The role of Treg lymphocytes in ischemic stroke is still unresolved, despite a growing number of studies. Are their presence and action beneficial to the course of a stroke, or are Tregs a double-edged sword? [11]. Nevertheless, the factors that determine which face of Tregs we observe in stroke need to be currently defined. Notably, most of the research is conducted on rodents. Unfortunately, the translation of these results into the human brain is limited, and none of the immunotherapies that have been effective in animals has had the expected effects in humans.

In the present study, no differences were found between the absolute number and percentage of Treg cells in the lymphocyte population (CD3+) in post-stroke patients compared to controls. We also did not observe any change in the studied parameters in time. However, an insignificant increase in the percentage of Tregs was observed on the third day, when the Treg value was highest. It was consistent with the results obtained in experimental studies [20, 21] and prospective clinical stroke studies conducted by Santamaría-Cadavid et al. and Jiang et al. [22, 23]. In addition, Hein et al. also confirmed in nonseptic shock—a different but sudden, life-threatening condition—the highest number of circulating Tregs on day 3 [24]. Not all clinical findings are unequivocal, and several studies have reported a reduction in Tregs in the CD4+ lymphocyte population in patients with acute ischemic stroke [25,26,27]. These discrepancies may be due to methodological differences (different definitions of Treg subpopulations), heterogeneous control groups (control groups consisting of healthy volunteers or patients with acute vascular diseases such as TIA and myocardial infarction), and biological diversity of Tregs.

The most important finding of our study, however, is that for the first time we described a significant, gradual (at least until day 10) decrease in the percentage of circulating Helios+ Tregs in the acute and subacute phases of stroke and a concomitant successive increase in both absolute values and the rate of Helios− Tregs on day 90 when these differences were less pronounced, although still significant. Due to the gap interval between examinations on day 10 and day 90, it is unclear at what time point these values began to normalize.

Such a marked shift in the H− and H+ Treg ratio is apparent as early as day 1 after the stroke. So far, such early quantitative changes in Tregs have not been observed. Thus, we suppose that changes in the ratio of H−/H+ Tregs in favor of H− precede changes in the number of Tregs. Simultaneously we did not observe significant differences in Treg kinetics and only observed a direction of changes consistent with those found in studies on larger populations. Then we can presume that such marked differences in the H−/H+ Treg ratio may be a sensitive marker of immune changes in acute stroke. Since the kinetics of H−/H+ Tregs in emergency conditions have not been studied, we cannot relate our results to other studies.

H+ and H− Tregs are currently under research, and their phenotypic and functional differences are not definitively known. Gene expression analysis showed that the two populations differ by approximately a thousand genes. H+ Tregs have more surface antigens characteristic for activated Treg lymphocytes with suppressor properties, while the H− Treg subpopulation secretes more IFN-γ. Then it is assumed that H+ Tregs play a primary suppressor function, while the H− Treg subpopulation is pro-inflammatory. Interestingly, H+ Tregs are produced from virgin cells, but under lymphopenic conditions, they may assume a phenotype with low Helios expression, and they may become less stable and lose FoxP3 [28]. Lymphopenia is a feature of the acute phase of stroke, so the shift between H+ and H− Treg subpopulations in this study confirms Treg lymphocytes’ behavior observed in other clinical situations.

It seems that the H−/H+ Treg ratio may be of clinical significance. We observed that on the third day after the stroke, patients who developed SAI had a significantly higher percentage of H+ Tregs and a considerably lower percentage of H− Tregs than SAI− subjects. This finding is consistent with the fact that the down-regulation of IFN-γ (preferentially secreted by H− Tregs) increases the prevalence of SAI [29]. Moreover, when adjusting for confounders that are significant for SAI (inter alia, severity of initial neurologic deficit, WBC on day 3, and infarct volume), the higher percentage of H+ Tregs on day 3 remains an independent risk factor of SAI. In longitudinal observation, we did not find any significant differences in the ratio of H+ and H− Tregs between studied time points within both SAI+ and SAI− groups. However, we noted on day 3 an insignificant increase of both: the H+ Tregs in SAI+ and H− Tregs in SAI− subjects. Most patients developed SAI as early as day 3, consistent with the previous finding that stroke-associated pneumonia (SAP) usually occurs on days 2–3 [19]. Since an increased percentage of H+ Tregs coincides with SAI, one cannot assign a predictive value. However, it is worth considering that an increased rate of circulating pro-suppressive H+ Tregs may be one of the etiological factors for SAI. Nevertheless, we found no differences between SAI+ and SAI− subjects in the percentage and the absolute number of circulating Tregs at any time point after stroke. According to our best knowledge, up to now the association between Treg cells and post-stroke immunodepression or infections has not been confirmed in humans although there are theoretical premises supporting this thesis [30, 31]. Since the correlation between H+ %Treg on D3 and SAI was confirmed in our study, we suppose that the transcription factor Helios with its suppressor function might be crucial and the changes in the number of Tregs might be less important than the qualitative shift (lost Helios expression) within a quantitatively stable population.

Although an association between a higher percentage of circulating H+ Tregs in the acute phase of stroke and a worse clinical condition in the convalescent stage as well as with final infarct volume were described, these correlations were not confirmed in multivariate logistic regression analysis. Differences in the immune regulatory response depending on the severity of the clinical symptoms of stroke have been noted before [32]; however, the results varied. Neither Urra et al. [25] nor Ruhnau et al. [26] found a correlation between the neurological deficit and the number of Tregs. In contrast, Santamaría-Cadavid et al. found that an increased level of circulating Tregs within 48 h after stroke was associated with better functional status [33]. Such a discrepancy may be due to different methodologies implemented by the Santamaría-Cadavid group, while in the study by Urra et al., patients with symptoms of infection were excluded, which made the results non-comparable. And finally, in the context of our results, the clinical outcome seems to be a weak correlation, if any.

On the first day after stroke (but on other days not), we observed a larger decrease in H+ Tregs in females than in males. Such a pro-inflammatory shift in females is of unknown clinical significance and might be random, especially since we observed this phenomenon only on the first day of stroke. On the other hand, it might exert a detrimental effect since it is well documented that women with stroke are at risk of worse clinical outcomes. Moreover, the limited suppressive function of Tregs and bias toward pro-inflammatory ones are observed in many autoimmune diseases characterized by female prevalence. This phenomenon in premenopausal women is related to estrogen E2. Its normal level suppresses the expression of FoxP3—the marker and major transcription factor of Tregs and increased secretion of anti-inflammatory IL-10 by Tregs was seen [34, 35]. It was associated with poorer recovery and post-stroke immunosuppression. However, the biology of Tregs in postmenopausal women seems to be more complex and remains unclear [36].

A disturbed H−/H+ ratio is not the only early marker of acute dysfunction detected within the first 24 h of symptoms onset, which raises the eternal “chicken or egg” causality dilemma. As a transcription factor, Helios probably reacts rapidly to environmental changes; however, the nature of Helios is not sufficiently well known to unequivocally state that its expression is the consequence of ischemia. It seems that very accurate Treg kinetics in the first minutes or hour in stroke (and even more so in TIA) might shed new light into processes preceding clinical symptoms of brain ischemia. Such clinical studies are strongly needed. Nevertheless, we realize that their logistics is troublesome.

Since stroke-induced immunodepression needs to be a better-understood phenomenon, even preliminary data are valuable. There is no information on changes in the expression of the Helios marker in Treg lymphocytes in the stroke literature. In this matter, the present study should be considered pioneering. It seems to be an important voice in the discussion about the Janus face of Treg lymphocytes in the acute phase of stroke, with arguments in favor of their damaging function and the role of Helios as a quarterback. This variable proportion between H− and H+ Tregs reflects the disturbed systemic balance between pro-suppressive and pro-inflammatory processes that “fight” after a stroke and might significantly hinder therapy. Nonetheless, several mechanisms of immunosuppressive and, to a lesser degree, the pro-inflammatory function of Tregs have been identified, and the Helios factor is only one of them. At this stage of the research, we can only assume a scenario in which lymphopenia is present from the first day of stroke (Table 2), and creates a microenvironment promoting destabilization of Tregs. From the first day of stroke the H− /H+ Treg shift is present as well, resulting in an increased percentage of pro-inflammatory H− Tregs. However, on the 2nd/3rd day, in some patients, the shift toward pro-inflammatory H− Tregs is not so prominent; pro-suppressor H+ Tregs predominate, and patients in this group develop SAI. Such an unfavorable shift was not confirmed on day 10 and beyond.

Further elucidation of the factors that control Helios expression would help identify potential therapeutic targets to manipulate Treg stability. For example, depletion of Helios expression in a selected patient population may help to avoid stroke-induced immunodepression, and in another one, enhancement of Helios expression may reduce post-stroke autoimmunity. Nevertheless, until now the experimental studies on animal immunotherapeutic models have failed [37].

There are many limitations of our research. Firstly, this is a single-center study with a limited sample size. Despite the initial group size estimation, a larger group would undoubtedly allow more relationships to be identified. Secondly, conducting observations in the subacute phase of stroke only up to day ten makes it impossible to clarify the time the H−/H+ Treg ratio begins to normalize and whether this is of prognostic significance. Thirdly, we assessed the expression of Helios in Tregs using cytometry as the only method, and our data are not supported indirectly with other strategies (the concentration of IFN-γ or IL-10 and TGF-β, which both promote the differentiation of Tregs [37]) confirming that higher expression of Helios translates into Treg suppressor function. And finally, there was some bias due to the limited number of patients on day 90. Most dropped-out subjects could not participate in the study due to disability; thus, we could not assess the patients with the most unfavorable outcomes. Nevertheless, we believe that the above observations are pioneering and wish to sow the seeds for further research into the involvement of immune cells in the pathophysiology and perhaps even the pathogenesis of stroke.