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4771 Identification of Microbiota-Reactive CD4+ T Cells in the Post-Transplant Setting

Program: Oral and Poster Abstracts
Session: 701. Experimental Transplantation: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, Immunology, Biological Processes, Microbiome, Study Population, Human
Monday, December 9, 2024, 6:00 PM-8:00 PM

Albert C Yeh, MD1, Tanya Cunningham2*, Saranya R. Chakka3*, R Graeme Black3*, Kate A. Markey, MBBS, PhD, FRACP, MPH3 and Marie Bleakley, PhD, MBBS, MMSc4

1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
2Fred Hutchinson Cancer Research Center, Seattle, WA
3Fred Hutchinson Cancer Center, Seattle, WA
4Translational Science and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA

Background:

The development of acute graft-vs.-host disease (GVHD) is primarily initiated by alloreactive donor T cells. However, we have recently shown using established murine transplant models that clonal expansion of donor CD4+ T cells post-transplant is also dictated by reactivity against the recipient microbiota which can act as a source of antigen to exacerbate GVHD (Yeh AC et al., Immunity 2024). To date, microbiota-targeting CD4+ T cells have been identified and cloned in the context of murine B6 background, which carries a single major histocompatibility antigen class II (MHCII) allele, I-A(b). However, there have been no microbiota-targeting CD4+ T cells identified in the patient setting thus far. Unlike in murine models, a major challenge in screening for reactive donor T cells in the clinic is due to the diversity of MHCII alleles across patients. Here, we develop a screening methodology that enables identification of microbiota-reactive donor T cells on the native MHCII background using both donor and recipient PBMC samples through detection of CD40L, a surface marker of T cell receptor (TCR) activation that allows rapid isolation of viable T cells for downstream characterization.

Methods:

Our platform includes three major components. First, given a limited number of cells in PBMC samples collected very early post-transplantation before the administration of post-transplantation cyclophosphamide (PTCy) and calcineurin inhibitors, we utilize an established rapid expansion protocol to cryopreserve a large and redundant pool of T cells (REP T cells) harvested from recipient PBMC post-transplantation. We next generate fresh monocyte-derived dendritic cells (moDCs) using a 5-day fast DC expansion protocol from donor PBMCs. We establish a co-culture incorporating a 10:1 ratio of REP T cells and moDCs on a 96-well plate along with bacterial lysate of interest. While REP T cells upregulate CD40L compared to naïve T cells, 24-hour lead-in co-culture with moDCs downregulates CD40L to baseline levels and enabled subsequent CD40L detection as an assay for TCR activation. On day 2, anti-CD40L antibody was added in co-culture along with monensin for optimal CD40L detection within 12-24 hours. Using this method, activated REP T cells are detected by flow cytometry by day 3 of co-culture.

Results:

As proof of concept, we utilized cryopreserved PBMCs isolated on day 2 from a patient undergoing a matched unrelated donor transplant prior to PTCy (NCT03970096). We subjected each cryopreserved sample to 1 round of REP to generate a pool of over 80M cells (>10x fold expansion). Upon co-culture of REP T cells with donor-derived moDCs, we demonstrated that CD40L was more sensitive at detecting TCR activation compared to IFNg, TNF, IL2, and CD107a using Staphylococcal enterotoxin B (SEB) (15-25% vs. 1-10%) while also preserving cell viability. We next conducted a co-culture comparing 3 different sterile bacterial lysate pools including oral strep (3 strains), enterococcus (7 strains), and commensal anaerobes (10 strains) at a lysate concentration of 10mg/mL REP T cells significantly upregulated CD40L within 24 hours when exposed to the commensal anaerobe pool but not to the other two pools when compared to buffered saline control. CD40L positive CD4+ T cells against the commensal anaerobe pool were subsequently flow-sorted and clonally expanded from a 96-well plate with 2 additional REP cycles. Subsequent rescreening for activity against the commensal anaerobe pool identified 1 out of 8 clones that were confirmed positive upon repeat assay.

Conclusions:

We developed a novel method that enables the detection of microbiota-reactive T cells post-transplantation in the context of native MHCII genotype. This tool can be used isolate T cell clonotypes of interest for downstream characterization and epitope validation.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH