Production of BM cytokines and reactive oxygen species (ROS) change with increased BMI
The BM microenvironment, which plays an important role in the maintenance of antigen-experienced adaptive immune cells, changes with age and CMV (12-13). To assess whether the BMI may also affect BM niches, the production of BM cytokines was measured in the BM of persons with different body weight (Figure 1). While the levels of IL-15 and IL-6 in BMMCs were not significantly different between persons with BMI below 25 and BMI between 25 and 30 (p=0.11 and p=0.47 respectively), the expression of both cytokines was the highest in the group BMI > 30 (Figure 1 A-B). The expression of IL-15 and IL-6 could was very variable in the group with BMI 25-30, but more homogeneous levels of both molecules were found in the other two groups.
Levels of ROS and the pro-inflammatory molecules IFNγ and TNF increase in the BM with age (12). When BMI was put in relationship with ROS levels in the BM, increased oxygen radicals were found in persons with higher body weight (Figure 1C). Furthermore, reduced production of IFNγ and TNF by CD8+ T cells was observed with higher BMI (Figure 1 D-E). No correlation was found between BMI and ROS levels, IFNγ+ and TNF+CD8+ T cells in PBMCs (Suppl. Fig. 1). Our results indicate that the expression of molecules supporting the maintenance of late differentiated adaptive immune cells in the BM and the production of T cell cytokines change in the BM with increased BMI.
Immune cell populations are affected by BMI in the BM
Associations between immune parameters and BMI have been described in the PB (8). Whether immune cell populations in the BM change with increased body weight is unknown. As CMV is known to affect the phenotype of immune cells, we assessed the frequency of populations in BMMCs from CMV seronegative and seropositive persons in correlation with BMI (Table 1). The gating strategy for the populations included in the analysis is shown in Figure 2. While natural killer (NK), natural killer T (NKT) cells, monocytes and plasma cells did not change, the overall B cell frequency in the BM increased with BMI in CMV- persons (Table 1). No differences were observed for CMV+ donors, although B cell levels were similar in CMV- and CMV+ persons (Suppl Fig.2 A). While the levels of T cells and CD8+ T cells did not differ when overweight and lean persons were compared, the frequency of CD4+ T cells increased with BMI in CMV- persons. Again, similar levels of CD4+ T cells were found in CMV- and CMV+ persons (Suppl Fig.2 B)., but no significant correlations between BMI and CD4+ T cells were observed for CMV+ donors.
In summary, although most of the immune cell populations in the BM were not affected, B cells and CD4+ T cells positively correlated with BMI, at least in CMV- persons.
CD8+ T cell subsets change with BMI
We next assessed whether CD8+ T cell subsets in the BM may change with increased body weight (Table 2). Using the markers CCR7 and CD45RA, we defined the four populations CCR7+CD45RA+ naïve (TN), CCR7+CD45RA- (TCM), CCR7-CD45RA- (TEM) and CCR7-CD45RA+ (TEMRA) within CD8+ T cells (Figure 3). While the frequency of CD8+ TCM cells in the BM negatively correlated with BMI in CMV- persons, CD8+ TEM cells decreased with increased body weight only in CMV+ donors (Table 2). No significant differences were observed for CD8+ TN and CD8+ TEMRA cells. When the levels of the four subsets were measured in paired PBMC samples, CD8+ TEM were highly reduced in the CMV- group with increased BMI, but CD8+ TCM cells, as well as CD8+ TEMRA cells did not change in overweight compared to lean persons (Table 3). A negative correlation between frequency of CD8+ TN cells and BMI in PB was also observed. With the markers CD28 and CD57, the populations CD28-, CD57+, as well as CD28+CD57-, CD28+CD57+, CD28-CD57- and CD28-CD57+ were gated within CD8+ T cells in BMMCs (Figure 2). Only in CMV- persons, the CD28+CD57- and CD28+CD57+ subsets negatively correlated, while CD28-CD57- were positively associated with BMI (Table 2). No significant differences were found for the CD28-CD57+ subset. Similar results were found in PBMCs for CD28+CD57+ and CD28-CD57- cells, while no changes were observed for the other two subsets (Table 3). Taken together, our data show that several CD8+ T cell populations change in the BM with increased BMI, particularly in the absence of CMV. Overall, the frequency of memory CD8+ T cells may be reduced in overweight compared to lean persons, while highly differentiated CD28-CD57- CD8+ T cells increase.
The phenotype of CD8+ T cell subsets change with BMI
To investigate whether, in parallel with their frequency, the phenotype of CD8+ T cell subsets may change with increased body weight, the expression of molecules involved in T cell activation/exhaustion, responsiveness to T cell cytokines and memory as well as senescence was measured in paired BMMC and PBMC samples using flow cytometry (Tables 4 and 5). Representative FACS plots are shown in Suppl. Fig. 3. PD-1 is overexpressed in activated and/or exhausted T cells (16). In the whole CD8+ T cell population in the BM, PD-1 was reduced in overweight compared to lean persons only in the CMV- group (Table 4). While no correlation was found between PD-1 expression within CD8+ TCM and CD8+ TEMRA cells and BMI, reduced levels of this molecule could be observed in the CD8+ TEM subset with higher body weight in CMV- persons. When the four CD28+/-CD57+/- subsets were considered, the negative correlation between PD-1 expression and BMI was shown in CD28+CD57- CD8+ T cells, but not in the other subpopulations. Similar results were obtained in the PB (Table 5). In this case, PD-1 levels were negatively associated to BMI, again in the CMV- group only, when the expression was measured within CD8+ TCM, CD8+ TEM, CD8+ TEMRA, and CD28+CD57-, CD28-CD57- and CD28+CD57+ CD8+ T cells. In all subsets, stronger and more significant correlations were obtained in comparison to the same populations in the BM. IL-7Rα expression on T cells is a marker for responsiveness to the T cell cytokine IL-7. In the whole CD8+ T cell population in the BM, the expression of the receptor negatively correlated with BMI in CMV- persons (Table 4). In CMV+ persons, overall IL-7Rα levels were lower in comparison to CMV- individuals (data not shown). Although the frequency of IL-7Rα+ cells within CD8+ TCM, CD8+ TEM and CD8+ TEMRA was not associated to BMI, its expression in both CD28+CD57- and CD28+CD57+ CD8+ T cells increased while in CD28-CD57+ CD8+ T cells decreased in overweight compared to lean CMV- persons. No differences were observed in the CMV+ group. The same results could be found in paired PBMC samples (Table 5). The combination of the markers IL-7Rα and KLRG-1 within CD8+ T cells allows the definition of IL-7Rα+KLRG-1- memory progenitor effector cells (MPEC), which are known to differentiate into memory cells, and IL-7Rα-KLRG-1+ short living effector cells (SLEC), which may either die or accumulate as senescent-like T cells (17,18). In the BM of both CMV- and CMV+ persons, MPEC were negatively associated with BMI, while the levels of SLEC did not change. In the PB, MPEC decreased in donors with higher BMI only in the CMV+ group (Table 5). Interestingly, the expression of KLRG-1, commonly associated with terminally differentiated cells (19), positively correlated with BMI when its expression was measured within CD8+CD28- and CD8+ TEMRA cells in CMV+ persons. Similar results could be obtained for the PB (Table 5).
In summary, our results indicate that responsiveness to IL-7 and expression of PD-1 and KLRG-1 change with increased BMI, in both BM and PB. In addition, memory CD8+ T cells may be reduced in overweight persons.
CD4+ T cell subsets change with BMI
We then aimed to investigate whether CD4+ T cell subpopulations may additionally change with increased BMI, in both BMMCs and PBMCs (Tables 6 and 7). Gating strategy used to define the subsets of interest using flow cytometry is shown in Figure 3. Again, CD4+ TN, TCM, TEM and TEMRA were gated using the markers CCR7 and CD45RA. Interestingly, in the BM but not in the PB of CMV+ persons, CD4+ TN, CD4+ TCM and CD4+ TEMRA were positively associated, while CD4+ TEM negatively correlated to BMI. In addition, in CMV- donors, the frequency of CD4+ TEMRA were higher in overweight persons. No differences were found for CD4+CD28- and CD4+CD57+ T cells, neither in BMMCs nor in PBMCs. Similarly to CD8+ T cells, PD-1 expression in the whole BM CD4+ T cell population and in CD4+ TEM cells negatively correlated with BMI in the BM of CMV- persons. In addition, differently from CD8+ T cells, IL-7Rα increased with higher BMI in CD4+ T cells. No differences were observed when the expression of PD-1 and IL-7Rα was assessed in the periphery (Table 7).
Taken together, our results indicate that, with increased BMI, the frequency and the phenotype of several CD4+ T cell subsets may change in the BM, although no differences are present in the PB.