untitled h1
16.4 appendix d complete list

Complete list of 123 peripheral immune cell subsets analyzed by flow **cytometry.** Nine classic subsets were identified as well as 114 refined subsets relating to maturation and function within the classic subsets.  
| 1.                    |    |
|-----------------------|----|
| Total                 |    |
| CD4                   |    |
| +                     |    |
| T                     |    |
| cells                 |    |
| -                     |    |
| PD-L1                 |    |
| +                     |    |
| CD4                   |    |
| -                     |    |
| PD-1                  |    |
| +                     |    |
| CD4                   |    |
| -                     |    |
| EOMES                 |    |
| +                     |    |
| CD4                   |    |
| -                     |    |
| TCR+                  |    |
| CD4                   |    |
| -                     |    |
| Tbet+                 |    |
| CD4                   |    |
| -                     |    |
| BATF                  |    |
| +                     |    |
| CD4                   |    |
| -                     |    |
| CTLA-4                |    |
| +                     |    |
| CD4                   |    |
| -                     |    |
| Tim-3                 |    |
| +                     |    |
| CD4                   |    |
| -                     |    |
| ICOS                  |    |
| +                     |    |
| CD4                   |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| CD4                   |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| ICOS                  |    |
| +                     |    |
| ICOS                  |    |
| +                     |    |
| CD4                   |    |
| -                     |    |
| Total                 |    |
| naïve                 |    |
| (CCR7                 |    |
| )                     |    |
| CD4                   |    |
| o                     |    |
| PD-L1                 |    |
| +CD45RA+              |    |
| +                     |    |
| naïve                 |    |
| CD4                   |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| naïve                 |    |
| CD4                   |    |
| o                     |    |
| CTLA-4                |    |
| +                     |    |
| naïve                 |    |
| CD4                   |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| naïve                 |    |
| CD4                   |    |
| -                     |    |
| Total                 |    |
| central               |    |
| memory                |    |
| (CCR7                 |    |
| +                     |    |
| CD45RA-)              |    |
| CD4                   |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| CM                    |    |
| CD4                   |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| CM                    |    |
| CD4                   |    |
| o                     |    |
| CTLA-4                |    |
| +                     |    |
| CM                    |    |
| CD4                   |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| CM                    |    |
| CD4                   |    |
| -                     |    |
| Total                 |    |
| effector              |    |
| memory                |    |
| (CCR7                 |    |
| -                     |    |
| CD45RA-)              |    |
| CD4                   |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| EM                    |    |
| CD4                   |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| EM                    |    |
| CD4                   |    |
| o                     |    |
| CTLA-4                |    |
| +                     |    |
| EM                    |    |
| CD4                   |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| EM                    |    |
| CD4                   |    |
| -                     |    |
| Total                 |    |
| EMRA                  |    |
| (CCR7                 |    |
| )                     |    |
| CD4                   |    |
| o                     |    |
| PD-L1                 |    |
| -CD45RA+              |    |
| +                     |    |
| EMRA                  |    |
| CD4                   |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| EMRA                  |    |
| CD4                   |    |
| o                     |    |
| CTLA-4                |    |
| +                     |    |
| EMRA                  |    |
| CD4                   |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| EMRA                  |    |
| CD4                   |    |
| 2.                    |    |
| Total                 |    |
| CD8                   |    |
| +                     |    |
| T                     |    |
| cells                 |    |
| -                     |    |
| PD-L1                 |    |
| +                     |    |
| CD8                   |    |
| -                     |    |
| PD-1                  |    |
| +                     |    |
| CD8                   |    |
| -                     |    |
| EOMES                 |    |
| +                     |    |
| CD8                   |    |
| -                     |    |
| TCR+                  |    |
| CD8                   |    |
| -                     |    |
| Tbet+                 |    |
| CD8                   |    |
| -                     |    |
| BATF                  |    |
| +                     |    |
| CD8                   |    |
| -                     |    |
| CTLA-4                |    |
| +                     |    |
| CD8                   |    |
| -                     |    |
| Tim-3                 |    |
| +                     |    |
| CD8                   | o  |
| PD-L1                 |    |
| +                     |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| -                     |    |
| Total                 |    |
| immature              |    |
| (CD16                 |    |
| -                     |    |
| CD56                  |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| immature              |    |
| NK                    |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| immature              |    |
| NK                    |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| immature              |    |
| NK                    |    |
| -                     |    |
| Total                 |    |
| unconventional        |    |
| (CD16                 |    |
| NK                    |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| unconventional        |    |
| NK                    |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| unconventional        |    |
| NK                    |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| unconventional        |    |
| NK                    |    |
| 6.                    |    |
| Total                 |    |
| NK-T                  |    |
| -                     |    |
| PD-L1                 |    |
| +                     |    |
| NK-T                  |    |
| -                     |    |
| PD-1                  |    |
| +                     |    |
| NK-T                  |    |
| -                     |    |
| Tim-3                 |    |
| +                     |    |
| NK-T                  |    |
| 7.                    |    |
| Total                 |    |
| cDC                   |    |
| -                     |    |
| PD-L1                 |    |
| +                     |    |
| cDC                   |    |
| -                     |    |
| PD-1                  |    |
| +                     |    |
| cDC                   |    |
| -                     |    |
| CD83                  |    |
| +                     |    |
| cDC                   |    |
| -                     |    |
| Tim-3                 |    |
| +                     |    |
| cDC                   |    |
| 8.                    |    |
| Total                 |    |
| pDC                   |    |
| -                     |    |
| PD-L1                 |    |
| +                     |    |
| pDC                   |    |
| -                     |    |
| PD-1                  |    |
| +                     |    |
| pDC                   |    |
| -                     |    |
| CD83                  |    |
| +                     |    |
| pDC                   |    |
| -                     |    |
| Tim-3                 |    |
| +                     |    |
| pDC                   |    |
| 9.                    |    |
| Total                 |    |
| MDSC                  |    |
| -                     |    |
| PD-L1                 |    |
| +                     |    |
| MDSC                  |    |
| -                     |    |
| PD-1                  |    |
| +                     |    |
| MDSC                  |    |
| -                     |    |
| CD16                  |    |
| +                     |    |
| MDSC                  |    |
| -                     |    |
| Total                 |    |
| monocytic             |    |
| (CD14                 |    |
| +                     |    |
| CD15                  |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| mMDSC                 |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| mMDSC                 |    |
| o                     |    |
| CD16                  |    |
| +                     |    |
| mMDSC                 |    |
| -                     |    |
| Total                 |    |
| granulocytic          |    |
| (CD14                 |    |
| -                     |    |
| CD15                  |    |
| +                     |    |
| )                     |    |
| MDSC                  |    |
| +                     |    |
| gMDSC                 |    |
| o                     |    |
| PD-L1                 |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| gMDSC                 |    |
| o                     |    |
| CD16                  |    |
| +                     |    |
| gMDSC                 |    |
| -                     |    |
| Total                 |    |
| lineage               |    |
| negative              |    |
| (CD14                 |    |
| MDSC                  |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| lin                   |    |
| neg                   |    |
| MDSC                  |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| lin                   |    |
| neg                   |    |
| MDSC                  |    |
| o                     |    |
| CD16                  |    |
| +                     |    |
| lin                   |    |
| neg                   |    |
| MDSC                  |    |
| BATF,                 |    |
| basic                 |    |
| leucine               |    |
| zipper                |    |
| transcription         |    |
| factor                |    |
| ATF-like;             |    |
| cDC,                  |    |
| conventional          |    |
| dendritic             |    |
| cells;                |    |
| CM,                   |    |
| central               |    |
| memory;               |    |
| CTLA-4,               |    |
| cytotoxic             |    |
| T                     |    |
| lymphocyte-associated |    |
| protein-4;            |    |
| EM,                   |    |
| effector              |    |
| memory;               |    |
| EMRA,                 |    |
| terminally            |    |
| differentiated        |    |
| effector              |    |
| memory;               |    |
| -                     |    |
| Total                 |    |
| naïve                 |    |
| (CCR7                 |    |
| )                     |    |
| CD8                   |    |
| o                     |    |
| PD-L1                 |    |
| +CD45RA+              |    |
| +                     |    |
| naïve                 |    |
| CD8                   |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| naïve                 |    |
| CD8                   |    |
| o                     |    |
| CTLA-4                |    |
| +                     |    |
| naïve                 |    |
| CD8                   |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| naïve                 |    |
| CD8                   |    |
| -                     |    |
| Total                 |    |
| central               |    |
| memory                |    |
| (CCR7                 |    |
| +CD45RA-)             |    |
| CD8                   |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| CM                    |    |
| CD8                   |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| CM                    |    |
| CD8                   |    |
| o                     |    |
| CTLA-4                |    |
| +                     |    |
| CM                    |    |
| CD8                   |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| CM                    |    |
| CD8                   |    |
| -                     |    |
| Total                 |    |
| effector              |    |
| memory                |    |
| (CCR7                 |    |
| -                     |    |
| CD45RA-)              |    |
| CD8                   |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| EM                    |    |
| CD8                   |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| EM                    |    |
| CD8                   |    |
| o                     |    |
| CTLA-4                |    |
| +                     |    |
| EM                    |    |
| CD8                   |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| EM                    |    |
| CD8                   |    |
| -                     |    |
| Total                 |    |
| EMRA                  |    |
| (CCR7                 |    |
| )                     |    |
| CD8                   |    |
| o                     |    |
| PD-L1                 |    |
| -CD45RA+              |    |
| +                     |    |
| EMRA                  |    |
| CD8                   |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| EMRA                  |    |
| CD8                   |    |
| o                     |    |
| CTLA-4                |    |
| +                     |    |
| EMRA                  |    |
| CD8                   |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| EMRA                  |    |
| CD8                   |    |
| 3.                    |    |
| Total                 |    |
| Tregs                 |    |
| -                     |    |
| PD-L1                 |    |
| +                     |    |
| Tregs                 |    |
| -                     |    |
| PD-1                  |    |
| +                     |    |
| Tregs                 |    |
| -                     |    |
| CTLA-4                |    |
| +                     |    |
| Tregs                 |    |
| -                     |    |
| ICOS                  |    |
| +                     |    |
| Tregs                 |    |
| -                     |    |
| CD45RA+               |    |
| Tregs                 |    |
| -                     |    |
| Tregs                 |    |
| 4.                    |    |
| Total                 |    |
| B                     |    |
| cells                 |    |
| -                     |    |
| CD49d                 |    |
| -                     |    |
| PD-L1                 |    |
| +                     |    |
| B                     |    |
| cells                 |    |
| -                     |    |
| PD-1                  |    |
| +                     |    |
| B                     |    |
| cells                 |    |
| -                     |    |
| CTLA-4                |    |
| +                     |    |
| B                     |    |
| cells                 |    |
| +                     |    |
| B                     |    |
| cells                 |    |
| 5.                    |    |
| Total                 |    |
| NK                    |    |
| -                     |    |
| Tim-3                 |    |
| -                     |    |
| PD-L1                 |    |
| +                     |    |
| NK                    |    |
| -                     |    |
| PD-1                  |    |
| +                     |    |
| NK                    |    |
| -                     |    |
| Tim-3                 |    |
| +                     |    |
| NK                    |    |
| -                     |    |
| Total                 |    |
| mature                |    |
| (CD16                 |    |
| +                     |    |
| CD56                  |    |
| dim)                  |    |
| NK                    |    |
| o                     |    |
| PD-L1                 |    |
| +                     |    |
| mature                |    |
| NK                    |    |
| o                     |    |
| PD-1                  |    |
| +                     |    |
| mature                |    |
| NK                    |    |
| o                     |    |
| Tim-3                 |    |
| +                     |    |
| mature                |    |
| NK                    |    |
| -                     |    |
| Total                 |    |
| functional            |    |
| intermediate          |    |
| (CD16                 |    |
| +                     |    |
| CD56                  |    |
| br)                   |    |
| NK                    |    |
untitled h1
urine protocol

A. Urine will be collected at room temperature from patients through voiding or instrumentation  
  
and transported to laboratory staff on ice. Collection will be 30-100 mL of urine in a sterile urine container.  
B. Centrifuge urine at 1200xg (2491 rpm for 20 minutes using our 5810R Eppendorf Centrifuge) at 4°C in 50 mL conical tube.  
C. Filter the supernatant (cell-free urine) with .45 μm filter to remove large debris. Save cell pellet.  
D. Store filtered urine as cell-free, filtered urine in 6 vials (1.8 mL cryovial each) at -80°C
without any cryopreservative.  
E. Resuspend urine cell pellet in 1 mL of 5% DMSO (95% MEM media) and transfer to 1.8 mL
cryovial tube.  
F. Spin this cryovial at 1200xg for 20 minutes at 4°C and leave cell pellet packed. G. Do slow freeze of cell pellet with Mr. Frosty and then store at -80. H. Schematic: