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. 2012 Jul 18;62(1):101–112. doi: 10.1007/s00262-012-1316-3

Distinct in vivo CD8 and CD4 T cell responses against normal and malignant tissues

David Coe 1, Caroline Addey 1, Matthew White 1, Nida Harwood 1, Julian Dyson 1, Jian-Guo Chai 1,✉
PMCID: PMC11028943  PMID: 22806093

Abstract

Normal tissue and tumour grafts expressing the same alloantigens often elicit distinct immune responses whereby only normal tissue is rejected. To investigate the mechanisms that underlie these distinct outcomes, we compared the responses of adoptively transferred HY-specific conventional (CD8 and CD4) or regulatory T (Treg) cells in mice bearing HY-expressing tumour, syngeneic male skin graft or both. For local T cell priming, T cell re-circulation, graft localization and retention, skin grafts were more efficient than tumours. Skin grafts were also capable of differentiating CD4 T cells into functional Th1 cells. Donor T cell responses were inversely correlated with tumour progression. When skin graft and tumour transplants were performed sequentially, contemporary graft and tumour burden enhanced CD8 but reduced CD4 T cell responses causing accelerated skin-graft rejection without influencing tumour growth. Although both skin grafts and tumours were able to expand HY-specific Treg cells in draining lymph node (dLN), the proportion of tumour-infiltrating Treg cells was significantly higher than that within skin grafts, correlating with accelerated tumour growth. Moreover, there was a higher level of HY antigen presentation by host APC in tumour-dLN than in graft-dLN. Finally, tumour tissues expressed a significant higher level of IDO, TGFβ, IL10 and Arginase I than skin grafts, indicating that malignant but not normal tissue represents a stronger immunosuppressive environment. These comparisons provide important insight into the in vivo mechanisms that conspire to compromise tumour-specific adaptive immunity and identify new targets for cancer immunotherapy.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-012-1316-3) contains supplementary material, which is available to authorized users.

Keywords: Tumour, Skin graft, T cell adoptive transfer, TCR transgenic, HY

Introduction

Host immune responses to allografts of normal tissue are generally productive leading to rejection [1]. Conversely, those against transplantable tumours are generally defective and fail to eliminate the developing tumour [2]. Where the normal and tumour allografts share alloantigens, outcome will be partly attributed to the distinct features of the immune responses. Comparison of antigen-specific T cell responses to normal and tumour allografts can provide insight into the cellular mechanisms underlying the failure of anti-tumour immunity.

The male-specific minor histocompatibility antigen, HY, is one of the best-characterized transplantation antigens [3, 4]. Identification of HY genes and peptides [5–8], subsequent development of tetramers [9, 10] and generation of TCR-transgenic mice [11, 12] have greatly facilitated the dissection of HY-specific T cell immunity and tolerance [13–18]. Recently, HY has been explored as a surrogate tumour antigen by us [19–21] and others [22–24], taking the advantages of a number of HY-expressing tumour cell lines. Additionally, HY provides a unique opportunity to evaluate T cell immunity to epitopes expressed on both malignant and non-malignant tissues, which is the aim of this study.

MB49 is a chemically induced, male C57BL/6 (H2b) murine bladder carcinoma expressing all HY epitopes [25]. MB49 tumours produce IL10 that inhibits (a) BCG-induced cytotoxicity by tumour-infiltrating macrophages [26] and (b) dendritic cell priming of HY-specific CD4 and CD8 T cells [27, 28]. Furthermore, we recently showed that MB49 cells express TGFβ1 [19], an immune suppressive cytokine often associated with tumour microenvironments [29]. Moreover, MB49 cells also express the complement inhibitor protein, Crry, which both protects cancer cells from killing via ADCC and prevents T cell priming via unknown mechanisms [30]. However, whether MB49 tumours also express other immunoregulatory molecules such as IDO [31] has not been investigated.

Here, we have applied adoptive transfer of defined populations of HY-specific effector and regulatory T cells to compare responses in mice bearing tumours, male skin grafts or both. In addition, we compared the expression of immunoregulatory genes and antigen burden in draining lymph node, tumour and skin grafts. These experiments have provided fresh insight into the cellular and molecular mechanisms that determine whether the immune responses lead to rejection of allografts but acceptance of tumours.

Materials and methods

Mice

Thy1.2 B6, Thy1.1 MataHari (Rag1−/− or Rag1+/−) and Thy1.1 Marilyn mice (Rag2−/− or Rag2+/−) have been described previously [11, 12]. Mice were housed under SPF conditions at the Central Biological Services Unit of Imperial College London. Procedures were conducted in accordance with the Home Office Animal (Scientific Procedures) Act of 1986.

Tumour cell line

MB49 is a chemically induced, male C57BL/6, H2b murine bladder carcinoma that constitutively expresses the endogenous HY genes [25]. MB49 cells were maintained in RPMI 1640 Medium (Gibco, UK) supplemented with 10 % FCS, 10 mM HEPES, penicillin (100 IU/ml) and streptomycin (100 μg/ml), 5 × 10−5 M 2-ME and 2 mM l-glutamine.

Purification of MataHari CD8, Marilyn CD4 and Marilyn Treg cells

This was performed as described previously [19–21]. In brief, CD8 and CD4 T cells were purified from pooled LN and spleen cells of Thy1.1 Rag2+/− MataHari and Thy1.1 Rag1−/− Marilyn females, respectively, by direct positive selection (anti-CD8 or anti-CD4 microbeads) using autoMACS (Miltenyi Biotec, Germany). For purifying Treg cells, CD25-expressing cells were enriched from pooled LN and spleen cells of Thy1.1 Rag1+/− Marilyn females by indirect positive selection (anti-CD25PE followed by anti-PE microbeads) using autoMACS before FACS sorting for Vβ6+CD4+CD25+ cells (typically with >98 % purity).

CFSE labelling and adoptive T cell transfer

These were performed as described previously [19–21]. CFSE labelling was conducted by incubating of purified CD8 or CD4 T cells (5–10 × 106/ml in PBS) with a final concentration of 5 μM of carboxyfluorescein succinimidyl ester (CFSE) in the dark at room temperature for 10 min. Reaction was terminated by adding 10 % FCS RPMI 1640. After extensive washing with PBS, the CFSE-labelled T cells (Thy1.1+) were adoptively transferred to Thy1.2+ B6 female recipients by intravenous injection. For Treg adoptive transfer experiments, CFSE labelling was omitted.

Tumour cell inoculation and skin grafting

These were performed as described previously [19–21]. Tumour inoculation was conducted by subcutaneous injection of Thy1.2 B6 female mice with 5 × 105 MB49 cells in 0.2 ml of PBS on the right flank. Skin grafting was conducted by the modified method of Billingham and Medawar using tail skin grafted onto the lateral thorax of Thy1.2+ B6 female mice. After removal of the plaster casts, grafts were monitored every 2–3 days and scored as rejected when less than 10 % viable tissue remained.

FACS analysis

This was performed as described previously [19–21]. For MB49-bearing and male graft-bearing mice, single-cell suspensions were prepared from tumour-draining (inguinal), graft-draining (axillary), non-draining lymph nodes (respective contralateral LN [ndLN]), MB49 tumour and skin-graft tissues. In control groups (the mice given T cells alone), inguinal and axillary LN were used as control for tumour-dLN and graft-dLN, respectively, and are referred to as peripheral LN (pLN). The cells were stained with anti-Vβ8.3PE (MataHari TCR) or anti-Vβ6PE (Marilyn TCR), anti-Thy1.1PerCP and anti-CD44APC. Donor CD8 and CD4 cells were identified by Thy1.1+Vβ8.3+ and Thy1.1+Vβ6+ and subjected to further analysis of the expression pattern of CFSE, CD44, Vβ8.3 or Vβ6. Intracellular Foxp3 staining was performed as described previously [19–21].

qPCR, isolation of CD11c+ cells, proliferation assay and measurement of intracellular cytokine production

These were performed as described previously [19–21]. RNA was prepared from skin grafts or tumour tissues using mechanical homogenization and TRIzol reagent. Test samples were assessed in triplicate for the expression of 18S ribosomal RNA and IDO, Arg. I, TGFβ1 and IL10 transcripts. Expression values were determined from standard curves generated from each RNA preparation, plotting cycle threshold values against log quantity. Normalized IDO, Arg. I, TGFβ1 and IL10 values were obtained by division with the corresponding 18S value and expressed as fold of increase. Isolation of CD11c+ cells from digested LN cells was performed by direct positive selection (anti-CD11c microbeads) using autoMACS. Purified CD11c+ cells were then co-cultured with purified Marilyn CD4 T cells in 96-well plates for 3 days. Proliferation of T cells was measured by thymidine uptake.

Intracellular staining was used to measure cytokine production by donor CD4 T cells. Draining LN cells were cultured with irradiated male B6 spleen cells in 24-well plates for 5 days. The cells were re-stimulated with PMA and ionomycin in the presence of Brefeldin B for 4 h. The cells were subjected to Ficoll centrifugation to remove dead cells before staining for surface CD4 and Thy1.1 expression. After fixing and permeabilizing, the cells were stained for intracellular IL10 and IFNγ.

Statistical analysis

Data are presented as mean ± SD. Comparisons between groups were performed using Student’s t test. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001.

Results

Rag1+/− MataHari females reject syngenic male skin grafts but not HY-expressing MB49 tumours

Although they efficiently reject syngeneic male skin grafts, Rag1+/− MataHari females cannot control MB49 tumours (Figure S1), indicating that the presence of a high percentage of functional HY-specific CD8 cells alone is sufficient to reject transplanted normal tissue but not for eliminating grafts of malignant tissue.

Distinct CD8 T cell responses to HY-expressing skin and tumour tissues

To better understand the cellular basis for these distinct outcomes, we performed CD8 T cell adoptive transfer experiments (Fig. 1a). The absolute numbers of donor cells recovered from graft-dLN were significantly higher than those in tumour-dLN (not shown), indicating that skin grafts are more potent than tumours for local priming of donor T cells. Likewise, the percentage of donor cells in graft-ndLN was significantly higher than that in tumour-ndLN and pLN of the control group (Fig. 1a and not shown), suggesting that skin grafts are also more efficient than tumours for inducing T cell re-circulation.

Fig. 1.

Fig. 1

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Distinct responses of MataHari CD8 T cells to tumours and male skin grafts. a Donor CD8 cells accumulate more efficiently in skin graft-dLN than tumour-dLN. 5 × 106 Thy1.1 CFSE-labelled MataHari CD8 cells were injected i.v. into three groups of Thy1.2 WT B6 females at day 1. On day 0, the mice in group 1 were left untreated, those in group 2 were s.c. inoculated with 5 × 105 MB49 cells, and those in group 3 were transplanted with syngenic B6 male skin grafts. All mice were culled on day 12, and the pLN cells of control, dLN of MB49 and dLN of graft were stained with anti-Vβ8.3PE and anti-Thy1.1PerCP. Panels show the percentage of donor CD8 T cells identified by co-expression of Thy1.1 and Vβ8.3. One representative experiment of three is shown. b Donor CD8 cells present in graft-dLN proliferate more than those in tumour-dLN. Comparison of CFSE and CD44 expression by donor CD8 cells. pLN of control, dLN of MB49 and dLN of graft were stained with anti-Vβ8.3PE, anti-Thy1.1PerCP and anti-CD44APC. Gated Thy1.1+Vβ8.3+ cells were further analysed for CFSE and CD44 expression. Data are representative of three independent experiments. c. Donor CD8 cells in tumour-dLN show greater downregulation of Vβ8.3 compared to those in graft-dLN. pLN of control, dLN of MB49 and dLN of graft were stained with anti-Vβ8.3PE and anti-Thy1.1PerCP. MFI of Vβ8.3 by gated Thy1.1+Vβ8.3+ cells in each group is showed in histogram. Data are pooled from three independent experiments with 6–9 mice per group. d. Graft-infiltrating but not tumour-infiltrating donor CD8 cells express high levels of CD44. Cells from either MB49 tumour mass or male skin grafts were stained with anti-Vβ8.3PE, anti-Thy1.1PerCP and anti-CD44APC. CD44 expression of donor CD8 cells in tumour and skin graft was shown. Data are representative of three independent experiments

To determine the relative extents of T cell proliferation occurring in the dLN of normal and tumour tissue, CFSE dilution profiles of donor CD8 cells were determined. >90 % donor cells recovered from pLN of control mice remained undivided due to lack of antigen (Fig. 1b). Donor cells in dLN of MB49 and dLN of graft both divided in response to antigen but displayed distinct CFSE profiles. The majority of donor CD8 cells in skin graft-dLN had divided at least 6 times. In contrast, most donor CD8 cells in the tumour-dLN underwent fewer than 6 divisions. In addition, the percentage of undivided donor cells in tumour-dLN (20 %) was higher than that in graft-dLN (7 %, Fig. 1b). Moreover, proliferating donor CD8 cells in skin graft-dLN expressed higher levels of CD44 than those in tumour-dLN (Fig. 1b).

As shown in Fig. 1b, CFSE dilution pattern by MataHari CD8 T cells in tumour-dLN was different with that in graft-dLN: there are more extensively divided cells present in graft-dLN than tumour-dLN. An optimal MataHari CD8 response requires persistent antigen presentation [16], which is distinct from other TCR-transgenic CD8 T cells for which activation can be achieved following just a single TCR stimulation.

Modulation of TCR on donor cells was also analysed. Donor CD8 cells in pLN of control mice maintained a relatively high level of Vβ8.3. In response to HY antigen from either tumour or skin graft, donor CD8 cells decreased Vβ8.3 expression (Fig. 1c). However, TCR downregulation was significantly greater on donor CD8 cells present in tumour-dLN than in graft-dLN (Fig. 1c and not shown).

Although donor CD8 cells were present in both grafts and tumours with a similar percentage (not shown), they displayed distinct expression of CD44: 96 % of skin-graft-infiltrating donor cells were CD44high compared to only 11 % in tumours (Fig. 1d).

Finally, the weak CD8 responses against tumour as shown in Fig. 1 at day 12 were not due to a delayed kinetic, as they had not improved by day 19 or 32 (not shown).

CD8 responses are inversely correlated with tumour progression

Figure 1 shows analysis of the CD8 response, at day12, against tumours inoculated 1 day after adoptive T cell transfer. Next, we investigated the kinetics of the CD8 response, at days 4, 7 and 14, against tumours inoculated at 3 or 7 days before, or 1 day after, CD8 transfer (Figure S2A). An important finding is that the proportion of tumour-infiltrating CD8 T cells in the most recently established tumours (G2) is significantly higher than that in well-established tumours (G3 and G4) (Figure S2B and S2D). Thus, there is a narrow time window when activated CD8 T cells can enter the tumour mass. The percentage (not shown) and CFSE dilution profile of donor CD8 cells in dLN of G2, G3 and G4 was comparable at day 14 (Figure S2A and S2C), although at day 4 and day 7, the proportion of divided donor CD8 T cells in G3 and G4 was higher than that in G2 (Figure S2A). The supply of additional, antigen-specific help by co-transferring Marilyn CD4 cells, however, has limited impact in correcting the diminished CD8 responses against established MB49 tumours (Figure S3).

Impact of concurrent male skin grafts on CD8 responses against tumours

We investigated potential immune cross-talk between tumour and skin grafts by comparing CD8 T cell responses between mice bearing tumour alone, a male skin graft alone or both (Fig. 2). Mice receiving only tumour or skin graft recapitulated the data shown in Fig. 1. Interestingly, anti-MB49 CD8 T cell responses were significantly enhanced by concurrent skin grafts as evidenced by the significantly increased percentage of donor CD8 T cells in ndLN (not shown) and tumour tissues (Fig. 2). In addition, anti-graft CD8 T cell responses were also significantly enhanced by concurrent tumour as shown by the significantly increased percentage of donor CD8 T cells in graft-dLN (Fig. 2). In this regard, we observed that male skin-graft rejection was significantly accelerated in the presence of MB49 tumours; however, the kinetic of MB49 tumour growth was not significantly altered in the presence of skin grafts (not shown). These different consequences highlight the impact of microenvironment on T cell responses.

Fig. 2.

Fig. 2

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CD8 T cell responses to MB49 tumours in the absence or presence of male skin grafts. MB49 inoculation, skin grafting, CD8 transfer and analysis were conducted as described in Fig. 1. Data are pooled from two independent experiments with 7–8 mice per group

Comparison of CD4 responses with MB49 tumours and male skin grafts

Using the same strategy, we extended the investigation to the behaviour of HY-specific Marilyn CD4 T cells. Donor CD4 cells showed an accelerated progression of cell division in graft-dLN in comparison with tumour-dLN (Fig. 3b). This resulted in a much greater accumulation of donor CD4 cells in graft-dLN in comparison with tumour-dLN (Fig. 3a). Activated donor T cells were present in skin-graft tissue but excluded from the tumour mass (Fig. 3c).

Fig. 3.

Fig. 3

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Differential responses of Marilyn CD4 T cells to tumours and male skin grafts. a Donor Marilyn CD4 cells accumulate more efficiently in skin graft-dLN than tumour-dLN. On day 7, Thy1.2+ WT B6 females were divided into 3 groups. The mice in group 1 were untreated, those in group 2 were s.c. inoculated with 5 × 105 MB49 cells, and those in group 3 were transplanted with male B6 skin grafts. On day 0, 1 × 106 Thy1.1+ CFSE-labelled Marilyn CD4 T cells were i.v. injected into all recipient mice. On day 5, the pLN cells of control, dLN of MB49 and dLN of graft were stained with anti-Vβ6PE and anti-Thy1.1PerCP. Panels show percentage of donor CD4 cells identified by co-expression of Thy1.1 and Vβ6. b Donor CD4 cells proliferate more extensively in graft-dLN than tumour-dLN. Donor CD4 cells (Thy1.1+Vβ6+) present in pLN of control, dLN of MB49 and dLN of graft were analysed for CFSE and CD44 expression. Data are representative of two independent experiments. c CD4 donor cells are present in grafts but not MB49 tumours. Donor cells in MB49 and grafts were identified by co-expression of Thy1.1 and Vβ6. Data are pooled from two independent experiments with 6–8 mice per group

CD4 responses are negatively related to tumour progression

Comparing Marilyn CD4 responses against very recently established tumours (Fig. 3) and more established tumours (Figure S4) showed the proportion of tumour-infiltrating donor CD4 T cells in established tumours (>11–15 days) was significantly lower than that in the most recently established tumours, indicating that the progression status of tumour is inversely correlated with the level of tumour infiltration or proliferation by donor CD4 cells. CFSE profiles showed that donor cell division in dLN of more established tumours (day 7) were already arrested at the first time point (day 4), and such arrest was sustained through later time points (day 8, 15). Thus, there is a very restricted time window for activated CD4 cells to enter the tumour mass.

The impact of male skin grafts on CD4 responses against tumours

The consequence of immune cross-talk between MB49 tumours and male allografts on CD4 T cell responses was also dissected (Fig. 4). The most important finding is that anti-MB49 CD4 responses are significantly enhanced by the presence of skin grafts, as evidenced by the significantly increased percentage of donor CD4 T cells in dLN and in tumour tissues (Fig. 4), and to a lesser extent, for donor CD4 T cells in ndLN (not shown). On the other hand, anti-graft CD4 responses appeared compromised by the concurrent presence of tumours, as evidenced by the significantly decreased percentage of donor CD4 T cells in the dLN and the graft (Fig. 4).

Fig. 4.

Fig. 4

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CD4 T cell response to MB49 tumours in the absence or presence of male skin grafts. MB49 inoculation, skin grafting, CD4 transfer and analysis were conducted as described in Fig. 1. Data are pooled from two independent experiments with 7–8 mice per group

HY antigens from male skin grafts but not those from MB49 tumours direct the differentiation of donor CD4 cells into functional Th1-like effectors

Figure 3 compares responses against tumours and skin grafts at day 5 after CD4 T cell transfer. We then extended the comparison to later stage responses including the analysis of donor T cell functionality (Figure S5, A and B). Anti-MB49 CD4 cell responses were initiated at day 5 (Fig. 3) but not sustained (Figure S5A), confirming our previous observations [19]. Importantly, we demonstrated that MB49 tumours induced anergy within the donor T cell population, whereas male skin grafts were capable of driving productive Th1 differentiation (Figure S5B).

MB49- and skin-graft-bearing mice display distinct microenvironments

Donor CD4 cells in MB49-dLN but not those in graft-dLN appeared to be able to spontaneously proliferate in vitro in the absence of exogenous HY peptide (not shown), indicating that there is a higher level of HY antigen presentation in MB49-dLN than in graft-dLN. To explore this possibility, we compared CD11c+ cells from both types of dLN for their ability to stimulate resting Marilyn CD4 T cells in vitro. CD11c+ cells from MB49-dLN but not male skin graft-dLN were capable of activating CD4 T cells (Fig. 5a), indicating elevated cross-presentation of tumour-derived antigen over skin-graft-derived antigen.

Fig. 5.

Fig. 5

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Comparison of antigen burden and microenvironment in the mice bearing a tumour or a skin graft. a CD11c+ cells in dLN of MB49 tumours display HY antigens. Two groups of B6 females (4 mice/group) were inoculated with MB49 cells or transplanted with male skin grafts. Two weeks later, CD11c+ cells (2 × 104/well), purified from pooled dLN cells in each group by MACS, were cultured without or with purified Marilyn CD4 T cells (2.5 × 104/well) in 96-well round-bottomed plates for 3 days. b–e MB49 inoculation and skin transplantation were performed as described in a. The cells of both types of tissue isolated at day 14 were analysed for the expression of IDO (b), Arg. I (c), TGFβ1 (d) and IL10 (e) by qPCR. MB49 cell line and fresh male tail skin were used as control for MB49 tumour and male skin graft, respectively. Data are pooled from two independent experiments with 5–6 mice per group

MB49 tumours but not male allografts express immunomodulatory products. Cultured MB49 cells expressed a low level of IDO mRNA, which was significantly increased after in vivo tumour formation (Fig. 5b). IDO expression by male allografts, however, was much lower (Fig. 5b). Furthermore, MB49 tumours induced IDO expression in the dLN but not in ndLN (not shown). Like IDO, Arg. I was also significantly upregulated following tumour formation but not after skin grafting (Fig. 5c). As reported previously [19], the MB49 cell line constitutively expresses TGFβ1, which was sustained after tumour formation and was higher than found in male grafts (Fig. 5d). Moreover, IL10 mRNA expression was significantly upregulated after in vivo MB49 tumour formation (Fig. 5e). Comparison of tumours isolated from WT and IL10KO mice confirmed MB49 tumour as the IL10 source (not shown). Interestingly, a significant increase of IL10 mRNA was also seen by skin grafts at day 14 compared with fresh tail skin (Fig. 5e), indicating that IL10 may involve in the process of tissue healing and remodelling.

Comparison of the fate of HY-specific Treg cells in MB49-bearing versus male skin-graft-bearing mice

Finally, adoptive transfer was extended to Treg cells purified from Rag2+/− female Marilyn mice (Fig. 6). The proportion of donor Treg cells in male graft-dLN was fourfold higher than that in female graft-dLN, confirming our previous conclusion that Treg cell expansion in vivo is antigen dependent [19]. There was no significant difference between frequencies of donor Treg cells in MB49-dLN and male graft-dLN, suggesting that both male skin and tumour were capable of expanding Treg cells.

Fig. 6.

Fig. 6

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Distinct fates of adoptively transferred Marilyn Treg cells in MB49-bearing and male skin-graft-bearing mice. Three groups of Thy1.2 B6 females (3 mice/group) were used, on day 0, the mice in group 2 were s.c. inoculated with 0.5 × 106 MB49 cells, and those in group 1 and group 3 were transplanted with female and male skin grafts, respectively. Next day, all mice were transferred with 105 FACS-sorted Thy1.1 Marilyn Treg cells. Analysis was performed at day 12 by surface staining of dLN, MB49 or graft tissue cells with anti-Vβ6PE, anti-Thy1.1PerCP and anti-CD4APC before intracellular Foxp3 staining. Data are pooled from four independent experiments with 10–12 mice per group

However, three notable differences between MB49-bearing and male graft-bearing mice emerged. Firstly, MB49-dLN (15 ± 0.3 %) contained proportionately fewer host CD4 T cells than MB49-ndLN (29 ± 2 %), whereas dLN (30 ± 2 %) and ndLN (31 ± 2 %) of male skin grafts had similar proportions, indicating that the MB49 tumour environment may be particularly supportive of Treg activity in compromising local CD4 help. Secondly, intragraft accumulation by host CD4 T cells (37 ± 4 %) was fourfold higher than that in MB49 (10 ± 2 %), although the percentage of donor Tregs in tumours was similar to that in grafts. As a result, the proportion of donor Treg cells in tumour (12 %) was fourfold higher than that in the male graft (3 %), and in other words, the ratio of donor Treg to host CD4 T cells in the MB49 tissues was significantly higher than that in skin grafts. These distinct cell distributions are likely to contribute to poor MB49 immunity. Finally, the majority of graft-infiltrated donor Treg cells maintained Foxp3 expression; however, a significant fraction of tumour-infiltrated donor Treg cells became Foxp3 negative (not shown), thus enforcing our previous results [19, 21].

Discussion

In all adoptive transfer experiments conducted in this study, WT B6 females have been exclusively utilized as recipients. Although the use of immune-competent mice is more much physiological than that of lymphopenic hosts, there is a concern over whether endogenous anti-HY T cells responses developed in recipients would impact on the behaviour of donor T cells transferred. To limit this possibility, we usually injected donor cells one day before (Figs. 1, 4), on the same day (Fig. 2), or 1 day after HY challenge (Fig. 6). Occasionally, donor cells were given 7 days (Fig. 3) after antigen exposure. The main reason for delayed CD4 transfer is that unlike 1-day-old tumours, 7-day-old MB49 tumours are not rejected by infused Marilyn CD4 T cells; thus, subsequent analysis of donor T cell representation can be performed using both dLN and tumour tissues.

Endogenous anti-male skin-graft T cell responses in B6 females appear to develop slowly. It takes an average of 40 days for a naïve B6 female to reject a syngeneic male skin graft [3–10, 13, 14]. Even after graft rejection, the frequencies of circulating HY-specific HY tetramer-positive CD8 [9, 13, 14] and CD4 T cells [10] are still very low and an optimal HY tetramer response usually requires a HY boost. Induction of HY-specific Treg cells in females by male skin grafts is a very inefficient process unless additional intervention is given [10, 13, 14]. Analysis of donor T cells (CD8, CD4 and Treg) in all experiments is conducted within 12 days after transfer. However, at this time point, endogenous HY-specific T cells clearly have not expanded into effector cells with sufficient number able to cause graft rejection. Therefore, it does not seem to be likely that donor T cells will be significantly affected by endogenous HY-specific T cells within this relative short time frame.

MB49 tumours are immunogenic, but the number of primed HY-specific T cells in MB49-bearing B6 females is very limited [42]. The impaired T cell responses may be related to a significant reduction in the percentage of endogenous CD4 T cells (Fig. 6). MB49 tumours neither expand endogenous polyclonal Treg nor convert conventional CD4 into induced Treg [unpublished observations]. Therefore, the impact of endogenous HY-specific T cell responses on the fate of donor T cells is likely to be very limited.

However, we cannot rule out the possibility that endogenous HY-specific T cells would influence donor T cells in the mice carrying a tumour and a male skin graft. This is because the presence of MB49 tumours leads to an accelerated rejection of male skin grafts by B6 females, although the presence of male skin grafts does not alter MB49 growth rate (unpublished data).

Multiple factors are likely to contribute to the poor anti-MB49 CD8 responses. Firstly, the higher level of HYDby peptide displayed by CD11c+ cells from MB49-dLN (Fig. 5a) leads to a weaker, semi-anergic CD4 response (Figure S5, A and B). In sharp contrast, although CD11c cells recovered from graft-dLN express a lower level of HYDby/Ab complexes (not shown), they induce a strong Th1 response (Figure S5, A and B). A common consequence of persistent antigen stimulation is the induction of T cell tolerance via various mechanisms. In this context, we have shown that both intranasal inhalation of HYDby peptide [14] and s.c. injection of dentritic cells pulsed with HYUty peptide [13] induce transplantation tolerance through the induction of Treg cells [32].

The second factor is an immunosuppressive microenvironment. MB49 tumours but not male skin grafts upregulate a number of immunoregulatory factors including IL10, IDO, TGFβ1 and Arginase I. IL10 KO mice control MB49 tumours more efficiently than WT mice showing it to be a potent immunosuppressive [27, 28]. Deficiencies of IDO, TGFβ and Arginase result in enhanced tumour clearance in a number of murine models, highlighting the importance of these molecules in suppression of anti-tumour immunity [29, 33]. Similarly, the IDO specific inhibitor 1MT can restore impaired anti-B16/F10 tumour T cell responses [34]. Whether in vivo blockade of IDO, TGFβ and Arginase activities would have a therapeutic effect for MB49 tumours has not been explored. The view that MB49 tumour represents a stronger immunosuppressive microenvironment than a male skin graft is supported by the findings that although adoptive CD4 therapy does not eliminate well-established MB49 tumours, long-term accepted male skin grafts in Rag−/− B6 females are rejected [41]. In the adoptive transfer experiments using FACS-sorted Rag1+/− MataHari CD8+Vb8.3+ cells, we found that in both dLN and tissues, the extent of TCR downregulation induced by tumours was significantly greater than by male skin grafts (not shown). However, an alternative interpretation for the lower level of TCR expression seen in the presence of tumour and male skin grafts is that Vb8.3-high cells are deleted leading to an enrichment of Vb8.3-low cells. The best approach to differentiate these two possibilities is to use Rag1−/− MataHari CD8 T cells as donor cells for adoptive transfer, as they are more sensitive to HY antigen and do not express endogenous TCR.

Thirdly, Treg cells, a key component of the immune suppressive network [35, 36], display a distinct tissue distribution. The percentage of donor Treg cells in MB49 tumour mass is significantly increased; in contrast, the percentage of conventional CD4 T cells in the same tissues is reduced by up to 50 % (Fig. 6). Importantly, an increased ratio between conventional CD4 and Treg cells in dLN and especially in tumour tissue has a significant consequence to the anti-tumour response. Growing and regressing tumours are usually associated with increased and decreased proportions of Treg cells, respectively [37–39]. In regard to the consequence of donor Treg cells on graft acceptance and tumour growth, we have previously shown that adoptively transferred HY-specific Treg cells can prevent skin-graft rejection and promote MB49 tumour growth [19].

Lastly, tumour and allograft display distinct patterns of antigen presentation. Skin contains three major populations of DC: Langerhans cells (LC) in the epidermis; and langerin+ and langerin- CD11c+ conventional DC populations in the dermis [40] which can mature and migrate to the dLN in response to inflammation caused by surgery. Consequently, these graft-derived professional APC can participate in direct antigen presentation resulting in rapid T cell activation. In contrast, as MB49 cells are non-migrating, non-professional APCs, antigen presentation will be indirect and delayed until host DCs have infiltrated the developing tumour. In addition, surgery-induced local inflammation is associated with skin transplantation [41], but not tumour inoculation. Tumour cells are injected in suspension, as a result, few tumour cells will survive the inoculation and go on to seed the tumour, hence, compared to the skin graft, there will be a delay in processing and presentation of the HY antigen and hence priming of CD8+ T cells. To overcome this, the implantation of tumour pieces or transplantation of tumour-bearing female body skin would be more appropriate. We are very keen to use these new approaches for the future studies.

Of note, MB49 tumours and male grafts have different potential for induction of re-circulating T cells. Detected CFSE-low cells in ndLN may represent re-circulating T cells that were initially activated in dLN. This is also applicable to the interpretation of donor T cell representation in mice bearing both tumour and male skin graft. For induction of re-circulating Marilyn CD4 and MataHari CD8 T cells in ndLN in different settings, male skin grafts are much more potent than MB49 tumours (Figs. 1, 2, 3, 4). Importantly, induction of re-circulating T cells not only represents a reliable indicator of a successful anti-tumour T cell response, but also is of therapeutic benefit as it can potentially target and eliminate metastases.

Whether MB49 induces systemic immune suppression [27, 28, 42] has been controversial. Our data indicate that MB49 does not cause obvious non-specific global immune suppression [42]. Nevertheless, the local microenvironments including dLN and tumour mass are clearly immune inhibitory [27]. This is best illustrated by the introduction of a male skin graft onto MB49-bearing mice, which led to a greater representation of donor CD8 T cells in ndLN and tumour and, to a lesser extent, in MB49-dLN (Fig. 1), while donor CD4 T cell representation increased only in MB49-dLN (Fig. 3). Thus, immunization by male skin graft principally benefits the donor CD8 T cell-mediated anti-MB49 response. On the other hand, when the reciprocal impact of MB49 tumours on the anti-graft T cell response was examined, we found that MataHari CD8 cells and Marilyn CD4 T cells have distinct susceptibilities to MB49-derived suppression. Anti-graft CD8 responses were significantly enhanced by a contemporary MB49 tumour, whereas anti-graft CD4 responses were reduced by at least 50 % (Figs. 2, 4). Therefore, CD4 T cells are more susceptible to MB49 tumour-mediated suppression.

Marilyn CD4 T cells have been found to be more effective than MataHari CD8 T cells in rejection in five HY+ tumour models [23]. Recent evidence suggests that this conclusion can be further generalized, as B16/F10 melanoma-specific TCR-transgenic CD4 T cells are also highly effective in mediating rejection of large, established tumours [43, 44]. However, why Marilyn CD4 T cells are superior to MataHari CD8 T cells in controlling MB49 is not fully understood [23]. Important insight is provided here. Firstly, MataHari CD8 T cells appear to be self-reactive, whereas Marilyn CD4 T cells are not (Manuscript under preparation). Secondly, of relevance to this unexpected cross-reactivity, MataHari CD8 T cells but not Marilyn CD4 T cells are very susceptible to TCR downregulation mediated by MB49 and to a lesser extent by male skin graft (Figs. 1, 2, 3, 4). Thirdly, in response to both MB49 and male skin-graft HY antigen, expansion of Marilyn CD4 T cells is more potent than MataHari CD8 T cells (Figs. 1, 2, 3, 4).

In summary, we have identified several important cellular mechanisms that are likely to contribute to (a) the inferior anti-MB49 responses by conventional T cells, (b) the superior response by CD4 over CD8 T cells and (c) the cross-talk between contemporary tumour and transplant immunity. The strength of this experimental model is that it enables a T cell response to the same antigen to be assessed in the context of tumour (progressively growing) and graft (rejected). One of key points established in this study is that the nature of the microenvironment, rather than antigen specificity, determines the outcome of a given immune response. Our analysis of the tissue microenvironment identifies roles in the induction and maintenance of tolerance and the control of T cell differentiation [45].

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 2772 kb) (2.7MB, pdf)

Acknowledgments

We thank Prof Liz Simpson for critically reading the manuscript. This study was supported by the Cancer Research UK Senior Cancer Research Fellowship (to JGC).

Conflict of interest

The authors declare that they have no conflict of interest.

Abbreviations

Arg. I

Arginase I

LN

Lymph node

dLN

Draining lymph node

ndLN

Non-draining lymph node

pLN

Peripheral lymph node

Tregs

Regulatory T cells

Mar

Marilyn

Mata

MataHari

CFSE

Carboxyfluorescein succinimidyl ester

IDO

Indoleamine 2,3-dioxygenase

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