Table 4.
Recommendations to: A) investigate CT product (upon release); B) monitor CART product after administration; C) investigate effects of CTs on immune system (efficacy).
| A) CT product characterization upon release | ||||
|---|---|---|---|---|
| Focus | Markers | Material/Technique | Other comments | |
| CARTa | Proportion of CART | CAR | Cell product/Cytometry | Commercially available reagents. |
| Differentiation | CD3, CD4, CD8, CD45RA, CCR7/CD27, CD69, CD25, CD137, CD127, Foxp3 | Cell product/Cytometry | ||
| Exhaustion and coinhibitory receptors | CD57, PD-1, LAG3, TIM3, TIGIT | Cell product/Cytometry | ||
| MSCf | Phenotype/viability | CD45, CD31, CD73, CD90, HLA-DR, | Cell product/Cytometry | |
| Alloreactivity | HLA I and II typing | Cell product/molecular biology | Only for allogeneic MSC | |
| Function/potency assays | Capacity to produce immunosuppressive and trophic factors | Cell product/quantification of soluble or membrane markers in steady state and in response to inflammatory stimuli at protein or at RNA levels Cell product/inhibition of T-cell proliferation |
||
| Tregs | Phenotype and activation status | CD3, CD4, CD127, CD25, FoxP3, Helios, (GARP, LAP, CTLA-4) | Cell product/Cytometry | |
| Function | T-cell suppression and/or suppressive cytokine production | Cell product/in vitro stimulation assays | ||
|
B) CART product monitoring after administration | ||||
|---|---|---|---|---|
| Focus | Marker | Technique | Time-pointse | |
| CARTd | Cell Count/viability | CAR (commercial reagents) | PBMC/Cytometry (done the day of blood collection) | d7, d10, d14, d28, mo3, mo6, mo9, mo12, mo18, mo24e |
| Basic characterization | CD4/CD8 TN/TEM/TCM/TEMRA |
PBMC/Cytometry | ||
| Extended phenotype | Treg (Foxp3, CD25, CD127) Activation (CD69, CD137, etc) Exhaustion (PD-1, LAG3, etc) |
PBMC/Cytometry | ||
| C) CTs effects on immune system | |||||
|---|---|---|---|---|---|
| Focus | Marker | Technique | Time-pointsc | ||
| Minimal | Serology | Ig Autoantibodies DSA (allogeneic MSC) |
Total levels of IgG, IgM, IgA AutoAb relevant to the AD Anti-donor HLA Ab |
Serum/ELISA +electrophoresis Serum/ELISA, IF Serum/Luminex |
Baselineb, d28, mo3, mo6, mo9, mo12, mo18, mo24 Baselineb, d28, mo3, mo6, mo9, mo12, mo18, mo24 Baseline and at mo3 |
| Immune status | Blood count Lymphocyte, NK cell, monocyte count |
WBC CD3, CD4, CD8, CD19, CD14, CD56/CD16 |
Blood count PBMC/Cytometry |
For CART: Baselineb, Day of infusion (pre), d7, d10, d14, d28, mo3, mo6, mo9, mo12, mo18, mo24 For MSC: Day of infusion (pre), d28, mo3, mo6, mo12. For Tregs: Day of infusion (pre), d7, d14, d28, mo3, mo6, mo12. |
|
| Extended | B cells | Differentiation | IgD, IgG, CD19, CD20, CD21, CD24, CD27, CD38, CXCR5, CD95, CD11c | PBMC/Cytometry | For CART: Baselineb, day of infusion (pre), d7, d10, d14, d28, mo3, mo6, mo9, mo12, mo18, mo24 For MSC: Day of infusion (pre), d28, mo3, mo6, mo12 For Tregs: Day of infusion (pre), d7, d14, d28, mo3, mo6, mo12 |
| Non-CART | Differentiation | CD3, CD4, CD8, CD45RA, CCR7, CD28, CD57, CXCR5, PD-1 | PBMC/Cytometry | ||
| Tregs | Differentiation | CD3, CD4, CD8, CD25, FoxP3, Helios, Ki-67 | PBMC/Cytometry | ||
| Myeloid cells | Differentiation | CD14, CD16, HLA-DR, CD123, CD1c, CD141, CD15 | PBMC/Cytometry | ||
| MAIT cells | Differentiation | CD3, CD4, CD8, CD161, TCRVa7.2, CCR6 and IL-18 R | PBMC/Cytometry | ||
| ILCs | Differentiation | CD45, lin (CD3, CD14, CD19), CD94, CD127, c-kit, CRTh2 | PBMC/Cytometry | ||
| Immune system reprogramming | Cell activation & differentiation | Soluble factors involved in AD activity (such as TGFb) and/or cell therapy product activity (such as inflammatory factors, BAFF, IL-7 …) | Plasma/ELISA or LUMINEX or PEA | ||
| Exploratory | Transcriptome and BCR/TCR repertoire | Renewal vs. Persistence | NA | PBMC/scRNAseq/TCRseq/BCRseq directly or frozen −80 °C or whole repertoire analysis (RACE PCR) | Baselineb, mo6, mo12 |
| Microbiome | Changes in microbiota profile | NA | Stool/salivary Instantly frozen −80 °C | Baselineb, d14 (upon discharge), mo3, mo12 | |
| Tissue-resident or infiltrating cells | Number and Phenotype | NA | Biopsy of bone marrow, skin, lymph node, CSF, or gut/RNASeq and/or scRNASeq | Baselineb, mo3, mo12 | |
Abbreviations: Ab antibody; AD autoimmune diseases; BAFF B-cell activating factor; BCR B cell repertoire; BCRseq B Cell Repertoire sequencing; CART chimeric antigen receptors T cells; CD cluster of differentiation; c-kit tyrosine kinase receptor; CRTh2 chemoattractant receptor-homologous molecule expressed on Th2 cells; CSF cerebrospinal fluid; CT cellular therapy; CTLA-4 cytotoxic T-lymphocyte-associated protein 4; d day; DSA donor specific antibodies anti HLA; ELISA enzyme-linked immunosorbent assay; FoxP3 forkhead box P3; GARP glycoprotein A repetitions predominant; HLA human leukocyte antigen; IF immunofluorescence; Ig immunoglobulin; IL interleukin; ILC innate lymphoid cells; LAG lymphocyte-activation gene; LAG3 lymphocyte-activation gene 3; LAP LAG-3-associated protein; MAIT mucosal-associated invariant T cells; mo month; MSC mesenchymal stromal cells; PBMC peripheral blood mononuclear cells; PCR polymerase chain reaction; PD-1 programmed cell death protein 1; PEA proximity extension assay; RACE rapid amplification of cDNA ends; RNA ribonucleic acid; RNASeq RNA-sequencing; scRNASeq single-cell RNA-sequencing; TCM naïve central memory T cell; TCR T cell repertoire; TCRseq T cell repertoire sequencing; TEM effector memory T cell; TEMRA effector memory T cells re-expressing CD45RA; TGFb transforming growth factor-β; TIGIT T-cell immunoreceptor with Ig and ITIM domains; TIM3 T-cell immunoglobulin and mucin-domain containing-3; TN naïve T cell; Tregs regulatory T cells; WBC white blood cells.
Biobanking should be performed, when possible, at the same time-points than phenotypic follow-up (frozen PBMC, serum, plasma).
This may be performed for any academic cell product. Commercial products may be measured according to local and national policy and in accordance with company regulations.
Baseline = before treatment and lymphodepletion.
To be repeated in case of repeated cell infusions.
CART and non-CAR T cells can be studied using the same flow cytometry panel.
Recommended time points ( ± 1 day up to day +14) but additional time points could be explored depending on specific scientific questions.
The preferred analytic methods for matrix assays evaluating the immunosuppressive and immunomodulatory capacities of MSC currently include12,15,16: a) flow cytometry analysis of functionally relevant surface markers; b) quantitative RNA analysis of selected gene products; c) protein-based assay of secretome; d) combination of transcriptome and secretome analyses. These parameters should be evaluated in steady-state and MSC activated by inflammatory stimuli.