Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Two Host Factors Regulate Persistence of H7a-Specific T Cells Injected in Tumor-Bearing Mice

  • Marie-Christine Meunier,

    Affiliations Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Quebec, Canada, Department of Medicine, University of Montreal, Montreal, Quebec, Canada, Division of Hematology, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada

  • Chantal Baron,

    Affiliations Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Quebec, Canada, Department of Medicine, University of Montreal, Montreal, Quebec, Canada, Division of Hematology, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada

  • Claude Perreault

    c.perreault@videotron.ca

    Affiliations Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Quebec, Canada, Department of Medicine, University of Montreal, Montreal, Quebec, Canada, Division of Hematology, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada

Two Host Factors Regulate Persistence of H7a-Specific T Cells Injected in Tumor-Bearing Mice

  • Marie-Christine Meunier, 
  • Chantal Baron, 
  • Claude Perreault
PLOS
x

Abstract

Background

Injection of CD8 T cells primed against immunodominant minor histocompatibility antigens (MiHA) such as H7a can eradicate leukemia and solid tumors. To understand why MiHA-targeted T cells have such a potent antitumor effect it is essential to evaluate their in vivo behavior. In the present work, we therefore addressed two specific questions: what is the proliferative dynamics of H7a-specifc T cells in tumors, and do H7a-specific T cells persist long-term after adoptive transfer?

Methodology/Principal Findings

By day 3 after adoptive transfer, we observed a selective infiltration of melanomas by anti-H7a T cells. Over the next five days, anti-H7a T cells expanded massively in the tumor but not in the spleen. Thus, by day 8 after injection, anti-H7a T cells in the tumor had undergone more cell divisions than those in the spleen. These data strongly suggest that anti-H7a T cells proliferate preferentially and extensively in the tumors. We also found that two host factors regulated long-term persistence of anti-H7a memory T cells: thymic function and expression of H7a by host cells. On day 100, anti-H7a memory T cells were abundant in euthymic H7a-negative (B10.H7b) mice, present in low numbers in thymectomized H7a-positive (B10) hosts, and undetectable in euthymic H7a-positive recipients.

Conclusions/Significance

Although in general the tumor environment is not propitious to T-cell invasion and expansion, the present work shows that this limitation may be overcome by adoptive transfer of primed CD8 T cells targeted to an immunodominant MiHA (here H7a). At least in some cases, prolonged persistence of adoptively transferred T cells may be valuable for prevention of late cancer relapse in adoptive hosts. Our findings therefore suggest that it may be advantageous to target MiHAs with a restricted tissue distribution in order to promote persistence of memory T cells and thereby minimize the risk of cancer recurrence.

Introduction

Adoptive transfer of allogeneic T lymphocytes, used primarily for treatment of hematopoietic malignancies, has met with a remarkable success rate [1], [2]. Accordingly, the so-called graft-versus-leukemia (GVL) effect represents the most conclusive documentation that the immune system can cure cancer in humans [1], [3], [4]. The GVL effect is due mainly, and perhaps exclusively, to recognition of minor histocompatibility antigens (MiHAs) [1], [2], [5]. We reported that injection of CD8 T cells primed against the model immunodominant H7a MiHA could eradicate not only leukemia but also melanoma in mouse [6], [7]. The chain of events leading to melanoma eradication by anti-H7a T cells involves the following steps [7]. First, primed T cells accumulate at the tumor site. This initial step depends on interaction between Vla-4 on T cells and Vcam-1 on tumor blood vessels. Second, local release of IFN-γ by anti-H7a T cells has two crucial effects: inhibition of tumor angiogenesis and upregulation of MHC I expression on tumor cells. Finally, anti-H7a CD8 T cells undergo antigen-specific granule exocytosis in the tumor and thereby kill tumor cells. Of note, T cells specific for a single MiHA, such as H7a, never elicit graft-versus-host disease even when their target MiHA is ubiquitously expressed in recipient tissues and organs [6][8].

To understand why MiHA-targeted T cells are so effective we deemed it essential to evaluate their in vivo behavior. In the present work, we therefore addressed two specific questions. First, what is the proliferative dynamics of H7a-specifc T cells in the tumor? This is a critical issue since cancer refractoriness to immunotherapy is commonly due to failure of T cells to penetrate and accumulate in the tumors [9][12]. Second, do H7a-specific T cells persist long-term, and is protracted T-cell reactivity to H7a necessary to prevent tumor recurrence? We report that the proliferation dynamics of anti-H7a CD8 T cells is dramatically different in the tumor compared with the spleen. We present evidence that the massive accumulation of anti-H7a in melanomas is largely due to extensive in situ proliferation. Moreover, we found that the long-term fate of H7a-specific T cells was dictated by two host factors: thymic function and expression of H7a by normal host cells. Notably, mice in which anti-H7a T cells did not persist long-term (day 100) nevertheless remained tumor-free. Thus, in this model, cure is probably due to eradication of clonogenic tumor cells rather than to induction of T-cell dependent tumor dormancy.

Materials and Methods

Mice, Tumor Cells and Statistics

We obtained B10.H7b(47N)/Sn (B10.H7b) and C57BL/10J (B10) mice from the Jackson Laboratory (Bar Harbor, ME) and the B16.F10 melanoma cell line from the American Type Culture Collection (Manassas, VA). Mouse care and experimental procedures were performed under approval from the Animal Care Committees of the University of Montreal and of the Maisonneuve-Rosemont Hospital. Mice were treated according to the guidelines of the Canadian Council on Animal Care. Differences between group means were tested using Student's t test.

Cell Transplantation and Thymectomy

On day 0, H7a-positive (B10) and H7a-negative (B10.H7b) recipients received 12 Gy total-body irradiation, 107 T cell-depleted bone marrow cells i.v. and 2×105 tumor cells s.c. in the right flank. On day 7, recipients were treated with 5×107 splenocytes from B10.H7b donors primed against H7a. Priming of B10.H7b donors against H7a was performed by i.p. injection of 2×107 B10 spleen cells 14 days prior to adoptive transfer. Tumor size was measured every 48 h and we sacrificed mice when the largest tumor diameter reached 17 mm. On day 100, we re-challenged cured mice with 2×105 B16.F10 melanoma cells s.c. Thymectomy were performed as previously described [13].

Cell Staining with Antibodies and Tetramers

We purchased antibodies specific for the following molecules: CD8 (53-6.7), from BD Pharmingen (San Jose, CA) and CD44 (11-0441), from eBioscience (San Diego, CA). We obtained phycoerythrin-labeled H7a-H2Db tetramers from the tetramer core facility of the Canadian Network for Vaccines and Immunotherapeutics (Montreal, QC, Canada). Cells were stained as previously described and were analyzed on a FACSCalibur using the CellQuest program (BD Biosciences, San Jose, CA) [14], [15].

Assessment of T-Cell Proliferative Dynamics from CFSE Profiles

We labeled splenocytes from B10.H7b female mice (immunized on day −7 against H7a) with CFSE (Molecular Probes, Burlington, ON, Canada) as previously described [16], [17]. Briefly, splenocytes were suspended at a concentration of 5×107 cells/ml in Hanks' balanced salt solution. After warming to 37°C, CFSE was added at a concentration of 5 µM for 15 minutes, followed by addition of ice-cold RPMI media and cell recovery by centrifugation. Donor cells were subsequently injected i.v. into the tail vein of recipient mice. Before analysis, we stained cell suspensions containing CFSE-labeled cells with anti-CD8 antibody and H7a-H2Db tetramers. Division peaks (as determined by CFSE-intensity) were labeled from 0 to n. Since a single T cell dividing n times will generate 2n daughter cells, if the total number of T cells which have divided three times (n = 3) is eight, then exactly one precursor had to divide three times to generate these eight cells (23 = 8) [17], [18]. Making use of this mathematical relationship, the number of T cells that have divided was extrapolated from the number of daughters under each division peak, and the total number of mitotic events was calculated as described [16], [19]. The proliferative burst size (number of daughter cells generated by a dividing T-cell “precursor”) was obtained by dividing the total number of mitoses by the number of precursors that had divided [19].

Results

Experimental Model (Fig. 1)

thumbnail
Figure 1. Experimental model and study design.

Melanoma-bearing mice received splenocytes primed against H7a on day 7. Short-term studies on T-cell proliferation kinetics were performed from day 10 to day 15, and long-term studies on memory T cells were initiated on day 100.

https://doi.org/10.1371/journal.pone.0004116.g001

On day 0, H7a-positive (B10) and H7a-negative (B10.H7b) mice received 12 Gy total-body irradiation, 107 T cell-depleted bone marrow cells i.v. and 2×105 B16.F10 melanoma cells (H7a-positive) s.c. On day 7, we treated mice by injection of 5×107 splenocytes from B10.H7b female immunized against H7a 14 days prior to adoptive transfer. Splenocyte suspensions (5×107 cells) from donors primed against H7a contained 2.5±0.3×105 H7a tetramer-positive CD8 T cells (data not shown). We have previously reported the gene expression profile and cell surface phenotype H7a tetramer-positive CD8 T cells primed under those conditions [7], [15]. CD8 T cells supplied with CD4 help at the time of initial priming generate more efficient effector cells upon secondary challenge [20], [21]. Thus, as in previous studies [6], [7], we used B10.H7b female mice primed against B10 male cells as a source of anti-H7a CD8 T cells. This immunization scheme leads to the expansion of anti-HY CD4 T cells and of anti-H7a CD8 T cells in donor mice (immunodomination prevents expansion of anti-HY CD8 T cells) [22][24]. We used only female mice as recipients. Thus, following adoptive transfer only anti-H7a CD8 T cells could encounter their cognate Ag in recipients because both recipients and tumor cells are HY-negative. Our model displays two features that make it relevant as a pre-clinical paradigm: i) our protocol involves a therapeutic rather than prophylactic setting [25]; ii) alike most spontaneous human tumors, B16.F10 cells do not express MHC II and display only low levels of MHC I molecules [26]. We displayed in Fig. 1 the overall study design and the timing of short-term and long-term studies whose results are presented in Fig. 23 and Fig. 4, 5, 6, respectively.

thumbnail
Figure 2. Anti-H7a T cells accumulate in the tumor.

We treated B10 recipient mice as described in Fig. 1. Cell suspensions from their spleen and tumor were obtained on day 10, 13 and 15, and stained with H7a tetramers and anti-CD8 antibody. A) Proportion and B) absolute numbers of H7a tetramer-positive CD8 T cells in 3–5 mice studied at each time point. * P<0.01; § P<0.001.

https://doi.org/10.1371/journal.pone.0004116.g002

thumbnail
Figure 3. Evaluation of T-cell proliferative dynamics from CFSE profiles.

B10 recipient were treated as in Fig. 1. A) One representative example of the CFSE profiles observed at each time point (gated on CD8+ H7a tetramer+ T cells) in the spleen and tumor. Numbers in the upper right corner of each panel represent the percentage of undivided cells (that is, cells showing the highest levels of CFSE). B) Mean (±SD) burst size, doubling time and expansion of H7a-specific T cells between day 10 and day 15 in the spleen and tumor. The proliferative burst size and doubling time of anti-H7a T cells were calculated as described in materials and methods. Expansion corresponds to the mean number of cells on day 15/mean number of cells on day 10. Each group contained 3–5 mice. * P<0.001 (spleen vs. tumor).

https://doi.org/10.1371/journal.pone.0004116.g003

thumbnail
Figure 4. Persistence of H7a-specific CD8+ memory T cells.

We studied four experimental groups: i) B10.H7b and B10 mice successfully treated for melanoma as described in Fig. 1 (+B16.F10); ii) euthymic and thymectomized (Tx) B10 mice that underwent the same protocol excepting injection of melanoma cells on day 0. On day 100, we sacrificed 3 mice per group and stained cell suspensions from spleen and bone marrow with H7a tetramers and antibodies against CD8 and CD44. A) Dot plots of one representative experiment out of three (gated on CD8 cells). B) Proportion and absolute number of H7a-specific T cells in recipients' spleen and bone marrow (2 tibiae and femurs). Histograms represent the mean±SD of 3 mice per group. Inserts depict CD44 expression on tetramer+ CD8 T cells found in euthymic B10.H7b and thymectomized B10 hosts. MFI: mean fluorescence intensity.

https://doi.org/10.1371/journal.pone.0004116.g004

thumbnail
Figure 5. Challenge of long-term chimeras with melanoma cells.

We studied mice from the four experimental groups described in Fig. 4. On day 100, mice were challenged with 2×105 B16.F10 melanoma cells s.c. A) Tumor surface area and B) survival of experimental groups (n≥10 mice per group). The mean survival times for the following groups were significantly different: white vs. black, white vs. red, and blue vs. black, P<0.0001; red vs. black, P<0.001. C) Examination of excised tumor injection site shows large tumor nodules (black) in B10 mice but only an inconspicuous micro-nodule in the B10.H7b mouse (one representative experiment out of 10). Tx, thymectomized.

https://doi.org/10.1371/journal.pone.0004116.g005

thumbnail
Figure 6. Anti-H7a T-cell response following tumor challenge.

We studied mice from the four experimental groups described in Fig. 4. On day 100, we challenged mice with 2×105 B16.F10 melanoma cells s.c. Mice were sacrificed on day 126 and cell suspensions from their spleen, bone marrow and tumor were stained with H7a tetramers and anti-CD8 antibody. A) Dot plots of one representative experiment out of three (gated on CD8 cells). B) Proportion and absolute number of H7a-specific T cells in recipients' spleen, bone marrow (2 tibiae and femurs) and tumor on day 126. Results are depicted as the mean±SD of 3 mice per group. Absolute numbers of H7a-specific T cells in the three sites were higher in thymectomized relative to euthymic B10 hosts (0.02<P<0.002), and were higher in B10.H7b than in euthymic (P<0.0005) or thymectomized (P<0.01) B10 hosts. Tx, thymectomized.

https://doi.org/10.1371/journal.pone.0004116.g006

Proliferation Kinetics of H7a-Specific CD8 T Cells

The outcome of adoptive immunotherapy is presumably dictated by the proliferative dynamics of both tumor cells and tumor infiltrating T cells. In the first set of experiments, we therefore sought to determine the rate of accumulation and proliferation of H7a-specific T cells in the tumor at early time points after adoptive transfer. To this end, anti-H7a donor cells were stained with CFSE before injection in B10 recipients on day 7. We sacrificed mice on day 10, 13 and 15 (that is, day 3, 6 and 8 after adoptive transfer) and prepared cells suspensions from tumors and spleens to evaluate T-cell accumulation and mitotic behavior. Non-specific labeling with H7a tetramers, as determined by staining naïve B10 or B10.H7b CD8 T cells, was ≤0.04% (data not shown). At each time point, the proportion of CD8 T cells that were H7a tetramer-positive was ≈1–2% in the spleen but ≈80–90% in the tumor (Fig. 2A). Thus, as early as day 10 (day 3 after adoptive transfer), we observed a selective accumulation of H7a-specific T cells in the tumors. Between day 10 and day 15, the absolute number of H7a-specific T cells remained relatively stable in the spleen but increased ≈8.5-fold in the tumor (Fig. 2B and 3B).

The expansion in the number of intratumoral H7a-specific CD8+ T cells could be due to their proliferation in situ or their recruitment from secondary lymphoid organs. To investigate this issue, we analyzed the CFSE content of H7a tetramer-positive T cells present in the spleen and the tumor. The CFSE content decreases by 50% after each cell division. The vast majority of H7a-specific T cells harvested on day 10 had underwent 0 or 1 mitosis following adoptive transfer (Fig. 3A). From day 10 to 15, the mean CFSE content of anti-H7a T cells decreased much more rapidly in the tumor than in the spleen (Fig. 3A). By day 15, all anti-H7a T cells harvested from the tumor had divided at least once while a substantial number of anti-H7a T cells in the spleen had not (Fig. 3A). Using equations developed by Turka et al. [17], [19] we calculated the proliferative burst size and the doubling time of H7a tetramer-positive T cells (Fig. 3). The proliferative burst size corresponds to the number of daughter cells generated by a dividing T-cell “precursor”, and the doubling time represents the time required for the average T cell to achieve a single cell division. Our calculations are based on the assumption that H7a-specific T cells present in one site (spleen or tumor) on day 15 derive from H7a-specific T cells that had seeded this particular site by day 10. On day 15, H7a-specific T cells in the tumor displayed a greater burst size (≈3.5-fold) and shorter doubling time (≈3-fold) than H7a-specific T cells in the spleen (Fig. 3B).

Our studies on the proliferation kinetics of H7a-specific CD8 T cells show that H7a-specific T cells found in the tumor and spleen have different mitotic histories: H7a-specific T cells in the tumor have undergone more cell divisions that those in the spleen (Fig. 3). These data suggest that the massive expansion H7a-specific T cells found in the tumor, but not the spleen, reflects their intratumoral proliferation.

Two Host Factors Regulate Long-Term Persistence of H7a-Specific Memory T Cells

In order to determine whether H7a-specific T cells persisted long-term following adoptive transfer, we assessed the numbers of H7a tetramer+ CD8 T cells present on day 100 in the spleen and bone marrow of euthymic mice successfully treated for melanoma (as shown in Fig. 1). We paid special attention to the bone marrow because it is a preferential homing site for memory T cells, and memory CD8 T cells proliferate more extensively in the bone marrow than they do in either secondary lymphoid or extra-lymphoid organs [27]. Since H7a expression by host cells might impinge on long-term persistence of H7a-specific T cells, we studied the fate of primed H7a-specific T cells transferred in B10 and B10.H7b hosts. The salient finding was that significant numbers of H7a-specific T cells were found in B10.H7b but not in B10 hosts (P<0.001; Fig. 4). The sole difference between these two strains of mice is that H7a MiHA is ubiquitously expressed in B10 mice, but absent in B10.H7b mice [6], [28], [29]. Thus, when confronted with ubiquitous expression of their cognate antigen, H7a-targeted T cells underwent apoptosis or replicative exhaustion. As expected for memory T cells, about 98% of anti-H7a T cells were CD44hi. Consistent with the fact that the bone marrow is a preferential homing site for memory T cells [27], [30], the frequency of H7a-specific T cells was higher in the bone marrow than the spleen of B10.H7b mice (P<0.01).

Naïve and memory CD8 T cells may compete for cytokines that regulate lymphocyte survival and proliferation [31]. We therefore asked whether in the absence of thymic output, anti-H7a memory T cells would persist long-term in B10 hosts. To test this, we evaluated the persistence of H7a-specific memory T cells in euthymic vs. thymectomized H7a-positive (B10) hosts. Significant numbers of H7a-specific memory T cells were present on day 100 in thymectomized B10 hosts (P<0.01 relative to euthymic B10 hosts; Fig. 4). Thus, anti-H7a T cells can persist long-term in B10 hosts in the absence of naïve T cell production by the thymus. We conclude that two host factors regulated persistence of anti-H7a memory T cells: thymic function and expression of H7a by normal host cells. H7a-specific memory T cells were abundant in euthymic B10.H7b hosts, present in low numbers in thymectomized B10 hosts, and undetectable in euthymic B10 hosts.

Challenge of Hematopoietic Chimeras with Tumor Cells

No melanoma recurrence was seen in B10 mice (n = 30) observed for 6 months after induction of complete remission by adoptive transfer of anti-H7a T cells ([7] and data not shown). To test whether lack of tumor relapse might be due to persistence of H7a-specific T cells at levels below the detection limit of tetramer-staining assays, cured B10 recipients were challenged on day 100, with 2×105 melanoma cells (as on day 0). In euthymic cured B10 recipients, tumors grew rapidly (Fig. 5) and no accumulation of H7a-specific T cells was detected in the spleen, bone marrow or tumor (Fig. 6). Thus, cured euthymic B10 mice did not show any functional evidence of immune reactivity to H7a or to any tumor-associated epitopes. In thymectomized B10 hosts, a minimal accumulation of H7a-specific T cells was found (Fig. 6) that entailed only a small delay in tumor growth (Fig. 5). Hence, H7a-specific memory T cells present in thymectomized B10 hosts were unable to provide a protective antitumor response. In contrast, H7a-specific memory T cells present in B10.H7b hosts generated a strong anamnestic response following tumor rechallenge (Fig. 6). In the latter mice, the tumor grew for a few days and then disappeared (Fig. 5). Thus, H7a-specific memory T cells found in B10.H7b hosts were perfectly functional.

Discussion

Intratumoral Accumulation of H7a-Specific T Cells

A major obstacle encountered in cancer immunotherapy trials, including those targeting MiHAs, is the failure of antigen-reactive T cells to invade tumors and persist long-term [12], [32][34]. Few studies have addressed the in vivo fate of MiHA-specific T cells using MHC-peptide tetramers [35], [36]. Moreover, to the best of our knowledge, no study has evaluated the in vivo fate of antigen-primed MiHA-specific T cells, nor their behavior in the tumor environment. We previously reported that adoptively transferred anti-H7a T cells were found in large numbers in regressing melanomas (day 19) [7]. Here, by looking at earlier time points, we found that selective tumor infiltration by anti-H7a T cells was established by day 3 after adoptive transfer. Preferential localization of adoptively transferred CD8 T cells to tumor sites has been reported in one clinical trial [37]. Homing to the tumor is probably due to interaction between activated T cells and antigen-independent inflammatory ligands such as Vcam-1 [7], [38]. We found that following initial seeding, numbers of H7a-specific T cells increased dramatically in the tumor but not the spleen. Furthermore, based on CFSE profiles, we found that H7a-specific T cells in the tumor had divided much more extensively than those in the spleen. These data suggest that the massive accumulation of H7a-specific T cells in the tumor between day 10 and day 15 is due to in situ proliferation. They are also consistent with the fact that differentiation and survival of primed CD8 (but not CD4) effector T cells are independent of secondary lymphoid organs in adoptive hosts [39]. However, we cannot formally discard the possibility that, in our model, H7a-specific T cells proliferate in the draining lymph node and then migrate to the tumor. Further studies are needed to understand the nature of signals that drive intratumoral expansion of MiHA-specific T cells. We speculate that IFN-γ may be an important player in this process. Indeed, the local release of IFN-γ by anti-H7a T cells upregulates expression of MHC I and H7a in the tumor [7], and may thereby stimulate proliferation of anti-H7a T cells. Nonetheless, though the tumor environment may not be particularly propitious to T-cell invasion and expansion [9][12], the present work illustrates that this limitation may be overcome by adoptive transfer of primed CD8 T cells targeted to an immunodominant MiHA. In line with this, new promising methods have been recently developed to generate high avidity MiHA-specific CD8 T cells for adoptive immunotherapy in human [40][42].

Relapse-Free Survival Did Not Necessitate Persistence of H7a-Reactive Memory T Cells

Whether long-term cancer remission induced by MiHA-targeted T cells is associated with total eradication of cancer cells or with persistence of low numbers of resting or slow growing tumor cells (tumor dormancy) is unknown. The distinction between these two outcomes is important since tumor persistence always entails the risk of relapse. Genuine cancer cure requires elimination of cancer stem cells which have a mostly quiescent cell cycle profile and are capable of self-renewal. The quiescent status of cancer stem cells renders them difficult to eradicate with chemotherapy that typically target proliferating cells. Nonetheless, T cells can eliminate quiescent cells as well as cycling cells. Thus, when cultured with acute myeloid leukemia cells, MiHA-specific T cells can wipe out leukemia stem cells [43]. Nevertheless, a substantial body of evidence suggests that remissions induced by various types of immunotherapy targeted to tumor specific antigens (but not to MiHAs) are usually associated with tumor dormancy [44] We found that recipients in which anti-H7a T cells disappeared (euthymic B10 mice) nevertheless remained tumor-free. The latter mice behave as naive mice when rechallenged with melanoma cells. Thus, in this model, cure is probably due to eradication of clonogenic tumor cells rather than to induction of T-cell dependent tumor dormancy. Notably, we have observed no late relapses in mice with EL4 leukemia treated with anti-H7a T cells ([6] and unpublished observations). This suggests that the findings reported herein may not be unique to melanoma and may be relevant to some rapidly growing tumors like B16.F10 and EL4. By no means, however, do we infer that persistence of memory T cells is generally irrelevant in adoptive cancer immunotherapy. The occurrence of late leukemia relapses following allogeneic hematopoietic cell transplantation argues against this view. Nevertheless, the lack of cancer relapse in mice devoid of anti-H7a memory T cells illustrates the potency of the acute anti-tumor effect that can be generated by anti-MiHA T cells.

Antigen Distribution and Thymus Function Regulate Long-Term Persistence of H7a-Specific Memory T Cells

Functional anti-H7a T cells persisted long-term in B10.H7b but not B10 mice. The ubiquitous expression of H7a on B10 host cells led to physical demise or functional impairment of anti-H7a T cells. The exhaustion of H7a-specific T cells in B10 recipients was similar to that of anti-viral T cells confronted with viruses that disseminate widely [45]. Thus, at least in euthymic subjects, adoptively transferred host-reactive T cells should have a longer life span when targeted to tissue-restricted (e.g., tumor-specific) as opposed to ubiquitous epitopes. Assuming that prolonged persistence of adoptively transferred T cells is probably relevant in preventing late cancer relapses, it would therefore be advantageous to target MiHAs with a restricted tissue distribution. Fortunately, several non ubiquitous MiHAs have been discovered in human [46][48]. Accordingly, MiHAs derived from oncoproteins, such as HA-1, represent particularly attractive targets for adoptive immunotherapy of hematopoietic malignancies and solid tumors [49], [50]. Finally, we found that thymic output had a negative impact on persistence of H7a-specific memory T cells. Many cancer patients, particularly those in older age groups, present thymic insufficiency [51]. MiHA-specific T cells may have a more protracted survival in these subjects. From a different perspective, the concept that thymic output mitigates the persistence of MiHA-reactive T cells could explain why graft-versus-host disease becomes more frequent with increasing recipient age following conventional allogeneic hematopoietic cell transplantation [52], [53].

Acknowledgments

We thank Marie-Ève Blais for technical assistance and thoughtful comments.

Author Contributions

Conceived and designed the experiments: MCM CB CP. Performed the experiments: MCM CB. Analyzed the data: MCM CB CP. Wrote the paper: MCM CB CP.

References

  1. 1. Bleakley M, Riddell SR (2004) Molecules and mechanisms of the graft-versus-leukaemia effect. Nat Rev Cancer 4: 371–380.M. BleakleySR Riddell2004Molecules and mechanisms of the graft-versus-leukaemia effect.Nat Rev Cancer4371380
  2. 2. Perreault C, Brochu S (2002) Adoptive cancer immunotherapy: discovering the best targets. J Mol Med 80: 212–218.C. PerreaultS. Brochu2002Adoptive cancer immunotherapy: discovering the best targets.J Mol Med80212218
  3. 3. Appelbaum FR (2001) Haematopoietic cell transplantation as immunotherapy. Nature 411: 385–389.FR Appelbaum2001Haematopoietic cell transplantation as immunotherapy.Nature411385389
  4. 4. Truitt RL (2004) The Mortimer M. Bortin Lecture: to destroy by the reaction of immunity: the search for separation of graft-versus-leukemia and graft-versus-host. Biol Blood Marrow Transplant 10: 505–523.RL Truitt2004The Mortimer M. Bortin Lecture: to destroy by the reaction of immunity: the search for separation of graft-versus-leukemia and graft-versus-host.Biol Blood Marrow Transplant10505523
  5. 5. Molldrem JJ, Lee PP, Wang C, Felio K, Kantarjian HM, et al. (2000) Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med 6: 1018–1023.JJ MolldremPP LeeC. WangK. FelioHM Kantarjian2000Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia.Nat Med610181023
  6. 6. Fontaine P, Roy-Proulx G, Knafo L, Baron C, Roy DC, et al. (2001) Adoptive transfer of T lymphocytes targeted to a single immunodominant minor histocompatibility antigen eradicates leukemia cells without causing graft-versus-host disease. Nat Med 7: 789–794.P. FontaineG. Roy-ProulxL. KnafoC. BaronDC Roy2001Adoptive transfer of T lymphocytes targeted to a single immunodominant minor histocompatibility antigen eradicates leukemia cells without causing graft-versus-host disease.Nat Med7789794
  7. 7. Meunier MC, Delisle JS, Bergeron J, Rineau V, Baron C, et al. (2005) T cells targeted against a single minor histocompatibility antigen can cure solid tumors. Nat Med 11: 1222–1229.MC MeunierJS DelisleJ. BergeronV. RineauC. Baron2005T cells targeted against a single minor histocompatibility antigen can cure solid tumors.Nat Med1112221229
  8. 8. Korngold R, Leighton C, Mobraaten LE, Berger MA (1997) Inter-strain graft-vs.-host disease T-cell responses to immunodominant minor histocompatibility antigens. Biol Blood Marrow Transplant 3: 57–64.R. KorngoldC. LeightonLE MobraatenMA Berger1997Inter-strain graft-vs.-host disease T-cell responses to immunodominant minor histocompatibility antigens.Biol Blood Marrow Transplant35764
  9. 9. Ochsenbein AF, Sierro S, Odermatt B, Pericin M, Karrer U, et al. (2001) Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction. Nature 411: 1058–1064.AF OchsenbeinS. SierroB. OdermattM. PericinU. Karrer2001Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction.Nature41110581064
  10. 10. Zou W (2005) Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 5: 263–274.W. Zou2005Immunosuppressive networks in the tumour environment and their therapeutic relevance.Nat Rev Cancer5263274
  11. 11. Yu P, Rowley DA, Fu YX, Schreiber H (2006) The role of stroma in immune recognition and destruction of well-established solid tumors. Curr Opin Immunol 18: 226–231.P. YuDA RowleyYX FuH. Schreiber2006The role of stroma in immune recognition and destruction of well-established solid tumors.Curr Opin Immunol18226231
  12. 12. Boon T, Coulie PG, Van den Eynde BJ, van der Bruggen P (2006) Human T cell responses against melanoma. Annu Rev Immunol 24: 175–208.T. BoonPG CoulieBJ Van den EyndeP. van der Bruggen2006Human T cell responses against melanoma.Annu Rev Immunol24175208
  13. 13. Dulude G, Roy DC, Perreault C (1999) The effect of graft-versus-host disease on T cell production and homeostasis. J Exp Med 189: 1329–1342.G. DuludeDC RoyC. Perreault1999The effect of graft-versus-host disease on T cell production and homeostasis.J Exp Med18913291342
  14. 14. Meunier MC, Roy-Proulx G, Labrecque N, Perreault C (2003) Tissue distribution of target antigen has a decisive influence on the outcome of adoptive cancer immunotherapy. Blood 101: 766–770.MC MeunierG. Roy-ProulxN. LabrecqueC. Perreault2003Tissue distribution of target antigen has a decisive influence on the outcome of adoptive cancer immunotherapy.Blood101766770
  15. 15. Baron C, Meunier MC, Caron E, Côté C, Cameron MJ, et al. (2006) Asynchronous differentiation of CD8 T cells that recognize dominant and cryptic antigens. J Immunol 177: 8466–8475.C. BaronMC MeunierE. CaronC. CôtéMJ Cameron2006Asynchronous differentiation of CD8 T cells that recognize dominant and cryptic antigens.J Immunol17784668475
  16. 16. Blais ME, Gérard G, Martinic MM, Roy-Proulx G, Zinkernagel RM, et al. (2004) Do thymically and strictly extrathymically developing T cells generate similar immune responses? Blood 103: 3102–3110.ME BlaisG. GérardMM MartinicG. Roy-ProulxRM Zinkernagel2004Do thymically and strictly extrathymically developing T cells generate similar immune responses?Blood10331023110
  17. 17. Wells AD, Gudmundsdottir H, Turka LA (1997) Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response. J Clin Invest 100: 3173–3183.AD WellsH. GudmundsdottirLA Turka1997Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response.J Clin Invest10031733183
  18. 18. Lyons AB, Parish CR (1994) Determination of lymphocyte division by flow cytometry. J Immunol Methods 171: 131–137.AB LyonsCR Parish1994Determination of lymphocyte division by flow cytometry.J Immunol Methods171131137
  19. 19. Gudmundsdottir H, Wells AD, Turka LA (1999) Dynamics and requirements of T cell clonal expansion in vivo at the single-cell level: effector function is linked to proliferative capacity. J Immunol 162: 5212–5223.H. GudmundsdottirAD WellsLA Turka1999Dynamics and requirements of T cell clonal expansion in vivo at the single-cell level: effector function is linked to proliferative capacity.J Immunol16252125223
  20. 20. Rocha B, Tanchot C (2004) Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses. Curr Opin Immunol 16: 259–263.B. RochaC. Tanchot2004Towards a cellular definition of CD8+ T-cell memory: the role of CD4+ T-cell help in CD8+ T-cell responses.Curr Opin Immunol16259263
  21. 21. Bevan MJ (2004) Helping the CD8+ T-cell response. Nat Rev Immunol 4: 595–602.MJ Bevan2004Helping the CD8+ T-cell response.Nat Rev Immunol4595602
  22. 22. Pion S, Fontaine P, Desaulniers M, Jutras J, Filep JG, et al. (1997) On the mechanisms of immunodominance in cytotoxic T lymphocyte responses to minor histocompatibility antigens. Eur J Immunol 27: 421–430.S. PionP. FontaineM. DesaulniersJ. JutrasJG Filep1997On the mechanisms of immunodominance in cytotoxic T lymphocyte responses to minor histocompatibility antigens.Eur J Immunol27421430
  23. 23. Roy-Proulx G, Meunier MC, Lanteigne AM, Brochu S, Perreault C (2001) Immunodomination results from functional differences between competing CTL. Eur J Immunol 31: 2284–2292.G. Roy-ProulxMC MeunierAM LanteigneS. BrochuC. Perreault2001Immunodomination results from functional differences between competing CTL.Eur J Immunol3122842292
  24. 24. Roy-Proulx G, Baron C, Perreault C (2005) CD8 T-cell ability to exert immunodomination correlates with T-cell receptor:epitope association rate. Biol Blood Marrow Transplant 11: 260–271.G. Roy-ProulxC. BaronC. Perreault2005CD8 T-cell ability to exert immunodomination correlates with T-cell receptor:epitope association rate.Biol Blood Marrow Transplant11260271
  25. 25. van Elsas A, Sutmuller RPM, Hurwitz AA, Ziskin J, Villasenor J, et al. (2001) Elucidating the autoimmune and antitumor effector mechanisms of a treatment based on cytotoxic T lymphocyte antigen-4 blockade in combination with a B16 melanoma vaccine: comparison of prophylaxis and therapy. J Exp Med 194: 481–490.A. van ElsasRPM SutmullerAA HurwitzJ. ZiskinJ. Villasenor2001Elucidating the autoimmune and antitumor effector mechanisms of a treatment based on cytotoxic T lymphocyte antigen-4 blockade in combination with a B16 melanoma vaccine: comparison of prophylaxis and therapy.J Exp Med194481490
  26. 26. Marincola FM, Jaffee EM, Hicklin DJ, Ferrone S (2000) Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance. Adv Immunol 74: 181–273.FM MarincolaEM JaffeeDJ HicklinS. Ferrone2000Escape of human solid tumors from T-cell recognition: molecular mechanisms and functional significance.Adv Immunol74181273
  27. 27. Di Rosa F, Pabst R (2005) The bone marrow: a nest for migratory memory T cells. Trends Immunol 26: 360–366.F. Di RosaR. Pabst2005The bone marrow: a nest for migratory memory T cells.Trends Immunol26360366
  28. 28. Eden PA, Christianson GJ, Fontaine P, Wettstein PJ, Perreault C, et al. (1999) Biochemical and immunogenetic analysis of an immunodominant peptide (B6dom1) encoded by the classical H7 minor histocompatibility locus. J Immunol 162: 4502–4510.PA EdenGJ ChristiansonP. FontainePJ WettsteinC. Perreault1999Biochemical and immunogenetic analysis of an immunodominant peptide (B6dom1) encoded by the classical H7 minor histocompatibility locus.J Immunol16245024510
  29. 29. McBride K, Baron C, Picard S, Martin S, Boismenu D, et al. (2002) The model B6dom1 minor histocompatibility antigen is encoded by a mouse homolog of the yeast STT3 gene. Immunogenetics 54: 562–569.K. McBrideC. BaronS. PicardS. MartinD. Boismenu2002The model B6dom1 minor histocompatibility antigen is encoded by a mouse homolog of the yeast STT3 gene.Immunogenetics54562569
  30. 30. Mazo IB, Honczarenko M, Leung H, Cavanagh LL, Bonasio R, et al. (2005) Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells. Immunity 22: 259–270.IB MazoM. HonczarenkoH. LeungLL CavanaghR. Bonasio2005Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells.Immunity22259270
  31. 31. Ge Q, Bai A, Jones B, Eisen HN, Chen J (2004) Competition for self-peptide-MHC complexes and cytokines between naive and memory CD8+ T cells expressing the same or different T cell receptors. Proc Natl Acad Sci U S A 101: 3041–3046.Q. GeA. BaiB. JonesHN EisenJ. Chen2004Competition for self-peptide-MHC complexes and cytokines between naive and memory CD8+ T cells expressing the same or different T cell receptors.Proc Natl Acad Sci U S A10130413046
  32. 32. Ho WY, Blattman JN, Dossett ML, Yee C, Greenberg PD (2003) Adoptive immunotherapy: engineering T cell responses as biologic weapons for tumor mass destruction. Cancer Cell 3: 431–437.WY HoJN BlattmanML DossettC. YeePD Greenberg2003Adoptive immunotherapy: engineering T cell responses as biologic weapons for tumor mass destruction.Cancer Cell3431437
  33. 33. Nijmeijer BA, van Schie ML, Verzaal P, Willemze R, Falkenburg JH (2005) Responses to donor lymphocyte infusion for acute lymphoblastic leukemia may be determined by both qualitative and quantitative limitations of antileukemic T-cell responses as observed in an animal model for human leukemia. Exp Hematol 33: 1172–1181.BA NijmeijerML van SchieP. VerzaalR. WillemzeJH Falkenburg2005Responses to donor lymphocyte infusion for acute lymphoblastic leukemia may be determined by both qualitative and quantitative limitations of antileukemic T-cell responses as observed in an animal model for human leukemia.Exp Hematol3311721181
  34. 34. Riddell SR, Bleakley M, Nishida T, Berger C, Warren EH (2006) Adoptive transfer of allogeneic antigen-specific T cells. Biol Blood Marrow Transplant 12: 9–12.SR RiddellM. BleakleyT. NishidaC. BergerEH Warren2006Adoptive transfer of allogeneic antigen-specific T cells.Biol Blood Marrow Transplant12912
  35. 35. Mutis T, Gillespie G, Schrama E, Falkenburg JHF, Moss P, et al. (1999) Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease. Nat Med 5: 839–842.T. MutisG. GillespieE. SchramaJHF FalkenburgP. Moss1999Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease.Nat Med5839842
  36. 36. Choi EY, Christianson GJ, Yoshimura Y, Jung N, Sproule TJ, et al. (2002) Real-time T-cell profiling identifies H60 as a major minor histocompatibility antigen in murine graft-versus-host disease. Blood 100: 4259–4264.EY ChoiGJ ChristiansonY. YoshimuraN. JungTJ Sproule2002Real-time T-cell profiling identifies H60 as a major minor histocompatibility antigen in murine graft-versus-host disease.Blood10042594264
  37. 37. Yee C, Thompson JA, Byrd D, Riddell SR, Roche P, et al. (2002) Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: In vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci U S A 99: 16168–16173.C. YeeJA ThompsonD. ByrdSR RiddellP. Roche2002Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: In vivo persistence, migration, and antitumor effect of transferred T cells.Proc Natl Acad Sci U S A991616816173
  38. 38. Lane C, Leitch J, Tan X, Hadjati J, Bramson JL, et al. (2004) Vaccination-induced autoimmune vitiligo is a consequence of secondary trauma to the skin. Cancer Res 64: 1509–1514.C. LaneJ. LeitchX. TanJ. HadjatiJL Bramson2004Vaccination-induced autoimmune vitiligo is a consequence of secondary trauma to the skin.Cancer Res6415091514
  39. 39. Obhrai JS, Oberbarnscheidt MH, Hand TW, Diggs L, Chalasani G, et al. (2006) Effector T cell differentiation and memory T cell maintenance outside secondary lymphoid organs. J Immunol 176: 4051–4058.JS ObhraiMH OberbarnscheidtTW HandL. DiggsG. Chalasani2006Effector T cell differentiation and memory T cell maintenance outside secondary lymphoid organs.J Immunol17640514058
  40. 40. Oosten LE, Blokland E, Van Halteren AG, Curtsinger J, Mescher MF, et al. (2004) Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen-specific cytotoxic T lymphocytes. Blood 104: 224–226.LE OostenE. BloklandAG Van HalterenJ. CurtsingerMF Mescher2004Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen-specific cytotoxic T lymphocytes.Blood104224226
  41. 41. Heemskerk MHM, Hoogeboom M, Hagedoorn R, Kester MGD, Willemze R, et al. (2004) Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer. J Exp Med 199: 885–894.MHM HeemskerkM. HoogeboomR. HagedoornMGD KesterR. Willemze2004Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer.J Exp Med199885894
  42. 42. Rice J, Dunn S, Piper K, Buchan SL, Moss PA, et al. (2006) DNA fusion vaccines induce epitope-specific cytotoxic CD8+ T cells against human leukemia-associated minor histocompatibility antigens. Cancer Res 66: 5436–5442.J. RiceS. DunnK. PiperSL BuchanPA Moss2006DNA fusion vaccines induce epitope-specific cytotoxic CD8+ T cells against human leukemia-associated minor histocompatibility antigens.Cancer Res6654365442
  43. 43. Bonnet D, Warren EH, Greenberg PD, Dick JE, Riddell SR (1999) CD8+ minor histocompatibility antigen-specific cytotoxic T lymphocyte clones eliminate human acute myeloid leukemia stem cells. Proc Natl Acad Sci U S A 96: 8639–8644.D. BonnetEH WarrenPD GreenbergJE DickSR Riddell1999CD8+ minor histocompatibility antigen-specific cytotoxic T lymphocyte clones eliminate human acute myeloid leukemia stem cells.Proc Natl Acad Sci U S A9686398644
  44. 44. Uhr JW, Scheuermann RH, Street NE, Vitetta ES (1997) Cancer dormancy: opportunities for new therapeutic approaches. Nat Med 3: 505–509.JW UhrRH ScheuermannNE StreetES Vitetta1997Cancer dormancy: opportunities for new therapeutic approaches.Nat Med3505509
  45. 45. Zinkernagel RM, Planz O, Ehl S, Battegay M, Odermatt B, et al. (1999) General and specific immunosuppression caused by antiviral T-cell responses. Immunol Rev 168: 305–315.RM ZinkernagelO. PlanzS. EhlM. BattegayB. Odermatt1999General and specific immunosuppression caused by antiviral T-cell responses.Immunol Rev168305315
  46. 46. Hambach L, Goulmy E (2005) Immunotherapy of cancer through targeting of minor histocompatibility antigens. Curr Opin Immunol 17: 202–210.L. HambachE. Goulmy2005Immunotherapy of cancer through targeting of minor histocompatibility antigens.Curr Opin Immunol17202210
  47. 47. Rijke BD, Horssen-Zoetbrood A, Beekman JM, Otterud B, Maas F, et al. (2005) A frameshift polymorphism in P2X5 elicits an allogeneic cytotoxic T lymphocyte response associated with remission of chronic myeloid leukemia. J Clin Invest 115: 3506–3516.BD RijkeA. Horssen-ZoetbroodJM BeekmanB. OtterudF. Maas2005A frameshift polymorphism in P2X5 elicits an allogeneic cytotoxic T lymphocyte response associated with remission of chronic myeloid leukemia.J Clin Invest11535063516
  48. 48. Brickner AG, Evans AM, Mito JK, Xuereb SM, Feng X, et al. (2006) The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL. Blood 107: 3779–3786.AG BricknerAM EvansJK MitoSM XuerebX. Feng2006The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL.Blood10737793786
  49. 49. Klein CA, Wilke M, Pool J, Vermeulen C, Blokland E, et al. (2002) The hematopoietic system-specific minor histocompatibility antigen HA-1 shows aberrant expression in epithelial cancer cells. J Exp Med 196: 359–368.CA KleinM. WilkeJ. PoolC. VermeulenE. Blokland2002The hematopoietic system-specific minor histocompatibility antigen HA-1 shows aberrant expression in epithelial cancer cells.J Exp Med196359368
  50. 50. Spierings E, Wieles B, Goulmy E (2004) Minor histocompatibility antigens - big in tumour therapy. Trends Immunol 25: 56–60.E. SpieringsB. WielesE. Goulmy2004Minor histocompatibility antigens - big in tumour therapy.Trends Immunol255660
  51. 51. Hakim FT, Memon SA, Cepeda R, Jones EC, Chow CK, et al. (2005) Age-dependent incidence, time course, and consequences of thymic renewal in adults. J Clin Invest 115: 930–939.FT HakimSA MemonR. CepedaEC JonesCK Chow2005Age-dependent incidence, time course, and consequences of thymic renewal in adults.J Clin Invest115930939
  52. 52. Storb R, Prentice RL, Sullivan KM, Shulman HM, Deeg HJ, et al. (1983) Predictive factors in chronic graft-versus-host disease in patients with aplastic anemia treated by marrow transplantation from HLA-identical siblings. Ann Intern Med 98: 461–466.R. StorbRL PrenticeKM SullivanHM ShulmanHJ Deeg1983Predictive factors in chronic graft-versus-host disease in patients with aplastic anemia treated by marrow transplantation from HLA-identical siblings.Ann Intern Med98461466
  53. 53. Zecca M, Prete A, Rondelli R, Lanino E, Balduzzi A, et al. (2002) Chronic graft-versus-host disease in children: incidence, risk factors, and impact on outcome. Blood 100: 1192–1200.M. ZeccaA. PreteR. RondelliE. LaninoA. Balduzzi2002Chronic graft-versus-host disease in children: incidence, risk factors, and impact on outcome.Blood10011921200