T cells, the component of the adaptive immune system, can recognize antigens via the highly specialized T-cell receptor (TCR), which is an αß heterodimer in the dominant type of T-cells with the unique structure in each individual clonotype. The peripheral TCR repertoire matures and undergoes selection within the thymus; it is capable of effective interaction with the molecules of the major histocompatibility complex (MHC) that present a foreign peptide or a peptide of an endogenous mutant protein [1]. At that, a significant portion, if not all, clones of peripheral T-cells are alloreactive [2]; that is, they can react to allogeneic MHC molecules and the associated peptides [3–5]. Up to 10% of T-cells of the recipient's T-cell repertoire can participate in the response to an individual allogeneic transplant [6].

Alloreactive T-cells are the key mediators of transplant immunity that mediate acute allograft rejection [7]. Current knowledge is inconsistent regarding whether the diversity of the TCR repertoire determines the intensity of the allogeneic response; the available data are conflicting [8]. Several studies have shown an association between baseline high diversity of the recipient’s TCR repertoire and an increased risk of acute rejection of allogeneic transplants [9, 10]. At the same time, it has been established that only a narrow oligoclonal repertoire is involved in graft rejection [11–13].

Experimental models involving transplantation of a tumor different in one transplant antigen [14, 15] enable in vivo exploration of the dynamics and features of development of allogeneic immune response. Direct recognition of allogeneic MHC class I molecules induces an intense cytotoxic CD8⁺ T-cell response, and the allogeneic tumor is rapidly rejected, with immunological memory formed in the process [14–16]. In this work, we sought to confirm our earlier findings about the dependence of the effectiveness of the allogeneic immune response on the diversity of the TCR repertoire [14]. For this purpose, we used mice with transgenic expression of the β-chain TCR (TCRß) of hybridoma 7, which was described earlier [17]. TCRß transgenesis allows reducing the diversity of T-lymphocyte receptors [14, 18]: under the allelic exclusion rule, the expression of transgenic TCRß blocks gene rearrangement and, consequently, the expression of endogenous β-chains in T-cells. As a result, the repertoire becomes mainly comprised of clonotypes with an invariant β-chain, and its diversity stems only from the endogenous α-chains in the receptor structure.

This study aimed to analyze the development of the immune response to tumor alloantigens in the TCRβ transgenic model in vivo, in which the T-cell clonal diversity is reduced.   

METHODS

We used female mice of the inbred lines CBA/Lac (H-2^k) and C57BL/6 (H-2^b) and transgenic mice 7B (6–8 weeks old, 18–20 g) bred at N.N. Blokhin National Medical Research Center of Oncology. The process of breeding the 7B transgenic mice was described earlier [18]. Initially, the transgene was transferred to the genetic background of C57BL/6 mice. Since the lifetime of the generated mice was limited, which hindered their breeding, the transgene was transferred to the genetic background of the CBA/Lac line; it is currently a part of the collection of the Laboratory of Regulatory Mechanisms in Immunity, Institute of Carcinogenesis (N.N. Blokhin National Medical Research Center of Oncology). 7B transgenic mice express the β-chain TCR, specific to the mutant MHC class I molecule K^bm3 and the MHC class II molecule I-A^b [17]. The animals were kept at 20–24 °C and 40% relative humidity, with a 12-hour light cycle, and had unrestricted access to feed and water. Transgenic 7B mice and control animals (non-transgenic wild-type siblings) were injected intraperitoneally with EL-4 (H-2K^b) lymphoma cells at a dose of 1 × 10⁶ cells per mouse in 500 µl of phosphate-buffered saline (PBS). After injection, we monitored the life span of mice with a follow-up period of more than 30 days. The Kaplan-Meier survival curves were plotted using SRplot [19]. Some of the animals were removed from the experiment by cervical dislocation on the 12th day after EL-4 cell transplantation, with subsequent extraction of peritoneal cells. The obtained lavage cell samples were analyzed by flow cytometry.

We used fluorescently labeled antibodies (BioLegend, USA; BD Bioscience, USA; BD Horizon, USA): CD3 — APC-Cy7, CD8 — Percp-Cy5.5, CD44 — Pacific blue, CD62L — APC, Vb8.3 — PE, Kb — FITC. Cell samples (0.5 – 1 × 10⁶) were incubated with Fc block antibodies (clone 2.4G2, BD Pharmingen, USA) for 10 minutes at 4 °C to prevent nonspecific binding of antibodies, then stained with labeled antibodies for 40 minutes at 4 °C.  For the analysis, we used a BD FACS Canto II flow cytometer (BD Bioscience) and the FACSDiva 6.0 software (BD Bioscience). Leukocytes were gated using forward scatter (FSC-A) and side scatter (SSC-A) parameters, and single cells were identified by plotting forward scatter height (FSC-H) against forward scatter area (FSC-A). The LIVE/DEAD fixable yellow dead cell stain kit (Invitrogen, USA) was used to stain dead cells; dead cells were excluded based on this staining and scatter parameters. The expression of surface markers was evaluated in a population of live single leukocytes. The relative number of T-lymphocytes expressing the transgenic TCR β-chain was assessed by staining with anti-Vb8.3 antibodies (the Vß family, to which the 7B transgenic β-chain belongs). The relative number of EL-4 tumor cells in the peritoneal cavity of experimental animals was determined by the expression of the MHC class I molecule H-2K^b (Kb). The populations of T-lymphocytes were analyzed after exclusion of Kb-positive cells. The data was processed in the FlowJo 7.6 software (TreeStar Inc., USA). Figure fig. 1 presents the cytofluorimetric analysis strategy.

The data are presented as the mean ± standard error of the mean. For statistical analysis, we used the Student's unpaired t-test, ANOVA, and the post-hoc Tukey test. The data samples were pre-checked for normality of the distribution using the Kolmogorov–Smirnov test, which confirms the validity of the selection of these statistical methods. For statistical analysis, we used the SRplot online service [19]. The differences were considered significant at p ≤ 0.05.

RESULTS

As noted above, the transgenesis of the TCR β-chain significantly reduces the diversity of the peripheral TCR repertoire. Seeking to investigate the features of functioning of the T-cell immune system when the diversity of clonotypes is reduced, we studied the immune response of 7B transgenic mice (H-2^k) to allogeneic EL-4 lymphoma cells (H-2K^b) in vivo. T-lymphocytes at the transplantation site (in the peritoneal cavity) were analyzed on the 12^th day after EL-4 injection. Normally, by this day, the response of cytotoxic T-cells to EL-4 lymphoma alloantigens peaks [14, 16].

Immunized 7B mice (TG+EL-4) had the absolute number of cells in the peritoneal cavity increased by two orders of magnitude compared with intact (non-immunized) transgenic  mice (TG) and 20-fold compared with immunized wild-type animals (WT+EL-4) (fig. 2A). The lavage from the immunized 7B animals contained over 90% EL-4 (Kb⁺) cells, whereas in control mice (WT+EL-4), the allogeneic tumor was almost completely eliminated by this time, and the proportion of Kb⁺ cells was only 5% (fig. 2B).

7B mice were unable to reject allogeneic EL-4 lymphoma; as shown in Figure 3, all of them died after an average of 19 days. It should be noted that these animals endured longer than C57BL/6 mice, as for the latter, EL-4 is syngeneic and typically kills them within 10–12 days (fig. 3). This finding indicates that in 7B transgenic mice, the response to allogeneic EL-4 lymphoma includes elimination and equilibrium phases — albeit insufficiently effective — before the tumor adapts to evade it.

The active immune response to EL-4 cells in wild-type mice (WT+EL-4) was characterized by an accumulation of T-cells, the number of which increased approximately fourfold (fig. 4A, B), and a fivefold growth of the proportion of cytotoxic CD8⁺ cells compared with the intact control (WT) (fig. 4C, D). Such developments were not registered in immunized transgenic mice (TG+EL-4): compared with the non-immunized control animals (TG), the relative number of T-lymphocytes (fig. 4A, B) and CD8⁺ T-cells (Figure 4C, D) did not increase in them. However, the ratio of CD8⁺ T-cells expressing the transgenic β-chain TCR (Vb8.3⁺) or endogenous β-chains TCR (Vb8.3^–) in the peritoneal cavity of immunized 7B mice (TG+EL-4) was approximately 1:1 compared with 2:1 in the control group (TG) (fig. 4D, E), which indicates the involvement of both subpopulations of cytotoxic T-cells in response to EL-4 cells.

Accordingly, immunized 7B mice showed a 1.8-fold increase in the proportion of effector (CD44⁺CD62L^–) CD8⁺ T-cells with transgenic TCRβ (fig. 5A, B) and a 1.5-fold increase in effector CD8⁺ T-cells with endogenous TCRβ (fig. 5C, D), compared with the corresponding CD44⁺CD62L^– T-cell subpopulations in intact transgenic mice (TG). Meanwhile, the relative number of Vb8.3⁺ and Vb8.3^– CD8⁺ effectors in the peritoneal cavity of immunized transgenic mice (TG+EL-4) was 1.5–1.7 times lower than in immunized wild-type animals (WT+EL-4) (50–56% vs. 84–86%) (fig. 5).

DISCUSSION

The results of this study indicate the development of a weak immune response to alloantigens in 7B transgenic mice in vivo with the formation of a small pool of effector cytotoxic T-cells, which is consistent with the data of our early work that used a different TCRβ-transgenic model [14].

Several studies have shown that TCRß-transgenic animals retain the ability to respond to various antigens, including allogeneic MHC molecules [20, 21]. These findings point to the high plasticity of the TCR repertoire, which allows compensating for the significant reduction in the diversity of clonotypes caused by the expression of the transgenic β-chain [20, 21]. However, the intensity of the immune response in TCRß-transgenic mice is lower than in wild-type animals [14, 20, 21] because of the low frequency of responding T-cell clones [20, 22]; consequently, these mice show a slower development of T-cell responses in vivo [21]. An important factor in this context is that the cytotoxic CD8⁺ immune response that TCRß-transgenic mice develop to the allogeneic MHC class I molecule (H-2K^b) is insufficient to reject the allogeneic tumor (fig. 4, fig. 5) [14]. Thus, the response even to a strong transplant antigen becomes ineffective due to the reduction of the diversity of the TCR repertoire. 

In this regard, recognition of allogeneic tumor antigens follows the same patterns as recognition of tumor antigens in combination with self MHC molecules. The effectiveness of the antitumor response and, consequently, elimination of the transformed cells is largely determined by the mutation load of the tumor — its immunogenicity [23, 24] — and the clonal diversity of T-cells capable of recognizing tumor neoantigens or tumor-associated antigens [24, 25]. It has been shown that a high diversity of T-cell receptors (TCRs) circulating in peripheral blood is a favorable prognostic factor in several cancers, such as melanoma [26], non-small cell lung cancer [27, 28], and breast cancer [29]. In contrast, a low TCR diversity of tumor-infiltrating T-cells is associated with a poor prognosis in stomach cancer and some other malignancies [30–32].

The natural aging of the immune system, especially thymus involution and clonal expansion of memory cells in the periphery, causes a reduction in the diversity of the repertoire of T-lymphocytes [33-35], which leads to a greater predisposition of the elderly to infections and cancer [33, 36]. In addition, experimental studies involving mice have shown that at 12 months of age, the animals exhibit decreased responses to alloantigens, which are partly attributable to an age-associated narrowing of the T-cell clonotype repertoire [34, 37].

The results of this study confirm that the effectiveness of the immune response to tumor alloantigens directly depends on the breadth of the repertoire of mature T lymphocytes, as previously established [14].

CONCLUSIONS

This study shows that 7B transgenic mice develop an in vivo immune response that is not effective enough to eliminate allogeneic tumor cells. Due to the expression of the transgenic β-chain TCR, the number and frequency of cytotoxic T-cell clonotypes capable of recognizing allogeneic MHC molecules decrease. However, the neoplastic process in such animals progresses through all stages of immunoediting, from ineffective stages of elimination and equilibrium to the tumor escaping from immune surveillance. The results of this study are consistent with our earlier findings; they confirm that decreased diversity of the TCR repertoire is a factor preventing rejection of the allogeneic tumor, allowing it to progress. In this case, the weak immune response that develops in the allogeneic recipient contributes to the selection of the least immunogenic malignant clones, thereby driving tumor immunoediting.