Memory T cells specific for murine CMV reemerge after multiple challenges and recapitulate immunity in various adoptive transfer scenarios

Abstract

Reconstitution of CMV-specific immunity following transplant remains a primary clinical objective to prevent CMV disease, and adoptive immunotherapy of CMV-specific T cells can be an effective therapeutic approach. Due to viral persistence, most CMV-specific CD8^pos T cells become terminally differentiated effector cells (T_EFF). A minor subset retains a memory-like phenotype (T_M), but it is unknown whether these cells retain memory function or persist over time. Interestingly, recent studies suggest that CMV-specific CD8^pos T cells with different phenotypes have different abilities to reconstitute sustained immunity following transfer. The immunology of human CMV (HCMV) infections is reflected in the murine model (MCMV). We found that HCMV- and MCMV-specific T cells displayed shared genetic programs, validating the MCMV model for studies of CMV-specific T cells in vivo. The MCMV-specific T_M population was stable over time and retained a proliferative capacity that was vastly superior to T_EFF cells. Strikingly, after transfer, T_M cells established sustained and diverse T cell populations even after multiple challenges. Although both T_EFF and T_M cells could protect Rag−/− mice, only T_M cells persisted after transfer into immune replete, latently-infected recipients and responded if recipient immunity was lost. Interestingly, transferred T_M cells did not expand until recipient immunity was lost, supporting that competition limits the antigen stimulation of T_M cells. Ultimately, these data show that CMV-specific T_M cells retain memory function during MCMV infection and can reestablish CMV immunity when necessary. Thus, T_M cells may be a critical component for consistent, long-term adoptive immunotherapy success.

Introduction

Latent Cytomegalovirus (CMV) is present within a large percentage of the population but is effectively controlled by the immune system (1-6). However, in transplant patients, immune suppression can allow CMV reactivations to progress to disease and increase mortality. Despite the advancements of anti-viral medications, long-term prevention of CMV disease is dependent on the reconstitution of CMV-specific immunity, which can be achieved through adoptive immunotherapy (5-18).

In adoptive immunotherapy, healthy CMV-seropositive donors provide CMV-specific T cells to an immune suppressed recipient. Due to the persistent nature of CMV infection, CMV-seropositive donors accumulate large numbers of CMV-specific CD8^pos T cells (approximately 5-10% of the total CD8^pos T cells), a process termed “memory inflation,” (19-28). Studies in humans and the well-characterized mouse model (MCMV) have shown that the majority of inflationary populations are composed of terminally differentiated effector phenotype (T_EFF) T cells that presumably develop as a result of repeated antigen stimulation and may not possess the proliferative or survival capacity necessary for long-term maintenance of CMV immunity (22, 27, 29-34). Interestingly however, a fraction of these inflationary T cells retain a memory-like (T_M) phenotype, despite sharing epitope specificity and T cell receptor sequences with the T_EFF subset (23, 25, 35-37). Studies with other infection models have shown that such a memory phenotype can identify cells that have “stem-cell like” characteristics (38, 39). If this model holds true for CMV immunity, the CMV-specific T_M cells would be ideal to use in an adoptive immunotherapy setting. Recent evidence supports this hypothesis. In a non-human primate model, CMV-specific effector T cells that were expanded in vitro from sorted T_M cell had a superior ability to survive after adoptive transfer (40). Moreover, a human study showed a positive correlation between the presence of CMV-specific T_M cells in a donor transfer and the long-term maintenance of donor derived cells (41).

The goal of our study was to utilize the mouse model (MCMV) to directly address the capacity of the CMV-specific T_M population to restore long-term CMV-specific immunity after transfer. Importantly, we found that the MCMV-specific T_M cells share a common genetic program with their human CMV-specific counterparts and that these cells could repeatedly restore long-term CMV-specific immunity under a spectrum of transfer scenarios. Our data suggest that adoptive immunotherapy with CMV-specific T_M cells will improve consistency and clinical outcomes in patients at-risk for developing CMV disease.

Materials and Methods

Mice

Unless otherwise indicated, C57BL/6 mice, CD45.1 mice (B6.SJL-Ptprc^a Pepc^b/BoyJ), Thy1.1 mice (B6.PL-Thy1^a/CyJ) and Rag−/− mice (B6.129S7-Rag1<tm1Mom>J) were purchased from Jackson Laboratory. OT-I transgenic mice (C57BL/6-Tg(TcraTcrb)1100Mjb/J) also purchased from Jackson, were bred with CD45.1 mice to produce double positive (CD45.2^pos/CD45.1^pos) OT-I mice. All protocols were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee.

Infections

Unless otherwise indicated, mice were infected intraperitoneally (i.p.) with 2 × 10⁵ pfu of MCMV strain MW97.01 (42). Mice were considered latently-infected at 8 weeks post-infection. Rag−/− mice were infected with 5 × 10⁴ pfu of MCMV-TK virus (43). OT-I T cell transfer recipients were challenged with 2 × 10⁵ pfu MCMV-SL8, which expresses the SIINFEKL peptide (44, 45).

Tetramer Staining, Antibodies and FACS Analysis

MHC-tetramers were provided by the NIH Tetramer Core Facility (http://tetramer.yerkes.emory.edu/) and have been described previously (27). Staining was performed as described previously (27) with tetramers and the following antibodies: [CD8(53-6.7); CD44(IM7); CD27(LG.3A10); CD127(A7R34); KLRG1(2F1); CD62L(MEL-14); CD45.1(A20); CD45.1(104); Thy1.1(OX-7); Thy1.2(30-H12); IFN-γ(XMG1.2); TNF−α(MP6-XT22); CD107a(1D4B)]. In all cases, samples were collected on an LSR II and analyzed with FlowJo software (Tree Star). The gating strategy for phenotypic characterization of tetramer^pos CD8^pos T cells involved first gating lymphocytes and then singlets. CD8^pos cells were gated as frequency of singlets. Tetramer^pos cells were identified as a frequency of CD8^pos cells. A B8R tetramer (specific for the B8R peptide from Vaccinia), was used as a negative tetramer control. Tetramer^pos cells were phenotypically defined by they expression of KLRG1, CD27, CD127 or CD62L.

Adoptive Transfers

CD8^pos splenocytes from latently-infected donors were enriched using EasySep Biotin selection kit (StemCell Technologies) and biotinylated antibodies against RBCs(Ter-119), CD4(GK1.5) and CD19(6D5) according to the recommended protocol. Enriched cells were stained to determine the frequency of tetramer^pos cells within the enriched fraction and then sorted on either a MoFlo (Dako Cytomation) or an ARIA II (BD Biosciences) cell sorter. Sorted cells were counted, and 5 × 10⁴ cells were transferred via the retro-orbital sinus. Sort purity was analyzed on an LSR II. The number of transferred tetramer-binding CD8^pos T cells was estimated using the tetramer frequency within the enriched CD8^pos population and the post-sort purity analysis. Fold change was calculated as the number of tetramer-binding T cells in the spleen 7 days post-challenge over the total number of tetramer^pos cells transferred (assuming 100% engraftment). The gating strategy for analyzing donor cells in the recipients was identical to that described above with antibodies specific for the relevant congenic marker (CD45.1 or Thy1.2).

For OT-I adoptive transfers, splenocytes from naive mice containing 600 OT-I T cells were transferred. Recipients were challenged with MCMV-SL8. To establish secondary and tertiary populations, OT-I T_M CD8^pos T cells were FACS sorted and transferred as described above. Following challenge with MCMV-SL8, the frequencies of donor OT-Is were determined in the blood of recipients using the strategy described above except that singlets were not identified and OT-I donors were identified by expression CD45.1 and Vα2.

Intracellular Stimulation (ICS)

ICS and staining was performed as previously described (27, 45), with minor modifications. Specifically, cells were incubated with 1 μg/mL peptide (Genemed Synthesis), 1 μg/mL brefeldin A (GolgiPlug, BD Biosciences) and CD107a-specific antibody for 3 hours.

CD70 Blocking Antibody Treatment

CD70 antibody blockade was performed as previously described (46), with minor modifications. Briefly, mice received either 150 μg of anti-CD70(FR70) or control rat IgG2b (both from BioXCell) via the i.p. route. Injections were administered at days -1, 0 and 3 post-infection.

Antibody Depletions

Antibody depletions were performed with Thy1.1(19E12), CD4(GK1.5) and NK1.1(PK136) antibodies. 300 μg of each antibody were administered i.p. in PBS. Three subsequent injections of 100 μg of each antibody were given at 7 day intervals.

Microarray

Splenocytes from latently-infected mice were co-stained with tetramers loaded with the antigenic peptides from M38, m139 and IE3 (25) and sorted on a MoFlo (Dako Cytomation) cell sorter. MCMV-specific T cells were identified as CD8^pos, CD44^hi and tetramer binding and then further segregated into T_M and T_EFF cells subsets by expression of KLRG1 and CD127. Naïve CD8^pos cells were CD44^lo. Total RNA was isolated using the Qiagen RNAeasy Plus Kit (Qiagen), quantified on a NanoDrop 2000c Spectrophotometer (Thermo Scientific) and processed at the Microarray Core Facility at Thomas Jefferson University. Briefly, 2.5 μg fragmented and biotinylated cDNA was hybridized to Mouse gene 1.0 ST array (Affymetrix). Chips were scanned on an Affymetrix Gene Chip Scanner 3000 and data were analyzed using the R programming language and various packages from Bioconductor (47). The oligo package (48) was used to extract expression data from the Affymterix CEL files and perform background and RMA normalization (49). Annotation information was added using the mogene10sttranscriptcluster.db (50) package. Probes without valid annotations (7,196 of 35,556 probes) were removed before differential expression analysis using the limma package's (51) linear modeling and Bayes methods (52). Genes showing up- or down-regulation of at least twofold and p-value < 0.05 in each of three contrasts (T_EFF vs. Naïve, T_M vs. Naïve, and T_EFF vs. T_M) were considered for gene set enrichment analysis (GSEA). Microarray data have been deposited in the Gene Expression Omnibus (GEO) database (53) (accession number: GSE61927 http://www.ncbi.nlm.nih.gov/genbank)

Gene Set Enrichment Analysis

Human data for series GSE24151 (54) was retrieved from NCBI's GEO database (53), extracted using Partek® Genomics Suite® software, version 6.6 (Partek Inc., St. Louis, MO) and curated for input into GSEA software (55) (http://www.broadinstitute.org/gsea/). Since the data for GSE24151 have been deposited in GEO as log₁₀ ratios of the reference pool to sample, each value was inverted by multiplying by -1. Gene names in the six mouse gene lists (up- or down-regulated in each of the three contrasts) were converted to human names using data from NCBI's Homologene database, Release 68 (http://www.ncbi.nlm.nih.gov/homologene). The converted gene lists along with genes specific to the liver and the TCR receptor pathway from the Molecular Signature Database (MSigDB) (55) were analyzed for enrichment in the human data using recommended settings for the GSEA command-line interface.

Results

MCMV-specific inflationary T_M populations are stable and share a common transcriptional program with HCMV-specific CD8^pos T cells in humans

In the mouse model, MCMV infection of C57BL/6 (B6) mice results in inflation of select MCMV-specific CD8^pos T cells specific for peptides from the viral proteins M38, m139 and IE3 (Fig. 1A and (25, 27, 28)). As in humans infected with human CMV (HCMV), the majority of MCMV-specific inflationary T cells express a T_EFF phenotype (often defined as T_EMRA in humans), while only a small fraction express a T_M-like phenotype, defined here as KLRG1^lo/CD27^hi and further sub-divided into central memory (T_CM – CD127^hi/CD62L^hi) and effector memory (T_EM – CD127^hi/CD62L^lo) subsets (Fig. 1A and (22, 23, 27, 29-33, 56)). In contrast, “non-inflationary” MCMV-specific CD8^pos T cell responses, represented by the response against the viral protein M45, contract after acute infection and are thought to be maintained by homeostatic mechanisms thereafter (Fig. 1A and (25, 27, 57)). As expected, noninflators express a predominately memory (T_M) phenotype, which also includes both T_CM and T_EM subsets (Fig. 1A and (23, 27)).

Figure 1. The non-inflationary and inflationary CD8^pos T cell populations retain similar numbers of T_M phenotype cells.

Cohorts of age-matched B6 female mice were infected with MCMV and sacrificed at the indicated time points (n = 4 per time point). Tetramer staining and phenotypic analyses were performed on blood and splenocytes. (A) Frequency of tetramer-binding CD8^pos T cells in the blood at indicated time points. The phenotypic analysis shown was performed at d326 post-infection. T_M cells were identified as CD27^hi/KLRG1^lo. T_CM and T_EM were further identified as CD127^hi and either CD62L^hi or CD62L^lo, respectively. (B) Absolute numbers of KLRG1^hi tetramer-binding CD8^pos T cells in the spleen. (C) Absolute numbers of T_CM and T_EM tetramer-binding CD8^pos T cells. Data are displayed as mean ± SEM and represent two independent experiments.

It remains unknown whether the constant immune stimulation needed to maintain memory inflation causes a decline of the T_M subset within inflationary populations over time. Using infection-matched cohorts, we found that the numbers of T_M cells that were specific for inflationary antigens were stable over time and remarkably similar to the numbers of non-inflationary T_M cells, despite great differences between the numbers of inflationary and non-inflationary T_EFF cells (Fig. 1B, 1C). Thus, although continuous antigen stimulation maintains memory inflation, the inflationary T_M population remains stable.

The MCMV model is well characterized and the T cell responses clearly recapitulate those seen in HCMV-infected people. To determine whether MCMV-specific T_M and T_EFF cells share a common transcriptional program with their human counterparts, we sorted MCMV-specific T_M (CD44^hi/CD127^hi/KLRG1^lo) and T_EFF (CD44^hi/CD127^lo/KLRG1^hi) cells specific for the M38, m139 and IE3 antigens. Microarray analyses were performed on these cells. Genes that were significantly up- or down-regulated in T_M and T_EFF subsets relative to each other or to naïve (CD44^low) T cells, were mapped to the corresponding human genes and compared with the profiles of HCMV-specific T cells, previously defined by the van Lier group as CD27^hi/CD45RA^lo (T_M) or CD27^lo/CD45RA^hi (T_EFF) (54). The CD27 and CD127 (IL-7Rα) molecules both mark CMV-specific T cells with a memory phenotype in mice and humans (27, 29, 32, 58, 59) and nearly all MCMV-specific KLRG1^lo/CD27^hi cells (T_M) co-expressed CD127 (either T_CM or T_EM, Figure 1A). Gene set enrichment analyses (GSEA) were used to measure the overall correlation between the mouse and human gene expression data. As shown in Fig. 2A, genes that distinguished mouse T_EFF and T_M cells from each other were highly enriched within the corresponding human data set. That is: genes up-regulated specifically in mouse T_M cells relative to mouse T_EFF cells were highly enriched within the genes that distinguish human T_M cells from human T_EFF and vice versa. Moreover, relative to naive T cells, mouse genes that were up and down-regulated by T_EFF or T_M cells were highly enriched within genes that distinguished their human counterparts from human naive T cells (Fig. 2B). The analyzed mouse genes and the core enrichment profiles of each comparison are listed in Supplemental Table 1. Importantly, several of these genes corresponded to our sorting parameters and the known phenotypes of T_M and T_EFF populations. As controls, identical analyses were performed with genes associated with the T cell receptor signaling pathway or liver and the data exhibited expected patterns (Fig. 2B).

Figure 2. Gene Set Enrichment Analyses reveal significant overlap between the transcriptional profile of CMV-specific T cells in humans and mice.

(A) Gene set enrichment was performed as described in the Methods. Shown are the enrichment plots for mouse genes that differed in a T_EFF vs. T_M comparison, plotted relative to human T_EFF and T_M cells. Values represent the normalized enrichment score (NES) and Family Wise Error Rate (FWER), which estimates the probability of a false positive NES. (B) Lists of significantly altered mouse genes (2-fold up or down and P<.05) were generated for T_EFF and T_M cells relative to each other and relative to naive (CD44^low) T cells. GSEA analyses were performed with these mouse gene sets relative to each of the indicated human data sets, rank ordered by expression (see methods). Stars indicate FWER corrected significance to control for multiple testing (* P<.05, ** P<.01, *** P<.001).

Overall, these data show that MCMV-specific and HCMV-specific T cells share a common genetic program, validating the use of the MCMV model to investigate the function of HCMV-specific T cells. To our knowledge, this is the first direct comparison of mouse and human CMV-specific T cell gene expression profiles.

The inflationary T_M population retains proliferative capacity

To test the proliferative capacity of the T_M and T_EFF cells, both populations were sorted from spleens of latently-infected B6 mice (>3 months post-infection) using their differential expression of KLRG1 and CD27. Sorted cells were transferred into naive congenic recipients and re-challenged. The M45- and M38-specific T_M cells proliferated robustly within 7 days after challenge, each expanding almost 1000-fold in the spleen alone, assuming 100% engraftment of the donor cells (Fig. 3A, 3B). In contrast, the M38-specific T_EFF population expanded less than 10-fold in the same time period. Importantly, while the T_EFF donor cells remained exclusively KLRG1^hi, the T_M donor cells produced large numbers of both T_EFF and T_M progeny (Fig. 3C). In fact, donor M45- and M38-specific T_M phenotype cells were present in the spleen 7 days after challenge at numbers that were approximately 50 to 100-fold higher than had been transferred (dotted line, Fig. 3D), indicating expansion of this subset without terminal differentiation. These data show that MCMV-specific T_M cells retain robust proliferative capacity and can produce phenotypically diverse progeny including new T_M-phenotype cells.

Figure 3. T_M cells dramatically expand 7 days post-challenge and produce both T_M and T_EFF progeny.

Age matched B6 mice received either T_M or T_EFF cells and were challenged with MCMV as described in the Methods. Spleens were collected 7 days later for analysis. (A) Representative FACS plots of tetramer^pos donors in the spleen 7 days post-challenge. Frequencies in the corner are relative to total CD8^pos cells. (B) Fold change of donor cells in the spleen, calculated as described in the Methods, 7 days after challenge. As antigen-specific T cells were not sorted, approximately equal numbers of M38- and M45-specific T_M cells were transferred but ~10-fold more M38-specific T_EFF cells were transferred compared with the T_M cells. Due to the extremely low number of M45-specific T_EFF transferred and the minimal expansion at day 7, it was not possible to calculate a comparable fold change value for the M45-specific T_EFF population. Data were collected from two independent experiments (T_M: n = 6 total; T_EFF: n = 5 total) are shown. Statistical significance was determined by a Student's t-test (*** P<.001; **** P<.0001). (C) Representative FACS plots of M38-specific CD8^pos T cell progeny from either T_M or T_EFF donors in the spleen at 7 days post-challenge. Frequencies in the corner are relative to M38-specific CD8^pos cells. (D) Absolute number of T_M and T_EFF phenotypic progeny that were produced from T_M donors. Data are from the same experiments described in (B). (E) Fold change of donor cells in the spleen following treatment with either isotype control or anti-CD70 antibody. Data were collected 7 days post-challenge and represent two independent experiments (n = 6 total). Statistical significance was determined by a Student's t-test (*** P<.001; **** P<.0001). All graphical data are displayed as mean ± SEM.

Recent work has shown that interaction between CD27 and its ligand CD70 plays a functional role in the proliferation of MCMV-specific inflationary T cells (46). To test the contribution of this interaction specifically within the T_M population, we sorted and transferred T_M cells as above and blocked the CD27-CD70 interaction as described in the Methods. Blocking the CD27-CD70 interaction significantly decreased the expansion of the M38- and M45-specific T_M cells 7 days post-challenge by approximately 4- to 6-fold (Fig. 3E), which is in line with the impact of CD70 blockade on unsorted (i.e. combined T_M and T_EFF populations) inflationary T cells (46). These data further suggest that the majority of proliferative potential of inflationary T cells is contained within the minor T_M subset. It should be noted that even in the presence of CD70 blockade, the T_M population retained a proliferative capacity that was greater than the T_EFF population, suggesting that additional pathways contribute to the total proliferative potential of these cells (Fig. 3B, 3E and unpublished observations).

The inflationary T_M population persists and can repeatedly recapitulate memory inflation

To determine the ability of the T_M donor cells to persist long-term, we tracked the progeny from T_M donor cells in the blood after re-challenge. M38-specific T cells from T_M-sorted donors persisted at high frequencies in recipients, while the M45-specific donor cells contracted after their initial expansion in the same mice (Fig. 4A, 4B). Despite their initial T_M phenotype, the donor M38-specific T cells largely expressed a T_EFF phenotype after challenge (Fig. 4C, 4D), consistent with a typical “inflationary” population. The population as a whole retained its ability produce IFN-γ, TNF-α and expose CD107a (Fig. 4E, 4F). Importantly, a small portion of donor T cells retained their T_M phenotype even after this secondary challenge (Fig. 4C, 4D).

Figure 4. T_M cells reinflate following re-challenge and retain function.

Age matched B6 mice received T_M cells and were challenged with MCMV as in the Methods. (A) Representative FACS plots of donor-derived T cells in the blood 126 days post-challenge. (B) Frequencies of tetramer-binding T cells in the blood over time. Data were collected from three independent experiments (n = 17 total). (C) Representative FACS plot of the phenotype of donor-derived M38-specific T cells in the blood 126 days post-challenge. (D) Frequencies of donor-derived, M38-specific T_M and T_EFF cells in the blood over time. Data are from the same experiments described in (B). Each line represents an individual mouse. The square datum point represents a mouse that appeared to lose the donor T cells after day 7 post-challenge, but effectors appeared ~20 weeks after challenge. (E-F) Intracellular cytokine staining was performed on splenocytes 221 days post-challenge. Shown are representative FACS plots of stimulated (with M38 peptide) and unstimulated cells (E) and the frequencies of IFN-γ positive cells that also express TNF-α and/or CD107a (F). Data were collected from a single experiment (n = 5) described above. All graphical data are displayed as mean ± SEM.

To understand whether these persistent T_M phenotype donors continued to be functional, we turned to the OT-I transgenic system to facilitate sorting and avoid the possible selection of different T cell clones (Fig. 5A). As shown previously, transferred naive OT-Is undergo inflation and produce both T_M and T_EFF progeny after primary challenge with SIINFEKL-expressing MCMV-SL8 (45). We sorted the T_M phenotype OT-I cells that formed after primary challenge, transferred these cells and challenged the recipients to establish secondary populations (Supplemental Fig. 1A). As with non-transgenic T cells (Fig. 4), secondary challenge of T_M OT-Is induced inflation and T_EFF formation as well as a persistent KLRG1^lo population (Supplemental Fig. 1B). These secondary T_M cells were again sorted (Supplemental Fig. 1C), transferred into a 3^rd set of naïve recipients and re-challenged. Incredibly, the donor secondary T_M population inflated and produced both KLRG1^hi and KLRG1^lo progeny following this tertiary challenge (Fig. 5B-5E).

Figure 5. T_M cells can reinflate following multiple re-challenges.

(A) Schematic of experimental design. To establish primary OT-I inflationary populations, 600 naïve OT-I T cells expressing CD45.1 were transferred into naïve B6 (CD45.2) recipients followed by infection with MCMV-SL8 (i.e. primary challenge). Thirteen weeks later, 6,000 T_M phenotype primary OT-Is, isolated by FACS sorting, were transferred into new B6 recipients followed by MCMV-SL8 challenge (i.e. secondary challenge). This process was repeated a third time, transferring 3,500 T_M OT-Is into naive mice and challenging with MCMV-SL8 (i.e. tertiary challenge). (B-C) Representative FACS plot of the donor stain 91 days post tertiary challenge (B) and frequencies of donor OT-Is (relative to total CD8s) in the blood at the indicated time points after tertiary challenge (C). Data were collected from two independent experiments (n = 12 total). Each line represents an individual mouse. (D-E) Phenotypic analyses of the mice described in (B-C). Representative FACS plot of the donor stain 30 weeks post-challenge Frequencies are relative to donor CD8s. (F-G) Intracellular cytokine staining was performed on splenocytes approximately 20 weeks after the tertiary challenge. Shown are representative FACS plots of stimulated (with SIINFEKL peptide) and unstimulated cells (F) and the frequencies of IFN-γ positive cells that also express TNF-α and/or CD107a (G). Data were collected from two independent experiments (n = 12). Data are displayed as mean ± SEM.

Repeated acute viral challenges of small numbers of T cells in naive mice drives T_EFF differentiation (60-63) and indeed the overall frequency of tertiary inflationary cells that retained a T_M phenotype was reduced (Fig. 5E and Supplemental Fig. 1B). However, these tertiary stimulated OT-Is remained functional, producing both IFN-γ and TNF-α, as well as exposing CD107a (Fig. 5F, 5G). These data show that T_M phenotype T cells specific for inflationary antigens can repeatedly recapitulate memory inflation upon viral challenge and produce functional T_EFF and T_M progeny.

Memory and Effector Subsets protect Rag−/− mice

To test the ability of transferred T_M cells to protect against a lethal MCMV challenge, T_M and T_EFF populations were sorted from latently-infected B6 mice as above and transferred into Rag−/− recipients. One day later, the Rag−/− recipients were challenged with MCMV-TK, which lacks the m157 gene and is therefore resistant to NK-mediated control (43). Both transferred T_M or T_EFF cells expanded following the challenge and were sufficient to protect the recipients (Fig. 6A, 6B). In contrast, Rag−/− mice that received no T cell therapy became moribund in 2-4 weeks and had to be sacrificed (Fig. 6B). Notably, the T_EFF population, which proliferated very poorly in immune replete mice (Fig. 3), expanded and persisted in immune deficient hosts for at least 11 weeks post-challenge (Fig. 6A, 6B). However, the T_EFF response in 5 out of 6 recipients M45-specific, non-inflationary T cells (Fig. 6A). These data show that MCMV-specific T_M cells are capable of protecting immune deficient mice and producing immune responses with broad specificities.

Figure 6. T_M and T_EFF cells protect Rag−/− mice following an acute MCMV challenge.

Age matched Rag−/− mice received either T_M or T_EFF cells and were challenged with MCMV-TK as described in the Methods. Mice were monitored daily for signs of morbidity (lethargy, raised hair and shaking) and sacrificed if they displayed clear signs of morbidity. Data were collected from two independent experiments. One experiment was carried out until 77 days post-challenge. A second experiment was censored at 33 days post-challenge. (A) Representative tetramer staining of T cells in Rag−/− that received either T_M or T_EFF transfers. Data were collected 11 weeks post-challenge. Frequencies are relative to total CD8s. (B) Survival curve (n = 7 for control group; n = 7 for T_M group; n = 6 for T_EFF group). Statistical significance was determined by a log-rank (Mantel-Cox) test (**** P<.0001).

The T_M population can persist long-term and respond when necessary

Patients undergoing hematopoietic stem cell transplantation (HSCT) are most susceptible to late-onset (>100 days) reactivating CMV, as opposed to an acute CMV infection ((12, 64-66) and reviewed in (67)). Furthermore, transferred CMV-specific T cells will need to compete with host immunity. Therefore, we developed a model to test whether T_M and T_EFF subsets could respond to viral reactivation after a long delay. To this end, T_M and T_EFF cells were sorted from latently-infected mice (> 3 months post-infection) and transferred into immune replete, infection matched or naive, congenic recipients differing at the Thy1 locus (Fig. 7A). Following the transfer, the latently-infected recipients were rested as described in the figure legend. Donor T cells did not expand dramatically in any animal following transfer (Supplemental Fig. 2A) supporting our previous conclusion that competition between T cells dictates MCMV-specific T cell expansion (45). Recipient T cells and NK cells were then eliminated in all mice using a cocktail of depletion antibodies that targeted the host cells (Thy1.1^pos), but left the donor cells (Thy1.2^pos) intact (Fig. 7B and Supplemental Fig. 2B). This depletion protocol did not induce detectible viral transcription in any animal as assessed by nested RT-PCR (unpublished observations), likely due to the presence of anti-viral antibodies (68). Despite the 9-12 week rest period, MCMV-specific donor T_M cells responded robustly in all infected recipients after host depletion (Fig. 7C and 7D). Importantly, donor T_M cells did not expand to detectible levels in depleted naive recipients (Supplemental Fig. 2C). However, viral challenge of naïve mice that received T_M donor cells 12 weeks previously induced a robust donor response in 3 of the 4 animals, indicating that the T_M cells persisted in these mice, even without any antigen (Supplemental Fig. 2C). Thus, antigen rather than homeostatic mechanisms accounts for the donor T_M response in infected recipients.

Figure 7. T_M cells persist in latently-infected, immune replete mice and expand when host immunity is lost.

(A) Schematic of experimental design. Age matched B6 and Thy1.1 mice were infected with 1×10⁶ pfu MCMV-Smith. Following the establishment of viral latency (>8 weeks post-infection), either T_M or T_EFF cells from the B6 donors were transferred, as described in the Methods, into the latently-infected Thy1.1 recipients or into naïve Thy1.1 mice. Latently-infected recipients were rested for 9-12 weeks, while the naïve recipients were rested for approximately 1.5 weeks. (B) Antibody depletion schedule. (C-E) The presence of tetramer^pos donors was analyzed by flow cytometry immediately following the depletion schedule. Data were collected from two independent experiments (n = 6 total). Three mice from each group were depleted 9 weeks after the transfer; three mice from each group were depleted 12 weeks after transfer. (C) Histograms of donor T cells within each individual recipient. (D) Representative FACS plots of tetramer^pos donors immediately following the depletion regimen. (E) Frequency within each individual recipient of each analyzed tetramer as a percent of total donor CD8+ cells. T_EFF recipients 3-6 are excluded because they did not have a donor population. (F) Tetramer staining was performed 11 weeks after depletion in one experiment described above (n = 3).

In marked contrast, after depletion, donor T cells were only detectible in 2 animals that had received T_EFF cells and then only at very low frequencies (Fig. 7C, 7D). Control experiments (Supplemental Fig. 3A-3C) supported previous work (69) suggesting that the KLRG1-specific antibody did not induce depletion of the transferred T_EFF subset. Thus, the failure of T_EFF cells to expand in this setting is not a sorting artifact, but rather the inability to persist and/or expand in response to low amounts of viral antigen.

After expansion, all infected mice that received T_M cells had a donor population specific for multiple epitopes and the progeny had differentiated to form new T_EFF populations (Fig. 7E and unpublished observations). Furthermore, the four tetramers used only stained ~60% of the total donor population in each animal (Fig. 7E), suggesting that the remaining 40% of each donor population contained cells specific for additional MCMV antigens. In contrast, in the two animals in which T_EFF donors expanded to detectible levels, each was skewed substantially towards a single inflationary epitope (Fig. 7E). Since these sorted T_EFF populations included large numbers of T cells specific for M38, m139 and IE3, this “hit-or-miss” expansion of donor T cells with select specificities implies that a very small number of non-T_EFF cells may have contaminated the transfer.

In the mice that received T_M donor cells, their diverse progeny persisted in recipients for more than 11 weeks after termination of the depletion regimen, even though host immunity had returned (Fig. 7F). These data suggest that T_M cells with inflationary specificities are capable of surviving in an environment with very little or no antigen stimulation and then responding as needed during a period in which the host is immune compromised and viral antigen becomes available.

In total, these data show that protective MCMV-specific T_M cells persist throughout infection, retain superior proliferative function, and can respond to viral antigen as needed, in contrast to the numerically dominant T_EFF cells. Since MCMV-specific T_M cells share a transcriptional program with HCMV-specific T_M cells, our data suggest that T_M cells may be ideal candidates to restore functional immune surveillance in patients at risk for CMV reactivation.

Discussion

Adoptive immunotherapy using CMV-specific CD8^pos T cells can be a successful therapeutic strategy for combating CMV reactivations (5-18). However, the majority of CMV-specific CD8^pos T cells isolated from healthy donors will express an effector-differentiated phenotype (CD27^lo/CD127^lo/CD45RA^hi/KLRG-1^hi/CD57^hi - reviewed in (70)), and in vitro expansion of CMV-specific T cells drives their differentiation towards an effector phenotype (40). We used the MCMV model to show that the ability to restore MCMV-immunity is contained almost entirely within the minor T_M subset that retains CD27. Although both T_M and T_EFF cells protected Rag−/− mice (Fig. 6), humans are unlikely to remain completely immune depleted like Rag−/− mice, and bolus CMV infections are of lesser concern than reactivation following transplantation. The inability of the T_EFF population to consistently expand after immune depletion in latently-infected hosts, suggests that these cells will only be protective under limited conditions. These data support a previous study in humans that correlated the transfer of CD27^hi CMV-specific T cells with an increased likelihood of T cell persistence and expansion (41).

To validate the use of the MCMV model, we compared human and mouse MCMV-specific T cells and show for the first time that T_M and T_EFF populations in mice and humans share a common transcriptional profile. The power of the GSEA analysis used for this comparison is that it identifies significant correlations across the entire transcriptional profile, rather than comparing individual genes. Nevertheless, we expect that future studies examining conserved and divergent genetic pathways will reveal significant and relevant information about CMV-specific immunity in mouse and man. These results highlight the usefulness of the MCMV model to: 1) perform CMV-specific CD8^pos T cell functional studies that are difficult or impossible to perform in humans and 2) provide translational insights into novel or improved therapeutic strategies.

Understanding how CMV-specific T cell immunity is maintained is critical for the improvement of CMV adoptive immunotherapy. Persistent antigen stimulation from CMV reactivations results in the majority of inflationary CD8^pos T cells developing a T_EFF phenotype and function. However, our previous work showed that unsorted inflationary CD8^pos T cells, containing primarily T_EFF cells, declined after transfer into congenic, latently-infected recipients (27). These data suggest that MCMV-specific T_EFF cells are unable to sustain themselves in an immune replete environment, even in the presence of antigen. It has been proposed that the accumulation of T_EFF cells is the result of continual antigen stimulation of the T_M population. Our data show that a small, stable MCMV-specific T_M population has strong functional similarities to classical memory T cells that develop following acute infections and can recapitulate memory inflation. For example, the ability to proliferate in response to antigen without terminal differentiation is hallmark of functional memory T cells (71). In addition to producing differentiated progeny that accumulated after MCMV challenge (Figure 4D), donor T_M cells also produced T_M phenotype progeny that outnumbered the cells transferred (Fig. 3C and D) and persisted throughout our observation period (Fig. 4C and D). These data suggest that MCMV-specific T_M cells have the ability to replace themselves even while producing differentiated progeny in response to antigen. Importantly, this was true through at least three rounds of stimulation using sorted splenic T_M cells (Figure 5). Thus, MCMV-specific T_M cells have the capacity to respond repeatedly to viral antigen during this persistent infection.

It is interesting that transferred T_M cells failed to expand in immune-replete, latently-infected hosts. Indeed, detectible numbers of donor T cells were only evident in one out of six mice prior to immune depletion (Supplemental Fig. 2A). In this case, the donors were not positive for any of the tetramers used in the analyses and made up less than 1% of the total CD8^pos population. However, loss of the host T cell populations led to rapid and robust expansion of donor T cells with diverse specificities and phenotypes in all T_M cell recipients (Figure 7). The failure of transferred T_M cells to expand in the presence of host MCMV-specific immunity may reflect the relative lack of available antigen during the latent phase of MCMV infection. Indeed, viral reactivations occur in only a fraction of latently-infected cells at any given time, and only rarely produce infectious viral particles (72, 73). Moreover, we have found that competition between T cells for access to this limited antigen regulates the expansion of individual T cell clones (45). Thus, the combination of low antigen and large numbers of MCMV-specific T cells in the recipients may have “shielded” the majority of the donor T_M cells from the ongoing infection – an idea we have proposed previously (45, 74). Importantly, MCMV antigen is not required for MCMV-specific T_M cell survival. We have previously shown that MCMV-specific T_M cells divide at a consistent rate with or without antigen (28) and our new data (Supplemental Fig. 2C) show that inflationary T_M cells can survive in naïve mice without any antigen. Thus, homeostatic mechanisms can support the inflationary T_M population when it does not have access to antigen, which may partially explain the preservation of memory function within the T_M subset. Taken together, these data suggest that the highly functional T_M population, which can persist without access to antigen, proliferates robustly and produces new T_M cells as well as more differentiated progeny upon antigen stimulation.

Overall, our data further support the model that the burden of maintaining memory inflation falls on the functional T_M population, which can provide a stable and consistent source of new T_EFF progeny whenever needed, over for prolonged periods of time. However, T cell competition for limited antigen appears to prevent the continuous stimulation of most T_M cells. Nonetheless, the T_M population is capable of robustly responding if T cell competition is lost - a conclusion with important clinical implications for adoptive immunotherapy. Variations in transplant protocols, patients and anti-viral therapy responses make it difficult to predict and standardize CMV prevention therapies. Our data suggest that the plasticity of the T_M population, transferred before any disease develops, may offer a “personalized” therapy, where the treatment adapts to the conditions of the patient and responds if and when antigen becomes available. Future studies will be needed to explore whether the addition of homeostatic cytokines (e.g. IL-15) or pharmacotherapeutics (e.g. rapamycin (75)) preserves the T_M phenotype either in vivo or during in vitro expansion.

Abbreviations

CMV

Cytomegalovirus

HCMV

Human Cytomegalovirus

MCMV

Murine Cytomegalovirus

T_M

memory phenotype CD8+ T cells

T_EFF

terminal differentiated effector phenotype CD8+ T cells

T_CM

central memory CD8+ T cells

T_EM

effector memory CD8+ T cells

T_EMRA

terminal differentiated CD8+ T cell phenotype in humans

i.p.

intraperitoneal route

GSEA

gene set enrichment analysis

GEO

gene expression omnibus

SEM

standard error of the mean