Table 1.
Clinical characteristics of patients.
Table 2.
MDR species in septic patients.
Fig 1.
Quantified mean white blood cells.
Cell counts (cells x 103/μL) are presented as Absolute Lymphocyte Count (a), Absolute Monocyte Count (b), Absolute Neutrophil Count (c) and White Blood Cell count (d) in Multidrug Resistant (MDR) Septic patients compared to Critically-Ill Non-Septic (CINS) patients. Bars represent mean +/- S.E.M. *p<0.05.
Fig 2.
Flow cytometric expression of PD-1 and PD-L1.
PD-1 was increased in patients with sepsis attributed to Multidrug Resistant (MDR) bacteria compared to Critically-Ill Non-Septic (CINS) patients on both CD4 and CD8 T cells, both in the percentage of cells bearing the receptor (a; % cells positive) and the intensity of staining (b; GMFI). GMFI = geo mean fluorescence intensity. Surface PD-L1 on monocytes showed increased staining intensity (d), however, there was insufficient evidence of a significant increase in the percentage of PD-L1 positive monocytes (c). Bars represent mean +/- S.E.M. *p<0.05, ** p<0.01, ***p<0.001.
Table 3.
Flow cytometric data.
Fig 3.
IL-7Rα surface levels on T cells.
There was a modest non-significant decrease in CD127 (IL-7Rα) positive CD4 T cells (p = 0.1) (a). There was no difference in IL-7Rα positive CD8 T cells (a). There was also no change in receptor intensity on either CD4 or CD8 T cells (b). Bars represent mean +/- S.E.M.
Fig 4.
Flow cytometric expression of monocyte HLA-DR.
Monocytes in the Multidrug resistant (MDR) Septic patients compared to Critically-Ill Non-Septic (CINS) patients had significantly decreased surface levels of HLA-DR. Gating strategy used for the Quantibrite Anti-HLADR/Anti-Monocyte stain is included (b). ABC = average number of HLA-DR antibodies bound per cell. Bars represent mean +/- S.E.M. **p<0.01.
Fig 5.
OX-40 surface levels on T cells.
There was no difference in OX-40 surface levels in the Multidrug resistant (MDR) Septic patients compared to Critically-Ill Non-Septic (CINS) patients in either the percent of positive cells (a) or receptor density (b). Staining on CD8 T cells was no greater than background.
Fig 6.
Samples displaying a response to treatments showed a greater than 20% increase in the number of IFN-γ producing cells (spots on the membrane). Here, ELISpot images from two representative IL-7 responders (R) are displayed along with a non-responder (NR).
Fig 7.
Multidrug resistant (MDR) septic patients’ response to immuno-adjuvant treatments.
Patients who displayed an increase in spot numbers of 20% or more in response to treatment were considered responders (Red), while patients who had an increase in spot number of less than 20% were considered non-responders (Blue). 15 of 24 patient samples responded to IL-7 (a), 10 of 24 responded to OX-40L (b), and 8 of 24 responded to PD-L1 antibody (c). Percent increase in IFN-γ was calculated by dividing the number of spots with treatment by the number of spots from sham (Isotype control) treatment.
Fig 8.
Critically-Ill Non-Septic (CINS) patients’ response to immuno-adjuvant treatments.
Patients who displayed an increase in spot numbers of 20% or more in response to treatment were considered responders (Red), while patients who had an increase in spot number of less than 20% were considered non-responders (Blue). 13 of 16 patient samples responded to IL-7, 8 of 16 responded to OX-40L, and 3 of 16 responded to PD-L1 antibody. Percent increase was calculated by dividing the number of spots with treatment by the number of spots from sham (Isotype control) treatment.
Table 4.
ELISpot % increase in IFN-γ producing cells after treatment.