Fig 1.
Complete blood cell counts (CBC).
CBC analysis was quantified from EDTA-treated whole blood obtained from each mouse in the following 4 groups at 4 weeks post T cell transfer. Mice in the–Abx/Syn and -Abx/Allo groups represent–NK/RAG mice engrafted with syngeneic or allogeneic CD4+ T cells and treated with aspartame alone whereas +Abx/Syn and +Abx/Allo mice represent–NK/RAG mice engrafted with syngeneic or allogeneic T cells and treated with aspartame plus antibiotics. Asterisks (*) designate significant differences between groups (*p < 0.05, **p <0.01).
Fig 2.
Acute GVHD-induced bone marrow damage.
Total bone marrow (BM) cell numbers as well as BM-residing immune cells were quantified at 4 weeks post T cell transfer for mice in the 4 groups described in Fig 1. Conventional (CD4+CD25-) T cells, regulatory T cells (Tregs; CD4+CD25+), myeloid (CD11b+) cells and NK (CD335+) cells were quantified by flow cytometry. Asterisks (*) designate significant differences between groups. (*p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001).
Fig 3.
Histopathological analysis of bone marrow and spleen.
A). Blinded histopathology scores were quantified for bone marrow and spleen obtained from mice in the 4 groups described in Fig 1 at 4 weeks post T cell transfer. B). Representative histological images of bone marrow and spleen from each group (40x total magnification). Asterisks (*) designate significant differences between groups. (**p <0.01; ***p<0.001).
Fig 4.
Acute GVHD-induced spleen hypoplasia.
Spleen weights, splenocyte numbers and spleen-residing immune cells were quantified at 4 weeks post T cell transfer for mice in the 4 groups described in Fig 1. Conventional (CD4+CD25-) T cells, regulatory T cells (Tregs; CD4+CD25+), myeloid (CD11b+) cells and NK (CD335+) cells were quantified by flow cytometry. Asterisks (*) designate significant differences between groups. (*p < 0.05, **p < 0.01, ****p<0.0001).
Table 1.
Plasma cytokine concentrations.
Fig 5.
Histopathological analysis of different tissue.
A). Blinded histopathology scores were quantified for the liver, lung, skin and colon obtained for mice in the 4 groups described in Fig 1 at 4 weeks post T cell transfer. Green circles and squares represent–Abx/Syn and +Abx/Syn whereas red triangles and diamonds represent–Abx/Allo and +Abx/Allo B). Representative histological images of the colon from each group (100x total magnification). Asterisks (*) designate significant differences between groups (*p < 0.05, **p < 0.01).
Table 2.
Summary of Inflammatory tissue injury in mice from each group.
Fig 6.
Alterations in fecal bacterial density and composition in untreated or Abx-treated mice.
A). Fecal bacterial density was quantified at 4 weeks post T cell transfer. Data are expressed as 16s rRNA gene copy numbers per ng of DNA. Asterisks (*) designate significant differences among the 3 groups (**p< 0.01). B). Relative abundance of the major phyla and genera were determined using 16S rDNA amplicon sequencing of fecal DNA. C). Amplicon Sequence Variants (ASVs) with Kruskal-Wallis pairwise comparisons was used to quantify α diversity within each group. ASVs analysis revealed that the microbial diversity within the +Abx/Syn and +Abx/Allo groups were significantly reduced when compared with the–Abx/Syn and–Abx/Allo groups. D). Principal Coordinate Analysis (PCoA) of weighted UniFrac distance was used to quantify differences between microbial communities (β Diversity). Using pairwise analysis of similarities, this plot reveals that the +Abx/Syn and +Abx/Allo groups were significantly different from each other as well as significantly different from the other two groups (p<0.05).
Table 3.
Alterations in the major bacterial phyla and genera in untreated or Abx-treated mice engrafted with T cells.
Table 4.
Alterations in the major bacterial species in untreated or Abx-treated mice engrafted with T cells.