Extensive clonally expanded CD8+ T cell populations in the bone marrow of AML patients
CDR3, a hypervariable region that consists of α and β chains and interacts with the MHC-peptide antigen complex, accounts for most of the antigen-reactive T cell diversity. We performed deep sequencing to assess the TCRβ CDR3 diversity from bone marrow (BM) and peripheral blood (PB) of AML patients and healthy donors. The distribution plot of the top 100 TCR clonotypes from bone marrow and peripheral blood of one AML patient and one healthy donor is shown in Fig. 1A. The graph demonstrates increased clonal expansion in the bone marrow of the AML patient compared to the other groups. Next, we sought to assess the extent of TCR repertoire diversity and clonal expansion by analyzing the total/unique clonotype ratio and highly expanded clones (HECs, clonotypes with a frequency of more than 0.1% in each sample[29]) ratio for all samples. Figure 1B shows that the total/unique clonotype ratios were higher in the bone marrow of AML patients than in the peripheral blood of AML patients and in the bone marrow and peripheral blood of healthy donors. A markedly higher frequency of highly expanded clones (HECs) was noted in the bone marrow and peripheral blood of AML patients than in those of healthy donors (Fig. 1C). In addition, the Shannon index and Gini index were used to evaluate the TCR repertoire diversity. Samples with expanded T cell clones of similar frequencies were found to be positively correlated with Shannon diversity index values. A lower value of the Gini index corresponded to a more uniform distribution of clone sizes. As shown in Fig. 2D, the Shannon index in bone marrow of AML patients was significantly higher than in peripheral blood of AML patients and bone marrow and peripheral blood of healthy donors; in contrast, the Gini index in the bone marrow of AML patients had a pronounced reduction compared with the other groups (Fig. 1E). Collectively, these findings showed that in CD8+ T cells from the bone marrow of AML patients, a decline in T cell repertoire diversity is closely associated with clonotypic expansion.
Comparison of overall usage of TCRβ V-J rearrangements in AML patients and healthy donors
The CDR3 region combines the junction of the V, D, and J gene segments and mainly accounts for the massive diversity of T cells. For several decades, it has been recognized that TCR diversity relies mainly on the hypervariable CDR3 region, and rearranged segments with the addition/subtraction of extra nucleotides at the recombination junctions contribute to T cell diversity and antigen binding. Thus, the overall usage profiles of V, D, and J gene segments were analyzed. We identified a total of 60 distinguishable gene transcription segments from the TCRβ V (TRBV) loci, 2 from the TRBD loci, 13 from the TRBJ loci, and 780 rearrangements in the TRBV-J region. First, the usage profiles of TRBV-J rearrangements present in samples from different groups were compared. As shown in the graph in Fig. 2A, each rearrangement event in TRBV-J is denoted by a dot, with its size indicating the average frequency of the rearrangement in the sample group. Similar overall usage profiles of the rearranged TRBV/J segments were noted between bone marrow and peripheral blood from healthy donors (81 differentially expressed rearrangements) and between peripheral blood from AML patients and peripheral blood from healthy donors (62 differentially expressed rearrangements); however, differences were greater between bone marrow and peripheral blood from AML patients (176 differentially expressed rearrangements) and between bone marrow of AML patients and bone marrow of healthy donors (202 differentially expressed rearrangements) (Fig. 2B-E). Similarly, we found comparable usage patterns of TRBV gene segments between PB of healthy donors and BM of healthy donors and between PB of healthy donors and PB of AML patients but found relatively different usage patterns between BM of healthy donors and BM of AML patients and between PB of AML patients and BM of AML patients (Figure S1). These data suggest that CD8+ T cells in the bone marrow of AML patients have specific expression of TRBV-J rearrangements, indicating that they may recognize bone marrow specific antigens.
Comparison of identical clonotypes in AML patients and healthy donors
We analyzed the CDR3 amino acid sequences of CD8+ T cells in peripheral blood and in bone marrow of 31 AML patients and 10 healthy donors to identify the presence of shared clones (i.e., shared CDR3 sequences) in the top 1,000 or 5,000 clones between any two sample pairs (Fig. 3A). Through intragroup analysis of the CDR3 sequences between different sample pairs, we found a higher ratio of identical T cell clones between sample pairs in the bone marrow of the AML patients (AML BM VS BM, n = 465) when compared to sample pairs in other groups (AML PB VS PB, n = 465 or healthy donor BM VS BM / PB VS PB, n = 45) (Fig. 3B). Intergroup analysis results indicated that there was no difference in the percent of identical T cell clones between sample pairs from different groups (AML patient BM vs. PB, n = 930; healthy donor BM vs. PB, n = 90; AML patient BM or PB vs. healthy donor BM or PB, n = 310) (Fig. 3C). By analyzing identical T cell clones in the peripheral blood and bone marrow of AML patients or healthy donors (i.e., comparison between BM and PB of the same individual, n = 31 or n = 10), we found that the percent of identical T cell clones between peripheral blood and bone marrow of the same individual was significantly higher than that between peripheral blood and bone marrow among different individuals. Moreover, the percentage of identical T cell clones between peripheral blood and paired bone marrow of the healthy donors was significantly higher than that in peripheral blood and paired bone marrow of the AML patients (Fig. 3D). These results suggested that clonal expansion of bone marrow CD8+ T cells in AML patients was highly specific.
TCRβ repertoire variety and stability among CR patients and relapsed patients
Thirty-one newly diagnosed patients were followed up after chemotherapy. In 12 patients who relapsed and 19 patients who had CR after chemotherapy, samples were collected for subsequent studies. Figure 4A shows bone marrow CD8+ T cell clonal expansion in one relapsed patient and one sustained CR patient at new diagnosis and after chemotherapy. Each square represents a clone. A larger square size indicates greater clone, and different colors of the squares indicate different V genes. After discontinuation of chemotherapy, markedly expanded T cell clones were noted in the bone marrow from the relapsed patient but not from the patient who achieved CR. Looking more deeply into the clonal diversity of the TCRβ repertoires, we found considerably less diverse in relapse post chemotherapy group compared to the other groups (Fig. 4B). We further calculated the V gene usage of CD8+ T cells in this relapse patient, and the results showed that compared to the new diagnosis, the preferential usage pattern of some V gene segments changed dramatically after relapse, and the number of different clones from the same V gene usage was also remarkably different (Figure S2). To better investigate the CD8+ T cell clone characteristics, we analyzed the Shannon and Gini indexes of bone marrow T cells in all 31 patients at new diagnosis and after chemotherapy. Compared to the new diagnosis, the Shannon entropy of the relapsed patients had a marked reduction, whereas the Gini index increased significantly. No difference was observed in the Shannon and Gini indexes in patients with complete remission (Fig. 4C-D). By assessing the ratio of identical clones in the top 1,000 T clones in the bone marrow of the patients with complete remission or in relapsed patients at new diagnosis and after treatment, we found that the ratio of identical T clones in the relapsed patients before and after treatment was significantly lower than that in the complete remission group. However, when we increased the number of clones assessed to 5,000, no difference was observed between the relapsed group and the complete remission group in terms of the ratio of identical T clones at new diagnosis and after chemotherapy (Fig. 4E). These results showed that compared to the AML patients who were in complete remission after chemotherapy, some of the CD8+ T cells of the patients who relapsed after treatment had massive clonal expansion and reduced clonal diversity and significant change in the T cell composition in the top 1,000 clones.
TCR repertoire distribution of CD8 + T cells based on PD-1 expression
As a dominant inhibitory receptor, PD-1 is the pivotal mediator that induces CD8+ T cell exhaustion and contributes to T cell impairment. Several previous reports have suggested the functional involvement of PD-1 in AML progression [2, 18, 30, 31]. Compared to the time of diagnosis, for PD-1-expressing CD8 T cells, a significantly increased tendency in the bone marrow of relapsed patients and a decreased trend in the bone marrow of patients who had CR were noted in our study (Fig. 5A). To determine whether CD8+PD-1+ cells exhibited more clonal expansion, TCRβ deep sequencing of CD8+PD-1+ and CD8+PD-1− T cells was performed. Figure 5B-E depicts the TCR repertoire distribution in 4 samples from one patient who relapsed and one patient who achieved sustained remission at new diagnosis and after chemotherapy. In all the samples analyzed, CD8+ PD-1+ T cells were found to be more oligoclonal than CD8+ PD-1− T cells. The top 50 clonotypes cumulative frequency made up 42.9%, 35.9%, 41.5%, and 50% of the PD-1+ T cells total frequency at the time of CR diagnosis, CR post chemotherapy, relapse diagnosis, and relapse post chemotherapy, respectively, but only 18.2%, 15.8%, 20.4%, and 29.6% of the PD-1− total frequency at the time of CR diagnosis, CR post chemotherapy, relapse diagnosis, and relapse post chemotherapy, respectively (Fig. 5B and D). Since the abundance of CDR3 based on TCR sequencing of bulk CD8 + T cells can better reflect T cell clonal expansion, we analyzed the distribution of the top 50 clonotypes in bulk CD8+ T cells in previous results of these two patients in CD8 + PD-1+ cells and CD8 + PD-1− T cells. At both diagnosis and post chemotherapy, the clones with higher frequency and the overall frequency of the top 50 clonotypes in the CD8+ PD-1+ group were higher than those in the CD8+ PD-1− counterpart (Fig. 5C and E, Table S2-S5). Moreover, the top 50 prevalent clonotypes in the CD8+ PD-1+ group were far less frequent in the PD-1− counterpart (Figure S3). Analysis of all samples from the 5 relapsed patients and 19 CR patients also revealed that the top 50 clonotypes in each CD8+ population were more frequently distributed in the CD8+ PD-1+ group than in the CD8+ PD-1− group (Fig. 5F). Regarding the Shannon index, there were significant reductions in the CD8+ PD-1+ group compared to the CD8+ PD-1− group, indicating lower TCR repertoire diversity in the CD8+ PD-1+ group (Fig. 5G).