Tuning of Human Mucosal Associated Invariant T (MAIT) Cell Function through Microbiota-Mediated T Cell Receptor Signals

Mucosal-associated invariant T (MAIT) cells are a subset of the T cell population defined by an invariant T cell receptor (TCR) that is stimulated by bacterial metabolites. These cells preferentially migrate to mucosal tissues such as gut, liver and skin. MAIT cells are activated by metabolites from the riboflavin (Vitamin B2) biosynthetic pathway that are presented by MHC class 1-related (MR1) molecules expressed on numerous of cell types including antigen presenting cells (APCs) and epithelial cells. The V alpha segment of MAIT TCRs is invariant and composed of Vα7.2, whereas the V beta portion can be variable. MAIT cells are present at very low levels in neonatal blood but significantly increase in adults and show very high variance in their frequency among individuals. While there is evidence that several pathogenic bacteria can activate MAIT cells and that one of their functions is to kill cells with intracellular bacteria, it remains unknown how they discriminate among thousands of commensal bacteria that can also produce Vitamin B2 metabolites. Further, the role of the TCR variable beta region in antigen recognition is still unclear. In this thesis project, we hypothesized that MAIT cells can be stimulated by bacteria that inhabit human mucosal tissues, and that this microbiota-MAIT cell interaction is one of the driving forces in their expansion and variation in the human population. In support of this hypothesis, we observed a reduced proportion of MAIT cells in the blood of perinatally HIV-infected children, which correlated with other microbiota-associated parameters including Th17 cells and inflammatory markers of microbiota alterations. However, it is unclear whether MAIT cells can discriminate bacterial species that reside in the human microbiota, which can produce the riboflavin intermediates. To address this, we developed an in vitro functional assay using human T cells engineered for MAIT-specific TCRs (eMAIT-TCRs) stimulated by MR1-expressing B cell lines presenting the bacterial metabolites. We then screened 47 microbiota-associated bacterial species from different phyla for their eMAIT-TCR stimulatory capacities. Only bacterial species that encoded a riboflavin biosynthesis pathway were stimulatory for MAIT-TCRs. Most species that were high-stimulators belonged to Bacteroidetes and Proteobacteria phyla, although with significant variance, whereas low/non-stimulator species were either Actinobacteria or Firmicutes. Furthermore, we determined a wide range of intra-species variation in eMAIT-TCR stimulation capacities of Staphylococcus epidermidis , a skin commensal, which suggests that bacteria can modulate the production capacities of MAIT-TCR stimulatory antigens. Remarkably, we also discovered that human T cells not only express low-levels of MR1 but can also present bacterial metabolites to MAIT cells in an MR1-restricted fashion and trigger TCR signaling to induce cytokine secretion (IFNγ and TNFα), albeit at lower levels. In conclusion, our findings revealed that MAIT cells could discriminate between MR1-restricted, bacteria-derived metabolites through their TCR signaling thresholds. This knowledge paves the way to elucidate the complexity of MAIT cell recognition and its response to the human microbiota, which establishes a framework to explore mechanistic or therapeutic approaches for maintenance of a healthy immunological equilibrium

CAR-T Cells with Phytohemagglutinin (PHA) Provide Anti-Cancer Capacity with Better Proliferation, Rejuvenated Effector Memory, and Reduced Exhausted T Cell Frequencies

The development of genetic modification techniques has led to a new era in cancer treatments that have been limited to conventional treatments such as chemotherapy. intensive efforts are being performed to develop cancer-targeted therapies to avoid the elimination of non-cancerous cells. One of the most promising approaches is genetically modified CAR-T cell therapy. The high central memory T cell (Tcm) and stem cell-like memory T cell (Tscm) ratios in the CAR-T cell population increase the effectiveness of immunotherapy. Therefore, it is important to increase the populations of CAR-expressing Tcm and Tscm cells to ensure that CAR-T cells remain long-term and have cytotoxic (anti-tumor) efficacy. In this study, we aimed to improve CAR-T cell therapy’s time-dependent efficacy and stability, increasing the survival time and reducing the probability of cancer cell growth. To increase the sub-population of Tcm and Tscm in CAR-T cells, we investigated the production of a long-term stable and efficient cytotoxic CAR-T cell by modifications in the cell activation-dependent production using Phytohemagglutinin (PHA). PHA, a lectin that binds to the membranes of T cells and increases metabolic activity and cell division, is studied to increase the Tcm and Tscm population. Although it is known that PHA significantly increases Tcm cells, B-lymphocyte antigen CD19-specific CAR-T cell expansion, its anti-cancer and memory capacity has not yet been tested compared with aCD3/aCD28-amplified CAR-T cells. Two different types of CARs (aCD19 scFv CD8-(CD28 or 4-1BB)-CD3z-EGFRt)-expressing T cells were generated and their immunogenic phenotype, exhausted phenotype, Tcm–Tscm populations, and cytotoxic activities were determined in this study. The proportion of T cell memory phenotype in the CAR-T cell populations generated by PHA was observed to be higher than that of aCD3/aCD28-amplified CAR-T cells with similar and higher proliferation capacity. Here, we show that PHA provides long-term and efficient CAR-T cell production, suggesting a potential alternative to aCD3/aCD28-amplified CAR-T cells

CAR-T Cells with Phytohemagglutinin (PHA) Provide Anti-Cancer Capacity with Better Proliferation, Rejuvenated Effector Memory, and Reduced Exhausted T Cell Frequencies

The development of genetic modification techniques has led to a new era in cancer treatments that have been limited to conventional treatments such as chemotherapy. intensive efforts are being performed to develop cancer-targeted therapies to avoid the elimination of non-cancerous cells. One of the most promising approaches is genetically modified CAR-T cell therapy. The high central memory T cell (Tcm) and stem cell-like memory T cell (Tscm) ratios in the CAR-T cell population increase the effectiveness of immunotherapy. Therefore, it is important to increase the populations of CAR-expressing Tcm and Tscm cells to ensure that CAR-T cells remain long-term and have cytotoxic (anti-tumor) efficacy. In this study, we aimed to improve CAR-T cell therapy’s time-dependent efficacy and stability, increasing the survival time and reducing the probability of cancer cell growth. To increase the sub-population of Tcm and Tscm in CAR-T cells, we investigated the production of a long-term stable and efficient cytotoxic CAR-T cell by modifications in the cell activation-dependent production using Phytohemagglutinin (PHA). PHA, a lectin that binds to the membranes of T cells and increases metabolic activity and cell division, is studied to increase the Tcm and Tscm population. Although it is known that PHA significantly increases Tcm cells, B-lymphocyte antigen CD19-specific CAR-T cell expansion, its anti-cancer and memory capacity has not yet been tested compared with aCD3/aCD28-amplified CAR-T cells. Two different types of CARs (aCD19 scFv CD8-(CD28 or 4-1BB)-CD3z-EGFRt)-expressing T cells were generated and their immunogenic phenotype, exhausted phenotype, Tcm–Tscm populations, and cytotoxic activities were determined in this study. The proportion of T cell memory phenotype in the CAR-T cell populations generated by PHA was observed to be higher than that of aCD3/aCD28-amplified CAR-T cells with similar and higher proliferation capacity. Here, we show that PHA provides long-term and efficient CAR-T cell production, suggesting a potential alternative to aCD3/aCD28-amplified CAR-T cells

Functional Interrogation of Primary Human T Cells via CRISPR Genetic Editing.

Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest. We showed that multiple genes

Tuning of human MAIT cell activation by commensal bacteria species and MR1-dependent T-cell presentation.

Human mucosal-associated invariant T (MAIT) cell receptors (TCRs) recognize bacterial riboflavin pathway metabolites through the MHC class 1-related molecule MR1. However, it is unclear whether MAIT cells discriminate between many species of the human microbiota. To address this, we developed an in vitro functional assay through human T cells engineered for MAIT-TCRs (eMAIT-TCRs) stimulated by MR1-expressing antigen-presenting cells (APCs). We then screened 47 microbiota-associated bacterial species from different phyla for their eMAIT-TCR stimulatory capacities. Only bacterial species that encoded the riboflavin pathway were stimulatory for MAIT-TCRs. Most species that were high stimulators belonged to Bacteroidetes and Proteobacteria phyla, whereas low/non-stimulator species were primarily Actinobacteria or Firmicutes. Activation of MAIT cells by high- vs low-stimulating bacteria also correlated with the level of riboflavin they secreted or after bacterial infection of macrophages. Remarkably, we found that human T-cell subsets can also present riboflavin metabolites to MAIT cells in a MR1-restricted fashion. This T-T cell-mediated signaling also induced IFNγ, TNF and granzyme B from MAIT cells, albeit at lower level than professional APC. These findings suggest that MAIT cells can discriminate and categorize complex human microbiota through computation of TCR signals depending on antigen load and presenting cells, and fine-tune their functional responses

Anti-cancer effect of metformin on the metastasis and invasion of primary breast cancer cells through mediating NF-kB activity

Current evidence strongly suggests that aberrant activation of the nuclear factor kappa B (NF-kB) signaling cascade is connected to carcinogenesis. The matrix metalloproteinases (MMP) which are also the key agents for tumor metastasis may be potent candidates for tumor diagnosis in clinics. In this in vitro study, we hypothesized that metformin with an effective dose can inhibit tumor cell proliferation and metastasis by modulating the expressions of MMP-2 and 9 and interfering with NF-kB signaling in primary breast cancer cells (PBCCs). 300 000 cells per ml were obtained from biopsies of breast tumors from five human donors. The cell viability and proliferation were tested. Immunocytochemistry was performed for MMP 2, MMP 9, and NF-kB, and enzyme-linked immunosorbent assay for NF-kB activity, quantitative real-time PCR for RELA/p65, IkBa, MMP-2, and MMP 9. Three different doses of metformin (5, 10, and 25 mM) (Met) reduced the viability and proliferation of PBCCs in a dose-dependent manner, maximum inhibition was observed at 25 mM Met. The expression of RELA/p65 was not affected by 25 mM Met. Nuclear immunoreactivity and activity of NF-kB reduced while cytoplasmic NF-kB (p65) elevated by 25 mM Met compared to non-treatment (P < 0.05). The expression and immunoreactivity of MMP 9 but not MMP 2 were decreased by 25 mM Met treatment, compared with the non-treatment (P < 0.05). Metformin may have an essential antitumor role in the invasion and metastasis pathways of PBCCs by downregulating the MMP 9 expression blocking both the activity and nuclear translocation of NF-kB

In Vitro FVIII-Encoding Transgenic Mesenchymal Stem Cells Maintain Successful Coagulation in FVIII-Deficient Plasma Mimicking Hemophilia A

Objective: Hemophilia A is an X-linked recessive bleeding disorder caused by a deficiency of plasma coagulation factor VIII (FVIII), and it accounts for about 80%-85% of all cases of hemophilia. Plasma-derived therapies or recombinant FVIII concentrates are used to prevent and treat the bleeding symptoms along with FVIIImimicking antibodies. Recently, the European Medicines Agency granted conditional marketing approval for the first gene therapy for hemophilia A. The aim of this study was to determine the effectiveness of coagulation in correcting FVIII deficiency with FVIII-secreting transgenic mesenchymal stem cells (MSCs). Materials and Methods: A lentiviral vector encoding a B domaindeleted FVIII cDNA sequence with CD45R0 truncated (CD45R0t) surface marker was designed to develop a transgenic FVIII-expressing primary cell line by transducing MSCs. The efficacy and functionality of the FVIII secreted from the MSCs was assessed with anti-FVIII ELISA, CD45R0t flow cytometry, FVIII western blot, and mixing test analysis in vitro. Results: The findings of this study showed that the transgenic MSCs maintained persistent FVIII secretion. There was no significant difference in FVIII secretion over time, suggesting stable FVIII expression from the MSCs. The functionality of the FVIII protein secreted in the MSC supernatant was demonstrated by applying a mixing test in coagulation analysis. In the mixing test analysis, FVIIIdeficient human plasma products were mixed with either a saline control or FVIII-secreted MSC supernatant. The mean FVIII level of the saline control group was 0.41±0.03 IU/dL, whereas the mean level was 25.41±33.38 IU/dL in the FVIII-secreting MSC supernatant mixed group (p<0.01). The mean activated partial thromboplastin time (aPTT) of the saline control group was 92.69±11.38 s, while in the FVIII-secreting MSC supernatant mixed group, the mean aPTT level decreased to 38.60±13.38 s (p<0.001). Conclusion: The findings of this in vitro study suggest that the new method presented here is promising as a possible treatment for hemophilia A. Accordingly, a study of FVIII-secreting transgenic MSCs will next be initiated in a FVIII-knockout animal model

Subject Characteristics.

<p>Subject Characteristics.</p

Lower CD8 MAIT cell frequencies correlate with loss of Th17 and Th22 subsets in HIV+ children.

<p>(A) FACS plot showing representative gating to identify Th17 and Th22 subsets in an HIV- and HIV+ subject. Plots shown are gated within CR45RO+ (memory) CD4+ T cells. (B) Comparisons of IL-17A⁺IFNγ^- and IL-17A⁺IFNγ⁺ memory CD4 T cells in HIV-, ART-, and ART+ children. (C) Comparisons of IL-22⁺IL-17A^- memory CD4 T cells in HIV-, ART-, and ART+ children. Correlations between CD8+ MAIT cells and (D) IL-17A⁺IFNγ^- and IL-17A⁺IFNγ⁺ memory CD4 T cells and (E) IL-22⁺IL-17A^- memory CD4 T cells in HIV+ (closed circles) and HIV- (open circles) children. All cytokine populations were gated within memory CD4+ T cells.</p