CD8⁺ memory T (T_M) cells play a critical role in immune defense against infection. Two common γ-chain family cytokines, IL-2 and IL-7, although triggering the same mTORC1–S6K pathway, distinctly induce effector T (T_E) cells and T_M cells, respectively, but the underlying mechanism(s) remains elusive. In this study, we generated IL-7R-/and AMPKα1-knockout (KO)/OTI mice. By using genetic and pharmaceutical tools, we demonstrate that IL-7 deficiency represses expression of FOXO1, TCF1, p-AMPKα1 (T₁₇₂), and p-ULK1 (S₅₅₅) and abolishes T cell memory differentiation in IL-7R KO T cells after Listeria monocytogenesis rLmOVA infection. IL-2– and IL-7–stimulated strong and weak S6K (IL-2/S6K^strong and IL-7/S6K^weak) signals control short-lived IL-7R⁻CD62L⁻KLRG1⁺ T_E and long-term IL-7R⁺CD62L⁺KLRG1⁻ T_M cell formations, respectively. To assess underlying molecular pathway(s), we performed flow cytometry, Western blotting, confocal microscopy, and Seahorse assay analyses by using the IL-7/S6K^weak–stimulated T_M (IL-7/T_M) and the control IL-2/S6K^strong–stimulated T_E (IL-2/T_E) cells. We determine that the IL-7/S6K^weak signal activates transcriptional FOXO1, TCF1, and Id3 and metabolic p-AMPKα1, p-ULK1, and ATG7 molecules in IL-7/T_M cells. IL-7/T_M cells upregulate IL-7R and CD62L, promote mitochondria biogenesis and fatty acid oxidation metabolism, and show long-term cell survival and functional recall responses. Interestingly, AMPKα1 deficiency abolishes the AMPKα1 but maintains the FOXO1 pathway and induces a metabolic switch from fatty acid oxidation to glycolysis in AMPKα1 KO IL-7/T_M cells, leading to loss of cell survival and recall responses. Taken together, our data demonstrate that IL-7–stimulated weak strength of mTORC1–S6K signaling controls T cell memory via activation of transcriptional FOXO1–TCF1–Id3 and metabolic AMPKα1–ULK1–ATG7 pathways. This (to our knowledge) novel finding provides a new mechanism for a distinct IL-2/IL-7 stimulation model in T cell memory and greatly impacts vaccine development. Key Points IL-2 and IL-7 induce S6K^strong and S6K^weak signals, leading to forming T_E and T_M cells. IL-7–stimulated S6K^weak signaling controls T cell memory via FOXO1 and AMPK pathways.

Energy sensors mTORC1 and AMPKα1 regulate T-cell metabolism and differentiation, while rapamycin (Rapa)-inhibition of mTORC1 (RIM) promotes T-cell memory. However, the underlying pathway and the role of AMPKα1 in Rapa-induced T-cell memory remain elusive. Using genetic and pharmaceutical tools, we demonstrate that Rapa promotes T-cell memory in mice in vivo post Listeria monocytogenesis rLmOVA infection and in vitro transition of effector T (T_E) to memory T (T_M) cells. IL-2- and IL-2+Rapa-stimulated T [IL-2/T and IL-2(Rapa+)/T] cells, when transferred into mice, differentiate into short-term IL-7R⁻CD62L⁻KLRG1⁺ T_E and long-lived IL-7R⁺CD62L⁺KLRG1⁻ T_M cells, respectively. To assess the underlying pathways, we performed Western blotting, confocal microscopy and Seahorse-assay analyses using IL-2/T and IL-2(Rapa+)/T-cells. We determined that IL-2(Rapa+)/T-cells activate transcription FOXO1, TCF1 and Eomes and metabolic pAMPKα1(T₁₇₂), pULK1(S₅₅₅) and ATG7 molecules and promote mitochondrial biogenesis and fatty-acid oxidation (FAO). We found that rapamycin-treated AMPKα-deficient AMPKα1-KO IL-2(Rapa+)/T_M cells up-regulate transcription factor HIF-1α and induce a metabolic switch from FAO to glycolysis. Interestingly, despite the rapamycin treatment, AMPKα-deficient T_M cells lost their cell survival capacity. Taken together, our data indicate that rapamycin promotes T-cell memory via transcriptional FOXO1-TCF1-Eomes programs and AMPKα1-ULK1-ATG7 metabolic axis, and that AMPKα1 plays a critical role in RIM-induced T-cell memory.

Two common γ-chain family cytokines IL-2 and IL-15 stimulate the same mammalian target of rapamycin complex-1 (mTORC1) signaling yet induce effector T (T_E) and memory T (T_M) cell differentiation via a poorly understood mechanism(s). Here, we prepared in vitro IL-2-stimulated T_E (IL-2/T_E) and IL-15-stimulated T_M (IL-15/T_M) cells for characterization by flow cytometry, Western blotting, confocal microscopy and Seahorse-assay analyses. We demonstrate that IL-2 and IL-15 stimulate strong and weak mTORC1 signals, respectively, which lead to the formation of CD62 ligand (CD62L)⁻ killer cell lectin-like receptor subfamily G member-1 (KLRG)⁺ IL-2/T_E and CD62L⁺KLRG⁻ IL-15/T_M cells with short- and long-term survival following their adoptive transfer into mice. The IL-15/mTORC1^Weak signal activates the forkhead box-O-1 (FOXO1), T cell factor-1 (TCF1) and Eomes transcriptional network and the metabolic adenosine monophosphate-activated protein kinase-α-1 (AMPKα1), Unc-51-like autophagy-activating kinase-1 (ULK1) and autophagy-related gene-7 (ATG7) axis, increasing the expression of mitochondrial regulators aquaporin-9 (AQP9), mitochondrial transcription factor-A (TFAM), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), carnitine palmitoyl transferase-1 (CPT1α), microtubule-associated protein light chain-3 II (LC3II), Complex I and ortic atrophy-1 (OPA1), leading to promoting mitochondrial biogenesis and fatty-acid oxidation (FAO). Interestingly, AMPKα1 deficiency abrogates these downstream responses to IL-15/mTORC1^Weak signaling, leading to the upregulation of mTORC1 and hypoxia-inducible factor-1α (HIF-1α), a metabolic switch from FAO to glycolysis and reduced cell survival. Taken together, our data demonstrate that IL-15/mTORC1^Weak signaling controls T-cell memory via activation of the transcriptional FOXO1-TCF1-Eomes and metabolic AMPKα1-ULK1-ATG7 pathways, a finding that may greatly impact the development of efficient vaccines and immunotherapies for the treatment of cancer and infectious diseases.

Fluorescence imaging is a well-known method for monitoring fluorescence emitted from the subject of interest and provides important insights about cell dynamics and molecules in mammalian cells. Currently, many solutions exist for measuring fluorescence, but the application methods are complex and the costs are high. This paper describes the design and development of a low-cost, smart and portable fluorimeter for the detection of colorectal cancer cell expressing IRFP702. A flashlight is used as a light source, which emits light in the visible range and acts as an excitation source, while a photodiode is used as a detector. It also uses a longpass filter to only allow the wavelength of interest to pass from the cultured cell. It eliminates the need of both the dichroic mirror and excitation filter, which makes the developed device low cost, compact and portable as well as lightweight. The custom-built sample chamber is black in color to minimize interference and is printed with a 3D printer to accommodate the detector circuitry. An established colorectal cancer cell line (human colorectal carcinoma (HCT116)) was cultured in the laboratory environment. A near-infrared fluorescent protein IRFP702 was expressed in the colorectal cancer cells that were used to test the proof-of-concept. The fluorescent cancer cells were first tested with a commercial imaging system (Odyssey^® CLx) and then with the developed prototype to validate the result in a preclinical setting. The developed fluorimeter is versatile as it can also be used to detect multiple types of cancer cells by simply replacing the filters based on the fluorophore.