CD161 Expression Identifies a Distinct Subset Of Drug-Effluxing Viral-Specific Memory CD4+ T Cells That Preferentially Survive Cytotoxic Chemotherapy
Authors: Muharrem Muftuoglu MD , Alsuliman Abdullah , Khoder Ahmad , Ahn Yong-Oon , Liu Enli , Chew Claude , Hamdi Amir , Sekine Takuya , Sarvaria Anushruti , Cooper Nichola , de Lavallade Hugues , Marin David , Stringaris Kate , Verneris Michael R. , Shpall Elizabeth J , Rezvani Katayoun 
Published: October 2019

Abstract
CD4+ T cells are important in the establishment of long-lived pathogen-specific immunity. However, the mechanisms by which antigen specific CD4 T resist insult by lymphocytotoxic agents and are sustained long-term is not well defined. A recent report described the existence of a subset of long-lived CD8+ memory T cells with stem-like properties (Turtle et al, 2009), including the ability to efflux cellular toxins through the ABC–superfamily multidrug efflux protein ABCB1. We hypothesized that a similar subset of T cells with drug-effluxing properties also exists within the CD4+ T cell compartment. We used multiparameter flow cytometry to measure the capacity of CD4+ T cells from donors to efflux the fluorescent substrate Rh123. We identified a subset of memory CD4+ T cells with rapid drug-effluxing ability, defined as CD161+CD95+CD45RA-CD127hiCD28+CD25int that shared remarkable phenotypic similarity to CD8+drug-effluxing memory T cells. The stem cell marker c-kit was preferentially expressed on Rh123 effluxing CD4+CD161+ T cells, whereas CD57, a marker of terminal differentiation, was exclusively expressed on non-effluxing CD4+CD161+ T cells. Rh123 effluxing CD4+CD161+ T cells also displayed differential expression of CD31, CD38, CD58, CD122 and IL-18RA.
Rh123 effluxing CD4+ CD161+ T cells were undetectable in cord blood, but found in adult blood, consistent with the emergence of this subset of memory T cells as a consequence of antigen exposure during childhood and adult life. We reasoned that this subset may be enriched within the viral-specific T cell repertoire. Indeed, CMV-specific CD4+ T cells were found to share the same phonotypic markers as Rh123 effluxing CD4+CD161+ T cells. We purified CMV-specific CD4+ T cells using the interferon gamma capture assay (Miltenyi), and showed that CMV-enriched CD4+T cells preferentially and rapidly efflux Rh123.
The high ABCB1-mediated drug efflux capacity of CD4+ CD161+ memory cells also facilitated their in vitro resistance to daunorubicin, which was abrogated by competitive inhibitors of ABCB1. In keeping with the in vitro data, we found a significant increase in the frequencies of CMV-specific CD4+ T cells in the peripheral blood of patients with AML after recovery from remission induction chemotherapy, suggesting that CMV-specific CD4+ T cells can preferentially survive and proliferate following chemotherapy. Since interleukin (IL)-7 and IL-15 drive the proliferation of T cells during lymphopenia to restore homeostasis, we assessed the response of CD4+CD161+ T cells to stimulation with CD3/CD28 +IL7 and IL15. Both effluxing and non-effluxing sort-purified central and effector memory CD4+CD161+ T cells proliferated and upregulated Ki67 in vitro. Whereas CD4+CD161+ T cells were able to differentiate into CD4+CD161- T cells, a subset retained CD161 expression. These data suggest that although CD4+CD161+ T cells share phenotypic similarities with terminally differentiated cells, they are able to fully proliferate, differentiate to CD161-ve cells and self-renew to preserve the pool of memory T cells
CD161 is also a hallmark of Th17 cells. We examined the cytokine profile of CD4+CD161+ T cells stimulated with a pool of overlapping MHC class II CMV pp65 peptides. After 6 and 24 hrs of in vitro stimulation we failed to detect significant IL-17 production. Furthermore, by real time qPCR, the Th1 transcription factor Tbet, rather than RORC2 (a Th17 hallmark), was found to be preferentially expressed in CMV enriched CD4+CD161+ T cells, indicating that CMV-specific CD4+CD161+T cells in fact represent a unique subset of Th1 cells, distinct from Th17 cells.
Our data delineate novel findings related to a distinct subset of drug-effluxing CD4+CD161+ viral-specific memory T cells. Signaling pathways leading to CD4+CD161+ABCB1+ differentiation, the role of this subset in drug resistance and the presence or absence of “stemness” which may impart this subset with extended longevity are being explored.
†Muharrem Muftuoglu and Abdullah Alsuliman contributed equally to this work.

Disclosures:
No relevant conflicts of interest to declare.