Why does Chemotherapy affect the proliferation of mature T cells?

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Why does Chemotherapy affect the proliferation of mature T cells?

Why does Chemotherapy affect the proliferation of mature T cells? Immune-based therapy is becoming a promising alternative or supplement to traditional chemotherapy…..

Introduction

Immune-based therapy is becoming a promising alternative or supplement to traditional chemotherapy. Most of these, such as chimeric antigen receptor (CAR) T cells or negative checkpoint receptor (NCR) inhibitors, are being used for relapsed or refractory chemotherapy. Transformation data from two milestone trials showed that the presence of early memory or naive T cells, or conversely the lack of depleted T cells, is associated with better expansion and persistence of CAR T cell products. However, these surface phenotypes, as independent biomarkers of CAR T cell performance, cannot provide complete sensitivity. This article mainly wants to explore the non-surface marker phenotypes of T cells that survive chemotherapy to investigate other features of potential dysfunction.

Previous reports in pediatric patients indicate that children with leukemia have lower absolute T cell counts at the time of diagnosis and throughout treatment, and that chemotherapy consumes T cells over time.

Importantly, T cells recovered from children after a chemotherapy cycle show an increased incidence of activation-induced cell death (AICD) in response to mitogens. The mechanism by which it responds to mitogens is unclear.

The internal energy potential of T cells, measured by oxygen consumption analysis and mitochondrial membrane potential (Dc), has also been shown to be critical to the effectiveness of T cell therapy. This is intuitive, because the mitochondrial mass and energy reserves of T cells change with the transition from the initial state to the memory state and then to the effect state.

We used in vitro chemotherapy exposure to characterize the effects of mature T cells and each subpopulation to help clarify the potential impact on T cell energy reserves, which is a possible mechanism of poor T cell function. Postoperative chemotherapy.

We found that the three types of chemotherapy impair T cell energy reserves in different ways at the mitochondrial level and by relying on glycolysis, and this also differs between T cell subpopulations. The ability of T cells and certain subpopulations to recover and expand varies depending on the nature and time of exposure.

We evaluated the use of N-acetylcysteine ​​(NAC), a known antidote to cyclophosphamide-induced reactive oxygen damage, to reverse the effects on proliferation. Using this model, we prepared CAR T cells from cyclophosphamide-exposed T cells, with and without NAC, indicating that NAC significantly increased the recovery of CAR1 cells and restored metabolic reserves.

Adaptive cell manufacturing, changing the medium or reagents according to the biomarkers of each product to cultivate T cells, is a subject of in-depth research.

These data reveal lingering defects in chemotherapy-exposed T cells whose metabolic characteristics may be targeted for reversing adaptive manufacturing to improve cell therapy.

Results:

Inspection of mitochondrial integrity after chemotherapy exposure

Mitochondrial damage after chemotherapy is a common way, and it is likely to be the main cause of cell death through apoptosis.

We hope to characterize the impact on the integrity and function of mitochondria in surviving cells after chemotherapy exposure, because these T cells will be collected for adoptive cell therapy. Cyclophosphamide (4HPCP used in in vitro studies) has a devastating effect on Dc, which is true in T cell naive, CM, or EM. It survives 24 hours after chemotherapy and remains viable for 72 hours (Figure 1) .

Cytarabine has no effect on the membrane potential, while doxorubicin seems to only increase (polarize) the membrane potential in TCM cells.

Using the mitochondrial matrix dye (Mitotracker Green), we saw a significant increase in the mitochondrial biomass exposed to cyclophosphamide, although follow-up inspections with TEM showed that this was not accurate.

We tried to correlate this with patient samples with sufficient T cells before and after cyclophosphamide chemotherapy for primary mediastinal B-cell lymphoma. In this patient, the mitochondria before T cell treatment appeared to have a normal long and narrow cristae, while the T cells collected 2 weeks after cyclophosphamide were similar to those exposed to cyclophosphamide in vitro, with small, round, Dense mitochondria and short and wide cristae (Figure 1).

Figure 1. Mitochondrial integrity after chemotherapy exposure. (A) A large number of T cells or T cells classified into 3 subsets (naive, CM, or EM) are exposed to chemotherapy for 24 hours, recovered, and then Dc is measured by JC1 dye. Cyclophosphamide (4HPCP) will ablate the membrane potential of all T cell subsets. 4HPCP, 2.5 mM cyclophosphamide; Cytarabine, 3 mM Cytarabine; Dox, 400 nM Doxorubicin. (B) Except for more dye uptake by cyclophosphamide-exposed T cells, there was no significant change in mitochondrial biomass after chemotherapy exposure. (C) Transmission electron microscopy (375 000) of T cells reveals the ultrastructural effects of cyclophosphamide. 1. T cells from a normal donor; 2. T cells from the same donor were exposed to cyclophosphamide for 24 hours; 3. T cells from a patient with non-Hodgkin’s lymphoma before any chemotherapy; and 4. The same patient had cyclophosphamide 2 weeks after amide chemotherapy. Short and widened ridges are characteristic of cyclophosphamide exposure in vitro or in vivo.

We can only evaluate one patient, because obtaining the required number of T cells from a child’s single peripheral blood usually does not produce enough numbers for TEM. Finally, we examined the presence of the anti-apoptotic factor BCL-2 in T cell subsets before and after chemotherapy. As expected from previous reports, naive T cells have higher levels of BCL-2 than TCM and T EM cells. These levels are still high in naive T cells that survived chemotherapy, while TCM and TEM cells that survived cyclophosphamide showed higher levels of BCL-2 compared to pre-chemotherapy treatment. Therefore, BCL-2 may be an important part of T cells to survive the mitochondrial damage caused by cyclophosphamide chemotherapy (Figure 2).

Figure 2. BCL-2 expression in T cells receiving chemotherapy. T cells from normal donors, whether in batches or classified into subsets, received chemotherapy and recovered. The expression of BCL-2 in living cells was checked by intracellular flow cytometry. Naive cells have a higher baseline BCL-2 than memory cells, and memory T cells that survive in cyclophosphamide have higher BCL-2 levels compared to controls.

Chemotherapy changes T cell mitochondrial respiration before and after stimulation

We tested OCR, Standby Respiratory Capacity (SRC) and ECAR on T cells exposed to doxorubicin, cyclophosphamide, and cytarabine for 24 hours, and then recovered them. Cytarabine damages all aspects in unstimulated T cells, while doxorubicin has no significant effect. Cyclophosphamide only reduces all three at higher doses (Figure 3).

Figure 3. Mitochondrial respiration analysis of T cells undergoing chemotherapy (short-term analysis). (A) OCR analysis of T cells exposed to chemotherapy and unstimulated or stimulated with CD3/28 beads for 48 hours. The short-term stimulation test revealed the shortcomings of OCR (B) ECAR analysis from the same experiment. Only Cytarabine (Cyta) will reduce ECAR in stimulated cells (C) SRC in the same experiment. In unstimulated cells, higher doses of cyclophosphamide and cytarabine will reduce this mitochondrial reserve, but these effects basically disappear after short-term stimulation. (D) Glucose is converted to lactic acid after chemotherapy exposure and short-term stimulation. Chemotherapy reduces the ability of T cells to produce lactic acid.

We evaluated the same parameters of T cells that were exposed to chemotherapy for 24 hours and then stimulated with CD3/28 beads for 24 hours. Compared to the control, cytarabine was again significantly reduced in all 3 parameters. Any dose of chemotherapy will impair the maximum OCR and moderately reduce ECAR. The effect on SRC varies, and it increases with the dose of cyclophosphamide. We also characterized glucose consumption and lactate conversion under the same conditions.

Regardless of whether chemotherapy is received, unstimulated cells will not produce lactic acid. In T cells stimulated with beads for 48 hours, doxorubicin reduced the production of lactic acid and was almost completely eliminated by cyclophosphamide and cytarabine (Figure 3).

It is expected that stimulated T cells will use glycolysis and increase lactic acid production. We hope to correlate these effects with the longer stimulation periods used in CAR T cell manufacturing. We exposed T cells to chemotherapy for 24 hours, followed by CD3/28 stimulation for 7 days, and then rested until the cell volume reached 350 fL. In control T cells that were exposed to chemotherapy but were not stimulated, chemotherapy was devastating for all aspects of mitochondrial respiration of surviving T cells in this time frame (13 days; Figure 4). T cells stimulated during CAR manufacturing changed the mitochondrial respiration curve. Cells exposed to doxorubicin and cytarabine showed a higher basal glycolysis rate, while doxorubicin showed a higher generation rate. Compensate glycolysis rate and ECAR. At this time point, compared with all other cells, the SRC of cyclophosphamide-exposed T cells was significantly reduced (Figure 4). These data not only indicate differences in the short-term and long-term consequences of chemotherapy exposure on T cell mitochondrial reserves and metabolism, but also that each chemotherapy category has different effects.

Figure 4. Mitochondrial respiration analysis of T cells exposed to chemotherapy (long-term stimulation). In this set of tests, T cells were exposed to chemotherapy for 24 hours, then recovered and remained unstimulated or exposed to CD3/28 beads for 7 days, and then rested for 6 days to simulate CAR manufacturing. The parameters evaluated by line are OCR (A), ECAR (B), SRC (C), basal glycolysis rate (D) and compensation glycolysis rate (E). Under unstimulated conditions, any amount of chemotherapy can disrupt mitochondrial respiration and glycolytic energy production. Under stimulation, surviving T cells have different changes. The most notable features are the increase in ECAR and glycolysis in T cells exposed to doxorubicin and cytarabine, and T cells exposed to cyclophosphamide. Loss of SRC in the cell.

T cell subsets respond differently to chemotherapy

Given the potential importance of naive and early memory cells in generating effective CAR T cells, and the clinical data showing that these cells are depleted by chemotherapy, we tried to classify the effects of chemotherapy on each T cell subpopulation.

Looking at the expansion potential first, we confirmed our previous report that naive, TSCM and TCM cells expand well under CD3/28 magnetic beads exposure. When stimulated with CD3/28 beads, EM cells will not proliferate or explode in large numbers (reflected by the maximum cell volume). As before, we exposed T cells to chemotherapy for 24 hours, then stimulated with CD3/28 magnetic beads for 7 days, then removed the beads and recovered within 6 days. Doxorubicin and cytarabine completely blocked the expansion ability of any T cell subpopulation (Table 1).

It is worth noting that chemotherapy also affects the ability of T cells to respond to stimuli, and TSCM cells retain this ability unless exposed to cytarabine. Cyclophosphamide had no significant effect on T SCM cells, which resulted in the expansion of these donors remaining unchanged. Due to the rarity of TSCM cells, we were unable to perform additional experiments on this subset because we did not recover enough cells for mitochondrial respiration studies. However, we analyzed the OCR, ECAR, and SRC of each subset after chemotherapy exposure and with or without stimulation (Figure 5). In unstimulated cells, all chemotherapy reduced OCR/ECAR/SRC stimulation.

Even after stimulation, cytarabine continues to inhibit these functions. In addition, we evaluated glucose uptake under these conditions and found that chemotherapy increased glucose uptake in unstimulated naive cells and decreased glucose uptake in EM cells. Again, these effects were alleviated by stimulating cells exposed to doxorubicin, on the contrary, the naïve exposure to cyclophosphamide and cytarabine and reduced glucose uptake in CM. TEM cells are basically unaffected (Figure 5).

Figure 5. Mitochondrial respiration analysis in T cell subsets. T cells from normal donors were divided into 3 subpopulations and exposed to chemotherapy for 24 hours to restore them, and then exposed to CD3/28 beads for 48 hours without stimulation. The parameters evaluated by line are OCR (A), ECAR (B), SRC (C) and glucose uptake (D). Doxorubicin at 400 nM reduces all aspects of unstimulated naive T cell respiration and increases glucose uptake accordingly. Cyclophosphamide and Cytara are harmful to almost all subgroups. The stimulation largely rescued the effects of doxorubicin, but revealed defects in T cells exposed to cyclophosphamide and cytarabine. Glucose uptake after stimulation is only affected by cytarabine in the initial and TCM cells.

NAC restores the proliferation of T cells exposed to cyclophosphamide

It is well known that cyclophosphamide induces reactive oxygen species as a method of damage, so we applied the ROS scavenger NAC to T cells exposed to cyclophosphamide. The purpose is to explore whether T cells exposed to chemotherapy are functionally defective after being made into CAR T cells, or whether the defect is less related to cytotoxicity, but is related to energy reserve and proliferation. Preliminary tests of doxorubicin, cyclophosphamide and cytarabine with NAC showed that only cyclophosphamide reversed cell death or mitochondrial function, so only cyclophosphamide was used for CAR experiments.

T cells from normal donors (n=53) were exposed to cyclophosphamide, rescued with NAC, and then manufactured into CAR T cells targeting CD19, as described above.

We noticed that the proliferation of T cells exposed to cyclophosphamide was reduced by nearly 10-fold, but the degranulation or cytokine production of those cells that survived the manufacturing process did not decrease (Figure 6). NAC rescue reversed the defects of proliferation and mitochondrial respiration (OCR and ECAR).

Figure 6. NAC restores the proliferation and metabolic reserve of T cells treated with cyclophosphamide. T cells from normal donors (n=53) were exposed to cyclophosphamide, rescued with NAC, and then manufactured into CAR T cells that target CD19. We targeted the CD19 target (Nalm-6) in a 24-hour assay (A) And IFNγ production (B) evaluated CD107α degranulation. T cells that survived after manufacturing did not have significant differences in short-term killing potential or cytokine release. The proliferation of cyclophosphamide (C) was observed to be significantly reduced during the manufacturing process, however, much fewer cells were recovered. The peak burst response measured by cell size (D) is also unaffected. (E-F) NAC restored ECAR and OCR to near normal levels. Treatment with NAC restores proliferation and metabolic reserves.

In conclusion

Although previous studies evaluated the effects of chemotherapy on T cells, these studies Focus on the patient’s consumption, rather than the impact on surviving cells. For most chemotherapy regimens, the potential of T cells to respond to in vitro stimuli declines over time, indicating that surviving T cells have lingering defects in chemotherapy exposure. The mechanism behind this has not yet been explored, whether the reasons behind dysfunction are also common or unique to each chemotherapeutic drug. This may have a potential impact on the participation of T cells in therapy or therapies that require collection of patient T cells for in vitro manipulation.

Most (if not all) CAR T cell manufacturing strategies involve T cell stimulation, whether using CD3/28 beads or alternative antigen presentation to the CAR, because stimulation is usually required to promote retroviral vector integration. Different centers and clinical products are made of different support media and reagents, highlighting that multiple support strategies are possible.

The strong energy response is related to T cell stimulation. Here we discussed the effects of three types of chemotherapy on mitochondrial energy storage; doxorubicin, cytarabine and cyclophosphamide. We have found profound effects on mitochondrial function and energy reserves in all three categories, although these effects vary by agent and individual T cell subsets. This applies to both short-term and long-term stimulation, although in most cases, the metabolic phenotype of chemotherapy-exposed T cells after stimulation is different from that before stimulation. Stimulation may lead to the induction of AICD, or damaged T cells may activate other pathways to survive the stress of the stimulus, thereby affecting long-term proliferation.

The presence of a high percentage of naive cells and SCM cells still does not guarantee expansion or clinical efficacy, which means that there may be quality defects that are not captured by the surface phenotype. With this in mind, we tried to characterize internal metabolic reserves that may be associated with better expansion potential. This prompted us to study the mitochondrial and metabolic reserves of T cells, including key subpopulations of naive T cells.

We focus on the effects of chemotherapy because this can be modeled in vitro. Chemotherapy has specific types of effects on T cells (including naive T cells), and these effects differ between unstimulated and stimulated T cells.

Cyclophosphamide has a devastating effect on all T cells, which is mediated by mitochondrial damage. T cells treated with cyclophosphamide showed small, damaged mitochondria, low membrane potential (depolarization), and loss of many mitochondrial reserve measures (including SRC).

It is worth noting that early stimulation after chemotherapy can alleviate many of these findings over time, although our data suggests that this may be due to the exceptional survival and proliferation of the SCM subset. This subset is rare, and it is difficult to study without artificial cultivation conditions, so we will focus on the further mechanism research of this subset in the future, and establish most of the baseline data here.

The mechanism behind doxorubicin is unclear, because cells explode in response to stimuli, increasing their volume, but then die in a process similar to activation-induced cell death. Doxorubicin has little effect on mitochondrial integrity or function. T cells exposed to doxorubicin showed impaired short-term lactic acid production, but increased use of glycolysis after long-term stimulation. Although the obvious final pathway is AICD, relatively complete mitochondrial membrane studies and normal OCR and ECAR rates indicate that the process may be more complicated and may be suitable for metabolic interventions for glucose use to reverse damage and maintain T cell capacity.

The lack of cytarabine provides a simple explanation of how it impairs T cell proliferation. Here, the pattern is that mitochondria are damaged, citrate synthase activity is low, and mitochondrial respiration measurement is impaired, but it has no effect on membrane potential or ultrastructural changes. Purine analogs have been reported to interfere with mitochondrial (mt) DNA, but we did not detect any mtDNA copy number changes in cytarabine-treated T cells (data not shown).

Previously, all three chemotherapeutics have been characterized for their ability to clear lymphocytes. The only focus of our attention is to assess the ability of lymphocytes that survive these drugs in the short term to respond to in vitro stimuli associated with adoptive cells.

As more clinical data are reported, the importance of “starting material”, that is, T cells collected from patients, becomes more and more obvious. The clinical data we reported earlier clearly show that cumulative chemotherapy consumes T cell potential and the difference between diagnosis and chemotherapy regimens. This has been explored in adult chronic lymphocytic leukemia, and found that the starting material from patients with poor clinical response in the CD19 CAR trial has higher glycolytic characteristics. Blocking the use of glucose with 2-deoxyglucose in a research-based remanufacturing process produces products with better in vitro amplification curves. Our data here shows that the defects caused by chemotherapy will persist, even if it succeeds, it will affect the final quality manufacturing of cell therapy products.

All in all, we examine here for the first time the effects of chemotherapy types on mature T cells and their subpopulations, with a focus on the relevance to pediatric cancer immunotherapy. We demonstrated the differences between the three types of chemotherapy in terms of stimulation and non-stimulation, as well as short-term and long-term differences in T cell mitochondrial energy storage and glycolytic function.

In addition, we plan to examine patients’ T cells in future trials to verify these characteristics. The time of chemotherapy exposure may be very important, because new thymus migration (recovering T cells that did not exist during chemotherapy) may increase over time and is important for the quality of the final cell therapy, while chemotherapy-exposed T cells are Has lingering mitochondrial defects. These studies have laid the foundation for the potential and rational adjustment of T cell collection or manufacturing strategies to ensure the highest activity of future cell therapies.

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