b v co  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panel B. P14 CD8 T cells (5 × 10 6 cells, CD45.1 + ) were adoptively transferred into B6 mice (CD45.2 + ), and their kinetics were monitored in the blood. (B) Kinetics of adoptively transferred P14 CD8 T cells (CD45.1 + DbGP33 tetramer + CD8 T cells) in the blood without infection. The frequency of P14 CD8 T cells at 1.5 days post-transfer was set as 100%. Each symbol represents the mean and error bars indicate SEM. (C) CD44, CD62L, and CD127 expression on splenic P14 CD8 T cells before transfer and at 35 days post-transfer. As a control, the expression of these markers on DbGP33 + memory CD8 T cells obtained from the spleen of LCMV-Arm infected mice (> day 100 post-infection) is shown. Histograms were gated on DbGP33 + CD8 T cells. (D) Experimental design for panels E to G. P14 CD8 T cells (long-lived P14, CD45.1 +/+ ), housed for 36 days after transfer in B6 mice, were mixed with an equal number of freshly isolated P14 CD8 T cells (fresh P14, CD45.1 + CD45.2 + ). The mixture (1 × 10 3 P14 CD8 T cells of each population) was adoptively transferred into CD45.2 +/+ B6 mice, followed by LCMV-Arm infection. (E and F) Percentages of progeny derived from fresh and long-lived P14 CD8 T cells within total P14 CD8 T cells (E) and their kinetics (F) in the blood post-infection. (G) The number of memory precursor effector P14 cells (CD127 + KLRG1 – ) in the blood on day 8 post-infection. (H and I) Equal numbers (1 × 10 3 cells each) of congenically distinct P14 CD8 T cells, housed for 1.5 days (short-housed P14) and 35 days (long-lived P14), were adoptively co-transferred into B6 mice, followed by LCMV-Arm infection. Percentages of progeny derived from each population within total P14 CD8 T cells (H) and their kinetics (I) in the blood post-infection. (J and K) Responses of long-lived P14 cells during chronic LCMV-Clone 13 infection. Long-lived P14 CD8 T cells (1 × 10 3 cells, CD45.1 +/+ ), housed for 74 days in B6 mice, were adoptively co-transferred with an equal number of fresh P14 CD8 T cells (1 × 10 3 cells, CD45.1 + CD45.2 + ) into B6 mice (CD45.2 +/+ ), followed by LCMV-Clone 13 infection. Percentages of progeny derived from each population within total P14 CD8 T cells (J) and their kinetics (K) in the blood post-infection. Data are representative of 2 or more independent experiments with 3 or more mice per group. In E–K, each symbol represents an individual mouse, and lines indicate paired comparisons within the same mice. Statistical analysis was performed using paired t test (G) and one-way ANOVA (E, F, H, I, J, and K). ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To prevent anti-CD44 antibody-mediated depletion of sorted cells after adoptive transfer, recombinant anti-CD44 antibody (clone: REA664, Miltenyi Biotec), that has a mutated Fc region, was used.

Techniques: Infection, Expressing, Control, Isolation, Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panels B–F. P14 CD8 T cells (CD45.1 + ) were adoptively transferred into B6 mice (CD45.2 + ). On 1.5 days (fresh) and 35 days (long-lived) post-transfer, P14 CD8 T cells (CD45.1 + DbGP33 + CD8 T cells) were sorted from the spleens of recipient mice ( n = 4 each) for RNA-seq analysis. Gene expression (read counts) was analyzed using DESeq2. (B) Heatmap of sample-to-sample distances using the Poisson Distance. (C) Fold changes and adjusted p -values of gene expression in long-lived P14 CD8 T cells relative to fresh P14 CD8 T cells. (D–F) Normalized read counts of Il18r1 (D), Cxcr3 (E), and Nt5e (F) in long-lived and fresh P14 CD8 T cells. Each symbol represents an individual mouse. (G–K) Protein expression levels of IL-18Rα, CXCR3, and CD73 on long-lived (>30 days post-transfer) and fresh P14 CD8 T cells in the spleens. (G) Representative histograms were gated on DbGP33 + P14 CD8 T cells. (H to J) Data were pooled from 6 independent experiments, and each symbol represents an individual mouse. (K) Heterogeneity of naïve P14 CD8 T cells (gated on DbGP33 + P14 CD8 T cells) based on the expression of IL-18Rα, CXCR3, and CD73. (L) Heterogeneity of endogenous bulk and DbGP33 + naïve CD8 T cells. Tetramer enrichment was performed using the cells isolated from the spleen and LNs (inguinal and brachial LNs) of naïve B6 mice, and the expression of IL-18Rα, CXCR3, and CD73 on either DbGP33 + or bulk CD44 Lo CD8 T cells was analyzed. Statistical analyses were performed using Walt’s test (D–F) and unpaired t test (H–J). *** p < 0.001; **** p < 0.0001.

Article Snippet: To prevent anti-CD44 antibody-mediated depletion of sorted cells after adoptive transfer, recombinant anti-CD44 antibody (clone: REA664, Miltenyi Biotec), that has a mutated Fc region, was used.

Techniques: RNA Sequencing, Gene Expression, Expressing, Isolation

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panels B and C. Congenically distinct B6 mice were conjoined by parabiosis to effectively transfer donor CD8 T cells into another mouse. Parabiotic mice were separated 5 weeks after conjoining, which allowed for the prolonged monitoring of donor-derived CD8 T cell phenotype. 1.5 to 2.5 months after fusion reversal, the phenotype of donor- and host-derived bulk CD44 Lo (B) and DbGP33 + CD44 Lo (C) CD8 T cells in the spleen was analyzed using tetramer enrichment. Data are pooled from 3 independent experiments with 10 mice. Each symbol represents an individual mouse and lines indicate paired comparisons within the same mouse. Statistical analysis was performed using paired t test. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To prevent anti-CD44 antibody-mediated depletion of sorted cells after adoptive transfer, recombinant anti-CD44 antibody (clone: REA664, Miltenyi Biotec), that has a mutated Fc region, was used.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panel B. Five naïve CD8 T cell subsets were sorted from the spleen of P14 TCR transgenic mice. Equal numbers (1 × 10 3 cells each) of congenically distinct P14 CD8 T cells, including each sorted subset and freshly isolated P14 CD8 T cells (control), were adoptively co-transferred into B6 mice, followed by LCMV-Arm infection. The responses of sorted and control P14 populations in the blood were monitored over time. (B) Kinetics of the frequency of sorted and control P14 CD8 T cells. The frequency of control P14 cells at day 8 post-infection was set as 100. Each symbol represents the mean, and error bars indicate the SEM. (C) Experimental design for panels D and E. IL18Rα Lo CD73 Lo (DN: double negative) or IL18Rα Hi CD73 Hi (DP: double positive) CD44 Lo naïve CD8 T cells were sorted from splenocytes of B6 mice (CD45.2 +/+ ). The number of DbGP33 tetramer + CD8 T cells was confirmed using a portion of the sorted cells via tetramer enrichment. Bulk sorted cells, containing equal numbers of DbGP33 tetramer + cells, were separately transferred into recipient mice (CD45.1 + CD45.2 + ), followed by LCMV-Arm infection. On days 7–8 post-infection, tetramer enrichment was performed to analyze the response of donor-derived DbGP33 + CD8 T cells in the spleen. (D) Representative flow plots. (E) Frequency of donor-derived DbGP33 + CD8 T cells among total DbGP33 + CD8 T cells in the spleen. Each symbol represents an individual mouse. Data were pooled from 2 independent experiments with 5 or 9 mice per group. Statistical analyses were performed using one-way ANOVA (B) and unpaired t test (E). * p < 0.05; ** p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: To prevent anti-CD44 antibody-mediated depletion of sorted cells after adoptive transfer, recombinant anti-CD44 antibody (clone: REA664, Miltenyi Biotec), that has a mutated Fc region, was used.

Techniques: Transgenic Assay, Isolation, Control, Infection, Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panel B. P14 CD8 T cells (5 × 10 6 cells, CD45.1 + ) were adoptively transferred into B6 mice (CD45.2 + ), and their kinetics were monitored in the blood. (B) Kinetics of adoptively transferred P14 CD8 T cells (CD45.1 + DbGP33 tetramer + CD8 T cells) in the blood without infection. The frequency of P14 CD8 T cells at 1.5 days post-transfer was set as 100%. Each symbol represents the mean and error bars indicate SEM. (C) CD44, CD62L, and CD127 expression on splenic P14 CD8 T cells before transfer and at 35 days post-transfer. As a control, the expression of these markers on DbGP33 + memory CD8 T cells obtained from the spleen of LCMV-Arm infected mice (> day 100 post-infection) is shown. Histograms were gated on DbGP33 + CD8 T cells. (D) Experimental design for panels E to G. P14 CD8 T cells (long-lived P14, CD45.1 +/+ ), housed for 36 days after transfer in B6 mice, were mixed with an equal number of freshly isolated P14 CD8 T cells (fresh P14, CD45.1 + CD45.2 + ). The mixture (1 × 10 3 P14 CD8 T cells of each population) was adoptively transferred into CD45.2 +/+ B6 mice, followed by LCMV-Arm infection. (E and F) Percentages of progeny derived from fresh and long-lived P14 CD8 T cells within total P14 CD8 T cells (E) and their kinetics (F) in the blood post-infection. (G) The number of memory precursor effector P14 cells (CD127 + KLRG1 − ) in the blood on day 8 post-infection. (H and I) Equal numbers (1 × 10 3 cells each) of congenically distinct P14 CD8 T cells, housed for 1.5 days (short-housed P14) and 35 days (long-lived P14), were adoptively co-transferred into B6 mice, followed by LCMV-Arm infection. Percentages of progeny derived from each population within total P14 CD8 T cells (H) and their kinetics (I) in the blood post-infection. (J and K) Responses of long-lived P14 cells during chronic LCMV-Clone 13 infection. Long-lived P14 CD8 T cells (1 × 10 3 cells, CD45.1 +/+ ), housed for 74 days in B6 mice, were adoptively co-transferred with an equal number of fresh P14 CD8 T cells (1 × 10 3 cells, CD45.1 + CD45.2 + ) into B6 mice (CD45.2 +/+ ), followed by LCMV-Clone 13 infection. Percentages of progeny derived from each population within total P14 CD8 T cells (J) and their kinetics (K) in the blood post-infection. Data are representative of 2 or more independent experiments with 3 or more mice per group. In E–K, each symbol represents an individual mouse, and lines indicate paired comparisons within the same mice. Statistical analysis was performed using paired t test (G) and one-way ANOVA (E, F, H, I, J, and K). ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques: Infection, Expressing, Control, Isolation, Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panels B–F. P14 CD8 T cells (CD45.1 + ) were adoptively transferred into B6 mice (CD45.2 + ). On 1.5 days (fresh) and 35 days (long-lived) post-transfer, P14 CD8 T cells (CD45.1 + DbGP33 + CD8 T cells) were sorted from the spleens of recipient mice ( n = 4 each) for RNA-seq analysis. Gene expression (read counts) was analyzed using DESeq2. (B) Heatmap of sample-to-sample distances using the Poisson Distance. (C) Fold changes and adjusted p -values of gene expression in long-lived P14 CD8 T cells relative to fresh P14 CD8 T cells. (D–F) Normalized read counts of Il18r1 (D), Cxcr3 (E), and Nt5e (F) in long-lived and fresh P14 CD8 T cells. Each symbol represents an individual mouse. (G–K) Protein expression levels of IL-18Rα, CXCR3, and CD73 on long-lived (>30 days post-transfer) and fresh P14 CD8 T cells in the spleens. (G) Representative histograms were gated on DbGP33 + P14 CD8 T cells. (H to J) Data were pooled from 6 independent experiments, and each symbol represents an individual mouse. (K) Heterogeneity of naïve P14 CD8 T cells (gated on DbGP33 + P14 CD8 T cells) based on the expression of IL-18Rα, CXCR3, and CD73. (L) Heterogeneity of endogenous bulk and DbGP33 + naïve CD8 T cells. Tetramer enrichment was performed using the cells isolated from the spleen and LNs (inguinal and brachial LNs) of naïve B6 mice, and the expression of IL-18Rα, CXCR3, and CD73 on either DbGP33 + or bulk CD44 Lo CD8 T cells was analyzed. Statistical analyses were performed using Walt’s test (D–F) and unpaired t test (H–J). *** p < 0.001; **** p < 0.0001.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques: RNA Sequencing, Gene Expression, Expressing, Isolation

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panels B and C. Congenically distinct B6 mice were conjoined by parabiosis to effectively transfer donor CD8 T cells into another mouse. Parabiotic mice were separated 5 weeks after conjoining, which allowed for the prolonged monitoring of donor-derived CD8 T cell phenotype. 1.5 to 2.5 months after fusion reversal, the phenotype of donor- and host-derived bulk CD44 Lo (B) and DbGP33 + CD44 Lo (C) CD8 T cells in the spleen was analyzed using tetramer enrichment. Data are pooled from 3 independent experiments with 10 mice. Each symbol represents an individual mouse and lines indicate paired comparisons within the same mouse. Statistical analysis was performed using paired t test. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panel B. Five naïve CD8 T cell subsets were sorted from the spleen of P14 TCR transgenic mice. Equal numbers (1 × 10 3 cells each) of congenically distinct P14 CD8 T cells, including each sorted subset and freshly isolated P14 CD8 T cells (control), were adoptively co-transferred into B6 mice, followed by LCMV-Arm infection. The responses of sorted and control P14 populations in the blood were monitored over time. (B) Kinetics of the frequency of sorted and control P14 CD8 T cells. The frequency of control P14 cells at day 8 post-infection was set as 100. Each symbol represents the mean, and error bars indicate the SEM. (C) Experimental design for panels D and E. IL18Rα Lo CD73 Lo (DN: double negative) or IL18Rα Hi CD73 Hi (DP: double positive) CD44 Lo naïve CD8 T cells were sorted from splenocytes of B6 mice (CD45.2 +/+ ). The number of DbGP33 tetramer + CD8 T cells was confirmed using a portion of the sorted cells via tetramer enrichment. Bulk sorted cells, containing equal numbers of DbGP33 tetramer + cells, were separately transferred into recipient mice (CD45.1 + CD45.2 + ), followed by LCMV-Arm infection. On days 7–8 post-infection, tetramer enrichment was performed to analyze the response of donor-derived DbGP33 + CD8 T cells in the spleen. (D) Representative flow plots. (E) Frequency of donor-derived DbGP33 + CD8 T cells among total DbGP33 + CD8 T cells in the spleen. Each symbol represents an individual mouse. Data were pooled from 2 independent experiments with 5 or 9 mice per group. Statistical analyses were performed using one-way ANOVA (B) and unpaired t test (E). * p < 0.05; ** p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques: Transgenic Assay, Isolation, Control, Infection, Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: IL-18Rα Hi CD73 Hi CD44 Lo and IL-18Rα Lo CD73 Lo CD44 Lo CD8 T cells were sorted from splenocytes of naïve B6 mice.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques: