Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity

This information is current as Wei-Zen Wei, Heather M. Gibson, Jennifer B. Jacob, Jeffrey of September 28, 2021. A. Frelinger, Jay A. Berzofsky, Hoyoung Maeng, Gregory Dyson, Joyce D. Reyes, Shari Pilon-Thomas, Stuart Ratner and Kuang-Chung Wei J Immunol published online 12 August 2020

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 12, 2020, doi:10.4049/jimmunol.2000466 The Journal of Immunology

Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity

Wei-Zen Wei,* Heather M. Gibson,* Jennifer B. Jacob,* Jeffrey A. Frelinger,† Jay A. Berzofsky,‡ Hoyoung Maeng,‡ Gregory Dyson,* Joyce D. Reyes,* Shari Pilon-Thomas,x Stuart Ratner,* and Kuang-Chung Wei*

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose- dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and Downloaded from IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in 17 that was responsible for regulating the response. A/J and NOD segments of in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC- IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to http://www.jimmunol.org/ our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies. The Journal of Immunology, 2020, 205: 000–000.

ancer immunotherapy such as active immunization and only 30% generated detectable IFN-g–producing T cells (5). Together, checkpoint blockade induces or strengthens endogenous these results indicate genetic regulation in HER2-specific immune C tumor immunity to render lasting protection in some pa- responses. Consistent with these observations, HER2 breast cancer tients while providing minimal benefit in others (1). Such disparity patients produced variable vaccine responses in our and others’ clin- in treatment outcome is due, in part, to different levels of neo- ical trials (6, 7). Our recent trial (NCT01730118; https://ascopubs.org/ by guest on September 28, 2021 antigens (2, 3). Although it has not received as much attention, doi/abs/10.1200/JCO.2019.37.15_suppl.2639) immunized patients the host genetic background can also dictate immune response who had not received prior HER2-specific therapy with an to tumor-associated Ags. We reported that ERBB2 (HER2) DNA adenovirus/HER2 (Ad/E2TM)–transduced dendritic cell (DC). electroimmunization induced markedly more Ab and IFN-g–producing Patients with HER2+ metastatic disease received 10–20 3 106 T cells in BALB/c (BALB) HER2 transgenic (Tg) mice than those DCs, and clinical benefit (complete response, partial response, or on C57BL/6 (B6) background, with (BALBxB6)F1 HER2 Tg stable disease) was seen in approximately half of the patients (6). mice producing an intermediate response (4). Similarly, in outbred Final results including dose expansion cohort (40 3 106 DCs) and domestic cats that received feline HER2 electroimmunization, in patients who received prior anti-HER2 therapy are under review. The specific nature of individual tumor can influence the vaccine response, but a genetic component of regulation is also likely. *Department of Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201; †Valley Fever Center of Excellence, Department of Immunobi- -wide association studies have been performed ology, University of Arizona, Tucson, AZ 85724; ‡Vaccine Branch, Center for Cancer x extensively to identify genetic variants that influence human traits Research, National Cancer Institute, Bethesda, MD 20892; and Department of Im- (8). Minor polymorphic alleles are expressed at very low fre- munology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612 quency in humans, requiring a large sample size for the analysis. ORCIDs: 0000-0001-6036-3610 (W.-Z.W.); 0000-0003-2652-8252 (H.M.G.); 0000- 0002-3173-2496 (J.B.J.); 0000-0002-0342-3194 (J.A.B.); 0000-0002-8194- Although thousands of loci have been confirmed as disease risk 6089 (H.M.); 0000-0002-1647-4572 (S.R.). factors, very few studies have investigated the mechanisms un- Received for publication April 29, 2020. Accepted for publication July 11, 2020. derlying particular associations (9). This work was supported by National Cancer Institute Grant CA76340 (to W.-Z.W.) A new Diversity Outbred (DO) mouse system arose after an ex- and the Herrick Endowment (to W.-Z.W.). tensive breeding effort (10, 11). DO mice were created by nonsibling Address correspondence and reprint requests to Dr. Wei-Zen Wei, Karmanos Cancer crossing of eight inbred founder strains (A/J, C57BL/6J, 129S1/ Institute, EL02, Wayne State University, 4100 John R. Street, Detroit, MI 48201. SvlmJ, NOD/HILtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/ E-mail address: [email protected] EiJ) to encompass .40 million single-nucleotide polymorphism The online version of this article contains supplemental material. (SNP), or .90% of all polymorphic alleles in mice. The genomes of Abbreviations used in this article: Chr, chromosome; CV, coefficient of variation; DC, dendritic cell; DO, Diversity Outbred; GigaMUGA, Giga Mouse Universal the founder strains have been fully sequenced so that the genetic Genotyping Array; LOD, logarithm of odds; QTL, quantitative trait locus; SC, composition of each DO mouse can be fully profiled using the Giga splenocyte; SNP, single-nucleotide polymorphism; Tg, transgenic; Treg, regulatory Mouse Universal Genotyping Array (GigaMUGA) that displays . 143,259 genetic markers, mostly SNPs, distributed evenly across Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 the 20 . This array allows the inference of small

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000466 2 DO MICE REVEAL QTL AND REGULATORS OF HER2 IMMUNITY chromosomal segments, haplotype blocks, by their inbred strain (Puresyn), and quantified by QuickTiter immunoassay (Cell Biolabs). Mice 8 of origin in each DO mouse by computational analysis. DO were twice immunized 2 wk apart by i.m. injection with 1 3 10 PFUs of mice express polymorphic alleles at high frequencies because Ad/E2TM in 50 ml of PBS. each allele comes from one of the eight founders. This model pE2TM electroimmunization enables robust mapping analysis with a manageable number of pE2TM and pEF-Bos/GM-CSF encoding murine GM-CSF were previously mice (12, 13). The ability to control the environment and described (20). Mice were anesthetized and 50 mg pE2TM in an admix treatment conditions of experimental mice further improves the with 20 mg pGM-CSF in 50 ml PBS was injected i.m. in the quadriceps accuracy of genetic mapping. muscle. Square wave electroporation using NEPA21 super electroporator In this study, syngeneic HER2 Tg mice were crossed with DO (Nepa ) was applied with pulses at 100 V with 50 ms duration de- livered eight times in two opposite orientations (4). mice to generate (B6xDO)F1 or (BALBxDO)F1 HER2 Tg mice. These F1 mice carry one chromosome from the syngeneic parent to Depletion of NK or regulatory T cell by Ab encompass the human HER2 gene in chromosome (Chr)5 and the To deplete regulatory T cell (Treg), mice were injected once i.p. with 500 mg DO genes in the other chromosome to provide individually dis- mAb PC61 directed at CD25 10 d prior to the first Ad/E2TM immuniza- tinct genetic backgrounds (14). Mice immunized with either Ad/ tion. To deplete NK cells, (NODxDO)F1 HER2 Tg mice received 1 mg E2TM or naked pE2TM developed a wide range of HER2-specific mAb anti-NK1.1 i.p., and (A/JxDO)F1 HER2 Tg mice received both 1 mg IgG and IFN-g–producing T cells, consistent with genetic regu- of anti-NK1.1 and 50 ml of rabbit anti-asialo GM1 serum on days 0, 2, and 4 before mice were immunized on day 7. lation of vaccine response. We analyzed the association between anti-HER2 IgG levels and genetic markers using DO quantitative HER2-specific Ab and T cell response trait locus (QTL) analysis. We found elevated logarithm of odds Sera and splenocytes (SC) were collected 2 wk following the last immu- Downloaded from (LOD) scores, an adjusted measurement of genomic association nization. Anti-HER2 IgG was measured by binding to HER2+ SKOV3 cells significance, with total IgG and IgG2a/b/c (IgG2) on Chr2 and using flow cytometry and Ab concentrations calculated by regression analysis Chr17. We further identified NK cells, which interact with MHC- using mAb TA-1 as the standard (21). Normal mouse serum or isotype- matched mAb was the control. Results were analyzed by Student t test. IB gene products on Chr17, as positive regulators of anti-HER2 HER2-reactive T cells were enumerated by IFN-g ELISpot assay (BD Ab response. Biosciences) (22). PBL or SC were incubated with recombinant HER2 or

DO mice have been used previously to identify genetic loci Neu (ecd–Fc fusion; Sino Biological). Results were expressed as http://www.jimmunol.org/ 6 regulating disease conditions, such as chemical toxicity (15), spot-forming units per 1 3 10 SC and analyzed using Student t test. atherosclerosis (16), and pain (17). In a DO F1 Tg adenocarci- Data standardization noma of the mouse prostate (TRAMP) model, QTLs regulating Ζ 2 prostate cancer development were identified, and RWDD4 and Ab levels were standardized by =(x m)/d, where x is the raw data, m is the mean, and d is the SD. The Z scores represent the number of SD above CENPU were implicated as increasing the aggressiveness of cancer or below the mean that a specific IgG level falls. cells (18). We show that the DO F1 model is effective for identifying key regulators of immunotherapy response as well as for investi- Mouse genotyping and QTL analysis gating the mechanism of action and new intervention strategies. The genotype of each test mouse is determined with the GigaMUGA,

built on the Illumina Infinium platform (23). The assay is performed by guest on September 28, 2021 centrally at Neogen (https://genomics.neogen.com/en/mouse-universal- Materials and Methods genotyping-array). Mice and cell line DO QTL or the later R/qtl2 software package calculates genotype All animal procedures were conducted in accordance with the U.S. Public probabilities from GigaMUGA with a hidden Markov model that generates Health Service Policy on Use of Laboratory Animals and with approval by a probabilistic estimate of the diplotype state at each marker locus in each Wayne State University Institutional Animal Care and Use Committee. DO animal and evaluates the association between genotype and phenotype C57BL/6 (B6) and BALB female mice were purchased from Charles River (https://rqtl.org/qtl2cran) (11–13, 24). Genotyping data for the DO foun- Laboratory. DO female mice (J:DO, stock number 009376) were pur- ders are available at ftp://ftp.jax.org/MUGA. DO mice carry 36 possible chased from The Jackson Laboratory. Heterozygous C57BL/6 HER-2 Tg diplotypes, but F1 mice have only eight possible haplotypes. With 7 df in mice (B6 HER-2 Tg) expressing wild-type human HER-2 under the whey the genome scan model, the F1 model has a greater power for detecting acidic protein promoter were generated in our laboratory (19) and available QTL peaks than the DO model with 35 df but does not detect recessive from The Jackson Laboratory [B6.Cg-Pds5bTg(Wap-ERBB2)229Wzw]. alleles. BALB/c HER-2 Tg (BALB HER-2 Tg) mice were generated by back- Support intervals for QTL localization were determined using a 95% crossing B6 HER-2 Tg mice with wild-type BALB/c mice. Bayesian credible interval (25). The LOD curve is transformed by Human breast cancer cell line SKBR3 was obtained from American Type raising it to the power of 10 and a region covering 95% of the area under Culture Collection (Manassas, VA) and cultured in the recommended the transformed curve defines the support interval. The area under the medium. Authentication of cell line by short tandem repeat profiling was curve is numerically approximated using trapezoids between the marker carried out with Promega’s Cell ID Systems as described by the supplier loci. Allele association was inferred by the strain influence on the QTL. before cell stocks were frozen. Authenticated frozen stocks were thawed Alleles were examined using the SNP viewer (www.sanger.ac.uk/sanger/ and maintained for ,10 passages for experiments. Mouse_SnpViewer) and filtered on the consensus regions. Ad/E2TM construction, titration, and immunization Results The Ad/E2TM was constructed from pE2TM (4) by inserting E2TM se- Humoral response to Ad/E2TM immunization in DO F1 HER2 quence in the E1a/E1b region of the adenovirus 5 vector that also has the E1a/E1b and E3 regions deleted and the Ad35 Knob and Fiber substituted Tg mice is more variable than inbred B6 or BALB HER2 for the corresponding Ad5 regions. The product is an Ad5f35 vector Tg mice expressing the ECD and TM domains of human HER2, as described by To verify that HER2 immune response is influenced by genetic Maeng et al. (H.M. Maeng, L.V. Wood, B. Moore, M.H. Bagheri, S. Webb, L. England, G. Martinez, S.M. Steinberg,S.Pack,D.Stroncek,J.C.Morris, background, we analyzed human HER2-expressing B6 HER2 or M. Terabe, and J.A. Berzofsky, manuscript in preparation). The vector BALB HER2 Tg mice after they received Ad/E2TM, which is modification changes the receptor tropism of the adenovirus from CAR to currently being tested in trial NCT01730118. HER2 Tg mice CD46 and allows more efficient transduction of human DCs and hema- express and are immune tolerant to wild-type human HER2 but topoietic cells. The adenoviral construct was developed by Dr. Malcolm Brenner at the Baylor Center for Cellular and Gene Therapy under contract do not develop tumors (19). Mice were immunized i.m. with Ad/ with the Berzofsky laboratory, Vaccine Branch, National Cancer Institute. E2TM twice 2 wk apart. HER2 IgG levels were measured 2 wk Ad/E2TM was propagated in HEK293 cells, Adenopure column purified after the second immunization (Fig. 1A, 1B) per our established The Journal of Immunology 3 protocol (21). BALB HER2 Tg mice produced 42.2 6 10.1 mg/ml IgG1 indicates Th2 dominance, which is more prominent in mice HER2 IgG (n = 14) (Fig. 1B) with a coefficient of variation (CV) from the BALB/c background (26). These differential IgG subclass of 23.9%, whereas B6 HER2 Tg mice produced ∼10-fold lower IgG trends are maintained in the F1 pups even though the variability is (4.3 6 1.1 mg/ml; n = 10; CV = 24.9%) (Fig. 1A). These findings greater. Nontransgenic littermates also showed the same Th1 or Th2 are consistent with our previous report that BALB HER2Tg mice polarization when immunized with Ad/E2TM (Fig. 1E). aremuchmoreresponsivethanB6HER2TgmicetoHER2DNA electroimmunization (4). Like Ab responses, the IFN-g–producing T cell response in DO The difference between BALB and B6 suggests a genetic control F1 mice is more variable than parental strains and skewed of the Ab response to self-antigen. To determine the extent and toward Th1 in (B6XDO)F1 HER2 Tg mechanism of Ab variability, we crossed syngeneic HER2 Tg We further examined the Th1/CD8 response directly by measuring males with DO females to generate (B6xDO)F1 or (BALBxDO)F1 T cell IFN-g production. T cell response was also variable in HER2 Tg mice, allowing the pups to display HER2 in individually (B6xDO)F1 HER2 Tg mice ranging from 0 to 348 spot-forming unique genetic backgrounds. HER2 IgG response induced by 23 units per 1 3 106 SC (45.3 6 84.5 spot-forming units per 1 3 106 Ad/E2TM immunization in (B6xDO)F1 HER2 Tg (n = 84) and SC) (n = 54; CV = 186.5%) (Fig. 2A). Half of the mice did not (BALBxDO)F1 HER2 Tg (n = 29) varied much more than their produce measurable IFN-g at all. Response in the (BALBxDO)F1 syngeneic inbred counterparts (Fig. 1A, 1B). (B6xDO)F1 HER2 HER2 Tg mice was similar, with just over half of the mice gen- Tg mice produced 0–128 mg/ml IgG (22.7 6 28.6 mg/ml; CV = erating highly variable T cell responses (Fig. 2B). These findings 125.6%) (Fig. 1A) and (BALBxDO)F1 HER2 Tg mice produced further indicate the genetic regulation of Th1/CD8 HER2 immu-

3–172 mg/ml IgG, with a higher mean value (51.3 6 49.6 mg/ml; nization response. Nontransgenic (B6xDO)F1 and (BALBxDO)F1 Downloaded from CV = 96.7%) (Fig. 1B), which is consistent with a higher HER2 littermates produced more consistent T cell response to foreign vaccine response in mice of BALB/c origin. In nontransgenic lit- HER2 (Fig. 2C). These results point to the conclusion that genetic termates in which human HER2 was a foreign Ag, Ad/E2TM im- polymorphism regulates immune response to self-antigen, but less munization resulted in consistently higher IgG response: (B6xDO) so to foreign Ags. F1 produced 93.7 6 37.1 mg/ml (n =58;CV=39.5%)and The percentage of CV for IgG response was 24.9 for syn-

(BALBxDO)F1 produced 67.5 6 43.8 mg/ml (n = 18; CV = 64.9%) geneic B6 HER2 Tg mice and 125.6 for all (B6xDO)F1 HER2 http://www.jimmunol.org/ HER2 IgG (Fig. 1C). Taken together, these data indicate that F1 Tg mice (Fig. 1A). The percentage of CV for siblings in each mice developed strong and more consistent HER2 IgG response of 14 litters of (B6xDO)F1 HER2 Tg mice ranged from 38 to when HER2 was a foreign Ag, but the response was highly het- 124, with an average of 80.1. Therefore, DO F1 siblings whose erogeneous when HER2 is a self-antigen. genetic heterozygosity falls between syngeneic mice and non- We also analyzed HER2-specific IgG subclasses. In (B6xDO) sibling DO F1 mice showed intermediate levels of variation, F1 HER2 Tg mice, the IgG2/IgG1 ratio was 1.8 6 1.0, whereas indicating a correlation between immune response variation and in (BALBxDO)F1 HER2 Tg mice, the ratio was 0.76 6 0.29 genetic heterozygosity. (p , 0.0001) (Fig. 1D). IgG2 is indicative of Th1 dominance, In one batch of (B6xDO) F1 HER2 Tg mice, Pearson correlation commonly detected in mice from the B6 background, whereas coefficient between the levels of IgG and IFN-g–producing T cells by guest on September 28, 2021

FIGURE 1. Ab response to Ad/E2TM immunization in DO F1 HER2 Tg mice. Mice were immunized twice with Ad/E2TM. Ab levels (micrograms per milliliter) are shown for (A)B6HER2and(B6xDO)F1 HER2 Tg mice, (B)BALBHER2,and (BALBxDO)F1 HER2 Tg mice, and (C) nontransgenic littermates. IgG2-to-IgG1 ratios are shown for (D) B6, (B6xDO)F1, BALB, and (BALBxDO)F1, HER2 Tg mice, and for (E) nontransgenic littermates. The number of mice and the CV are shown below each figure. 4 DO MICE REVEAL QTL AND REGULATORS OF HER2 IMMUNITY

FIGURE 2. T cell response to 23 Ad/E2TM im- munization in DO F1 HER2 Tg mice. IFN-g T cell response was measured by ELISPOT assay for (A)B6 and (B6xDO)F1 HER2 Tg mice, (B) BALB and (BALBxDO)F1 HER2 Tg mice, and (C) nontransgenic littermates. The number of mice in each group and CV are shown below each figure. Downloaded from http://www.jimmunol.org/

was calculated to show strong to moderate correlation (data not arrows), suggesting that genes in these regions regulate HER2 shown), consistent with genetic regulation of both humoral and IgGresponse.GenomescansofIgG1andIgG2levelsareshownin cellular immunity. It will be necessary to further analyze Th2 cells Fig. 3F, 3G, respectively. There are several peaks in each genome by guest on September 28, 2021 that produce IL-4 or IL-5 to fully elucidate the relationship be- scan, consistent with many regulatory mechanisms for Ab production. tween Ab and T cell responses. Because the LOD scores are relatively modest, we have chosen to focus on the regions that are identified in multiple scans and Defining QTL regulating HER2 IgG response in shared among the total IgG, IgG1, and IgG2 responses. Com- Ad/E2TM-immunized mice paring LOD profiles of total IgG (Fig. 3E) and IgG2 (Fig. 3G), For QTL analysis, the immunization of and tissue collection from the dominant IgG isotype in (B6xDO) HER2 F1 mice, common 84 (B6xDO)F1 HER2 Tg mice were performed in three batches. QTLsemergedinChr2andChr17(redarrows).Meanwhile,the Tail tissue DNA samples were submitted for haplotype analysis QTL in Chr15 (green arrows) was shared between the total using GigaMUGA, which encompasses 143,259 chromosomal IgG and IgG1 datasets (Fig. 3F). We focused on the QTL map- markers (23). Genotype probabilities/reconstructions and kinship ping to Chr2 and Chr17 because IgG2 was the most prominent matrices were generated using the DO QTL package (https:// isotype. github.com/rqtl/qtl2) (12). Kinship is expressed in color with a Total IgG and IgG2 responses were further analyzed to value of 1 (white), representing complete identity or relatedness to evaluate founder strain QTL effects at the Chr2 locus (Fig. 4A, self, and 0 (red) denoting no genetic relatedness at all (Fig. 3A). 4B, respectively). The eight founder strains are color coded. The shades between red and white indicate degrees of relatedness. The founder strain haplotype at each marker is analyzed against Of 84 (B6xDO) F1 mice, 69 mice had kinship values ,0.4, or Ab response (Fig. 4A, 4B, upper panels). Positive values in- ,40%, genetic identity with any other mouse in the cohort. These dicate higher-than-average response. The corresponding ge- were chosen for further analysis to reduce bias from closely re- nome scan is displayed just below the founder strain effect plot lated mice. HER2-binding Ab in immune sera were measured by (Fig. 4A, 4B, lower panels). Three regions were noted. For total flow cytometry. Ab levels from these 69 mice were subjected to DO IgG, Chr2:137,958,080 (LOD = 5.6) and Chr2:153,912,130 QTL analysis. To minimize batch effects, the Ab levels in each (LOD = 5.5) were prominent QTLs, although the LOD scores batch were converted to a standardized Z score: Ζ =(x 2 m)/d, were modest. In the IgG2 dataset, the LOD scores for Chr2:137,207,420 where x is the IgG level of individual mice, m is the mean, and d (LOD = 7.4) and Chr2:153,912,130 (LOD = 7.8) were higher, is the SD. The Z scores of the 69 samples ranged from +3.2 to 21 generating more confidence. Importantly, despite the relatively (Fig. 3B). Similar Z score ranges were observed for IgG1 and low LOD scores, these two regions largely overlap in total IgG2(a/b/c) (IgG2) (Fig. 3C, 3D). IgG and IgG2. Comparing the 95% confidence interval of the The association between HER2-binding Abs and genetic QTLs in total IgG and IgG2, Chr2:136,565,356–137,954,640 markers was calculated, and LOD values are presented in the and Chr2:152,765,413–153,917,380 represent the shared re- genome scan Manhattan plot (Fig. 3E). The most prominent gions. Another prominent QTL appeared in Chr2:64,912,590 QTLs were found in Chr2, Chr15, and Chr17 (red and green (LOD = 9.33) for IgG2 and in Chr2:64,912,850 (LOD = 5.1) The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Kinship and QTL analysis of standardized HER2-specific Ab. (A) Kinship analysis of (B6xDO)F1 HER2 Tg mice. (B–D) Standardized Z scores of anti-HER2 total IgG, IgG1, or IgG2. (E–G) Genome scan Manhattan plots show candidate QTLs for total IgG, IgG1, or IgG2. Green arrows indicate QTLs shared between total IgG and IgG1. Red arrows indicate QTLs shared between total IgG and IgG2. for total IgG, with overlapping 95% confidence interval immunization, and HER2 Ab was measured. As expected, total (Chr2:64,670,932–65,972,260). IgG (Fig. 5A) and IgG2 levels (Fig. 5B) were highly variable. Meanwhile, cancer immunogens can be delivered with viral A genome scan Manhattan plot for pE2TM-induced total IgG vectors by DNA electroporation or in other formulations. Each (Fig. 5C) identified an LOD peak in Chr17 adjacent to that in Ad/ vaccine formulation could induce shared or unique regulatory E2TM total IgG plot (Fig. 3F, Supplemental Table I). IgG2, again mechanisms. To identify QTL that regulate immunity to HER2 rather the most abundant IgG, was also measured and an LOD peak than the vector, we also analyzed HER2 IgG response induced by identified (Figs. 3G, 5D) within 1.5 megabase (Supplemental naked DNA electroimmunization. Table I). Based on the 95% confidence interval for LOD peaks, Chr17:34,962,626–36,638,254 was the most stringent denomina- HER2 IgG response induced by pE2TM electroimmuniztion in tor shared by all four datasets (Supplemental Table I). The founder (B6xDO)F1 HER2 Tg mice strain effects for Ad/E2TM and pE2TM at this QTL (Fig. 5E–H) (B6xDO)F1 HER2 Tg mice were electroimmunized with naked show A/J (yellow) or NOD (dark blue) haplotypes associating DNA encoding E2TM, the same recombinant HER2 protein with elevated total IgG and IgG2. B6 (gray) haplotype at this QTL expressed by Ad/E2TM. Sixty-eight mice with kinship ,0.4 were is associated with poor total IgG and IgG2 consistent with low analyzed. Immune serum was collected 2 wk after the second HER2 IgG found in B6 HER2 Tg mice (Fig. 1A). 6 DO MICE REVEAL QTL AND REGULATORS OF HER2 IMMUNITY

FIGURE 4. Founder strain QTL effects at Chr2 for total IgG and IgG2. Founder QTL effects for (A) total IgG and (B) IgG2 are shown in the top panel. LOD scores are shown in the lower panel. Each founder Downloaded from strain is shown in the designated color code. Positive values indicate higher-than- average response. Negative values indicate the opposite. http://www.jimmunol.org/ by guest on September 28, 2021

We tested if anti-HER2 IgG in (B6xDO)F1 HER2 Tg mice is polymorphisms in this region but has one unique structural alteration functional by incubating HER2+ breast cancer cell line SKBR3 at 65,284,693–695 because of insertion. cells with sera collected before and after immunization (Supplemental The region of Chr17 (Chr17:34,962,626–36,638,254) is a Fig. 1). Immune serum was diluted to 3, 1.5, and 0.75 mg/ml based large region that encompasses many genes (83 polymorphic on HER2-binding IgG level measured by flow cytometry. We ob- genes). The major driver strain of elevated total IgG and IgG2 in served a dosage-dependent inhibition of SKBR3 cell survival by the all four datasets is A/J. Strain NOD contributes in three of four immune serum, showing antitumor activity of immunization induced datasets. There are 23 genes with A/J unique substitutions in anti-HER2 IgG. this region, including the genes Atat1, Cdsn, Ddx39b, Gm8909, Gm9573, Gtf2h4, H2-Bl, H2-D1, H2-M10.1, H2-Q1, Identification of candidate genes in QTL regions H2-Q2, H2-Q5, H2-Q7, H2-Q10, H2-T22, H2-T23, Ly-6g6e, We evaluated the polymorphic genes in the shared QTL regions. Ly-6g6f, Msh5, Prrc2a, Sapcd1, Vars2,andVwa7. These are shown We coupled that information with the strongest strain inference in Supplemental Table III. (Figs. 4, 5) to identify unique polymorphisms in those regions. The genes in the region of Chr2:136,565,356–137,954,640 (region 2) MHC-IB and NK cells regulate HER2 IgG response were identified. In this region Ab response was driven by NZO and These 23 genes encompass several important biological and im- CAST, and these two strains share several unique missense poly- munological mechanisms. A similar gene cluster is found in human morphisms in Mkks and Slx4ip genes (Supplemental Table II). In Chr6, suggesting a preservation of immune regulatory genes region 3, Chr2:152,765,413–153,917,380, the response was driven by through evolution. Of note are H2-Q7, H2-Q10, and H2-T23, all PWK/CAST. PWK has unique missense SNPs in Mylk2, Ttll9,and encoding that interact with inhibitory receptors of NK cells Pofut1. CAST has missense variants in Tpx2, Mylk2, Foxs1, Ttll9, (Supplemental Table I). Both H2-Q7 and H2-Q10 proteins are Ccm2l, Tspyl3, Commd7, Bpifb2,andBpifb6. The genes in these ligands of inhibitory receptor Ly-49C (27). H2-T23 (Qa1b), a regions are potential regulators of Ab response to self-antigen and homolog of human HLA-E, binds another inhibitory receptor their missense SNPs are shown in Supplemental Table II. For the first CD94/NKG2A (28, 29). Interestingly, A/J and NOD haplotypes QTL region in Chr2 (Chr2:64,670,932–65,972,260), the Ab response share 17 missense mutations that are distinct from all other appeared driven by 129. Strain 129 did not have any unique missense founder strains within Chr17:36,031,051–36,032,420 of H2-T23 The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Shared QTLs and genes in HER2 Ab response induced by pE2TM and Ad/E2TM. HER2-specific (A) total IgG and (B) IgG2 in (B6xDO)F1 HER2 Tg mice after two-time pE2TM DNA electroimmunization. Genome scans of test mice show QTLs for (C)totalIgGor(D) IgG2. Founder QTL effects of pE2TM induced (E)totalIgGor(F) IgG2, and Ad/E2TM-induced (G)totalIgGor(H) IgG2 are shown. The red arrow identifies the LOD peak in Chr17. The blue arrow identifies A/J (yellow) and NOD (blue) as drivers at this locus. Founder QTL effects (upper panels) and LOD scores (lower panels) are shown. gene (Supplemental Table III). We proceeded to test the hypoth- mice. Given that NK cells produce copious amount of IFN-g,one esis that NK cells regulate HER2 vaccine response in HER2 Tg might expect greater impact on IgG2 production. The reduction of mice. We crossed B6 HER2 Tg mice with A/J and NOD mice and IgG1 may suggest mechanisms beyond IFN-g. It should be noted tested the effect of depleting NK cells in their HER2 Ab response. that anti-NK1.1 would also deplete a portion of NKT cells, which Test mice were immunized with Ad/E2TM twice. Before immu- can also produce IFN-g, but because their restriction element CD1d nization, half of the mice received mAb NK1.1 or NK1.1 plus is on mouse Chr3, not Chr17, the genetics do not point to this anti-asialo GM1 to deplete NK cells. HER2 binding total IgG, lineage as playing a major role. IgG1, and IgG2 were measured after each immunization. HER2 IgG production was reduced by 5–10-fold in NK-depleted (B6xA/J) Depletion of Treg amplifies and normalizes HER2 F1 (Fig. 6A) and (B6xNOD)F1 (Fig. 6B) HER2 Tg mice, showing IgG response NK cells as positive regulators of HER2 Ab response. Similar re- Immune reactivity is often regulated by both positive and negative ductions were observed in IgG1 and IgG2 (p , 0.05). Furthermore, mechanisms. Because NK cells positively regulate HER2 immune the reduction in IgG1 is near complete in (B6xNOD)F1 HER2 Tg response, negative regulation is to be expected. We previously 8 DO MICE REVEAL QTL AND REGULATORS OF HER2 IMMUNITY

FIGURE 7. Treg negatively regulate Ad/E2TM-induced HER2 IgG re- Downloaded from sponse. Treg in (B6xDO)F1 HER2 Tg mice and nontransgenic littermates were partially depleted with anti-CD25 mAb PC61 prior to Ad/E2TM immunization. HER2-specific total IgG were measured following 23 Ad/E2TM immunization.

genetic markers on GigaMUGA with standardized Ab levels.

Chr17:34,962,626–36,638,254, home to the MHC-IB gene http://www.jimmunol.org/ family, emerged as a common denominator. H2-Q7, H2-Q10, and H2-T23 in this region interact with inhibitory receptors on NK cells (27, 29). NK cell depletion before immunization reduced HER2 IgG response, demonstrating NK cells as a positive regulator. We further described in DO F1 HER2 Tg mice a negative regulation of HER2 IgG response by Treg. FIGURE 6. NK cells positively regulate Ad/E2TM-induced HER2 IgG response. HER2-specific total IgG, IgG1, and IgG2 in (A) (B6xA/J)F1 and QTL analysis did not pull out Treg-associated genetic loci, (B) (B6xNOD)F1 HER2 Tg mice were measured after 23 immunization likely because Treg suppress tumor immunity regardless of with Ad/E2TM with or without prior NK cell depletion. genetic background. Thus, by combining the new DO QTL by guest on September 28, 2021 analysis with conventional tumor immunology experience, we discovered that NK and Treg regulate HER2 Ab response from reported enhanced HER2 vaccine response by Treg depletion in opposite directions, setting a platform for modulating HER2 syngeneic HER2 Tg mice (4, 30). To test if Treg also control immune response by balancing NK and Treg or their func- HER2 immune response in outbred mice, (B6xDO)F1 HER2 Tg tional receptors. We do note that each candidate gene identi- mice were treated with anti-CD25 mAb 10 d prior to the first Ad/ fied through DO QTL still needs to be individually validated to E2TM immunization per our standard protocol (4). HER2 IgG establish its role and mechanism of action. Murine QTL, or the response was elevated and became more uniform (Fig. 7, gray parallel human genome-wide association studies, reveals ge- circles). Treg depletion of nontransgenic (B6xDO)F1 littermates netic association, not causation, of the phenotype. Deciphering had little impact on their response to HER2 as a foreign Ag (Fig. 7, the functional QTL and causative genes requires some con- white circles). Therefore, Treg negatively regulate vaccine humoral sideration. F1 mice have eight possible haplotypes, or 7 df. response to self-HER2 regardless of the genetic background. With If DO mice with 36 possible diplotypes were used, many these results, we conclude that NK cells and Treg are opposing more mice would be required to reach the necessary power regulators in HER2 immunization response with strong implica- of analysis. Furthermore, we had the opportunity to cross- tions on future planning of immunotherapy. reference genome scan results from two different immuno- gens encoding the same Ag, as well as cross-referencing two Discussion independent Ig measurements: total IgG and IgG2 in the same Our HER2 DNA constructs have advanced to clinical trials in- serum sample. This allowed us the opportunity to identify a cluding the ongoing phase I autologous AdHER2 DC trial (https:// QTL in Chr17 without an excessive number of mice or ex- ascopubs.org/doi/abs/10.1200/JCO.2019.37.15_suppl.2639) (6, 7). ceptional LOD value. Interestingly, MHC is the single region Preclinical studies indicated direct tumor inhibitory effects of most often associated with autoimmunity (31, 32), reinforcing immune sera (6). Clinical benefit has been observed in selected the validity of the Chr17 QTL in self-HER2 immunity. It was patients suffering from HER2-expressing solid tumors. To expand somewhat surprising to map the QTL to a region rich in the benefit of HER2 immunization in heterogeneous human pa- nonconventional MHC-IB and to have the subsequent confir- tients, the regulatory mechanisms of HER2 Ab response was mation that NK cells regulated by MHC-IB contribute directly sought. We tested HER2 vaccines in (B6xDO)F1 and (BALBxDO) to HER2 vaccine response. NK cells are recognized for their F1 HER2 Tg mice to recapitulate immunization response in outbred prompt secretion of IFN-g when encountering immune stim- populations and to identify candidate regulatory genes. Ad/E2TM ulation. We reported that NK cells were important in Ad/E2- and pE2TM immunization were compared side-by-side. Using induced, Ab-dependent, cell-mediated cytotoxicity against the DO QTL package in the R environment, we associated mammary tumor growth (33). However, this activity was at the The Journal of Immunology 9 effector, not priming, phase. NK cell or IFN have been asso- B10 mice (46). Interestingly, we also observed that A/J back- ciated with both positive or negative impact on viral ground led to greater Ab production compared with B6. Similarly, or autoimmunity (20, 34–36). Treatment with NKG2A inhib- although HER2 IgG levels are highly variable in DO F1 HER2 Tg itory mAb in cancer therapy may shed on whether mice, the average Ab level is higher in mice with a BALB/c blocking this checkpoint receptor will be beneficial (37). parent, consistent with greater HER2 Ab response in BALB NKT cells, which recognize lipid Ags in the context of CD1d, can HER2 than B6 HER2 Tg mice (4). This is also true when the ratios also be depleted by anti-NK1.1 mAb (38, 39). Inhibitory NK cell of IgG2/IgG1 were measured to reflect the impact of Th1 and Th2. receptors CD94/NKG2A and Ly-49c, which interact with H2-T23 and Introduction of DO genetic composition did not fully override Th1 H2-Q7/Q10, respectively, can also regulate NKT cell activation (40, versus Th2 preference. 41). It is possible that depletion of NK cells with mAb NK1.1 also In nontransgenic mice, the response to human HER2 as a foreign depleted a portion of NKT cells, and these are also known for pro- Ag is relatively uniform. Regardless of the parental strains or ducing IFN-g and could thus promote IgG2 Ab responses as well as genetic polymorphisms, high levels of HER2 IgG and T cell re- activate DCs. However, CD1d, the restriction element for NKT cells, sponses were induced, showing effective immunity against foreign is on mouse Chr3, not Chr17, with other class IB genes, so the genetic Ag even though their T and B cell and Treg composition in the PBL mapping does not point to NKT cells. More in-depth analysis could demonstrated a high degree of diversity (data not shown), sug- elucidate if NKT cells contribute to HER2 immunity even though gesting the adaptation of genetically diverse individuals to survive HER2 is not a lipid Ag. Still, modulation of inhibitory receptors environmental assaults. shared by NK and NKT cells may enhance immune response if Studies using the eight founder strains of DO mice showed

NKT cells also contribute to the regulation of HER2 immunity. strain-driven differences in enteric microbial structure, indicating Downloaded from Ag delivery formulations have been developed empirically, and genetic influence of intestinal microbiome (47). When nearly 400 they evoke different regulatory mechanisms. The same HER2 ECD DO mice were subjected to DO QTL analysis, slc10a2 was andTMdomainswereexpressedbybothAd/E2TMandpE2TM,but identified as a possible regulator of both a gut microbe and a bile adenoviral vector introduces foreign viral components that may tilt acid (48). In other studies, vaccine response to infectious patho- HER2 response. The uptake and processing of viral particles involves gens was associated with HLA polymorphism (47, 49). One could

more cellular mechanisms than direct delivery of naked DNA by ask whether the same genetic elements could regulate both http://www.jimmunol.org/ electroporation. QTL analysis indicates common and unique regulatory microbiome and vaccine response and could be identified through mechanisms in the Ad/E2TM and pE2TM vaccine delivery system, DO QTL. Although our DO QTL results did not indicate a direct providing additional insights to immunotherapy considerations. association between microbiota and vaccine response, the DO and Additional QTL shared by selected datasets merit attention (e.g., DO F1 platforms would be most suitable for addressing this and Chr2:136,565,356–137,954,640 and Chr2:152,765,413–153,917,380 many other unanswered biological questions. appeared in both total IgG and IgG2 following Ad/E2TM immuni- One important question in any DO mouse study is whether QTL zation). Genes encompassed in these regions may lead to improved identified in mice can be translated to humans because not every understanding of Ad/E2TM immune response. mouse gene has a human homolog, and the regulation mechanisms One QTL in Chr15 was found in Ad/E2TM-induced total IgG of mouse and human phenotypes are not always the same. We show by guest on September 28, 2021 and IgG1, but not IgG2, datasets. The LOD peak for total IgG that identification of a QTL rich in MHC-IB gene combined with (chr15: 26,932,660; LOD = 6.6) and IgG1 (chr15:27,255,750; LOD conventional tumor immunology knowledge led to the discovery = 7.0) overlap in their 95% confidence intervals. March11 and that NK cells positively regulate HER2 humoral immunity. With FBXL7, both involved in protein ubiquitination, are found in this extensive conventional effort, blockade of NKG2A checkpoint is region. Further verification will be required to determine if being tested in clinical trials in combination with cetuximab (37). ubiquitination contributes to Ad/E2TM-induced IgG1 production. Our findings argue that DO mice are a strong representation of In addition to assessing genetic regulation of vaccine response, outbred humans and a powerful tool for discovering and validating HER2 DO mice are a powerful platform for testing novel cancer clinically important genetic variables. The richness of information immunotherapies. Reduction in total IgG, IgG1, and IgG2 was from DO F1 QTL analysis will withstand repeated query and observed in Ad/E2TM-immunized, NK-depleted DO F1 mice. On provide new leads for scientific discoveries and disease control. the contrary, a striking elevation and more consistent production of HER2 IgG was observed when mice received anti-CD25 mAb prior Acknowledgments to Ad/E2TM immunization. This is consistent with our report that This article is prepared in proud memory of Richard F. Jones, who dedicated Treg suppress HER2 immunity and autoimmunity in HER2 Tg his professional life to cancer research. We thank Daniel M. Gatti and Pen- mice of BALB, B6, and HLA-DR3 background (42, 43). Inter- Yuan Hsing for invaluable guidance in DO QTL analysis. We thank Claire estingly, a direct antagonism of Treg and NK cells in mammary McCarthy and Soumith Inturi for assistance in performing the analysis. We tumor lung metastases has been previously reported (44). It will be thank Malcolm Brenner and other members of the Center for Cell and Gene of interest and importance to determine if NK and Treg interact Therapy, Baylor College of Medicine, Texas Children’s Hospital, and Hous- directly in HER2 immunization. The current finding supports the ton Methodist Hospital, Houston, Texas for help in Ad/E2TM construction. We also recognize Karmanos Cancer Institute Microscopy, Imaging and likely benefit of modulating Treg and NK cells during immuno- Cytometry Shared Resources for flow cytometry support. therapy in genetically diverse human patients. Because anti-CD25 mAb can deplete regulatory as well as activated effector T cells, mAb PC61 was given to mice 10 d before the first Disclosures immunization and not after any immunization. If Treg depletion The authors have no financial conflicts of interest. should be required after the initiation of immunization, the study could be conducted in mice of DEREG background (45), in which Treg can References be removed specifically by administration of diphtheria toxin. 1. Zappasodi, R., T. Merghoub, and J. D. Wolchok. 2018. Emerging concepts for Previous work demonstrated that genetic background can in- immune checkpoint blockade-based combination therapies. [Published erratum appears in 2018 Cancer Cell 34: 690.] Cancer Cell 33: 581–598. fluence Ab production in an MHC-independent manner, with A/J 2. Thorsson, V., D. L. Gibbs, S. D. Brown, D. Wolf, D. S. Bortone, T. H. Ou Yang, mice producing more Abs but with lower affinity compared with E. Porta-Pardo, G. F. Gao, C. L. Plaisier, J. A. Eddy, et al; Cancer Genome Atlas 10 DO MICE REVEAL QTL AND REGULATORS OF HER2 IMMUNITY

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Post Immune Sera Pre-immune Sera

100 90 80 70 60

PercentViability 50 40 (compared to Untreated Control) Pg/ml 3 1.5 0.75 3 1.5 0.75 3 1.5 0.75 3 1.5 0.75 3 1.5 0.75 3 1.5 0.75 Sample #108 #126 #129 # 142 #147 #150 (50Pg/ml) (65Pg/ml) (208Pg/ml) (167Pg/ml) (230Pg/ml) (49Pg/ml)

Supplemental Figure 1 Immune serum inhibits HER2+ breast cancer cell survival in vitro. SKBR3 cells were plated at 4000 cells/well and were treated with serum from (B6xDO)F1 HER2Tg mice before and after 2X pE2TM electroimmunization. Immune serum was diluted to 3, 1.5 and 0.75 Pg/ml of HER2 binding IgG level as measured by flow cytometry. Cell viability was assessed by Alamar Blue assay 24hrs post treatment. Number in parenthesis shows the total HER2 binding IgG level in each sample. Six representative samples with IgG levels 49-230 Pg/ml are shown.

Supplemental Table I Chromosome 17 QTL and MHC IB genes Chromosome 17 QTL location Vaccine Antibody Chromosome Peak position LOD CI* low CI high subclasses Ad/E2TM Total IgG 17 35,916,030 6.0 34,962,626 41,976,160 IgG2(a/b/c) 17 35,961,290 5.9 33,920,577 41,976,160 pE2TM Total IgG 17 35,667,881 4.6 8,800,201 36,974,560 IgG2(a/b/c) 17 34,535,980 5.1 33,920,577 36,638,254

MHC IB genes in Chromosome 17 QTL interact with NK cell receptors Gene Protein Receptor cell Location Bordering pE2TM Ad/E2TM markers LOD LOD H2-Q10 H2-Q10 Ly49C NK chr17:35,470,089-35,474,563 UNCHS044208 4.7 5.0 UNC27818801 4.4 5.0 H2-Q7/9 Qa-2 Ly49C NK chr17:35,439,155-35,443,734 same same same H2-T23 Qa-1b CD94- NK chr17:36,029,976-36,032,855 UNC27824779 4.3 5.4 NKG2A/C/E JAX00437506 4.3 5.3 * CI – 95% confidence interval