell (HLA-A2þMART-1þ) or with A375melanomacells (HLA-A2þ) pulsed or unpulsed with 10 mg of MART-127-35peptide at a ratio of 1:1 T cell to tumor cell ratio. For thephenotyping screen, transduced T cells were cocultured with0.12 mg/mL of hgp10025-33-pulsed splenocytes at a 1:2 splenocyteto T-cell ratio for 48 hours, stainedwith the Zombie Aqua viabilitydye (BioLegend), anti-CD45.2 and anti-CD8a antibodies, andstained with the LegendScreen Mouse Cell Screening (PE) Kit(BioLegend). Malme-3M and A375 cell lines were obtained fromATCC, used, and were tested for mycoplasma within 3 years ofpurchasing them. All flow cytometry experiments were per-formed on the BD LSRII at the Greenebaum ComprehensiveCancer Center Flow Cytometry Shared Service Lab and analyzedby FlowJo (TreeStar).

Statistical analysisProliferation and ELISA experiments were performed in

triplicate in at least two independent experiments and analyzedby one-way ANOVA. Animal studies contained 8 to 10 animalsper group for growth and survival, and 5 per group for ex vivoflow cytometry analysis. For flow cytometry and the cytokinearrays, the values and error bars represent mean� SEM (�, P �0.05; ��, P � 0.01; ��, P � 0.001; one-way ANOVA with Tukeymultiple comparison test; n ¼ 3 experimental replicates and arerepresentative of at least two independent experiments). Tumorsizes were analyzed by a mixed model repeated measures withAR(1) covariance structure (yielding smallest BIC), followedby Sidak-adjusted comparisons at each time.

ResultsCD8a:MyD88 lowers the threshold of T-cell activation,resulting in increased proliferation and cytokine production

The extracellular and transmembrane domain of CD8a werefused to the human MYD88 death and intermediate domain togenerate a CD8a:MyD88 coreceptor (Supplementary Fig. S1A).As controls, we generated CD8a lacking the intracellulardomain (CD8aDIC), or an empty vector control. Each vectoralso contained a GFP reporter to distinguish transduced T cells(Supplementary Fig. S1B). CD8a:MyD88 expression was con-firmed by Western blot and imaging flow cytometry (Supple-mentary Fig. S1C and S1D).

Gp10025-33–specific TCR transgenic pmel CD8þ T cells were

engineered to express CD8a:MyD88, CD8aDIC, or only GFPusing retroviral vectors and stimulated with gp10025-33-pulsedsplenocytes. Pmel T cells expressing CD8a:MyD88 exhibited alower activation threshold as demonstrated by increased pro-liferation in response to lower tumor antigen (TAg) levelscompared with control T-cell groups (Fig. 1A). Notably,although only half of the CD8þ T cells expressed CD8a:MyD88,proliferation was doubled at most antigen concentrations(Supplementary Fig. S1B; Fig. 1A). The ability for T cells to

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recognize exceedingly low TAg levels is relevant as tumor cellscan downregulate TAg or MHC I expression to evade T-celldetection. In the absence of TAg, CD8a:MyD88 T cells dis-played little to no baseline proliferation, indicating that theenhancement of TCR responses by MyD88 is TCR dependent.

To determine whether the increased proliferation observedin CD8a:MyD88 T cells was due to their ability to promotethe division of nontransduced T cells, we compared cell divi-sion of CD8a:MyD88 (GFPþ) and nontransduced (GFP�)T cells. CD8a:MyD88 T cells loaded with a proliferation dye

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Figure 1.

CD8a:MyD88 expression enhances T-cell responses to tumor antigen. A, Pmel T cells were engineered with the empty vector control, CD8aDIC, orCD8a:MyD88. T-cell proliferation was measured by 3H-thymidine incorporation after stimulation with gp10025-33-pulsed splenocytes for 48 hours.B, Flow cytometry of pmel T cells cocultured with splenocytes pulsed with 0.12 mg/mL gp10025-33 peptide at 72 hours. C, Proliferation of OT-I T cellscocultured with OVA (SIINFEKL)-pulsed splenocytes measured by 3H-thymidine incorporation at 48 hours. D and E, DMF5 empty vector control, DMF5 CD8a,or DMF5 CD8a:MyD88 human T cells were labeled with proliferation dye and cultured with HLA-A2þ MART-1þ Malme-3M or with HLA-A2þ MART-1� A375melanoma pulsed or not with 10 mg/mL/106 cells MART-127-35 cells at a 1:1 ratio. T-cell proliferation was measured by flow cytometry after 5 days. F,Intracellular staining of phosphorylated proteins in transduced T cells stimulated with peptide-pulsed splenocytes and fixed at given time points. Valuesand error bars represent mean� SEM. � , P � 0.05; �� , P � 0.01; �� , P � 0.001 (A and C), one-way ANOVA with Tukey multiple comparison test; n ¼ 3experimental replicates; representative of at least two independent experiments. D and E are representative of three independent experiments.

Universal Approach to Enhance Antitumor T-cell Responses

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expanded to a greater degree than nontransduced T cells, asindicated by a lower median fluorescence intensity in GFPþ

cells (Fig. 1B, right). The increased proliferation by CD8a:MyD88 T cells was more pronounced at suboptimal TAg con-centrations that were too low to activate control T cells (Sup-plementary Fig. S1E). In addition, the degree of cell divisionbetween the GFP� T cells from each of the groups was com-parable. These data indicate that the costimulatory effects ofCD8a:MyD88 occur within engineered T cells and do notimpact the proliferation of neighboring T cells.

One of the potential advantages to using CD8a:MyD88 is theability to boost responses with any given TCR. This was con-firmed using OT-I TCR transgenic CD8þ T cells specific for anovalbumin (OVA) peptide presented on MHC I, which alsoshowed increased proliferation to antigen stimulation in adose-dependent manner (Fig. 1C). Likewise, human T cellsengineered to express fully human CD8a:MyD88 and the DMF5TCR, which recognizes the MART-127-35 TAg (12), demonstratedgreater proliferation than control T cells following stimulationwith MART-1þ Malme-3M melanoma cells (Fig. 1D). In addi-tion, DMF5 CD8a:MyD88–expressing T cells proliferated inresponse to MART-127-35 peptide-pulsed but not unpulsedA375 (HLA-A2þ MART-1�) melanoma cells (Fig. 1E), furtherhighlighting that the enhancing effects of CD8a:MyD88 arestrictly regulated by tumor antigen expression and are effectivein multiple tumor and antigen models.

MyD88 transduces signals by recruiting IL1 receptor associ-ated kinase-4 (IRAK-4). In the absence of functional IRAK-4,CD8a:MyD88 did not enhance proliferation (SupplementaryFig. S1F), demonstrating the dependence of CD8a:MyD88costimulation on the canonical MyD88 pathway. CD8a:MyD88 led to the enhanced and more rapid activation (phos-phorylation) of downstream signaling proteins ERK1/2, JNK,and p38 (Fig. 1F). Increased ERK1/2 activation is advantageousas ERK2 drives CD8þ T-cell proliferation and survival (13).CD8a:MyD88 also enhanced the activation of the proximalTCR protein ZAP-70, a CD3 receptor–associated protein tyro-sine kinase (Fig. 1F).

CD8a:MyD88 expression in pmel and OT-I T cells alsoincreased IFNg and TNFa production over CD8aDIC and emptyvector T cells (Fig. 2A and B) and occurred in an antigen concen-tration-dependent fashion (Fig. 2C). Increased cytokine levelswere in part due to the ability of CD8a:MyD88 to increase thefrequency of T cells capable of producing both IFNg and TNFa orTNFa alone (Fig. 2D). In addition, increases in IFNg-producingCD8a:MyD88 T cells were more apparent at lower TAg levels(Supplementary Fig. S2A). MART-127-35–specific human DMF5CD8þT cells also showed enhanced IFNg andTNFaproduction inresponse to stimulation with TAg (Fig. 2E).

CD8a:MyD88 upregulates cytokine receptors andcostimulatory molecules while reducing moleculesassociated with T-cell exhaustion

To better understand changes induced by CD8a:MyD88,we conducted a flow cytometry–based protein expression arrayevaluating the levels of 252 surface proteins. Changes in theexpression of 89 surface proteins were identified comparing thefold change between vector control andCD8a:MyD88orCD8a:DIC T cells (Fig. 2F). CD8a:MyD88 increased expression of IL2receptor chains CD25 and CD132, costimulatory moleculesinvolved in T-cell proliferation and cytolytic function, such as

CD71, CD26, andCD137 (4-1BB; ref. 14), and various activation-associated adhesion molecules, including CD44 and CD69.CD8a:MyD88 also decreased the expression of coinhibitorymolecules including CD160, CD73, Tim-3, and CD39 (Fig. 2F).Notably, CD39 has been reported to be a marker of terminallyexhausted CD8þ T cells and was considerably reduced in CD8a:MyD88 T cells (15). The most differentially expressed proteins fellinto a wide variety of classes, but changes by CD8a:MyD88universally favored improved T-cell function (Fig. 2F, bottom;Supplementary Fig. S2B).

CD8a:MyD88 increases T-cell responses to weaklyimmunogenic B16-F1 melanoma cells

Cancer cells, including the melanoma cell line B16-F1, oftencarry alterations in antigen presentation machinery (16, 17) thatmake them poor T-cell targets. Despite having nearly undetect-able MHC I levels (Supplementary Fig. S3A), CD8a:MyD88substantially enhanced T-cell proliferation following coculturewith B16-F1 as compared with control T cells (Fig. 3A). Prolif-eration occurred in a tumor cell number–dependent manner,and T cells did not proliferate in the absence of tumor cells.Notably, CD8a:MyD88–expressing T cells required nearly10-fold fewer melanoma cells to achieve the same level of pro-liferation as control T cells (Fig. 3A, hashed lines). CD8a:MyD88 T cells produced higher levels of IFNg and granzymeB than control T cells in response to tumor cells (Fig. 3B).Furthermore, CD8a:MyD88 T cells produced IFNg at tumor cellnumbers at which control T cells could not, emphasizing thepropensity of CD8a:MyD88 to lower the TCR activation thresh-old, a beneficial feature for destroying weakly immunogenictumor cells. CD8a:MyD88 also induced multiple chemokinesimportant for immune cell recruitment, including RANTES,CXCL9, and CCL3 (Fig. 3C; Supplementary Fig. S3B). In addi-tion, we evaluated changes in the levels of other cytokines/chemokines following stimulation with B16-F1 melanoma cellsand found that CD8a:MyD88 increased CXCL1, CXCL2, CCL4,CXCL10, GM-CSF, IL3, and LIF levels in tumors and serum(Supplementary Fig. S4A and S4B).

CD8a:MyD88 expression increases the infiltration of T cellsinto the tumor and reduces T-cell exhaustion phenotype

On the basis that MyD88 activation in T cells enhancedproliferation and production of various cytokines/chemokines,we evaluated T cells in the tumor and spleen 7 days after adop-tive transfer into tumor-bearing mice. CD8a:MyD88–expres-sing T cells comprised a greater proportion of the CD8þ T-cellpopulation within the tumor as compared with vector controlT cells (Fig. 3D). However, the numbers of control and CD8a:MyD88–expressing T cells were similar in the spleen (Fig. 3D).These data suggest that the costimulatory effect of CD8a:MyD88 signaling occurs in a manner that depends strictly onTCR engagement in the tumor and does not occur in a non-specific manner in other tissues. Furthermore, in agreementwith data demonstrating a reduction of exhaustion markers onCD8a:MyD88 T cells (Fig. 2F), there were nearly four timesfewer Lag3þTim3þ CD8a:MyD88–expressing T cells in tumorsas compared with control T cells, but similar proportions inspleens (Fig. 3E). Lag3þTim3þ expression on T cells is associ-ated with a reduced ability to produce effector molecules,including IFNg . Fittingly, CD8a:MyD88 T cells induced higherlevels of IFNg in tumors, whereas IFNg was nearly undetectable

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CD8a:MyD88 T cells exhibit enhanced cytokine production and alter the expression of costimulatory and coinhibitory molecules in response to tumor antigen.A and B, IFNg and TNFa levels measured by ELISA in supernatants from pmel T cells cocultured with gp10025-33-pulsed splenocytes (A) and OT-I T cells withOVA (SIINFEKL)-pulsed splenocytes at 24 hours (B). C, IFNg production by engineered pmel T cells cocultured with gp10025-33-pulsed splenocytes.D, Intracellular cytokine staining of pmel T cells stimulated with splenocyte pulsed with 5 mg/mL gp10025-33. E, Cytokine production by DMF5 empty vector,DMF5 CD8a, or DMF5 CD8a:MyD88 human T cells in response to stimulation with MART-127-35–pulsed PBMCs as measured by ELISA 4 days later. Datafrom three independent experiments with three replicates per reaction are shown. Values, mean � SEM. �� , P � 0.001, one-way ANOVA. F, Flowcytometry screen of pmel T cells cocultured for 48 hours with splenocytes pulsed with 0.12 mg/mL gp100. The fold change of the median fluorescenceintensity of CD8aDIC and CD8a:MyD88 T cells over control vector T cells from three independent experiments is displayed in a heatmap. Gray boxes representdata not available (na). Representative histograms are shown of the expression of most upregulated and downregulated molecules. Values and errorbars represent mean � SEM. � , P � 0.05; �� , P � 0.01; �� , P � 0.001 (A–C), one-way ANOVA with Tukey multiple comparison test at each concentration;n ¼ 3 experimental replicates; representative of at least two independent experiments.






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Figure 3.

CD8a:MyD88 T cells display improved responses to melanoma in vitro and in vivo. A, Proliferation of engineered pmel T cells stimulated with irradiated B16-F1tumor cells for 72 hours. B and C, ELISA and Luminex analysis of cytokines and chemokines in the supernatant of T cells cocultured with B16-F1 tumorcells for 48 hours. D–I,Mice bearing established B16-F1 tumors were exposed to a sublethal dose of irradiation (550 rads), followed by transfer of 3� 106 T cellsby intravenous injection one day later. Tissues were harvested and analyzed by flow cytometry one week after T-cell transfer. D, The frequency of GFPþ

cells in the tumor and spleen. E, The percentage of CD8þGFPþ cells expressing exhaustion markers Tim-3 and Lag-3. F, IFNg levels in tumor tissuemeasured by ELISA of tumor homogenate. G, Protein levels of CXCL9 in the tumor and serum. H, Frequency of CD8þ T cells in the tumor and spleen.I, Frequency of CD45þ cells, CD19þ cells, and NK1.1þ cells in the tumor and spleen. Values and error bars represent mean� SEM. � , P � 0.05; �� , P � 0.01;�� , P � 0.001 (A–C), one-way ANOVA with Tukey multiple comparison test at each concentration; n ¼ 3 experimental replicates or n ¼ 5 for D–I.

Kaczanowska et al.





in tumors of control-treated or untreated mice (Fig. 3F). IFNg isknown to induce CXCL9, a potent chemoattractant that recruitsT cells to the tumor and skews responses toward a Th1 phe-notype (18, 19). Indeed, CXCL9 was elevated in tumors andserum of CD8a:MyD88 T cell–treated mice (Fig. 3G). Accord-ingly, the percentage of CD8þ T cells was higher in the tumors,but not the spleens, of mice treated with CD8a:MyD88 T cells(Fig. 3H). Differences were not detected in the number ofCD45þ hematopoietic cells, B cells, or NK cells in the tumorsor spleens of mice (Fig. 3I).

CD8a:MyD88 T cells alter the TME and induce tumorregression

In accordance with increased IFNg levels, MHC I expressionon nonlymphoid (CD45�) cells was increased in mice treatedwith CD8a:MyD88 T cells (Fig. 4A and B). Treatment with

CD8a:MyD88 T cells also increased the expression of MHC IIand CD86 on dendritic cells (DC; Fig. 4A and B). The percent-age of activated DCs coexpressing MHC II and CD86 wasalso elevated in the tumors, but not spleens, of CD8a:MyD88T cell–treated mice (Fig. 4C).

The percentage of intratumoral CD45þCD11c�CD11bþF4/80þ macrophages was decreased in mice treated with CD8a:MyD88 T cells as compared with control-treated or untreatedmice (Fig. 4D). Further analysis of the macrophage populationrevealed an increase in the percentage of macrophages withan M1-like phenotype (CD38þCD206�), typically associatedwith antitumor responses, following administration of CD8a:MyD88 T cells (Fig. 4E, left). The rise of M1-like macrophages inthe tumor was associated with reduced M2-like protumormacrophages (CD38�CD206þ) (Fig. 4E, right). In contrast, thepercentage of M1-like and M2-like macrophages remained

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CD8αΔIC CD8α-MyD88

Figure 4.

CD8a:MyD88 T cells alter the tumor microenvironment. A–G, Mice bearing an established B16-F1 tumor (�50 mm2) were exposed to a sublethal dose ofirradiation (550 rads), followed by transfer of the CD8a:MyD88 or vector control pmel T cells (3 � 106) by intravenous injection one day later. Tissues wereharvested and analyzed by flow cytometry one week after T-cell transfer. Transduction efficiency of CD8a:MyD88 T cells was 50%. Each data pointrepresented one mouse. A, Median fluorescence intensity (MFI) of MHC I on CD45� cells and MHC II and CD86 on CD11cþ DCs. B and C, Frequency ofCD11cþ DCs coexpressing activation markers CD86 and MHC class II in the tumor and spleen. D, Frequency of macrophages in the tumor and spleen asdefined by CD11c�CD11bþF4/80þ cells. E, Frequency of M1 (CD38þCD206�) and M2 (CD38�CD206þ) macrophages in the tumor and spleen. F, Frequencyof myeloid-derived suppressor cells (MDSC) in the tumor and spleen as defined by coexpression of Gr1 and CD11b. G, Luminex analysis of CCL2, IL4,and IL5 in tumor homogenate. ns, nonsignificant.






similar in the spleens of all groups. We did not detect anydifferences in the number of CD45þGr1þCD11bþ myeloid-derived suppressor cells (Fig. 4F).

We characterized the levels of cytokines and chemokines asso-ciated with myeloid cell migration and differentiation. CCL2,which is involved inmonocyte recruitment, Th2polarization, andtumor progression (20, 21), were decreased nearly 3-fold in thetumors of CD8a:MyD88 T cell–treated mice (Fig. 4G, left).Likewise, IL5, which has been associated with Th2 responses andprotumor effects (22), was decreased following treatment withCD8a:MyD88 T cells (Fig. 4G, middle). IL4 levels remainedsimilar in all groups (Fig. 4G, middle). These studies highlightthe propensity for CD8a:MyD88 to alter different facets of theTME, including the cellular composition, cell surface proteinexpression, and cytokine profile toward one that can supportCD8þ T-cell expansion and antitumor activity.

Given these findings, we assessed the antitumor activity ofCD8a:MyD88–expressing T cells in tumor-bearing mice. Miceharboring established B16-F1 tumors (�30 mm2) were treatedwith control pmel or CD8a:MyD88–expressing pmel T cells.Two injections of CD8a:MyD88 pmel T cells were sufficient todelay tumor growth as compared with mice treated with controlT cells or untreated mice. Importantly, although control T cellstemporarily delayed or reversed tumor growth, CD8a:MyD88 Tcells induced and maintained tumor regression of large estab-lished tumors (Fig. 5A and B). All mice treated with CD8a:MyD88 T cells exhibited tumor regression and 50% (4/8) ofmice cleared tumor. Notably, these antitumor responses wereaccomplished in the absence of additional T-cell support suchas TAg vaccination, cytokines, or checkpoint blockade, empha-sizing the potent antitumor effects of activating MyD88 signal-

ing in T cells. A single injection of CD8a:MyD88 T cellsmoderately delayed tumor growth as compared with micetreated with vector control T cells or untreated mice (Supple-mentary Fig. S5A). Furthermore, we observed tumor control,and tumor regression in some animals lasting up to one weekafter cell transfer (Supplementary Fig. S5B). In sharp contrast,tumor growth rates and mouse survival of the control T cell–treated group were similar with those of the untreated group(Supplementary Fig. S5A–S5C), indicating that control tumor-specific T cells were ineffective at the given dose, exemplifyingthe potent antitumor effects of CD8a:MyD88 T cells. We alsoevaluated whether checkpoint blockade using anti–PD-L1 anti-bodies or antibodies to stimulate 4-1BB signaling would poten-tiate antitumor T-cell responses. We observed that althoughblocking PD-L1 had a negligible effect, stimulating 4-1BBincreased the antitumor activity of control T cells, but it didnot improve CD8a:MyD88 T-cell responses (SupplementaryFig. S6A and S6B). These data suggest that the costimulatorysignals activated by 4-1BB signaling might be redundant tothose activated by CD8a:MyD88.

DiscussionThrough these studies, we present a novel strategy in which

expressing the fusion protein CD8a:MyD88 activated MyD88signaling in cytotoxic CD8þ T cells in a TLR-independent butTCR-dependent fashion, resulting in enhanced responses to sub-optimal TAg levels and in an ability to reshape the TME towardone favoring antitumor immune responses. Notably, theenhancement of TCR responses byMyD88was strictly dependenton TCR stimulation with the tumor antigen. To the best of ourknowledge, this is the first strategy designed to boost TCRresponses against weakly immunogenic or lowly expressed anti-gens without the need to alter the TCR sequence or identifystronger MHC I epitopes.

The use of CD8a:MyD88 offers several unique strategies tocontrol tumor growth. First, the costimulatory effects of CD8a:MyD88 are not limited to a single tumor antigen nor are theyMHC restricted, as CD8a interacts with the a3 domain on allhuman MHC I complexes. Therefore, immunotherapy usingCD8a:MyD88–expressing T cells could be utilized as a methodto enhance TCR responses against a variety of tumor antigens andthus could be applied to patients with various malignanciesregardless of HLA haplotype or tumor antigen specificity. Fur-thermore, ongoing studies in our laboratory suggest that CD4:MyD88 extends CD4 T cells a similar costimulatory effect andcould be used as a strategy to potentiate responses against tumorantigens expressed on MHC II molecules.

One limitation to T cell–based therapies is low TCR affinityand/or avidity (3, 4). T cells with high affinity toward self-antigens, such as tumor antigens, are eliminated in the thymusduring development, leaving behind low-affinity or low-aviditytumor-reactive T cells that recognize subdominant epitopes andweakly immunogenic antigens including neoantigens. There-fore, a second benefit of expressing CD8a:MyD88 is that itlowers the TCR activation threshold, providing the potential toexpand both high and low affinity TCR T cells while preservingendogenous TCR signaling. The ability to enhance weak TCRresponses is especially relevant when redirecting T cells towardneoantigens that can be expressed at low levels and/or havelower affinity than nonmutated self-antigens. Amplifying T-cell

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Figure 5.

CD8a:MyD88 T cells induce tumor regression of established melanomatumors. A and B, Mice bearing an established B16-F1 tumors wereexposed to a sublethal dose of irradiation (550 rads), followed by transferof 6 � 106 pmel T cells engineered to express CD8a:MyD88 or vectorcontrol-GFP by intravenous injection one day and 8 days later. Averagetumor volume as well as tumor volumes for individual mice are shown.Values and error bars represent mean� SEM. � , P � 0.05 starting on day 28and onward. Tumor growth was analyzed by a mixed model repeatedmeasures with AR(1) covariance structure, followed by Sidak-adjustedcomparisons at each time.

Kaczanowska et al.





responses toward neoantigens is important for developingeffective cancer therapies, as they offer the ability to targetbona fide tumor-specific antigens. Furthermore, it is estimatedthat up to 20% of cancers are associated with oncogenic viruses.Therefore, another attractive strategy to specifically target cancerwhile eliminating off-target effects is to develop CD8a:MyD88T cells reactive toward viral antigens expressed by tumor cells.

Another important and potentially clinically valuable use ofCD8a:MyD88 is its ability to enhance the function of T cellsengineered to express tumor-reactive TCRs, such as those targetingMART-1 or Ny-Eso (3, 4). For example, our data demonstratingthat CD8a:MyD88 expression augmented the responses of DMF5TCR–engineered T cells (Fig. 1D and E) highlight the opportunityto use CD8a:MyD88 to increase responses to a variety of engi-neered TCRs currently in clinical testing (3, 4). Furthermore, theability for T cells to recognize tumor cells expressing suboptimalMHC levels (in addition to lowTAg levels) is an important feature,as tumor cells can downregulate MHC I expression or antigenexpression to evade detection by T cells. A third advantage offeredby CD8a:MyD88 is the ability to activate MyD88 signalingspecifically in transduced T cells. Tumor-specific cells can be

isolated and activated against the tumor prior to introduction ofthe CD8a:MyD88 to limit immune-related adverse events. This isan important quality as it indicates that the boosting effects ofCD8a:MyD88 occur only in gene-modified T cells and do notnonspecifically costimulate nontransduced T cells, such as auto-reactive T cells. Importantly, the ability to activate MyD88 sig-naling in engineered T cells would eliminate the need to providesystemic TLR agonists, which could have the undesirable effect ofpromoting tumor growthby stimulating TLRson cancer cells (10).

Fourth, CD8a:MyD88–expressing T cells exhibited a reducedability to become exhausted in vitro and in vivo. This was exem-plified by reduced frequencies of Lag3þTim3þCD8þ T cells and anability for CD8a:MyD88–expressing T cells to produce higheramounts of IFNg (Fig. 3B and F) and/or granzyme B (Fig. 3B) thancontrol T cells, which did not produce detectable IFNg levelsunder those stimulation conditions. At present, we are unsure asto themolecularmechanisms underpinning this important obser-vation, which is an important part of our ongoing investigations.In addition to demonstrating a reduced tendency to becomeexhausted, CD8a:MyD88–expressing T cells altered various facetsof the TME toward one that favors antitumor immune responses.

Endogenous CD8+ T cells

DC

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Figure 6.

Proposed cellular processes impactedby CD8a:MyD88–expressing T cells inthe tumor microenvironment. A, Weobserved an increased frequency ofCD8a:MyD88 T cells in the tumor,indicating enhanced infiltration,expansion, and/or survival of CD8a:MyD88–transduced T cells. B, CD8a:MyD88 T cells exhibit enhancedproliferation in response to tumorantigen and tumor cells in culture, aneffect that we speculate might beoccurring in vivo. C, CD8a:MyD88 Tcells produce increased levels of IFNg .D, Increased levels of the IFNg can leadto induction of the T-cell chemokineCXCL9, which were detected in thetumor and could contribute to theenhanced CD8þ T-cell numbersobserved in the tumor. E and F, IFNghas the ability to influence the antigenpresentation machinery and isreflected by an enhanced expressionof MHC I on nonhematopoietic(CD45�) cells, presumably tumor cells(E), and increased MHC II and CD86expression on DCs (F). G, There weredecreased levels of CCL2 in the tumor,which is a monocyte chemoattractantprotein. Monocytes are the precursorsof macrophages, and accordingly, anoverall decreased number ofmacrophages was detected in thetumor of CD8a:MyD88 T cell–treatedmice. H, The macrophages thatwere present was skewed from aprotumorigenic phenotype toward aphenotype favoring antitumorimmunity.






These changes included increased T-cell infiltrates, an alteredcytokine/chemokine milieu, increased expression of stimulatorymolecules on DCs, and a skewing of the macrophage populationfrom a protumor to an antitumor population. The proposedmechanisms by which CD8a:MyD88 T cells enhance antitumorresponses are provided in Fig. 6.

In summary, the use of CD8a:MyD88 represents a universalapproach to potentiate T-cell responses in cancer patients,regardless of the HLA type. Moreover, that the TCR-enhancingeffects of CD8a:MyD88 occur strictly upon antigen recogni-tion is innovative and highly suitable for self-regulating T-cellresponses.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Kaczanowska, E. DavilaDevelopment of methodology: S. Kaczanowska, A.M. Joseph, E. DavilaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Kaczanowska, A.M. Joseph, J. Guo, A.K. Tsai,J.J. Lasola

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Kaczanowska, J. Guo, A.K. Tsai, K. Younger,Y. Zhang, E. DavilaWriting, review, and/or revision of themanuscript: S. Kaczanowska, A.K. Tsai,Y. Zhang, E. DavilaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. YoungerStudy supervision: S. Kaczanowska, Y. Zhang, E. DavilaOther (statistical analysis of additional mice studies): C.V. Gonzalez

AcknowledgmentsWe thank S. Vogel for providing us IRAK-4 kinase dead mice.

Grant SupportThis research was supported by the University of Maryland Marlene and

Stewart Greenebaum Comprehensive Cancer Center, NCIR01CA140917,P30CA134274, VA Merit Award BX002142, and National Institute of Allergyand Infectious Diseases 2T32AI007540-17.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 3, 2017; revised September 14, 2017; accepted October 17,2017; published OnlineFirst October 20, 2017.

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2017;77:7049-7058. Published OnlineFirst October 20, 2017.Cancer Res Sabina Kaczanowska, Ann Mary Joseph, Jitao Guo, et al. AntigensResponses to Weakly Immunogenic and Lowly Expressed Tumor

T-cell+:MyD88 Coreceptor Enhances CD8αA Synthetic CD8

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