• Tumor infiltrating lymphocyte - TIL
• Tumor microenvironment - TME
• T cell Receptor - TCR
• Lung Cancer

The composition, characteristics and response effects of tumor-infiltrating T cell subsets in the tumor microenvironment (TME) are key targets for analyzing tumor cell immune escape mechanisms and informing tumor diagnosis, prognosis and immunotherapy. However, the high heterogeneity of tumor-infiltrating T cells has been puzzling immunologists.1 The successful application of single-cell RNA+TCR-sequencing (scRNA+TCR seq) in tumor-infiltrating T cell research can annotate the status of each T cell subset in the TME and their clone expansion, analyze the expression of cytokines, cytokine receptors and transcription factors of each T cell, and analyze the composition and characteristics of the pairing sequence of TCR (T Cell Receptor) CDR3 of each T cell. This enables identification of potential tumor antigen epitopes that may drive TME responses, providing a transformative advance for TIL (Tumor infiltrating lymphocyte) research.2–5

The adaptive immune response of T cells to antigenic epitopes is based on the clonal selection theory of “one lymphocyte expresses only one type of TCR” which is mainly achieved through the rearrangement of germ line V(D)J allelic exclusion.6 However, dual TCR-expressing lymphocytes caused by incomplete allelic exclusion rearrangement were first reported in 1988,7–9 and their role in autoimmunity,10 infection11 and transplant12 are supported by extensive experimental evidence. At present, the antitumor effect of dual TCR T cells has been confirmed in a mouse model, where dual TCR T cells inhibited the growth of melanoma cells (B16-ova and B16-gp33).13 Dual TCR CD8+T cells (recognition of tumor-specific Ag and recognition of endogenous Ag in the pancreas) are effective against tumors without serious side effects.14 Dual TCR T cells were increased and showed antitumor effects in B16F10 melanoma-specific tumor-infiltrating T cells.15 Although a large number of experiments have confirmed the existence of a high proportion of dual TCR T cells in human peripheral blood,16 17there is currently a paucity of TIL studies from human clinical tumors assessing whether dual TCR T cells in peripheral blood can enter the TME and exert anti-tumor effects.

He et al recently sequenced scRNA+TCR seq through tumor tissue, tumor adjacent tissue, normal tissue, and peripheral blood samples of 5 non-small cell lung cancer (NSCLC) patients. This innovative discovery confirmed the antitumor effects of two major subsets,CD4+CXCL13+T cells (T) and CD8+CXCL13+T cells (T).18 Based on the scRNA+TCR seq data of 20 samples shared in the original article (cloned proliferation T cells with clone count ≥2), we calculated the number of dual TCR CD4+T and dual TCR CD8+T in each tissue from each patient, their proportion, clonability, overlap, VJ usage, TCR characteristics, and so on. The distribution and differences of dual TCR CD4+T and dual TCR CD8+T in tumor tissue, tumor adjacent tissue, normal tissue, and peripheral blood were compared and statistically analyzed and the proportion differences between 10 CD4+T subsets and 9 CD8+T subsets as defined in the original article were calculated. The composition of TCR CDR3 of dual TCR CD4+T and dual TCR CD8+T was compared with that of antitumor response T cells which was identified in the original study. The summary diagram of this experimental analysis is shown in online supplemental figure 1. For specific sharing data information, analysis steps and methods, see the “Materials and methods” section in online supplemental material 1. The number and proportion of dual TCR T cells analyzed are shown in online supplemental table 1. We compared and analyzed the main composition and characteristics of dual TCR CD4+T and dual TCR CD8+T in 20 samples from 5 NSCLC patients, as shown in figure 1.

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Figure 1

Single-cell RNA+TCR-sequencing (scRNA+TCR seq) revealed dual TCR T cells antitumor response in the TME of NSCLC. (A) Dual TCR CD4+T and dual TCR CD8+T ratios in blood, normal, peri, and tumor tissue of each patient. (B) Dual TCR CD4+T CDR3 overlap and dual TCR CD8+T CDR3 overlap in blood, normal, peri, tumor tissue of each patient. (C) TRAV, TRAJ, TRBV, TRBJ usage of single TCR T cells and dual TCR T cells. (D) Dual TCR T cells accounted for the proportion of total dual TCR T cells in blood, normal, peri, and tumor tissue. (E) Ratios of dual TCR CD4+T and dual TCR CD8+T in each subset. (F) Single TCR T and dual TCR T diversity (1/DS). (G) Single TCR T cells and dual TCR T cells clonality (clone count:2–5, 6–9,≥10). (Original clones proliferating to cell numbe r≥2 in online supplemental table 2), including ‘single TCR T cell+dual TCR T cell’ ≥2 clones. For instance, P1_TCR_286 clone type (5 cells) in CD4+T cells in online supplemental table 2)=single TCR CD4+T (1 cell)+dual TCR CD4+T (4 cells), but in our analysis, single TCR and dual TCR were separated for comparative analysis. Therefore, single TCR or dual TCR T with cell number 1 was not analyzed in clonal amplification analysis.) (H) Number, sequence composition, source patient and tissue, and subsets of dual TCR CD4+T and dual TCR CD8+T from the top five high-frequency clones. TCR, T Cell Receptor; TRAV, T cell receptor alpha chain Variable gene; TRAJ, T cell receptor alpha chain Jointing gene; TRBV, T cell receptor beta chain Variable gene; TRBJ, T cell receptor beta chain Jointing gene; NSCLC, non-small cell lung cancer; TME, tumor microenvironment.

(1) A certain proportion (1.04%–14.78%) of dual TCR CD4+T and dual TCR CD8+T (figure 1A) were present in blood, normal, peritumor, and tumor tissues of each patient, except for CD8+T cells in P3. Additionally, CDR3 overlap of dual TCR CD4+T and CDR3 overlap of dual TCR CD8+T occurred between blood, normal, peri, and tumor tissues in each patient (figure 1B). The results showed that both dual TCR CD4+T and dual TCR CD8+T existed in 2–4 types of tissues. It is suggested that dual TCR T cells may migrate from peripheral blood to local normal tissue, adjacent to tumor tissue, and into tumor tissue, They may be involved in specific antitumor responses.

(2) The V and J usage of single TCR T cells and dual TCR T cells was basically the same (figure 1C). However, in the high-frequency usage family of dual TCR T cells, TRBV5-6/6-5/9, TRAV22/23. The usage of TRAJ6/27/37/42 was significantly higher than that of single TCR T cells, suggesting that dual TCR T cells may respond to unique tumor epitopes.

(3) The proportion of dual TCR CD4+T was higher in normal while the proportion of dual TCR CD8+T was significantly increased in tumor (figure 1D), suggesting that dual TCR CD8+T played a stronger tumor killing effect than dual TCR CD4+T.

(4) Dual TCR CD4+T was mainly derived from CD5+T (47.11%) and LMNA+T (28.78%) subsets, suggesting that dual TCR CD4+T may play a major role in immune regulation. Dual TCR CD8+T was mainly derived from CXCL13+T subset (51.61%) (figure 1E), suggesting that dual TCR CD8+T may mainly play a tumor-killing role.

(5) The number of dual TCR T cells with clone count ≥2 was much less than that of single TCR T cells with clone count ≥2. The diversity of dual TCR T cells was significantly lower than that of single TCR T cells (figure 1F). It is suggested that dual TCR T cells are not the dominant effector cell population in response. Dual CD4+T clonality (ratio greater than equal 10) was higher than single TCR T, dual TCR CD8+T clonality (ratio greater than equal 10) was lower than single TCR T(figure 1G), dual TCR T cells had lower diversity and higher percentage of clonal proliferation. These results suggest that, like single TCR T cells, dual TCR T cells may be involved in antitumor response.

(6) The top five dual TCR CD4+T subsets of high-frequency clonal hyperplasia (56, 33, 31,2 9, 27 cloned cells, respectively) were mainly derived from normal and peri tissue sites of patients with P1 but belonged to the CD5+, LMNA+, MKI67+ and CCL4+ subsets (figure 1H). It is suggested they play the role in chemotaxis and immune regulation. The top five high-frequency clones of dual TCR CD8+T subsets (99, 56, 48, 41, 28 cloned cells, respectively) were mainly derived from tumor tissue sites of P5, P1, and P4 patients (few clones in normal and peri). The main class belongs to CXCL13+T subset while a few belong to MKI67, ANXA1, GZMK subsets (figure 1H). It is suggested that they mainly play an immune-killing effect.

(7) In the antitumor T cells of P4 or P5 patients verified in the original paper, five clones of CXCL13+proliferated in the CD4+T (TCR-1 to TCR-5) or CD8+T (TCR-1 to TCR-5) response subsets (original text, online supplemental table 2 and online supplemental table 8). Our in-depth analysis found that effective P5-CD8+CXCL13+T cells (TCR-1, the same CDR3 was found in dual TCR CD8+T(P5_TCR_3, 99 cells) cloned at high frequency (α1: TRAV22/TRAJ27, CAVGHNTNAGKSTF; α2:TRAV23DV6/TRAJ44, CAASNTGTASKLTF; β:TRBV9/TRBJ2-2, CASSVVRGGELFF); Effective P5-CD8+CXCL13+T cells (TCR-3), the same CDR3 was found in dual TCR CD8+T (P5_TCR_10, 11 cells) cloned at high frequency (α1:TRAV12-1/TRAJ42: CAVNFYGGSQGNLIF; α2: TRAV12-3/TRAJ33, CAMSASNYQLIW; β:TRBV3-1/TRBJ2-1, CASSPSPGYYNEQFF); Play to the effect of P4 - CXCL13+CD8 + T cells (TCR-2), the same CDR3 was found in dual TCR CD8+T (P4_TCR_9, 28 cells) cloned at high frequency (α 1: TRAV21/TRAJ49, CAVLYTGNQFY; α2:TRAV24/TRAJ45, CAFYTGGGADGLTF; β:TRBV6-4/TRBJ2-6, CASSPGRPSSGANVLTF); At the same time, the only effective P4-CD4+CXCL13+T cells (TCR-1), the same CDR3 was found in dual TCR CD4+T (P4_TCR_229, 2 cells, 1 CXCL13+T cells, 1 MKI67+T cells) cloned at high frequency (α1:TRAV12-1/TRAJ3, CVVNMDSSASKIIF; α2: TRAV2/TRAJ5, CAVRDMDTGRRALTF; β:TRBV5−4/TRBJ2-1, CASSLPGTGENEQFF). The results indicate that dual TCR CD4+T and dual TCR CD8+T with high-frequency clone proliferation are both involved in the antitumor response effect.

In summary, we report for the first time the existence of dual TCR T cells in the blood, normal, peri, and tumor tissues of each patient, as confirmed by strictly controlling the composition characteristics of T cell subsets in scRNA-seq and scTCR-seq data. These unique dual TCR T cells exhibit the ability to migrate across four tissue types. Dual TCR T cells display high clonal proliferation, especially dual TCR CD8+CXCL13+T in tumor tissue, suggesting a potential antitumor response effect. Five CD8+CXCL13+T cells validated in the original text demonstrate antitumor effects in P4 or P5 patients, respectively. Three of them were found to be highly cloned dual TCR T sources (sharing TCR β CDR3 with single TCR T cells). Two CD4+CXCL13+T cells validated in the original text to exert effect. One of them is a highly cloned dual TCR T source (sharing TCR β CDR3 with single TCR T cells), suggesting dual TCR T cells represent a “new subgroup TIL” for antitumor response.

The detailed mechanism of the allelic exclusion and incomplete allelic exclusion V (D) J rearrangement is unclear, the proportion and self-tolerance selection mechanism of single TCR T or dual TCR T cells rearranged by the germ line V (D) J gene according to two rearrangement mechanisms have not been elucidated.19–22 However, under physiological conditions, there is a high proportion of dual receptor lymphocytes in both central and peripheral regions of individuals, as well as evidence of dual receptor T cell effects in autoimmune, antitumor, transplant rejection, and aging.23–25

Does the TCR of dual TCR T cells confer an advantage in recognizing novel tumor antigen epitopes? Do dual TCR T cells mount a stronger response than single TCR T cells? Are dual TCR T cells prevalent across different types of TME? These are worthy questions for further research and requiring greater researcher participation. This study, for the first time, identified a new responsive TIL subgroup using scRNA+TCR seq analysis of the TME in NSCLC. Identification and characterization of dual TCR T cells provides a potential novel area of exploration within TIL research.