The Hypothesis of the Human iNKT/Innate CD8(+) T-Cell Axis Applied to Cancer: Evidence for a Deficiency in Chronic Myeloid Leukemia

We recently identified a new human subset of NK-like [KIR/NKG2A(+)] CD8(+) T cells with a marked/memory phenotype, high Eomesodermin expression, potent antigen-independent cytotoxic activity, and the capacity to generate IFN-γ rapidly after exposure to pro-inflammatory cytokines. These features support the hypothesis that this new member of the innate T cell family in humans, hereafter referred to as innate CD8(+) T cells, has a role in cancer immune surveillance analogous to invariant natural killer T (iNKT) cells. Here, we report the first quantitative and functional analysis of innate CD8(+) T cells in a physiopathological context in humans, namely chronic myeloid leukemia (CML), a well-characterized myeloproliferative disorder. We have chosen CML based on our previous report that IL-4 production by iNKT cells was deficient in CML patients at diagnosis and considering the recent evidence in mice that IL-4 promotes the generation/differentiation of innate CD8(+) T cells. We found that the pool of innate CD8(+) T cells was severely reduced in the blood of CML patients at diagnosis. Moreover, like iNKT and NK cells, innate CD8(+) T cells were functionally impaired, as attested by their loss of antigen-independent cytotoxic activity and IFN-γ production in response to innate-like stimulation with IL-12 + IL-18. Remarkably, as previously reported for IL-4 production by iNKT cells, both quantitative and functional deficiencies of innate CD8(+) T cells were at least partially corrected in patients having achieved complete cytogenetic remission following tyrosine kinase inhibitor therapy. Finally, direct correlation between the functional potential of innate CD8(+) T and iNKT cells was found when considering all healthy donors and CML patients in diagnosis and remission, in accordance with the iNKT cell-dependent generation of innate CD8(+) T cells reported in mice. All in all, our data demonstrate that CML is associated with deficiencies of innate CD8(+) T cells that are restored upon remission, thereby suggesting their possible contribution to disease control. More generally, our study strongly supports the existence of an innate iNKT/innate CD8(+) T-cell axis in humans and reveals its potential contribution to the restoration of tumor immune surveillance.

We recently identified a new human subset of NK-like [KIR/NKG2A(+)] CD8(+) T cells with a marked/memory phenotype, high Eomesodermin expression, potent antigenindependent cytotoxic activity, and the capacity to generate IFN-γ rapidly after exposure to pro-inflammatory cytokines. These features support the hypothesis that this new member of the innate T cell family in humans, hereafter referred to as innate CD8(+) T cells, has a role in cancer immune surveillance analogous to invariant natural killer T (iNKT) cells. Here, we report the first quantitative and functional analysis of innate CD8(+) T cells in a physiopathological context in humans, namely chronic myeloid leukemia (CML), a well-characterized myeloproliferative disorder. We have chosen CML based on our previous report that IL-4 production by iNKT cells was deficient in CML patients at diagnosis and considering the recent evidence in mice that IL-4 promotes the generation/differentiation of innate CD8(+) T cells. We found that the pool of innate CD8(+) T cells was severely reduced in the blood of CML patients at diagnosis. Moreover, like iNKT and NK cells, innate CD8(+) T cells were functionally impaired, as attested by their loss of antigen-independent cytotoxic activity and IFN-γ production in response to innate-like stimulation with IL-12 + IL-18. Remarkably, as previously reported for IL-4 production by iNKT cells, both quantitative and functional deficiencies of innate CD8(+) T cells were at least partially corrected in patients having achieved complete cytogenetic remission following tyrosine kinase inhibitor therapy. Finally, direct correlation between the functional potential of innate CD8(+) T and iNKT cells was found when considering all healthy donors and CML patients in diagnosis and remission, in accordance with the iNKT cell-dependent generation of innate CD8(+) T cells reported in mice. All in all, our data demonstrate that CML is associated Innate-CD8(+) T-Cell Deficiency in CML Frontiers in Immunology | www.frontiersin.org January 2017 | Volume 7 | Article 688 inTrODUcTiOn A hallmark of the antigen-specific T lymphocytes of the adaptive immune system is their capacity to "remember" foreign pathogens long after they are first encountered. Indeed, as far as the CD8 T cell pool is concerned, the main features that provide antigen-specific memory CD8(+) T cells with a protective advantage over naive CD8+ T cells are that memory CD8+ T cells persist for extended periods of time, are present in larger numbers, and respond more rapidly to foreign antigen than naive CD8(+) T cells do. However, studies in mice have provided clear evidence that some naive CD8(+) T cells can acquire the characteristics and functions of memory CD8(+) T cells in the absence of foreign antigen encounters [reviewed in Ref. (1,2)]. Such cells, hence called innate/memory CD8(+) T cells, may develop in response to alterations in the environment or the presence of high levels of the cytokine IL-4, and have also been identified in unmanipulated animals. A hallmark of innate/memory CD8(+) T cells is their marked NK-like/memory phenotype associated with pronounced expression of the transcription factor eomesodermin (Eomes) (3,4). Even though the actual physiological significance of innate/memory CD8(+) T cells remains to be established, these cells have enhanced response potential, including the ability to efficiently combat pathogens (5,6). Moreover, because these cells can rapidly produce large amounts of IFN-γ in response to innate-like stimulation by IL-12 + IL-18 (7,8), they might contribute to the inflammation milieu during an early stage of immune response. The existence of innate/memory CD8(+) T cells in mice raises the question whether an equivalent to these innate T cells exists in humans. Earlier studies identified a subset of CD8(+) T cells in human peripheral blood that express NK-cell receptors such as killer cell Ig-like receptors (KIRs) (9)(10)(11). We further demonstrated that the majority of KIR/NKG2A(+) CD8(+) T cells have a memory phenotype and share functional and phenotypic features (12), see commentary (13): with innate/ memory T CD8(+) cells in mice (3,4,7,8). Indeed, they express high levels of Eomes and display innate functions, such as cytolytic capacity and rapid TCR-independent IFN-γ production in response to the innate cytokines IL-12 and IL-18. Regardless of their origin, KIR/NKG2A(+) Eomes(+) CD8(+) T cells harboring memory phenotype and innate-like functions have also been identified in human cord blood, suggesting that their development did not depend on cognate antigens. Additionally, the presence of these cells correlated well with expression of promyelocytic leukemia zinc finger (PLZF) among NKT cells in cord blood, a finding that is consistent with the hypothesis of a contribution of IL-4-producing, PLZF-expressing cells to their generation, as reported for their equivalents in mice (14,15). These features validate the existence of a new member of the innate T cell family in humans, which we have termed innate CD8(+) T cells. However, to date, even though evidence accounts for their potential physiological relevance, whether innate CD8(+) T cells can mediate potent immunity in humans remains to be investigated.
Chronic myeloid leukemia (CML) is a well-characterized myeloproliferative disorder, which results from dysregulated tyrosine kinase (TK) activity of the fusion oncoprotein BCR-ABL (16). Imatinib mesylate (IM), a competitive inhibitor of the BCR-ABL TK activity, is currently used as a first-line therapy for newly diagnosed patients in the chronic phase (CML-CP) (17). Overall, TK inhibitor (TKI) therapies have led to deep molecular responses in CML, dramatically improved life expectancy, and more recently, successful treatment-free survival has become a new goal (18). However, a significant proportion of patients will not fulfill the conditions allowing for treatment discontinuation while more than 40% of those eligible for treatment discontinuation relapse during the first 6 months after therapy cessation. Additional therapeutic strategies aiming at a potentiation and/ or restoration of immune antitumor functions, therefore, remain a main avenue of research, which may be helpful in long-term control of CML.
A sizable number of clinical and experimental data underscore the dysregulation of innate immune components in CML. These regulatory elements comprise dendritic cells (19), NK cells (20,21), and invariant natural killer T (iNKT) cells (22), an innate T cell subset co-expressing activated/memory and NK markers, which is well-recognized for its antitumor activity (23)(24)(25)(26). In accordance with this notion, we recently reported CML immune subversion of iNKT-cell activities in CML patients at diagnosis (22,27), including reduced or suppressed expression of perforin, CD95L, and (PLZF), a transcription factor required for maintenance of iNKT cell functions (28,29). Remarkably, these functional deficiencies were shown to have been partially repaired in patients having achieved complete cytogenetic remission (CCyR) following TKI therapy (22).
In mice, IL-4-producing/PLZF-expressing cells, including iNKT cell, have been demonstrated to regulate the generation of innate/memory CD8(+) T cells (14,15). Considering the functional deficiencies of iNKT cells in CML-CP patients at diagnosis and the fact that IL-4 production by iNKT cells was partially restored in patients having achieved CCyR after TKI therapy (22), we surmised that innate CD8(+) T cells might also be altered during CML and be restored in CML patients with CCyR. We further attempted to correlate the presence of innate CD8(+) T cells with that of iNKT cells to estimate the possible implication in humans of the peripheral iNKT cell pool in the development of its CD8(+) T-cell counterpart.
with deficiencies of innate CD8(+) T cells that are restored upon remission, thereby suggesting their possible contribution to disease control. More generally, our study strongly supports the existence of an innate iNKT/innate CD8(+) T-cell axis in humans and reveals its potential contribution to the restoration of tumor immune surveillance.

Peripheral Blood Mononuclear cells (PBMcs)
Venous blood from CML-CP patients at diagnosis or having achieved a major molecular response and currently treated with IM (CML-IM) was collected on heparin (Oncology-Hematology Department, Poitiers, France). All patients gave informed consent in accordance with the Declaration of Helsinki for participation in the study, which was approved by the scientific committee of the INSERM CIC-1402 (Poitiers, France). Healthy donors (HDs) were volunteers from the Pôle Biologie Santé (Poitiers, France). PBMCs were isolated from blood samples by density gradient centrifugation (Histopaque ® -1077, Sigma-Aldrich), resuspended in 90% fetal calf serum with 10% DMSO, and placed in a controlled rate freezer for cryopreservation at −80°C until use.

cell culture and Functional assays
All cell cultures (1 × 10 6 cells/mL) were performed in RPMI 1640 medium supplemented with 10% heat-inactivated FCS and antibiotics. For IL-12 + IL-18 stimulation, PBMCs from HD or CML patients were seeded at 1 × 10 6 cells/mL into 24-well plates and incubated for 48 h with 20 ng/mL of each cytokine (R&D Systems). Golgistop (BD Biosciences) was added for the last 5 h of culture. CD107a degranulation assays were performed as previously described (12). Briefly, PBMCs were seeded into 96-well round (U) bottom culture plates, preincubated for 48 h with IL-15 (20 ng/mL, R&D Systems) prior to CD16 triggering, and Golgistop (BD Biosciences) was added in the last 4 h of culture. For IL-4 stimulation, PBMCs from HDs were seeded at 0.5 × 10 6 cells/mL into 24-well plates and incubated for 7 days with 20 ng/mL of recombinant human IL-4 (R&D Systems).

statistical analysis
Statistical analyses were performed using GraphPad Prism version 6.0 (GraphPad Software). The statistical significance of differences in mean values was analyzed by the Mann-Whitney or Wilcoxon non-parametric test. The correlation Spearman test was used to test the association between the ranked variables Eomes and PLZF. Results were considered to be statistically significant when p < 0.05.

Positive correlation between inKT cell PlZF expression and innate cD8(+) T cell eomes expression
We have previously reported that IL-4 production by iNKT cells returned to normal in patients having achieved complete CML remission with TKI therapy (22). Given that, in mice, the differentiation of innate CD8(+) T cells depends mainly on PLZF-expressing iNKT cells via their IL-4 production (14, 15); we reasoned that the same phenomenon might be applied to humans. In accordance with this notion, we found a significant positive correlation between the levels of Eomes in KIR/NKG2A(+) CD8(+) T cells and of PLZF in iNKT cells including all the HD, CML-CP, and CML-IM samples available ( Figure 4A). Moreover, we found that after 7 days of culture in the presence of IL-4, recovery of CD8(+) T cells was slightly, but significantly, increased both in terms of frequency and numbers as compared to the total CD3(+) CD8(+) cells (Figures 4B,C). We also confirmed in humans that IL-4 strongly enhances Eomes expression both in total CD3(+) CD8(+) cells and in innate CD8(+) T cells (Figure 4D). Taken together, these findings support the possible involvement of iNKT cells through their IL-4 production in the generation/maintaining of innate CD8(+) T cells in CML patients.

DiscUssiOn
Although the concept of innate memory CD8(+) T cells is now well established in mice, whether an equivalent T-cell population exists in humans remains under debate. We recently reported that CD8(+) T cells co-expressing Eomes and KIR/NKG2A may represent a new, functionally distinct innate T cell subset in humans. Here, by extending our study to CML, we report that this disease is associated with an acquired and reversible defect of innate CD8(+) T cells. To the best of our knowledge, these findings are the first providing insights into the potential role of innate CD8(+) T cells in a physiopathological context in humans.
The finding that CML, at diagnosis is closely associated with a profound quantitative and functional deficiency in the innate CD8(+), T cell pool is in line with our hypothesis that this new subset may be involved during tumorigenesis in humans. This assumption was earlier based on our demonstration of terminally differentiated effector features of innate CD8(+) T cells in humans, such as rapid production of IFN-γ and induction of cytolytic function upon stimulation in vitro. In the present study, our revelation of the profound impact of CML on these potential antitumoral functions of innate CD8(+) T cells, together with those of NK (see Figure S2 in Supplementary Material) and iNKT cells (22,27), support a role in cancer immune surveillance of innate CD8(+) T cells analogous to the two other innate cell pools. Even though these findings corroborate the concept that innate CD8(+) T cells, like iNKT cells, may act against tumors in a TCR-independent and NK-like manner, the possibility that these cells recognize leukemia cells cannot be excluded and deserves further investigation.
Remarkably, deficiencies of innate CD8(+) T cells found at diagnosis in CML patients were significantly reversed upon remission following TKI therapy. These data are consistent with the hypothesis that reconstitution of the pool of innate CD8(+) T cells and its partial functional restoration together with those of NK cells and iNKT cells effectively contribute to disease control of CML during TKI therapy. To definitively confirm this assumption, it will be important to further evaluate whether the emergence of innate CD8(+) T cells in terms of both number and functional potential is closely related to long-term remission after treatment discontinuation.
To date, in mice, even though the effector functions of innate/ memory CD8(+) T cells have been extensively tested in vitro, their actual role during immune responses remains poorly documented. Testing the in vivo response following infection with the bacteria Listeria monocytogenes, innate/memory CD8(+) T cells were found to have a protective function (5,6). As far as immune responses against tumors are concerned, only in lymphodepleted mice, does there exist evidence that these cells can enhance response to tumors (30)(31)(32)(33). Given our findings, it remains to be directly determined, using experimental tumor models in mice, whether or not innate CD8(+) T cells exert protective functions.
The precise mechanisms for the generation and/or maintenance of innate CD8(+) T cells in humans remain to be determined. In the mouse, a key role in Eomes upregulation has been attributed to PLZF-expressing T cells. Similarly, our data in peripheral blood show a close correlation between the levels of Eomes in innate CD8(+) T cells and of PLZF in iNKT cells. Moreover, expression of PLZF and Eomes in iNKT cells and innate CD8(+) T cells, respectively, were both found to be reduced in patients at CML diagnosis, and reversed after disease remission, as were innate CD8(+) T cells in terms of both number and functions. Taken together, these findings are consistent with the existence of a PLZF-dependent mechanism in humans, similar to that reported in mice. It remains to determine whether, as in mice, PLZF-expressing T cells, especially iNKT cells, promote the development of blood peripheral innate T CD8(+) T cells by providing IL-4. In accordance with this view, we recently demonstrated a functional deficiency of IL-4 production by iNKT cells in CML patients at diagnosis and its partial restoration in patients having achieved remission after TKI therapy (22).
Further investigations are required to elucidate the exact mechanisms accounting for the deficiency of innate CD8(+) T cells at diagnosis and the beneficial effect of TKI therapy on this pool of cells. Nonetheless, from our data, it is tempting to speculate that the dysfunctions of innate CD8(+) T cells in CML patients at diagnosis that are remedied by TKI therapy originate from antigen APC-dependent dysfunctions, which in turn might contribute to iNKT cell deficiencies. This hypothesis arises from our recent findings showing that BCR-ABL from CML myeloid DCs mediates iNKT-cell immune subversion by downregulating cell-surface CD1d expression (27). Another non-exclusive mechanism may involve IL-15, which is required for maintenance not only of conventional memory CD8(+) T cells (34,35) but also innate/memory CD8(+) T cells (32,36). Indeed, CD8α(+) DC trans-presentation of IL-15 contributes to development of innatelike CD8(+) T cells in the periphery (37). For these reasons, the possible existence of a deficit in IL-15 expression and/or in IL-15 trans-presentation by mDCs from CML-CP patients is deserving of special attention.
All in all, by revealing an iNKT/innate CD8(+) T-cell axis with expected physiopathological relevance in CML, our findings should enhance understanding of the T cell components restored in CML treatment that contribute to disease control. From a more general standpoint, our study underscores the potential role of innate CD8(+) T cells against tumors in conjunction with iNKT cells and may contribute significantly to our understanding of the role of these innate T cell subsets in the development of protective immunity in humans.
aUThOr cOnTriBUTiOns FJ, EC, and AB designed the experiments, performed the experiments, analyzed and interpreted the data, and wrote the manuscript. DD, AL, and SB designed the experiments, performed the experiments, and analyzed and interpreted the data. AR, NP, and LL provided assistance with cell cultures. CG contributed to PBMC preparation from patients and healthy controls. FG and LR provided clinical samples and contributed to the interpretation of data. AH and J-MG together were responsible for the overall study design, supervised the project, and took primary responsibility for writing the manuscript.