3.1. Treatment of PBMCs with Tag7 Results in the Formation of Cytotoxic Subpopulations of NK (CD16+CD56+), CD3+CD4+ and CD3+CD8+ Lymphocytes, But Only If the PBMC Pool Contains Monocytes
As we have previously shown, Tag7 can induce in PBMCs of at least three distinct subpopulations of cytotoxic lymphocytes that alternately exhibit their activity against HLA-negative tumor cell lines during a 6-day incubation period [7
]. In this study, Tag7 also proved to induce the cytotoxic fraction of NK cells (CD16+
), which reached a peak of activity on day 3–4. The number of these cells subsequently decreased significantly, and cytotoxicity associated with them was no longer observed (Figure S1
). The cytotoxic activity of CD3+
lymphocytes was detected on days 4 and 6 of incubation with Tag7, while that of CD3+
lymphocytes was only detected on day 6 (Figure S1
According to our previous data, Tag7 treatment of PBMCs depleted of monocytes does not result in the induction of cytotoxic activity [7
]. Here, we tested whether monocytes are necessary for the formation of each cytotoxic lymphocyte subpopulation. Monocytes were removed from PBMCs by magnetic cell separation, and the resulting PBMC mc (–) cells were incubated with Tag7. Using the same method, NK cells and CD3+
lymphocytes were isolated from the Tag7-treated PBMC mc (–) cell pool on incubation day 4, and CD3+
lymphocytes were isolated on day 6. Cytotoxicity tests showed that the removal of monocytes prevented the induction of cytotoxic activity in either NK cells or CD3+
lymphocytes (Figure 1
a). As a control, we also used an inhibitory peptide to TREM-1 receptor LP17, which was added to PBMC 1 h before activation Tag7. The data demonstrated the absence of the cytotoxic activity of PBMC during 6-day co-incubation of the LP17 peptide and Tag7 (Figure 1
a). Thus, monocytes appear to be the first link in the chain of signal transmission from Tag7 to effector lymphocytes.
It is known that Tag7 is a ligand for the innate immune receptor TREM-1 [16
]. We continued to study the interaction of Tag7 with TREM-1 by affinity chromatography. We detected the binding of a soluble form of TREM-1 immobilized on Sepharose with Tag7 (Figure 1
b). An excess amount of Tag7 was passed through the column with TREM-1 immobilized on CNBr-Sepharose. Elution of Tag7 bound to TREM-1 was performed using triethylamine, and the material was analyzed by SDS-PAGE and WB. The elution material containing Tag7 was detected with specific antibodies (Figure 1
b (1)). Recombinant Tag7 was used as a control for the obtained results, which were analyzed by SDS-PAGE and WB and developed with specific antibodies (Figure 1
3.2. Tag7 Stimulates Secretion of Cytokines TNFα, IFNγ and IL-2
Taking into account that monocytes produce lymphocyte-activating factors [17
], our next task was to analyze the profile of cytokines secreted to the medium by Tag7-activated PBMCs. First, PBMCs were incubated with Tag7 for 3 days, and samples of the conditioned medium were taken every 24 h for quantitative determination of proinflammatory cytokines TNFα и IFNγ by ELISA. As shown in Figure 2
a, the level of TNFα reached a peak on day 2 and then decreased, while the level of IFNγ consistently increased during the incubation period. Thus, PBMCs treated with Tag7 secrete not only the proinflammatory cytokine TNFα but also IFNγ, which is known for its role in antiviral defense and the ability to activate lymphocytes, acting together with IL-2.
We then evaluated the profile of IL-2 secretion by Tag7-activated PBMCs and the involvement of monocytes in its induction. In this case, PBMCs were incubated with Tag7 for 6 days. In view of the data that TREM-1 activation may lead to the induction of genes coding for proinflammatory cytokines [7
], the incubation was also performed in the presence of specific TREM-1 inhibitor LP17. Conditioned medium from untreated PBMC was used as additional control. The medium conditioned by PBMCs was sampled every 24 h. The results showed that the level of IL-2 consistently increased during the incubation period in both variants but was generally lower when TREM-1 activation was blocked or when untreated PBMC was used (Figure 2
b). Despite the presence of a certain amount of IL-2 in conditioned medium, we did not observe cytotoxic activity after 6 days of co-incubation of LP17 and Tag7 with PBMC (Figure 1
a). This is evidence that the interaction of Tag7 with TREM-1 is necessary for inducing PBMCs to produce and secrete a sufficient amount of IL-2 into the medium to a mature lymphocytes subpopulation.
3.3. CD3+CD4+ Lymphocytes Are the Main Source of IL-2 and Are Necessary for the Formation of Each Cytotoxic Subpopulation
The appearance of TNFα and IFNγ in the conditioned medium in the first days of Tag7 incubation with PBMC plays an important role in the formation of an activation signal. We hypothesized that these cytokines promote the activation of CD3+
-lymphocytes and the secretion of IL-2. In view of this hypothesis, we then analyzed the changes in the expression of mRNA of IL-2, IFNγ and TNFα in CD3+
-lymphocytes under the treatment of TNFα and IFNγ. A subpopulation of CD3+
-lymphocytes was isolated by magnetic separation from PBMCs and incubated with recombinant TNFα or IFNγ for 24 h. As shown in Figure 3
a, incubation of CD3+
-lymphocytes with recombinant TNFα led to increased levels of IL-2 mRNA (45-fold), IFNγ (36-fold) and TNFα. Comparable results were detected in CD3+
-lymphocytes incubated with IFNγ. The level of IL-2 mRNA increases greater (more than 97-fold) than IFNγ mRNA (15-fold) in the case of incubation of CD3+
-lymphocytes with recombinant IFNγ. In both cases, under the action of TNFα and IFNγ in lymphocytes, there is a slight change in TNFα mRNA expression by 2–4 folds. To prove the effect of TNFα and IFNγ cytokines on the secretion of IL-2 CD3+
-lymphocytes, we analyzed the conditioned medium of pure CD3+
-lymphocytes after TNFα or IFNγ treatment. We demonstrated that under TNFα or IFNγ treatment, CD3+
-lymphocytes secrete IL-2 into conditioned medium (Figure S2
). It was also shown that specific antibodies against cytokines TNFα, IFNγ and IL-2, which were co-incubated with Tag7-PBMC, decrease the cytotoxic effect of Tag7-PBMC against tumor cells (Figure S3
Thus, we can point out that secretion of TNFα and IFNγ after incubation of Tag7 with PBMC in the first days triggers the activation of CD3+ CD4+-lymphocytes.
-lymphocytes are the main source of IL-2 in the immune response system [18
], it was relevant to assess the role of CD3+
cells in the transmission of activation signal from Tag7 and the production of IL-2 in the Tag7–PBMC system. Therefore, we evaluated IL-2 secretion in PBMCs depleted of CD3+
-lymphocytes by magnetic separation and incubated with Tag7 for 6 days. As shown in Figure 2
b, no IL-2 was detected in the conditioned medium. Moreover, PBMCs depleted of CD3+
-lymphocytes showed no cytotoxic activity (Figure 3
b). It appears that the cytotoxic activity of lymphocytes is stimulated by IL-2 produced by CD3+
-cells under the effect of Tag7 and secreted into the medium.
Our next task was to estimate the effect of IL-2 secreted into the medium by Tag7-treated PBMCs on the formation of individual cytotoxic lymphocyte subpopulations. To this end, subpopulations of NK (CD16+
cells were isolated from PBMCs by magnetic separation and incubated with recombinant IL-2 for 4 or 6 days. The results showed that all these subpopulations acquired the ability to kill K562 tumor cells after treatment with IL-2. To test whether Tag7 directly induces cytotoxicity in effector cells, the same lymphocyte subpopulations were treated with this protein. No cytotoxic activity was detected in this case, indicating that Tag7 has no direct effect on this activity (Figure 3
b). This is evidence that IL-2 produced by CD3+
lymphocytes plays a key role in inducing the cytotoxic activity of NK, CD3+
We then analyzed changes in the expression of genes encoding IFNγ, IL-2 and IL-2 receptor subunits—IL-2Rα (CD25), IL-2Rβ (CD122) and IL-2Rγ (CD132)—in CD3+
lymphocytes under the effect of PBMC treatment with Tag7. PBMCs were incubated with Tag7 for 22 and 46 h, and the CD3+
subpopulation was then isolated by magnetic separation to evaluate changes in the levels of mRNAs from the above genes. After 22 h incubation, the mRNA levels for IL-2 and CD25 receptor subunit were enhanced most strongly (more than 130-fold), while enhancement in the mRNA levels for other receptor subunits and IFNγ was an order of magnitude lower, with a similar pattern being observed after 46 h (Figure 3
c). Thus, CD3+
cells from the Tag-7-treated PBMC pool showed an enhanced gene expression for cytokines IL-2 and IFNγ, which stimulate lymphocyte activation, and for IL-2 receptor subunits (especially CD25), which improve IL-2 signal transmission, such that CD25 (IL-2Rα) ensures high affinity of IL-2 binding.
These results confirm that IL-2 produced by CD3+CD4+ cells plays a key role in inducing the cytotoxic activity of lymphocytes: if these cells are removed from the Tag7–PBMC system, IL-2 is absent in the conditioned medium, and active cytotoxic lymphocytes are not generated.
3.4. Role of IL-2 Receptor in the Induction of Cytotoxic Lymphocytes
Cells expressing individual IL-2R subunits were removed from PBMCs by magnetic separation, and the remaining cells were treated with Tag7 and tested for cytotoxic activity. As shown in Figure 4
a, no cytotoxicity was observed after the removal of cells expressing IL-2Rα (CD25) or IL-2Rγ (CD132) subunits, whereas the removal of IL-2Rβ (CD122)-positive cells did not prevent the induction of cytotoxic lymphocytes. Therefore, two IL-2 receptor subunits, CD25 and CD132, are necessary for the formation of cytotoxic lymphocyte subpopulations.
Cytokine IL-2 secreted to the medium during PBMC incubation with Tag7 is the main factor responsible for activation of cytotoxic lymphocyte subpopulations. Therefore, it was relevant to estimate what stages of signal transduction from the IL-2 receptor are indispensable for the induction of lymphocyte cytotoxicity. It is known that cytoplasmic kinases JAK1 и JAK3 are functionally coupled to the IL-2 receptor and required for signal transduction [19
]. To block their activity, we used specific JAK1 and JAK3 inhibitors, which were added to PBMCs 1 h before their treatment with Tag7. No cytotoxic effect was observed after 6 days of incubation with each of the inhibitors (Figure 4
b), indicating that the induction of cytotoxicity depends on activation of JAK1 and JAK3 kinases upon IL-2 binding to the receptor. These kinases, in turn, activate STAT3 and STAT5 proteins, which are translocated as dimers into the nucleus and initiate gene transcription [20
]. The role of this process in signal transduction was evaluated using STAT3 and STAT5 inhibitors that block their dimerization. The experimental procedure was the same as above, but the results showed that treatment with these inhibitors did not affect the induction of cytotoxic lymphocytes. Therefore, the IL-2–JAK signaling pathway does not depend on dimerization of STAT3 and STAT5 transcription factors.