FIGURE 1. B2M-deficient T cells evade alloreactive T cells but are susceptible to NK cell–mediated lysis. ( A ) Schematic demonstrating that the status of HLA class I expression dictates susceptibility of allogeneic T cells to rejection by T cells or NK cells from an unrelated donor. ( B ) Schematic showing B2M-specific single-guide RNA (sgRNA) disrupting the splice donor site at the exon/intron junction (B2M^KO). ( C ) Frequency of on-target A > G nucleotide conversion by next-generation sequencing in primary human T cells base-edited with ABE8.20m mRNA and B2M-specific sgRNA. Symbols indicate four independent T cell donors. ( D ) Histogram shows cell surface expression of HLA class I in B2M^KO and unmodified T cells. ( E and F ) T cell mixed leukocyte assay as described in Materials and Methods. FACS plots (E) and summarized data (F) are shown for the frequency of B2M^KO and unmodified T cells at 48 h after culture in triplicate at the indicated E:T ratios with HLA-mismatched CD8⁺ T cells that were labeled with CellTrace Violet. ( G ) Frequency of CD107a⁺ NK cells after stimulation with B2M^KO and unmodified T cells. Symbols represent three independent NK cell donors in duplicate. ( H and I ) NK cell cytotoxicity assay as described in Materials and Methods. FACS plots indicate frequency (H) and summarized data indicate NK cell–dependent lysis (I) of B2M^KO and unmodified T cells at 48 h after culture with NK cells at the indicated E:T ratios. Symbols represent four independent NK cell donors in duplicate. For all data, bars represent mean and error bars indicate ±SEM. More about this image found in B2M-deficient T cells evade alloreactive T cells but are s...

FIGURE 2. scHLA-E⁴ exhibits improved cell surface expression and inhibits NK cell–driven lysis. ( A ) Schematic demonstrating that expression of an HLA-E single-chain (sc) molecule in B2M^KO T cells inhibits NK cells by engaging the NKG2A/CD94 heterodimer. ( B ) Schematic detailing components of scHLA-E fusion constructs, including scHLA-E dimer (D), scHLA-E trimer (T), and scHLA-E molecules containing the CD8α (8) and CD4 (4) transmembrane domain and truncated cytoplasmic regions. ( C and D ) B2M^KO T cells were transduced with an equivalent titer of lentivirus that encoded a unique scHLA-E variant and mDHFR. (C) FACS plots demonstrate the cell surface expression of the scHLA-E variants. (D) Summarized data show the geometric median fluorescence intensity (GMFI) of HLA-E on total nontransduced B2M^KO T cells, and HLA-E–positive unmodified T cells and B2M^KO T cells that were transduced with scHLA-E⁴ variants. Nontransduced B2M^KO T cells served as a gating control to discriminate positive and negative HLA-E expression. Symbols represent four independent T cell donors in duplicate at 11 d posttransduction. ( E and F ) Histograms (E) and summarized data (F) indicate GMFI of scHLA-E variants bound to recombinant human NKG2A/CD94 heterodimer. Symbols represent three independent T cell donors. ( G and H ) Frequency of NKG2A⁺ NK cells and total CD56⁺ NK cells that upregulated expression of CD107a (G) and TNF-α (H) after stimulation with B2M^KO T cells from three independent donors that were nontransduced or expressed scHLA-E⁴. Symbols represent eight independent NK cell donors in duplicate. ( I ) Correlation between frequency of NKG2A⁺ NK cells and percentage change in frequency of total responding NK cells after stimulation with scHLA-E⁴B2M^KO T cells from stimulation with nontransduced B2M^KO T cells. Symbols represent eight independent NK cell donors. ( J ) NK cell cytotoxicity assay as described in Materials and Methods. NK cell–dependent lysis of B2M^KO T cells from three independent donors that were nontransduced or expressed scHLA-E⁴ at 48 h after culture with NK cells at the 4:1 E:T ratio. Symbols represent five independent NK cell donors in duplicate. For all data, bars represent mean and error bars indicate ±SEM. Statistical significance was calculated by a Wilcoxon matched-pairs signed rank test (D, F, G, H, and J) and Spearman correlation (I). More about this image found in scHLA-E⁴ exhibits improved cell surface expression and inhibits ...

FIGURE 3. VL9 epitope variants modulate surface expression and NK cell inhibitory potential of scHLA-E⁴ molecules. ( A ) Schematic of a scHLA-E⁴ molecule and VL9 epitope variants derived from signal sequences of HLA-G*01 and representative HLA class Ia alleles. ( B and C ) B2M^KO T cells were transduced with an equivalent titer of lentivirus that encoded a unique scHLA-E⁴ VL9 variant, and HLA-E cell surface expression was quantified at 11 d posttransduction. (B) FACS plots indicate cell surface expression of each scHLA-E⁴ VL9 variant and corresponding GMFI of HLA-E on transduced B2M^KO T cells. (C) Heatmap shows GMFI of HLA-E on total nontransduced B2M^KO T cells and HLA-E–positive unmodified T cells and B2M^KO T cells expressing each scHLA-E⁴ VL9 variant at 11 d posttransduction. Nontransduced B2M^KO T cells served as a gating control to discriminate positive and negative HLA-E expression. Data represent the mean of four independent T cell donors in duplicate. ( D – H ) NK cells from unrelated donors were stimulated with unmodified T cells or B2M^KO T cells from three independent donors that were nontransduced or expressed unique scHLA-E⁴ VL9 variants. (D) FACS plots indicate frequency of NKG2A⁺ NK cells that express CD107a and TNF-α poststimulation with B2M^KO T cells that were nontransduced or expressed scHLA-E^4v4. (E–H) Summarized data indicate the frequency of CD107a⁺ (E) and TNF-α⁺ (F) NKG2A⁺ NK cells, and CD107a⁺ (G) and TNF-α⁺ (H) total CD56⁺ NK cells. Symbols represent data from eight (E and F) and seven (G and H) independent NK cell donors in duplicate. ( I ) NK cell cytotoxicity assay as described in Materials and Methods. NK cell–dependent lysis of B2M^KO T cells from four independent donors that were nontransduced or expressed unique scHLA-E⁴ VL9 variants at 48 h after culture with NK cells at the 4:1 E:T ratio. Symbols represent seven independent NK cell donors in duplicate. For all data, bars represent mean and error bars indicate ±SEM. For (C), a Wilcoxon matched-pairs signed rank test was used to calculate significance. For (E)–(I), statistical significance between B2M^KO T cells that were nontransduced and each scHLA-E⁴ VL9 variant was calculated using a Wilcoxon matched-pairs signed rank test, whereas the Friedman test with a Dunn test for multiple comparisons calculated statistical significance between the scHLA-E⁴ VL9 variants. More about this image found in VL9 epitope variants modulate surface expression and NK cell inhibitory pot...

FIGURE 4. Disulfide trap mutations improve surface expression and NK cell inhibitory potential of scHLA-E⁴ variants. ( A ) Schematic of scHLA-E⁴ molecule that is either nonmutated or comprises the disulfide trap (dt) mutations in the (G₄S)₃ flexible linker and HLA-E H chain. ( B ) B2M^KO T cells were transduced with an equivalent titer of lentivirus that encoded a unique scHLA-E⁴ and dtHLA-E⁴ VL9 variant. HLA-E cell surface expression was quantified as GMFI of HLA-E on HLA-E–positive B2M^KO T cells that were transduced. Nontransduced B2M^KO T cells served as a gating control to discriminate positive and negative HLA-E expression. Symbols represent four independent T cell donors in duplicate at 11 d posttransduction. ( C and D ) Frequency of CD107a⁺ (C) and TNF-α⁺ (D) NKG2A⁺ NK cells poststimulation with unmodified T cells or B2M^KO T cells that were nontransduced or expressed a unique dtHLA-E⁴ VL9 variant from five (C) and three (D) independent donors. Symbols represent 12 (C) and 8 (D) independent NK cell donors in duplicate. ( E ) NK cell cytotoxicity assay as described in Materials and Methods. NK cell–dependent lysis of B2M^KO T cells that were nontransduced or expressed a unique dtHLA-E⁴ VL9 variant from four independent donors at 48 h after culture with NK cells at the 4:1 E:T ratio. Symbols represent seven independent NK cell donors in duplicate. ( F and G ) Frequency of CD107a⁺ (F) and TNF-α⁺ (G) NKG2A⁺ NK cells poststimulation with B2M^KO T cells that expressed a unique scHLA-E⁴ VL9 variant or the corresponding dtHLA-E⁴ VL9 variant from three independent donors. Symbols represent eight independent NK cell donors in duplicate. ( H ) NK cell cytotoxicity assay as described in Materials and Methods. NK cell–dependent lysis of B2M^KO T cells that expressed a unique scHLA-E⁴ VL9 variant or the corresponding dtHLA-E⁴ VL9 variant from four independent donors at 48 h after culture with NK cells at the 4:1 E:T ratio. Symbols represent six independent NK cell donors in duplicate. For all data, bars represent mean and error bars indicate ±SEM. For (B)–(H), a Wilcoxon matched-pairs signed rank test was used to calculate significance. For (E), a Friedman test with a Dunn test for multiple comparisons calculated statistical significance between dtHLA-E⁴ VL9 variants. More about this image found in Disulfide trap mutations improve surface expression and NK cell inhibitory ...

FIGURE 5. dtHLA-E⁴ molecules bound to HLA-A and HLA-C–derived VL9 epitopes mitigate activation of NKG2C⁺ NK cells. ( A ) FACS plot indicates binding of recombinant human NKG2C/CD94 heterodimer to unmodified T cells and B2M^KO T cells that expressed dtHLA-E^4v1. ( B and C ) FACS plots (B) and summarized data (C) show the frequency of CD107a⁺NKG2C⁺ NK cells after stimulation with B2M^KO T cells that were nontransduced or expressed a unique dtHLA-E⁴ VL9 variant from three independent donors. Symbols represent six independent NK cell donors in duplicate. ( D and E ) FACS plots (D) and summarized data (E) indicate the frequency of NKG2A and NKG2C expression of total NK cells from donors classified as NKG2C^low and NKG2C^high based on frequency of NKG2C⁺ NK cells being less than or greater than 10%, respectively. Each symbol represents an independent NK cell donor. ( F ) NK cell cytotoxicity assay as described in Materials and Methods. NK cell–dependent lysis of B2M^KO T cells that were nontransduced or expressed a unique dtHLA-E⁴ VL9 variant from three independent donors at 48 h after culture with NK cells from NKG2C^high donors at the 4:1 E:T ratio. Symbols represent four independent NK cell donors in duplicate. For all data, bars represent mean and error bars indicate ±SEM. For (C) and (F), a Wilcoxon matched-pairs signed rank test was used to calculate significance between nontransduced and dtHLA-E⁴ variant–expressing B2M^KO T cells. For (C), a Friedman test with a Dunn test for multiple comparisons calculated statistical significance between dtHLA-E⁴ VL9 variants. More about this image found in dtHLA-E⁴ molecules bound to HLA-A and HLA-C–derived VL9 epitopes...

FIGURE 6. Hypoimmunogenic dtHLA-E^4v4 evades HLA-E–reactive CD8⁺ T cell responses. ( A ) Frequency of responding CD107a⁺ and TNF-α⁺ CD8⁺ T cells after in vitro stimulation with the indicated aAPC.dtHLA-E⁴ variant. ( B and C ) T cells were stimulated with aAPCs expressing a unique dtHLA-E⁴ variant and cultured for 1 wk. FACS plots (B) and summarized data (C) indicate the frequency of CD107a⁺ and TNFα⁺ CD8⁺ T cells after restimulation with the appropriate aAPC and K.86 cell lines. ( D–F ) T cells were stimulated with K.86 cells, aAPC.hypoA2, and aAPC.scA2 cells and then cultured for 1 wk. (D) Total CD8⁺ T cells were enumerated at 1 wk poststimulation. Dotted line indicates average CD8⁺ T cell count when cultured in the absence of K-562 cells. (E and F) Frequency of CD107a⁺ (E) and TNF-α⁺ (F) CD8⁺ T cells after restimulation with aAPC.scA2 and aAPC.hypoA2 cells. Symbols represents nine independent T cell donors. ( G ) Amino acid alignment of the α3 domain region for the indicated HLA class I alleles. ( H and I ) T cells were stimulated with aAPC.dtHLA-E^4v4 cells for 1 wk in culture. FACS plots (H) and summarized data (I) indicate frequency of CD107a⁺ and TNF-α⁺ CD8⁺ T cells after restimulation with aAPC.dtHLA-E^4v4 and aAPC.hypoHLA-E^4v4 cells. ( J ) NK cell cytotoxicity assay as described in Materials and Methods. NK cell–dependent lysis of B2M^KO T cells that were nontransduced or expressed dtHLA-E^4v4 or hypoHLA-E^4v4. Symbols represent mean of three independent NK cell donors in duplicate. For (A), (C), and (I), symbols represent nine independent T cell donors in duplicate. For (A), (C), (E), (F), and (I), values are background subtracted from restimulation with K.86 cells. For (E), (F), and (I), a Wilcoxon matched-pairs signed rank test was used to calculate significance. For all data, bars represent mean and error bars indicate ±SEM. More about this image found in Hypoimmunogenic dtHLA-E^4v4 evades HLA-E–reactive CD8⁺...

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FIGURE 1. An overview of IL-10 signaling in macrophages. When IL-10 binds to the receptor complex, JAK1 activates STAT3 and represses proinflammatory cytokines. An IL-10–responsive gene, SOCS3 negatively regulates the IL-10/TYK2/STAT pathway. Inducing IL-10 through TLR activation and adaptor molec... More about this image found in An overview of IL-10 signaling in macrophages. When IL-10 binds to the rece...

FIGURE 2. Role of IL-10 in foamy macrophage formation. IL-10 increases CD36 and ACAT cell-surface expression and promotes FM synthesis and lipid body formation via an IL-10/STAT3 axis. Pathogens such as Mtb, M. leprae, and M. avium-containing phagosomes consume these lipid compounds and allow bacilli persistence. By activating TLR2, Chlamydia sp. increase IL-10 cytokine in host macrophages, causing FMs by blocking LXR signaling and limiting cholesterol efflux through ABCA1 and ABCG1 transporters. Protozoan parasites like T. gondi, L. major, and H. capsulatum induce FM via TLR2/TLR4 through IL-10–dependent pathway by upregulating SR-A and CD36, which facilitate uptake of modified LDL and cholesteryl ester accumulation. HLA-DR, human leukocyte Ag – DR isotype; CD163, cluster of differentiation 163; PD-L1, programmed death ligand 1; LB, lipid body; AA, arachidonic acid; LOX, lipoxygenase; COX, cyclooxygenase; LTB₄, leukotriene B₄; RXR, retinoid X receptor; LXR, liver X receptor; FXR, farnesoid X receptor; FXRE, FXR-responsive elements; miR-33, microRNA-33; SREBP-2, sterol regulatory element-binding protein 2; HMG-CoA reductase, β-hydroxy β-methylglutaryl-CoA reductase; Chol, cholesterol; FA, fatty acid; TG, triglyceride; ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette, subfamily G member 1; ApoA1, apolipoprotein AI; HDL, high-density lipoprotein. More about this image found in Role of IL-10 in foamy macrophage formation. IL-10 increases CD36 and ACAT ...

FIGURE 1. IgG subclass and glycosylation variant complexity and impact on IgG effector functions. ( A ) Schematic representations of the four human IgG subclasses. Intermolecular disulfide bonds connecting IgG H or L chains are represented in yellow. ( B ) Different sugar structures attached to Ig... More about this image found in IgG subclass and glycosylation variant complexity and impact on IgG effecto...

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