T-cell frequencies determined by flow cytometry before and during pomalidomide treatment

Our patient group was preliminary compared with a group of sex and age matched healthy people before starting Pomalidomide. We first assessed the frequencies of CD3+, CD4+, CD8+, and CD16-56 + cells, as illustrated in Fig. 1A-E. Intriguingly, we found a significant decrease in CD4 + frequency (mean 40.9% vs. 27.9%, p = 0.00055) in MM patients, while CD8 + frequency (mean 22.2% vs. 35.8%, p = 0.00023) were significantly higher in patients when compared to controls. The frequency of CD3+ (mean 67.6% vs. 63.3%, p = 0.27), CD16+/56+ (mean 21.4% vs. 25.62%, p = 0.21) and Treg (mean 0.94% vs. 0.56%, p = 0.12) cells was substantially stable, without showing any significant difference.

Our patients underwent a median of three assessments during their treatment with Pomalidomide, corresponding to t2, t3 and t4. We tracked the frequencies of CD3+, CD4+, CD8+, CD19+, CD3+/16+/56+, CD3+/TCRαβ, CD3+/TCRδλ, and Treg, during therapy, as depicted in Fig. 1F-M. Most cell populations remained stable across all time points, except for CD4 + frequency, which notably decreased from t1 to subsequent assessments (t2, t3, and t4; p = 0.0329). A similar declining trend was observed in CD3+/TCRαβ frequency (p = 0.0115), suggesting that TCR rearrangements indeed occurred during the course of Pomalidomide treatment. However, this trend was not evident in B-lymphocytes (CD19 + cells; p = 0.0538). The frequencies of natural killer T cells (CD3+/16+/56+, p = 0.9444) and Treg (p = 0.9282) exhibited an upward trend, despite the absence of statistical significance.

Fig. 1

T-cell frequencies determined by flow cytometry before and during pomalidomide treatment. (A-E) T-cell frequencies of a pool of age- and sex- matched healthy control (ctr) that were compared with MM patients (pts) prior to initiating Pomalidomide treatment. The lymphocytes subpopulations are reported as percentage over total lymphocyte population from peripheral blood samples collected at t = 1. Statistical difference between the two groups (Ctl vs. Pts) is evaluated with the Mann Whitney test (two-tailed, non-parametric test for non-matched samples). (F-M) The percentages of lymphocyte subpopulations in MM patients are shown relative to the total lymphocyte population in peripheral blood samples collected before Pomalidomide treatment and at 3, 6, and 12 months (t1, t2, t3, and t4, respectively). Statistical difference within these groups is evaluated with the Friedman test (non-parametric test for repeated measures)

Kinetic of expanded T-cell subpopulations in MM patients during pomalidomide treatment

We further examined the expansion of T-cell subpopulations in MM group during Pomalidomide treatment. We noted that the BV expansion percentages in the CD4 + subpopulation relative to the total BV examined were 8%, 13%, 14%, and 24% for t1, t2, t3, and t4, respectively (Fig. 2A). In more detail, seven out of ten patients showed an overall increasing trend of BV expansions, with their proportion ranging from 4 to 38%. This trend was also consistent with their response to treatment, as we collected in Supplementary Table 1. However, three expansions disappeared in subsequent evaluations, while three patients displayed stable or slightly decreased BV expansion during treatment. While in the CD8 + subpopulation, the percentages of BV expansions relative to the total BV examined were 2%, 3%, 4%, and 5%, also corresponding to t1, t2, t3, and t4, respectively (Fig. 2B). Six out of ten patients with baseline BV expansions displayed an increased proportion, ranging from 2 to 8%. Unlike CD4+, BV expansions in CD8 + remained relatively stable during treatment, with three patients maintaining similar levels compared to previous assessments. This suggests that BV expansion in CD8 + cells during treatment is typically transient. In general, there was a consistent upward trend in BV expansions in both CD4 + and CD8 + subpopulations, aligning with the expected induction of CD4 + and CD8 + cells by Pomalidomide. The very limited numbers of patients would be one of the key reasons that we could not show any relevant difference in both CD4 + and CD8 + cells during treatment.

Fig. 2

Kinetic of expanded T-cell subpopulations in MM patients during Pomalidomide treatment. (A, B) Percentages of BV expansions over total BV examinate from CD4 + subpopulation and from CD8 + subpopulation of MM patients. Peripheral blood samples were collected before Pomalidomide treatment and after 3-, 6- and 12-months therapy including Pomalidomide (t1, t2, t3 and t4 on x-axis, respectively) (C, D) Percentages of MM patient that show none, a single or at least two expansions of BVs from CD4 + subpopulation and from CD8 + subpopulation. Peripheral blood samples were collected before Pomalidomide treatment and after 3-, 6- and 12-months therapy including Pomalidomide (t1, t2, t3 and t4 on the x-axe, respectively)

Next, we observed the distribution of patients with none (0), a single (1), or 2 or more BV expansions over time in CD4 + and CD8 + cells (Fig. 2C, D). A key observation emerging from our study was the notable increase in CD4 + expansions and the rising incidence of patients demonstrating these expansions. Specifically, in relation to CD4 + cells, the distribution was as follows: single or multiple BV expansions accounting for all patients at t1. At t2, the distribution shifted to single expansions for the minority (22%), while the majority exhibited multiple expansions (78%). Remarkably, at t3 and t4, all patients displayed multiple expansions (100%). Similarly, for CD8 + cells, the proportions were as follows: multiple expansions for 60% of patients, single expansions for 30% of patients, and multiple expansions for the rest at t1. By t2, the scenario changed, with a higher occurrence of single expansions in almost 80% of patients. Following this trend, BV expansions were observed to experience a more dramatic increase, from nearly 60% (t3) to 100% of patients (t4).

Overall, our analysis revealed a consistent trend that an increasing fraction of patients harboring at least one expansion by the end of the monitoring period. Notably, for CD4 + cells, all patients manifested at least two expansions starting from t3, prompting speculation about the potential involvement of these lymphocyte expansions in mechanisms associated with antitumor immunity.

Percentages of CDR3 skewing (skewed) and oligoclonality (oligo) in both CD4 + and CD8 + cells

During Pomalidomide treatment, we employed spectratyping to monitor changes in CDR3 profiles in our patients, with more evident changes observed in the CD4 + subpopulation. Initially, we conducted a comparative analysis between patients and controls (Fig. 3A). In the CD4 + population, we observed a higher degree of CDR3 skewing in CD4 + cells among patients compared to controls (mean 25.8% vs. 55.3%, p < 0.0001). Conversely, the percentage of skewed BVs was significantly decreased in patients in CD8 + population (mean 75.9% vs. 55.2%, p < 0.0001), indicating a reduction in skewing profiles in the CD8 + population among patients compared to controls. However, as indicated in Fig. 3E, the percentage of oligoclonal BV subfamilies was consistent between patients and controls, both in the CD4+ (mean 1.97% vs. 6.22%, p = 0.1798) and in the CD8 + subset (mean 11.1% vs. 6.67%, p = 0.5340).

Fig. 3

Percentages of CDR3 skewing (Skewed) and oligoclonality (oligo) in both CD4 + and CD8 + cells. (A, E) Percentages of skewed and oligo BVs were determined by spectratyping of a pool of age- and sex- matched healthy controls (ctr) with MM patients (pts) before starting Pomalidomide therapy. Statistical difference between groups is calculated with Mann Whitney test (non-parametric test for unpaired measures) (B, C, F, G) Percentages of skewed and oligo BVs were determined by spectratyping of samples before Pomalidomide treatment (t1) and after 3-, 6- and 12-months therapy including Pomalidomide (t2, t3 and t4 on x-axis, respectively). Statistical difference within groups is calculated with Friedman test (non-parametric test for repeated measures) (D, H) Percentages of CDR3 skewing (S), oligoclonality (O) and Gaussian profile (G) were found among BV expanded from both CD4 + and CD8 + cells. Data are reported for samples were collected before Pomalidomide treatment (t1) and after 3, 6 and 12 months of therapy including Pomalidomide (t2, t3 and t4 on x-axis, respectively). Percentages were obtained by formulae: % G on exp: (Number of Gaussian/number of total expanded BVs) * 100; % O on exp: (Number of oligo/number of total expanded BVs) *100; % S on exp: (Number of Skewed/number of total expanded BVs) * 100

We continued to observe the kinetics of CDR3 skewing and CDR3 oligoclonality in CD4 + and CD8 + cells at various assessment points before and during their treatment with Pomalidomide (t1, t2, t3, and t4). In CD4 + cells, CDR3 skewing values were 53.2%, 50.5%, 64.0%, and 48.0%, respectively (Fig. 3B). Meanwhile, the CDR3 skewing values in CD8 + cells were 55.2%, 63.5%, 53.1%, and 57.4%, respectively (Fig. 3C). The kinetics of CDR3 oligoclonality in CD4 + cells were 6.23%, 5.73%, 4.87%, and 7.67%, respectively, while in CD8 + cells, they were 6.70%, 2.60%, 9.9%, and 16.0%, respectively (Fig. 3F, G). We also conducted a comparison of these spectratyping data before and after treatment, once again demonstrating a relatively stable pattern throughout the course of Pomalidomide treatment.

Finally, the study specifically examined clonal expansions within CD4 + and CD8 + cells, categorized into Skewed, Oligoclonal, and Gaussian. Across the treatment phases, notable shifts in proportions were observed. In CD4 + cells, the prevalence of Skewed increased from 43% (t1) to 52% (t3), then decreased to 36% (t4). Concurrently, the proportion of Gaussian decreased from 24% (t1) to 12% (t3) and further to 8% (t4). Oligoclonal expansions exhibited variability, declining from 29% (t1) to 0% (t3), followed by a marginal resurgence to 4% (t4) (Fig. 3D). Regarding CD8 + cells, the prevalence of Skewed ascended from 14% (t1) to 46% (t2), subsequently stabilizing at 25% at both t3 and t4. Oligoclonal expansions remained absent except for a resurgence to 12.5% at t4 (Fig. 3H). It is crucial to acknowledge the presence of data gaps and expansions that do not neatly fit into the designated categories (S, O, or G), thus resulting in percentages that do not sum up to 100%.