Previous studies of TREG cells in human AML have included patients with untreated disease and patients in complete hematological remission. AML patients with chemotherapy-induced cytopenia have not been examined, even though clinical studies suggest that immunological events early after chemotherapy are important for the antileukemic effect of chemotherapy. In the present study we therefore compared T cell subset distribution in AML patients with severe chemotherapy-induced cytopenia with (i) untreated patients or patients during regeneration after intensive chemotherapy; and (ii) healthy controls. The cytopenic patients had increased relative levels of circulating TREG cells, whereas levels of TC1 and TH1 cells were decreased while TH17 cells were not altered.
Early lymphoid reconstitution after chemotherapy is associated with decreased risk of leukemia relapse [4–7, 28], and these observations suggest that immunological events early after chemotherapy are clinically important. The immunological status during treatment-induced cytopenia are determined by pre-existing disease-induced abnormalities and chemotherapy-induced defects . Previous studies have demonstrated that even patients with severe therapy-induced lymphopenia have an operative T cell system , and in this context we investigated the balance between various proinflammatory and suppressive T cell subsets in AML patients.
As expected the relative levels of CD3+ T lymphocytes to total leukocytes were dependent on the degree of leukemization in patients with untreated AML. More important, the frequencies of circulating CD3+ T cells among total lymphocytes were decreased in patients with untreated disease compared with healthy controls, these levels were increased during chemotherapy-induced pancytopenia before normalization during reconstitution. Even though our cytopenic patients had lymphopenia, our results suggest that CD3+ T cells are less sensitive to intensive chemotherapy than myeloid cells and other lymphocyte subsets (e.g. B cells, NK cells).
We did not find any significant differences in relative CD8+ and CD4+ T cell levels between healthy controls and patients examined before or following intensive chemotherapy. In contrast, the relative levels of circulating IFNγ secreting CD4+ (TH1) and CD8+ (TC1) T cells were decreased in cytopenic patients before normalization during reconstitution (Figure 3). This decrease is also reflected in the decreased TC1:TREG and TH1:TREG ratios during cytopenia (Figure 6). Taken together these observations strongly suggest that treatment-induced lymphopenia is not a random process and the susceptibility to intensive chemotherapy differ between T cell subsets.
TH17 cells constitute a separate proinflammatory T helper (TH) cell subset [29–33]. Studies in animals as well as humans suggest that TH17 cells are important for anticancer immune reactivity [34, 35]. A previous study described increased frequencies of circulating TH17 cells in patients with untreated AML and with a normalization when patients achieved complete remission after chemotherapy . In contrast, we observed normal TH17 levels for patients with untreated AML. In addition we observed that functional TH17 cells could be detected during severe therapy-induced cytopenia, and these frequencies did not differ from healthy controls although the variation range was broader. Studies of absolute and relative TH17 levels in a second patient cohort confirmed that only minor variations were seen during therapy-induced cytopenia. The decreased TH17:TREG ratio during cytopenia is thus caused by relatively stable TH17 absolute levels with only minor decreases and maintained or increasing absolute levels of TREG cells (Figure 6). Finally, we observed increased IL17-A release by circulating cells in response to stimulation by T cell specific antibodies (anti-CD3 combined with anti-CD28); this is probably caused by T cell release because IL17-A release from other cells in response to these T cell specific stimulatory signals seems less likely. Our overall results thereby strongly suggest that neither the leukemia nor the chemotherapy has any selective suppressive effect on TH17 cells compared with other T cell subsets. Release of IL17-A from other cells than T cells activated in response to these stimulatory signals seems less likely.
Immunosuppressive CD4+ TREG cells inhibit effector T cells and NK cells [36, 37]. Depletion of TREG cells enhances murine anti-cancer immunity [38, 39] as well as vaccine-induced anticancer reactivity in humans . Increased levels of circulating TREG cells seem to occur in untreated AML [9, 10], although these previous studies did not analyze FoxP3+ cells. We observed increased levels of circulating FoxP3+ TREG cells in patients with untreated AML. Even though previous studies suggest that at least certain cytotoxic drugs can reduce the levels of circulating TREG cells , our present results showed that the frequencies of TREG cells were increased before and following intensive AML chemotherapy. Studies in a second cohort of patients demonstrated that the absolute levels of Treg cells even increased during cytopenia for certain patients. This was true even for patients regenerating after therapy, an observation that is also supported by another study . The relative levels of CD4+ CD25HIGH TREG cell were also increased in untreated AML and during cytopenia. The variations of various T cell subsets relative to TREG cells then suggest that the various subsets are differentially affected, and the increased TREG levels may then be caused either by TREG proliferation or development from T cell progenitors or other T cell subsets.
We observed a correlation between the levels of TH1 and TC1 cells both for AML patients and healthy controls, and this correlation was maintained during severe therapy-induced cytopenia. In contrast, the correlation between TH1 and TH17 levels was detected only for AML patients with untreated disease and therapy-induced cytopenia. These observations further support the hypothesis that various T cell subsets are differentially affected in AML patients.
The levels of circulating TH17 cells were significantly higher in males than females for healthy controls, and TH1 cells were also higher in males for elderly controls. In a recent gene expression study of activated T cells the responsiveness was generally higher in women than in men, but IL17-A was the only effector gene that showed highest expression in men . Estrogen response elements in the promoter regions of several immune genes could possibly explain the gender differences . This gender differences reached only borderline statistical significance for patients with untreated AML and could not be detected in our cytopenic patients. Thus, the influence of gender differences on circulating T cell subsets becomes less important after chemotherapy.
Exogenous IL17-A had no or only weak effects on functional AML cell characteristics; even though some differences were statistically significant their biological significance can be questioned. Even though the relative levels of TH17 cells are maintained after chemotherapy, we conclude that TH17 cells may affect AML cells indirectly through their immunoregulatory effects; whereas direct effects of IL17-A on the leukemic cells probably do not have any major impact.