High expression of PD-1 in CD4+ T cell subsets in MM
It is well established that immune checkpoint receptors play an essential role in the immune surveillance and anti-tumor response . We and others have previously demonstrated the dysregulated expression of CTLA-4, PD-1, and BTLA suppressors in tumors [4, 14-22]. We also reported that CTLA-4 blocking antibody might be a beneficial form of immunotherapy for a proportion of chronic lymphocytic leukemia (CLL) patients depending on the level of CTLA-4 expression on leukemic cells; for those with high CTLA-4 expression, it could be an unfavorable strategy leading to induction of pro-survival signals in CLL cells . We therefore aimed to analyze the involvement of a particular immune checkpoint in the development of systemic T-cell immunosuppression in MM.
In patients, we found a significant increase in PD-1 expressing cells within different PB T cell subsets – CD3+CD4+ (15.92% [11.75% - 19.25%], p =0.025), CD4+CD127+ (Teff) (23.53% [15.84%-30.05%], p = 0.025), and CD4+CD127- (Treg) (10.62% [6.44%-13.66%], p = 0.008) – when we compared them to corresponding healthy cells (12.55% [8.37%-15.29%], 16.87 [10.52%-21.75%], and 6.14 [3.90%-7.62%], respectively) (Fig. 1a, suppl. 1-2). Regarding tumor stage, we observed no marked differences among the patients with respect to studied cell subpopulations (Table 2).
Analysis of PD-1 fluorescence intensity in CD4+ and CD4+CD127+ Teff cells revealed no statistically significant differences between patients and healthy controls (Tables 3 and 4). In contrast, we noted diminished PD-1 amounts in MM CD4+CD127- Treg cells (Table 3), more pronounced in stage I/II of the disease when compared to healthy individuals (Table 4).
Although the median PD-1 intensity in Tregs from patients at stage III remained at a comparable level to that found in healthy Tregs, it was significantly up-regulated when compared to patients at stage I/II (Table 4).
Unaffected frequency of BTLA+ T cells in MM patients
In contrast, we found no significant differences in the percentages of BTLA+ cells within CD3+CD4+, CD4+CD127+ Teff and CD4+CD127- Treg cells between patients (27.20% [21.61%-55.72%]; 42.62% [22.93%-67.43%]); 5.30% [2.00%-9.64%], respectively) and healthy individuals (23.85% [17.04%-40.16%]; 32.50% [20.75%-54.43%]; 2.87% [1.85%-3.40%], respectively) (Fig. 1b, Additional file 1 and 2).
Also, we did not find any differences in BTLA expression in CD4+ T cells, CD4+CD127+ Teff and CD4+CD127- Treg cells among patients regarding tumor stage (Tables 2 and 4).
In contrast, a decrease in the MFI values of BTLA in the MM CD4+ and CD4+CD127+ Teff cell subsets and no significant difference in BTLA levels in CD4+CD127- Treg cells compared to controls were seen (Table 3).
Unaffected frequency of CTLA-4+ T cells in MM patients
The proportions of CTLA-4-expressing CD3+CD4+, CD4+CD127+ Teff and CD4+CD127- Treg cells did not significantly differ between patients (1.51% [0.89% - 2.51%]; 3.28% [1.75% - 6.00%]; 1.82% [0.75% - 2.32%], respectively) and controls (1.27% [0.78% - 2.00%]; 3.55% [1.64% - 4.22%]; 1.14% [0.56% - 1.37%], respectively) (Fig. 1c, suppl. 1-2). Similarly to BTLA, CTLA-4 expression in the examined T cells did not differ among patients regarding MM stage (Tables 2 and 4).
Of note, the MFI values of CTLA‑4 on CD4+CD127+ Teff and CD4+CD127- Tregs were lower in MM compared to corresponding healthy cells (Table 3).
Taken together, the results show that PD-1 is the only immune T cell inhibitor significantly increased among PB CD4+ T cell subsets of MM patients.
PD-1 determines clinical course of MM
There is growing body of evidence, including ours, that alterations in the immune checkpoints’ expression in T cells affect the clinical outcome of cancer [4, 14, 16, 18-22]. Therefore, we wanted to find out whether the dysregulated expression of T cell inhibitors in MM might be associated with clinicopathologic features, such as albumin and beta2-microglobulin level, myeloma isotype, PLC numbers in the BM, light chain, renal function, and tumor stage.
Some associations were found between both the proportions of the CD4+CD127+PD-1+ cells or tumor clinical stage and the serum levels of beta2-microglobulin (Fig. 2a and c). Although quantitative analysis revealed no significant differences in PD-1 amounts in the CD4+CD127+ Teff subset between patients and healthy controls, worthy of note was a correlation of borderline significance of PD-1 levels in these cells with tumor stage (Fig. 2d). Similarly, we observed that the PD‑1 intensity values in CD4+CD127- Treg cells were also correlated with tumor stage (Fig. 2e). In contrast, there was no correlation of PD-1 expression with other indices of severe disease.
We observed no significant association of BTLA and CTLA-4 expression in CD4+, CD4+CD127+ Teff, and CD4+CD127- Treg cells with any of the clinicopathological indices studied (not shown).
PD-1 rather than BTLA or CTLA-4 affects MM patient survival
We also analyzed our patient cohort regarding the possible dependence of the patients’ overall survival (OS) on the studied immune checkpoint receptors. We stratified for low and high expression of each factor according to median split.
In the group of studied patients, we did not find any association between PD-1+, BTLA+, and CTLA‑4+ cell frequencies in the whole PB CD4+ T cell population and patients survival (Additional file 3a-c). However, further analysis of CD4+CD127+ Teff and CD4+CD127- Treg subsets revealed that patients with high abundance of PB PD-1+ Teff cells have worse survival than patients with a low percentage of these cells (Fig. 3a). In contrast, the frequency of PD-1+ Tregs (Fig. 3b) as well as BTLA+ and CTLA-4+ cells within circulating CD4+CD127+ Teff and CD4+CD127- Treg cells did not influence patients’ survival (Additional file 3d-g).
The above results indicate clear involvement of PD-1 rather than the other inhibitory receptors in MM clinical outcome.
Activated phenotype of PB CD4+ T cells affects MM clinical outcome
In the light of demonstration that the expression of immune checkpoints is induced upon cell stimulation in order to suppress the ongoing immune response, we analyzed the state of systemic activation of MM CD4+ T cells.
As expected, in MM patients, the median proportion of circulating CD4+CD69+ T cells were significantly higher than in healthy controls (0.58% [0.25% - 0.96%] vs. 0.33% [0.22% - 0.44%] p = 0.01), primarily at stage III (Table 2). In consequence, the abundance of activated PB CD4+ T cells was correlated with MM stage (Fig. 2b). In addition, a higher proportion of MM CD4+CD69+ T cells in PB showed some association with poor survival (Fig. 3c).
The in vitro stimulation revealed that patients’ cultured CD4+ T cells exhibited a lower proportion of CD69+ cells than corresponding healthy cells (44.29% [26.22% - 54.72%] vs. 55.73% [53.00% - 73.16%], p = 0.044) under the same stimulation conditions.
Regarding MFI values, there was no marked difference in the levels of CD69 expression in either freshly isolated or in vitro stimulated T cells between the groups (Tables 3 and 4).
This part of the data shows that MM CD4+ T cells are maximally activated in vivo most likely by myeloma antigens, thereby supporting strengthened inhibition.
Lower abundance of Th1 cells in PB of MM patients
Recently, the involvement of PD-1 expression in conversion of conventional T cells, primarily Th1, into regulatory T cells has been demonstrated . Therefore, we wanted to verify whether overexpression of PD-1 as the only inhibitor dysregulated in the different PB CD4+ T cell subtypes might contribute to peripheral imbalance in Th1/Th17/Treg distribution in our cohort of patients.
Th1 cells were detected as CD194-CD183+IFN‑γ+ and CD3+CD8-IFN‑γ+ cells. In the MM group, we observed significantly diminished proportions of Th1 cells presented with respective phenotypes compared to corresponding cells from healthy donors (3.45% [2.22% - 5.80%] vs. 8.77% [5.13% - 9.71%], p = 0.002 and 4.49% [3.22% - 7.00%] vs. 9.00% [7.66% - 10.07%], p = 0.0004, respectively) (Fig. 4a).
Although we did not find any significant differences in the abundance of Th1 cells in PB between patients at different stages of the disease, the largest CD3+CD8-IFN‑γ+ cell deficit was observed in the most advanced MM when compared to the control group only (Table 5).
Also, we found markedly lower values of IFN-γ fluorescence intensity in the respective Th1 populations than those seen in corresponding control cells (52.49 [26.23-79.14] vs. 83.24 [64-97-102.43], p= 0.003 and 31.86 [20.72-37.10] vs. 58.79 [36.41-69.87], p = 0.01, respectively).
Elevated Th17 cells in PB of MM patients
Simultaneously, we assessed the number of PB Th17 cells, which were identified as CD194+CD196+IL-17+ and CD3+CD8-IL-17+. In patients, the median frequencies of PB Th17 cells were significantly higher than in healthy controls (0.56% [0.30% - 0.83%] vs. 0.36% [0.26% - 0.45%], p = 0.042 and 0.51% [0.25% - 0.65%] vs. 0.32% [0.22% - 0.45%], p = 0.021, respectively) (Fig. 4b).
There was no difference in the phenotyped Th17 cell abundance between patients’ groups characterized by clinical stage (Table 5). However, we observed that subjects at stage I/II exhibited markedly increased proportions of CD3+CD8-IL-17+ cells compared to controls only; patients at stage III demonstrated normal frequency of these cells, however, some trend toward a higher level was seen as well (Table 5).
The MFI values of IL-17 in respective Th17 subpopulations were lower or comparable to those seen in controls (43.14 [38.55-52.85] vs. 63.57 [47.48-78.94], p = 0.002 and 23.39 [13.14-39.00] vs. 21.98 [16.37-52.11], p > 0.05, respectively).
Elevated Treg cells in PB of MM patients
We next determined Treg cells as CD4+CD25+CD127-, CD4+CD25highCD127-, CD4+CD25+FOXP3+, CD4+CD25++FOXP3+, and CD4+FOXP3+CD127- cells. The median percentages of studied Treg subsets were significantly higher in MM patients in comparison to the healthy controls (Table 6, Fig. 5a and b).
We found no differences between Treg proportions in PB of MM patients at the different stages of the disease, except for CD4+CD25+FOXP3+ and CD4+FOXP3+CD127- cell subsets, which were significantly lower at stage III of MM compared to the values observed at stage I/II, although remaining still increased compared to those seen in controls (Table 5).
Altogether, our data suggest that MM development is related to a clear imbalance in PB Th1, Th17, and Treg cells.
Correlation of the frequencies of PB Treg cells with prognostic factors
Given the demonstrated correlation of T-cell immunosuppression with tumor progression, we sought to evaluate possible relationships between PB Th1/Th17/Treg alteration and laboratory or clinical signs of MM progression. We found no associations of Th1 and Th17 with the level of albumin or beta2-microglobulin, type of Ig, light chain, renal function or tumor progression (not shown).
In contrast, among examined subtypes of PB Treg cells, the abundance of CD4+CD25+CD127- and CD4+FOXP3+CD127- cells was observed to negatively correlate with some clinicopathological features (Fig. 2f-h). In particular, CD4+CD25+CD127- cells were associated with β2-microglobulin level as well as MM stage (Fig. 2f and g); however, the latter correlation was of borderline significance only. Similarly, we observed that the frequency of CD4+FOXP3+CD127- cells was associated with tumor stage as well (Fig. 2h). There was no other correlation of Treg frequency with albumin concentration, type of Ig, light chain or renal function (not shown).
No association of PB Th1, Th17, and Treg cell abundance with patient survival
None of the phenotyped PB Th1, Th17 or Treg subpopulations contributed to patients’ overall survival (Additional file 4).
Taking the evidence together, progression of MM is accompanied by dysregulated peripheral abundance of the CD4+ T cell subsets involved in the tumor responses, which could not affect patient survival.