*For medical professionals only
The birth of CAR-T cell therapy has changed the fate of many children with leukemia.
However, the effect of CAR-T cell therapy is difficult to predict. Some children have long-term remission after treatment, and even achieve clinical cure; More children are quickly returning due to the disappearance of CAR-T cells and other reasons.
Although the persistence of CAR-T cells has been found to be closely related to the effectiveness of treatment [1], scientists and doctors know little about the characteristics of CAR-T cells that persist.
At the beginning of last year, the Carl H. June team of the University of Pennsylvania analyzed CAR-T cells in two adult patients with chronic lymphocytic leukemia (CLL) who had been in remission for more than a decade, and found that the last group of cytotoxic CD4+ CAR-T cells that persisted was actually a group of cytotoxic CD4+ CAR-T cells [2]. However, this study was small, and it is unknown whether other types of patients have similar conditions.
Recently, a research team led by Sara Ghorashian of Great Ormond Street Children's Hospital of University College London and Sam Behjati of the Sanger Institute published the largest persistent CAR-T cell study to date in the famous journal Nature Medicine [3], further revealing the characteristics of CAR-T cells in patients who achieve sustained remission after receiving CAR-T therapy.
It is worth noting that in this study, the researchers found that persistent CAR-T cells were not CD4-positive, but CD4/CD8 double-negative, a conclusion different from June's team, highlighting the complexity of persistent CAR-T. However, the good news is that the two teams found similar transcriptional signatures in persistent CAR T cells, a discovery that may leave a window for scientists studying persistent CAR T cells.
Screenshot of the first page of the paper
Next, let's take a look at how the Ghorashian team's research unfolded and what they found.
To further characterize persistent CAR-T cells, Ghorashian's team used CD19 CAR-T cell samples from 10 children with R/R B-ALL who participated in the CARPALL trial. The CARPALL study initially enrolled 15 children, 13 of whom achieved complete remission; Six of these children with complete remission subsequently relapsed, and the other seven were shown to achieve long-term remission.
They obtained enough CAR-T cells from blood samples from 10 of the children and divided the samples into early (1-3 months, time window to achieve complete remission), intermediate (4-6 months), and late (7-60 months, time window for relapse) based on the sampling distance. CAR-T cells are then isolated from peripheral blood or bone marrow using CD3 and CAR expression by flow cytometry.
The patient's disease profile and research process
In total, Ghorashian's team collected 264827 single cells that had passed quality control, of which about 50,000 were CAR-T cells. After analyzing markers on the surface of all CAR T cells, they found that CD8+ T cells were the primary CAR-T cells in most cases; However, at this late time point, CAR-T cells lack the expression of CD4 and CD8A transcripts.
It is worth noting that at all time points, the proportion of CD4+ CAR-T cells is small. In summary, those persistent CAR-T cells are mainly CD4/CD8 double-negative T cells. This finding is diametrically opposed to what June's team found (more CD4+ CAR-T cells and fewer double-negative T cells) [2].
To confirm these findings, Ghorashian's team obtained peripheral blood (PB) and bone marrow (BM) samples from seven additional children who received sustained remission, which were collected at advanced time points (7-72 months after infusion).
They isolated and analyzed these CAR-T cells, confirming that most cells at advanced time points were double-negative. In contrast, the proportion of non-CAR-T cells in these children is low. In addition, they found that these persistently present CAR-T cells also exhibited a lack of CD45RA and CCR7 expression, suggesting that they have an effector memory phenotype.
Based on the above research data, the researchers believe that most persistently present CAR-T cells are double-negative alpha T cells.
Different T dynamically change
The next question is, are there any similarities between persistent CD4-CD8-CAR T cells in different patients at the transcriptional level, and whether they are different from CAR-T cells in other time periods?
There really is.
Ghorashian's team used single-cell sequencing technology to analyze transcriptome data from early, middle, and late CAR-T cells, as well as pre-infusion CAR T cells, and found that there were large differences, and that late-stage persistent CAR-T cells exhibited a common transcriptome signature.
Transcriptome features
Specific to late-stage persistent CAR-T cells, they are highly expressed in immune-related genes such as TIGIT and GPR183, as well as genes with unknown or emerging roles in immunobiology, such as FXYD2, HMOX1, DENND2D, and ISG20.
The emergence of TIGIT, a marker of T cell depletion, caught the attention of Ghorashian's team. Further analysis found that persistent CAR-T cells did express some typical co-inhibitory receptors, such as HAVCR2 and LAG3; PDCD1 expression also increased slightly. In addition, similar to precursor-depleted T cells (related to immunotherapy efficacy), advanced CAR-T cells also have relevant effector genes (GZMK and PRF1), as well as the expression of transcription factors associated with T cell depletion (TOX, NFATC1, BATF, and PRDM1, etc.).
Although persistent CAR-T cells exhibit depletion characteristics, they do not show terminal differentiation characteristics. In addition, the expression of JUN in persistent CAR-T cells is strong, and JUN can mediate the reversal of T cell failure, as well as maintain those cells with stem cell memory properties. Based on the above results, the researchers believe that persistent CAR-T cells have depletion-like memory states and unique transcriptional characteristics.
Transcriptional characteristics of CAR-T cells at different stages
As for whether these persistent CAR-T cells originated from clonal expansion, Ghorashian's team's results suggest that this functional convergence is not due to clonal expansion, because the CAR-T cell population is genetically diverse at all time points, and there is no evidence that one or more clones dominate at late time points.
In particular, Ghorashian's team compared this transcriptional signature with the June team's data, and the results were surprisingly similar, and although the persistent T cells found in the two studies were very different (one double-negative and one CD4-positive), the transcriptional characteristics of persistent CAR-T cells in the two studies were highly similar.
This similarity led Ghorashian's team to doubt the universality of this transcriptional signature, but after scoring T cells from a variety of healthy tissues, other CAR-T studies to date, and single-cell atlas of cancer cells, they dispelled this suspicion because they did not find this transcriptional signature for persistent CAR-T cells. That is, this transcriptional feature is specific to persistent CAR-T cells.
Transcriptional characteristics of different T cells
Overall, Ghorashian's team believes that the most important finding of their study was the discovery of unique transcriptional signatures of persistent CAR-T cells. And this feature also exists in different populations and different CAR-T therapies. So this suggests that this unique transcriptional signature has the potential to represent a universal feature of persistent T cells.
In the future, the transcriptome changes found in this study deserve further exploration. If a breakthrough can be made, it may help scientists solve the puzzle of persistent CAR-T and find ways to make CAR-T cells persist and better treat various cancers.
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Bibliography:
[1]. Finney OC, Brakke HM, Rawlings-Rhea S, et al. CD19 CAR T cell product and disease attributes predict leukemia remission durability. J Clin Invest. 2019;129(5):2123-2132. doi:10.1172/JCI125423
[2]. Melenhorst JJ, Chen GM, Wang M, et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature. 2022;602(7897):503-509. doi:10.1038/s41586-021-04390-6
[3]. Anderson, N.D., Birch, J., Accogli, T. et al. Transcriptional signatures associated with persisting CD19 CAR-T cells in children with leukemia. Nat Med, 2023. https://doi.org/10.1038/s41591-023-02415-3
The author of this article 丨BioTalker