The metabolic profile of reconstituting T-cells, NK-cells, and monocytes following autologous stem cell transplantation and its impact on outcome

Previous studies indicated a role of the reconstituting immune system for disease outcome upon high-dose chemotherapy (HDCT) and autologous stem cell transplantation (auto-SCT) in multiple myeloma (MM) and lymphoma patients. Since immune cell metabolism and function are closely interconnected, we used flow-cytometry techniques to analyze key components and functions of the metabolic machinery in reconstituting immune cells upon HDCT/auto-SCT. We observed increased proliferative activity and an upregulation of the glycolytic and fatty acid oxidation (FAO) machinery in immune cells during engraftment. Metabolic activation was more pronounced in T-cells of advanced differentiation stages, in CD56bright NK-cells, and CD14++CD16+ intermediate monocytes. Next, we investigated a potential correlation between the immune cells’ metabolic profile and early progression or relapse in lymphoma patients within the first twelve months following auto-SCT. Here, persistently increased metabolic parameters correlated with a rather poor disease course. Taken together, reconstituting immune cells display an upregulated bioenergetic machinery following auto-SCT. Interestingly, a persistently enhanced metabolic immune cell phenotype correlated with reduced PFS. However, it remains to be elucidated, if the clinical data can be confirmed within a larger set of patients and if residual malignant cells not detected by conventional means possibly caused the metabolic activation.


Results
Absolute T-and NK-cell but not monocyte count is significantly reduced at engraftment time following HDCT/auto-SCT. Several studies in different malignancies indicated that a stable lymphocyte reconstitution upon auto-SCT and a lymphocyte to monocytes ratio ≥ 1 are both associated with improved PFS and OS 10 . Therefore, we monitored the lymphocyte and monocyte count within our patient population, thereby excluding B-cells since they were usually not detected in lymphoma patients due to prior rituximab therapy. The number of CD3 + T-cells was significantly reduced at the time of engraftment (746 vs. 180 per µl; p < 0.0001) and fully recovered at the restaging appointment (Fig. 1A). Interestingly, we noticed a skewing towards CD8 + T-cells over the course of hematopoietic reconstitution (Fig. 1B). Next, we investigated the dynamics of T-cell differentiation including naturally occurring regulatory (T REG ), naïve (T N ), recent thymic emigrant (T RTE ), stem cell memory (T SCM ), central memory (T CM ), transitional memory (T TM ), effector memory (T EM ), and effector memory re-expressing CD45RA (T EMRA ) T-cells (Supplementary Table S1 and Figure S1 online) 20,21 . Within the CD4 + T-cell compartment, early differentiation stages (i.e., T N , T SCM , and T RTE ) decreased. In contrast, proportion of all memory phenotypes (except T CM ) and of T REG increased to higher levels as compared to the pre-HDCT situation. Among CD8 + T-cells we observed a decrease of T N and T SCM cells. At engraftment T CM and T TM frequencies increased and T EMRA decreased while T EM remained stable, which was followed by a contraction of T CM , and T TM , and a remarkable expansion of T EM (Fig. 1C and Supplementary Table S2 online).
Since the duration from auto-SCT until the restaging appointment significantly differed between myeloma and lymphoma patients (Table 1), we further compared absolute cell numbers between the two patients groups at this time point. However, no significant differences were observed for neither of the three cell subsets (Supplementary Figure S2 online).
T-cells, NK-cells, and monocytes maintain their ability to produce cytokines following HDCT/ auto-SCT. Next, we were interested whether reconstituting immune cells were capable of cytokine production. In CD4 + T-cells IL-2 production was stable, whereas IL-4, IFN-γ, and TNF displayed elevated levels. Which was similarly observed for IFN-γ and TNF in their CD8 + counterparts. However, only TNF production in CD8 + T-cells reached statistical significance, probably due to the low number of tested patients ( Fig. 2A-F). Similarly, pro-inflammatory cytokine production by NK-cells (i.e., IFN-γ) and monocytes (i.e., IL-6) was not negatively affected. Rather, production of IFN-γ by NK-cells was highest at the time of engraftment (Fig. 2G,H).

T-cells, NK-cells, and monocytes display increased proliferative activity and an enhanced glycolytic machinery during reconstitution upon HDCT/auto-SCT.
Early and robust immune cell reconstitution upon auto-SCT is associated with less infectious complications and improved tumor surveillance 10 . During engraftment T-cells, NK-cells, and monocytes were highly proliferative as indicated by the high frequency of Ki-67 + cells (Fig. 3A). It is well established, that proliferating immune cells maintain high levels of (aerobic) glycolysis 11 . As anticipated, we found in all investigated types of cells an increased expression of hexokinase 2 (HK2), the rate-limiting enzyme of glycolysis (Fig. 3B). This was accompanied by an abundance of glucose transporter 1 (GLUT1, Fig. 3C) and an increased glucose uptake (Fig. 3D). Over time, all glycolytic parameters examined normalized together with the cells' proliferative activity (Fig. 3A-D).  www.nature.com/scientificreports/

FAO and mitochondrial parameters are increased in T-cells, NK-cells, and monocytes upon
HDCT/auto-SCT. Beyond glycolysis, FAO and mitochondrial metabolism are essential elements of the immune cells' homeostasis and function 23 . In analogy to HK2, carnitine palmitoyl-transferase I alpha (CPT1α), the rate-limiting enzyme of the FAO pathway, was significantly increased during engraftment in all three cell populations. Of note, while CPT1α levels decreased back to baseline levels in NK-cells, they remained elevated in T-cells and monocytes (Fig. 4A). Increased CPT1α expression during engraftment was accompanied by elevated uptake of fluorescent fatty acid analogues (Bodipy FL C16 ) (Fig. 4B). Mitochondria represent central metabolic hubs. Mitochondrial mass was increased in T-and NK-cells but not monocytes during engraftment and returned to baseline levels at later time points (Fig. 4C). Mitochondrial potential as a surrogate for mitochondrial fitness and an indicator for mitochondrial activity was elevated in all cell types during engraftment (Fig. 4D). There are several sources for reactive oxygen species (ROS) in a cell. Mitochondria are a major site for ROS production, which takes place at the electron transport chain during OXPHOS. We found elevated global ROS levels in all cell types during engraftment, which is consistent with their increased metabolic activity (Fig. 4E). However, mitochondrial ROS production was only significantly increased in NK-cells during engraftment, but not in T-cells or monocytes (Supplementary Figure S3 online).
Proliferative activity and metabolic armamentarium are not homogenous within T-cell, NK-cell, and monocyte subpopulations following HDCT/auto-SCT. Next, we assessed, whether the observed alterations in terms of proliferation and expression of glycolysis or FAO pacemaker enzymes following HDCT/auto-SCT are consistent among the major subsets of T-cells, NK-cells, and monocytes. Proliferation was increased at engraftment compared to pre-HDCT in all investigated major subsets. The proliferating fraction of CD4 + and CD8 + T-cells was comparable at engraftment. CD56 bright NK-cells displayed a superior proliferative activity as compared to their CD56 dim counterpart. Finally, fraction of proliferative cells was highest amongst intermediate monocytes at all investigated time points (Fig. 5A). In terms of HK2, reconstituting CD8 + T-cells had significantly higher levels as compared to CD4 + T-cells. No differences could be observed for CD56 bright and CD56 dim NK-cells. In addition, classical and intermediate monocytes demonstrated significantly higher HK2 levels during engraftment and restaging as compared to non-classical ones, which did not demonstrate elevated HK2 levels at restaging as compared to the pre-HDCT timepoint (Fig. 5B). CPT1α levels increased upon auto-SCT and were similar in the analyzed T-and NK-cell compartments during engraftment. www.nature.com/scientificreports/ In fact, CPT1α content was higher in reconstituted CD4 + T-cells and lower in CD56 dim NK-cells prior to HDCT. Intermediate monocytes displayed the highest CPT1α expression during all investigated time points (Fig. 5C). Taken together, proliferative activity and expression of metabolic pacemaker molecules display a heterogeneous pattern amongst T-cell, NK-cell, and monocyte subsets following auto-SCT, with proliferation not always positively correlating with HK2 and/or CPT1α expression (as an expression of an increased energetic demand).
CD4 + and CD8 + T-cells at early differentiation stages were less proliferative and less metabolically active. After characterizing the two major T-cell subsets, we aimed to extend our assessment to the area of T-cell differentiation including T REG , T N , T SCM , T TM , T EM , T EMRA , and T CM cells (Supplementary Figure S1 online) 21 . In fact, during engraftment early differentiation stages such as T N and T SCM cells had low proliferation rates (Fig. 6A) as well as low HK2 (Fig. 6B) and CPT1α (Fig. 6C) expression levels as compared to later differentiation stages (i.e., T CM , T TM and T EM CD4 + /CD8 + cells). CD4 + and CD8 + T TM were strongly proliferative and were HK2 high during engraftment. High CPT1α levels were observed within CD4 + and CD8 + T EM as well as within CD8 + T CM cells at this time point. In addition, CD4 + T REG cells were (like CD4 + T TM cells) Ki-67 high , HK2 high , and CPT1α high . Despite that CD8 + T EMRA cells did not show a strong proliferation rate as compared to CD8 + T TM and T CM cells, they demonstrated a HK2 high and CPT1α high phenotype (Fig. 6).
In summary, our data indicates that early differentiation stages of both CD4 + and CD8 + conventional T-cells are less metabolically active, whereas CD4 + T REG cells have an activated metabolic profile during engraftment.
Persistently increased metabolic activity upon HDCT/auto-SCT is associated with refractory disease or early relapse in lymphoma. Finally, we were interested in whether the metabolic profile of T-cells, NK-cells or monocytes can be correlated with the clinical outcome in lymphoma patients [8][9][10] . Therefore, we compared the cells' Ki-67, HK2, and CPT1α expression levels in patients in remission versus patients with  www.nature.com/scientificreports/ refractory disease or early relapse during the first year upon HDCT/auto-SCT. By applying multiple unpaired Mann-Whitney tests and False-Discovery-Rate adjustments we identified HK2 for NK-cells, Ki-67 for T-and NK-cells, and CPT1α for all three analyzed cell types at the time point of the restaging (i.e., post-SCT) as discriminators for both patient groups (Fig. 7A). Interestingly, NK-cells displayed upregulated Ki-67, HK2, and CPT1α levels during engraftment that remained elevated in the patient group with r/r lymphoma (Fig. 7B-D). Similar patterns were also noticed for CPT1α and Ki-67 in T-cells and for CPT1α in monocytes respectively ( Fig. 7E-G).
To obtain the optimal cut-off values of the continuous variables Ki-67, HK2, and CPT1α that correlates with PFS, area under the ROC curve analyses were performed (Supplementary Figure S4-9 online). In fact, we were able to identify expression (cut-off) levels for HK2 in NK-cells, for Ki-67 in T-and NK-cells, and for CPT1α in T-cells, NK-cells, and monocytes significantly correlating with PFS ( Fig. 7H-M). Since we observed a significant skewing of the CD8 + :CD4 + T-cell ratio during reconstitution, we separately analyzed the impact of the metabolic profile of CD8 + and CD4 + T-cells on the clinical outcome of lymphoma patients. We identified Ki-67 for CD8 + T-cells, and CPT1α for CD4 + and CD8 + T-cells to be significantly different between the two patient groups during To exclude confounding clinical parameters, we compared time from last therapy to HDCT and from auto-SCT to engraftment and restaging in the remission versus the r/r group and could not detect any differences. Moreover, no significant differences were observed concerning gender distribution, age, number of previous therapies or the number of infused CD34 + hematopoietic stem cells. Finally, although it has been previously reported that lymphoma patients with lower absolute lymphocyte counts (ALC) upon auto-SCT had an inferior PFS and OS 10 , we observed no difference between the two groups. In addition, the time of relapse from auto-SCT ranged from several days before restaging until 99 days afterwards in our patient cohort, with at least two patients who have already relapsed during the last analysis. The clinical data is summarized in Supplementary  Table S3 online.

Discussion
HDCT/auto-SCT can induce long-term remission in several hematological malignancies. A rapid reconstitution of immune cells including lymphocytes and NK-cells is associated with an improved PFS and overall survival (OS) 10,24-26 . Mounting evidence suggests an interrelation of immune cell proliferation, function, and metabolism. In fact, following HDCT/auto-SCT we observed an upregulation of the glycolytic key enzyme HK2 and of glucose transporters as well as an enhanced glucose uptake in T-cells, NK-cells, and monocytes. CPT1α that is critical for FAO was also found increased in all tested immune cell types. Furthermore, T-and NK-cells demonstrated enhanced mitochondrial biogenesis during engraftment. These changes of the metabolic phenotype were accompanied by an increased proliferative activity and a superior production of pro-inflammatory cytokines by CD8 + T-cells.
T-cells undergo metabolic reprograming upon activation to be able to rapidly proliferate and to carry out their effector functions. They activate glycolysis to rapidly generate bioenergetic substrates and biosynthetic precursors, which is in line with our observations 27 . We also observed increased CPT1α expression during engraftment, a feature that is typically seen in memory T-cells 27 . Fittingly, frequency of memory T-cells was increased at this www.nature.com/scientificreports/ time point. Moreover, mitochondrial biogenesis was enhanced as well, which has been observed in memory and effector T-cells 28 . In addition, IL-15 levels are elevated at engraftment, which triggers the metabolic master regulator mammalian target of rapamycin (mTOR) leading amongst others to enhanced glycolysis, FAO, and mitochondrial biogenesis [29][30][31] . Furthermore, robust IFN-γ production by NK-cells early after auto-SCT is in line with our previous observations that NK-cells are active early upon auto-SCT with preserved degranulation and production of cytokines and chemokines in response to target cell encounter 32 . The link between cytokine-induced metabolic reprogramming of NK-cells and functionality has been well described. IFN-γ production by CD56 bright NK-cells upon cytokine stimulation depends on both glycolysis and oxidative phosphorylation 33 . Blocking glycolysis abolishes the NKcells' ability to control CMV infections, while treatment with the IL-15 super-agonist ALT-803 promotes CMV clearance and even prevents CMV reactivation upon allogeneic SCT 34 . However, the exact role of FAO in NKcells is not unambiguously clarified. Increased lipid uptake can inhibit degranulation and IFN-γ production in NK-cells and blocking fatty acid transport into the mitochondria can restore NK-cell cytotoxicity 35 . Contrariwise, it has been demonstrated that NK-cells treated intermittently with IL-15 possess a superior capacity for degranulation and IFN-γ production together with high levels of CPT1α expression 36 .
In line with previous reports, we observed a significant drop of the T-and NK-cell count during engraftment, which normalized over time 37 . Moreover, it has been shown that CD4 + T-cell recovery is delayed, which is partly explained by a reduced number of naïve precoursors 37,38 . Accordingly, we document a reduced CD4 to CD8 ratio, which is paralleled by reduced early differentiation stages. T-cell reconstitution upon auto-SCT is achieved by mainly two mechanisms: homeostatic proliferation and thymopoiesis 39 . While thymopoiesis restores the T-cell repertoire at around 6 months upon auto-SCT, homeostatic proliferation is responsible for early reconstitution. However, it favors expansion of memory T-cells and reduces T-cell receptor diversity 40,41 . Fittingly, T-cells at early differentiation stages were less proliferative and less equipped with metabolic key proteins during engraftment. In addition, Ki-67 levels remained elevated for a longer period in CD4 + T-cells, which could be due to their protracted recovery. In this regard, CD8 + T-cells demonstrated a stronger upregulation of their glycolytic machinery, which could support their rapid reconstitution by glycolysis-derived carbon equivalents 42 .
CD56 bright NK-cells displayed a higher proliferation rate and enhanced metabolic parameters as compared to their CD56 dim counterparts. Both subsets are known to upregulate glycolysis and OPXHOS upon cytokine treatment with this effect being more pronounced in CD56 bright NK-cells 33 . These differences (together with our findings) could be explained by the increased expression of cytokine receptors on CD56 bright NK-cells 43 .
In contrast to T-and NK-cells, monocytes were not impacted quantitatively, which is in accordance with previous reports [44][45][46] . The monocyte composition was also not affected in contrast to findings that intermediate monocytes are less susceptible towards HDCT in MM patients 44 . IL-6 production was also preserved at all tested time points as a further indication of robustness of monocytes. Like T-and NK-cells, monocytes upregulated their metabolic machinery following auto-SCT, which is an established prerequisite for their expansion and funcionality 18,47 . Overall, proliferative activity and endowment with key metabolic molecules was stronger in intermediate monocytes upon auto-SCT, which is in opposition to findings during steady state conditions. Here, classical monocytes display the highest expression of glycolytic and non-classical monocytes of OXPHOSrelated genes 48 . Interestingly, intermediate monocytes are known to expand during pro-inflammatory conditions like graft-versus-host disease 49,50 , rheumatoid arthritis 51 , and sepsis 52 , which could explain their increased proliferation and HK2 upregulation during engraftment. However, despite increased proliferation rates, we did not observe a significant proportional increase. This might be due to an increased susceptibility of CD16 + monocytes (including intermediate monocytes) towards spontaneous and ROS-induced apoptosis 53 , since we observed increased ROS levels in bulk monocytes during engraftment. Moreover, classical monocytes are known to upregulate CD16 on their surface under inflammatory conditions 54 , so the intermediate monocyte subset might be contaminated with activated classical monocyte leading to the observed increased proliferation and HK2 expression levels.
Finally, we observed that r/r lymphoma patients had persistently enhanced metabolic machinery in T-cells, NK-cells, and monocytes during first restaging.
Residual malignant cells below or above the conventional detection limit and/or defective cell intrinsic mechanisms that allow the activation to subside might cause these phenomena. In this context, at least two of the r/r lymphoma patients have already relapsed during our last analysis. In fact, chronic stimulation is known to cause exhaustion and senescence in immune cells, which results in inferior tumor control 55,56 . Within a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection exhausted Eomes high CD8 + T-cells had higher expression levels of Ki-67 compared to non-exhausted T-bet high CD8 + T-cells. However, when transferred into infection-matched mice Eomes high CD8 + T-cells proliferated less than T-bet high ones 57 . Similar results were obtained when analyzing CD8 + tumor-infiltrating lymphocytes (TILs) exhibiting high expression levels of Ki-67 in terminal exhausted CD8 + T-cells 58 . In addition, microarray analysis of early exhausted T-cells within the LCMV mouse model demonstrated increased CPT1α and HIF1α gene expression levels in exhausted T-cells compared to memory ones early during infection. However, these differences were lost during infection persistence 59 .
Therefore, it would be of interest to study expression of immune checkpoint receptors like PD1, TIGIT or NKG2A, exhaustion markers, and the functionality of lymphocytes and monocytes in patients with r/r lymphoma in comparison to patients in remission.
Notably, increased proliferation of T REG at day 28 upon auto-SCT is associated with a poor 5-year PFS in lymphoma patients 60 . We observed increased T REG frequencies during engraftment with a consecutive decrease until the first restaging. We cannot rule out that the persistently increased metabolic T-cell activity in r/r lymphoma patient is due to proliferating T REG . However CD4 + T-cell counts decreased upon auto-SCT and their Ki-67 expression levels did not significantly differ between r/r lymphoma patients and those in remission in contrast to Ki-67 expression levels in CD8 + T-cells. www.nature.com/scientificreports/ Strikingly, ALC did not differ in both groups, although lower ALC upon auto-SCT has been associated with reduced PFS and OS in lymphoma patients 10 .
Taken together, we characterized the metabolic profile of reconstituting immune cells following HDCT/auto-SCT for the first time. We were able to show that a persistently upregulated metabolic machinery in immune cells correlates with reduced PFS. However, the number of analyzed lymphoma patients in our study was too low (due to limited patient material and cell numbers) to appropriately establish a predictive pattern of the immune cell profile for PFS. Therefore, this data will require to be evaluated within a greater validation cohort and additional investigations are needed for understanding the underlying pathophysiology and to develop novel means of therapeutic interventions.

Methods
Study design and Patients' characteristics. Healthy donors and patients with MM or lymphoma who underwent HDCT/auto-SCT between 2018 and 2020 were enrolled upon informed written consent in accordance with the Declaration of Helsinki. This study was approved by the local ethics committee of the University of Erlangen (no.: 313_17B and 200_12). We analyzed 53 patients (35 male, 18 female) with a median age of 60 years (range: 32-76 years). 30 patients suffered from MM and 23 from lymphoma including DLBCL (n = 13), TCL (n = 5), MCL (n = 2), Hodgkin's Disease (HD) (n = 1), marginal zone lymphoma (MZoL) (n = 1), and follicular lymphoma (FL) (n = 1). Unfortunately, due to limited patient material and cell numbers we were not able to perform all of the listed experiments within all included patients. Blood from healthy donors was obtained from the blood bank of the Department of Transfusion Medicine and Haemostaseology of the University Hospital in Erlangen.
Peripheral blood (PB) was drawn at three time points: time point 1 (pre-HDCT) was before HDCT and at least two weeks after the start of the last therapy, time point 2 was during engraftment defined as the first (± 1) day after leukocyte regeneration (> 1000 leukocytes/µl), time point 3 was at restaging (post-SCT) (Supplementary Figure S14 online). Time between last conventional treatment and pre-HDCT time point was significantly longer in MM as compared to lymphoma patients (41 vs. 25 days; p < 0.0001). Time to engraftment was similar while the average time to restaging was different between myeloma and lymphoma patients based on our institutional guidelines. As anticipated the number of previous lines of therapy differs between diagnoses. The patients' characteristics are summarized in Table 1. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood or leukocyte concentrates by density gravity centrifugation with Ficoll (General Electric/GE healthcare, Chicago, Illinois, USA) or Pancoll (PAN-Biotech, Aidenbach, Germany) using Leucosep tubes (Greiner Bio-One GmbH, Kremsmünster, Austria). Flow cytometry. Cell viability was assessed using the zombie aqua fixable viability dye (Biolegend, San Diego, California, USA). PBMCs were than either barcoded (procedure described later) or immediately stained following the manufacturers' recommendations. For intracellular staining cells were fixed with BD Cytofix solution and washed in BD Perm/Wash solution (BD Bioscience).
Samples were measured with a BD FACSCanto II or BD LSR Fortessa II flow cytometer using the BD FAC-SDiva Software. Data was analysed with the FlowJo V10 software (BD Bioscience).
A list of all antibodies and dyes can be found in Supplementary Table S4 online.
Fluorescent cell barcoding technique. For optimizing comparability between different patient samples and to minimize interfering factors, fluorescent cell barcoding technique was used to stain PBMCs from all three time points of one donor together with an additional healthy donor (for standardization) in one well 62 . Briefly, cells were fixed with 2% paraformaldehyde (PFA) or BD Cytofix. Cells were then either carefully re-suspended in ice-cold 50% methanol (diluted in 0,9% NaCl) and incubated over night at -20 °C or washed twice with BD Perm/Wash solution (the latter method was needed in case surface epitopes of interest were denaturated by methanol). Subsequently, cells were labeled using different dilutions of the dye pacific blue (ThermoFisher Scientific). After three additional washing steps, all cells were re-suspended into one well and stained for additional surface or intracellular markers. Barcoding was deconvoluted by gating on the corresponding fluorescent cell barcoding dilution out of the whole cell population (Supplementary Figure S1 online).

Intracellular cytokine detection.
For intracellular cytokine staining cells were incubated for 6 h in complete media in a 96-well U-bottom plate with either 250 ng/ml Phorbol-12-myristat-13-acetat (PMA) and 1000 ng/mL Ionomycin or 1 µg/mL lipopolysaccharides (LPS). After the first two hours Brefeldin A was added, cells were harvested, labeled with fluorescent cell barcoding, and stained with antibodies against intracellular cytokines. A p-value less than 0.05 was regarded statistically significant, statistical significance is indicated by the p-values (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Data availability
The datasets generated during and/ or analyzed during the current study are available from the corresponding author on reasonable request.