Percentage Of CD34 Research Articles

Prospective Validation of CD34+CD38- Leukemic Stem Cell Frequency in the HOVON-SAKK 132 Trial: Perspectives for Future Improvements

Clinical implementation of measurable residual disease (MRD) requires accurate MRD detection to assess relapse risk for acute myeloid leukemia (AML) patients. One possibility to further improve the prognostic ability of the current MRD assay is to include the quantification of relapse initiating cells such as the CD34+CD38- leukemic stem cells (LSC) in the assay. In the HOVON-SAKK 102 trial, detection of LSC by multiparameter (MFC) flow MRD at diagnosis and after 2nd chemotherapy cycle (C2) has shown prognostic value (Zeijlemaker et al. Leukemia 2019). In addition, together with MRD detection, LSC could identify a category of patients with very high risk of relapse. Based on those data, improvements for LSC assessment measurements have been suggested, such as measuring more white blood cells (WBC) with the purpose of enhancing the sensitivity for the low frequency of CD34+CD38- cells. In the HOVON-SAKK 132 trial (Lowenberg et al. Blood Adv 2021), the one tube LSC assay was used that included LSC markers CD45RA, CD123, CD33, CD44, CLL-1, TIM-3, CD56, CD7, CD22 and CD11b (Zeijlemaker et al. Leukemia 2016). The one tube approach allows acquiring the suggested 4 million WBC for LSC-load estimation. In the current study, we prospectively validated the prognostic value of LSC burden at the following time points, i.e. at diagnosis (764/905 patients) and after two cycles of chemotherapy (C2; 357/706 patients). The median WBC acquired was 2x106 (range: 3x104- 6x106) at diagnosis and 3x106 (range: 4x105-5x106) after C2. Median percentages of LSC at diagnosis and after C2 were respectively 0.004% (range: 0.0000%-26.85%) and 0.00002% (range: 0.0000%-1.36%). Similar to the HOVON-SAKK102 study, three prognostic groups based on the presence or absence of CD34 and/or LSC, showed significant associations between LSC burden at diagnosis and overall survival (OS) or relapse- free survival (RFS) (Figure 1A): 1. CD34negative patients without any measurable LSCs and Leukemia Associated Immunophenotypes on the CD34+ blasts fraction (CD34negLSC=0 3 years (yrs) OS: 76.4% standard error (SE) 5; median OS not reached). 2. Patients with low percentages of CD34+CD38-LSCs < 0.03% (LSClow; 3 yrs OS 62.8% SE 2; median OS not reached) 3. Patients with high percentages of CD34+CD38-LSCs ≥0.03% (LSChigh, 3 yrs OS 44.5% SE 3; median 25.6 months; 95% confidence interval (CI): 18.7-49.1). Multivariate analysis adjusted for age, WBC at diagnosis, AML type, ELN2017 risk group and -only for RFS- the number of chemotherapy cycles to attain complete remission, showed that LSC burden at diagnosis is a strong independent prognostic factor for OS and RFS (Figure 1B). After C2, before the consolidation treatment, a cut-off value of 0.000075% of LSC showed the best prognostic value of LSC (univariate, LSCneg versus LSCpos: OS, HR 1.79 95% CI: 1.25-2.56, P < 0.01; RFS, HR 1.55, 95% CI: 1.12-2.13, P < 0.01). This cut-off appeared also highly significant as a prognostic factor in former trials (Terwijn et al. PLoS One 2014; Zeijlemaker et al. Leukemia 2018). Multivariate analysis showed that also LSC status after C2 was an independent prognostic factor for OS and RFS (Figure 1B). By combining the LSC status after C2 (≥0.000075%) together with the MRD status as used in the HOVON-SAKK 132 (based on ≥0.1% MFC-MRD and/or NPM1-MRD), MRDpos patients who are LSCpos have worse prognosis in OS and RFS (univariate: MRDnegLSCneg vs MRDposLSCpos; OS HR 3.2, 95% CI: 1.94-5.28, P < 0.01, RFS: HR 2.61 95% CI: 1.65-4.12, P < 0.01, multivariate: Figure 1B). Further analyses of the data are now directed towards potential influences of the cell amount of acquisition, ELN risk groups and MRD guidance in this trial. In conclusion, this prospective validation confirmed that assessing LSC burden, at diagnosis and after C2, remains to have additional prognostic value to MRD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD82 Is Sufficient to Uniquely Identify Pluripotent Stem Cell-Derived Hemogenic Endothelium with the Hematopoietic Lineage Potency to Give Rise to Bona Fide Lymphocytes

Chimeric antigen receptor (CAR)-T and natural killer (NK) cells represent compelling opportunities for the treatment of hematological malignancies and solid tumors. However, autologous and donor-derived allogeneic CAR-T and NK cell therapies are limited based on the quantity and quality of the starting engineered cells. We previously described our off-the-shelf induced pluripotent stem cell (iPSC)-derived CAR-T (CAR-iT) and CAR-NK (CAR-iNK) cell platforms that eliminate some of the hurdles associated with patient-derived cells by creating a homogenous cell product that is derived from a renewable bank of iPSC master cell lines. A major challenge in the manufacturing of iPSC derived immune cells is the derivation of hemogenic endothelium (HE), which is the rare and transient direct precursor of hematopoietic cells, making its in vitro specification from iPSCs inefficient and challenging. Furthermore, there is not a reliable marker or set of markers for the identification of HE in culture. HE identification has traditionally relied on detection of the transcription factor RUNX1 and the establishment of reporter cell lines, neither of which are suitable for the manufacture of HE cells destined to become therapeutic immune cells. We have established a cytokine driven protocol for the differentiation of iPSCs that improves the efficiency of the derivation of RUNX1+ HE cells. To identify candidate HE surface markers, we compared iPSC-derived cells containing a high percentage of HE, based on RUNX1 expression, to control differentiated cells containing a low percentage of HE, via a cell surface antibody array. Through the array we identified several surface markers that were expressed by a higher percentage of CD34+ endothelial cells compared to control CD34+ cells. Several of the candidates identified, including CD44, CD61, CD226 and CD143 have been previously identified by other groups as candidate HE markers, thereby validating our screening method. We identified CD82 as a novel candidate HE marker, with expression uniquely restricted to the CD34+ endothelial population and positively correlating with RUNX1 expression (Fig. 1A). Furthermore, we demonstrated via limiting dilution assays that HE is enriched in the CD34+CD43-CD82+ population (HE frequency, 1/38) compared to the CD34+CD43-CD82- population(HE frequency, 1/736) (Fig. 1B). Even more strikingly, isolation of cells based on CD82 alone was sufficient to enrich for HE cells (HE frequency, 1/44) and outcompeted CD34 enrichment alone, suggestive of its unique ability to identify the HE population (Fig. 1B). To determine if CD82+ HE cells could give rise to lymphocytes we carried them forward into T cell differentiation and showed thatthey efficiently gave rise to CAR-iT cells (i.e. >95% CD7+ T lymphocytes). The identification of CD82 for the enrichment of iPSC derived HE is a critical step to the development of definitive hematopoiesis with ability to give rise to all lineages of the hematopoietic compartment and facilitates the scaled and consistent manufacture of high-quality T and NK cells for off-the-shelf therapeutic applications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bone Marrow WT1 mRNA Upregulation in Acute Myeloid Leukemia (AML) in Complete Molecular Remission (mCR): Understanding the Limitations and Advantages of WT1 As a Measurable Residual Disease (MRD) Target

Introduction WT1 mRNA levels assessed by q-PCR have been employed as a marker of remission in AML patients undergoing intensive chemotherapy (Nomdedeu J. et al. Leukemia 2013;27:2157) and hematopoietic cell transplantation (HCT) (Nomdedeu J et al. Biol Blood Marrow Transplant 2018;24:55). Patients with adjusted bone marrow WT1 mRNA levels greater than 100 copies showed a high probability of relapse. Despite the usefulness of WT1 quantification in this and other settings, this marker is scarcely employed as an MRD target because levels greater than >100 copies can also be observed in normal bone marrows. Furthermore, 10-20% of AML at diagnosis and most chronic myelomonocytic leukemia (CMML) cases lack WT1 upregulation. The high levels of bone marrow WT1 copy numbers observed in most AML, decline in responding patients, although the utility, if any, of WT1 levels in long-term survivors of AML is currently unknown. We investigated the frequency and biological findings of mCR AML samples showing WT1 upregulation(>100 copies) compared to that mCR AML with concordant low WT1 levels. Patients and methods The study included 717 bone marrow samples from 80 AML patients (23 patients with RUNX1-RUNX1T1, 18 pts with CBFb-MYH11, four pts with NPM1 mutations, and 35 pts with PML-RARa). Samples were obtained at diagnosis (58 samples) and the follow-up (659 samples, all in mCR and without relapses) from one month to 219 months after diagnosis. Chimeric transcripts and WT1 levels were investigated by standardized real-time methods (ELN). Hemoglobin and leukocyte counts were also recorded. Flow cytometry immunophenotyping was also performed using four-color flow cytometry. We analyzed the percentage of CD34+, CD117+, CD123+ cells, and the Myeloid/Lymphoid CD34 ratio. nCounter Stem cell panel (Nanostring) was used to investigate the differences between that AML in mCR, showing high (>100) and low (<100) WT1 levels. In an additional cohort (11 samples: 4 with WT1>100 and 7 with WT1 <100), we performed bulk RNAseq (Novogene) to investigate enriched genes in samples with high WT1 levels. Results Thirty-five patients had at least one sample with WT1 levels greater than 100 copies (44%) despite being in mCR. Twelve samples had WT1 levels greater than 300 copies. In one case, this level reached 770 copies. These WT1 "peak" samples were distributed as follows: 49/306 PML-RARa, 25/161 RUNX1-RUNX1T1, 10/174 CBFb-MYH11, and 1/18 NPM1. Patients showing WT1 peaks were older than those without elevations (47 years vs. 36 years, p:0.003) For PML-RARa samples, "peaking" was associated with low levels of hemoglobin (133 g/L for <100 copies vs. 125 g/L >100 copies, p:0.009). For RUNX1-RUNX1T1, "peaking" was linked to less platelets (132x109 vs. 197x109 p:0.05) , higher CD34 (1.5% vs. 1% p:0.05) and also higher CD117+ cells (1.6 vs.1%, p:0.01). Lastly, CBFb-MYH11+ samples with WT1 peaks had higher leukocyte counts that those showing low WT1 levels (4.5x109 vs. 2.9x109, p:0.006). Nanostring comparisons between mCR WT1 high vs. low samples showed upregulation of TOR1A, PIK3CB, PTPN6, and PGM1 with downregulation of HMGA1, HOXB2 and CDC6. RNA seq analysis detected an enrichment of SUZ12 targets in those samples with high WT1 levels. Conclusions Bone marrow mRNA WT1 levels commonly exceed the clinical relevant value of 100 copies in AML samples in mCR (13%). Peak samples were related to peripheral blood values and/or raised numbers of immature bone marrow cells (CD34, CD117). Bone marrow WT1 although unable to accurately predict relapses in long-term survivors of AML may be a non-invasive sensor of a yet undefined myeloid state.

Potentially Probiotic Fermented Glutinous Rice (Oryza sativa L.) with Lactiplantibacillus plantarum Improved Immune System Response in a Small Sample of BALB/cByJ Mice

L. plantarum strains displayed different abilities to exhibit high survivability to acid (pH 3.0), bile salts (3%), enzyme (pepsin), and temperature (40 °C) and good antibiotic susceptibility. The isolates were further supplemented in traditional tapai pulut to study the immunomodulation properties of tapai pulut based on the splenic T- and B-cell populations. The mice groups were divided into group one (unfermented glutinous rice), group two (tapai pulut group), and group three (probiotic tapai pulut group). Group one showed consistent body weight gain, with the highest observed after four weeks. Group three exhibited the most significant reduction in the percentage of CD19+ B-cells. The CD3+ T-cells population of Group three increased significantly compared with the control mice, followed by Group two. The results suggest that traditional tapai pulut supplemented with L. plantarum has a high potential for supporting the immune system’s immunomodulatory effect.

UM171 EXPANDS IMMATURE BONE MARROW CD34+ CELLS FROM PATIENTS WITH TELOMEROPATHIES

Telomeropathies, also referred to as telomere biology disorders (TBD), correspond to a spectrum of diseases characterized by genetic defects in the maintenance mechanisms of telomeres and telomerase. Individuals carrying variants in the genes involved in telomeres machinery may have critically short or dysfunctional telomeres, which leads to poor cell regeneration and predisposition to cancer. The commonly affected tissues are those that renew rapidly, such as the bone marrow, lungs, and skin. The most effective therapeutical approach to treat bone marrow failure in patients with TBD is allogeneic bone marrow transplantation, restricted by the low availability of compatible donors and complications of the conditioning regimen. Fares et al. (2013) demonstrated that the small molecule UM171 expands the CD34 + cell subpopulations from cord blood with superior engraftment potential. We demonstrated that UM171 expands primitive CD34 + cells from patients with immune aplastic anemia. The expanded hematopoietic progenitors showed no observable chromosomal, genetic, or telomeric changes. Here we assessed the potential of the HSC agonist UM171 to expand the HSPC compartment of patients with telomeropathies. Bone marrow aspirate samples were collected from five patients, and CD34 + cells were enriched using immunomagnetic labeling with human CD34 MicroBeads and a magnetic separator. Cells were cultured for 7 days in ACF medium supplemented with cytokines that support expansion of HSPC and UM171 or DMSO (negative control). To assess the capacity of the cells cultured either with DMSO or UM171 to generate hematopoietic progenitors, 1,000 cells/mL were resuspended in Methocult and seeded onto 35 mm dishes in triplicate. After 14 days, the colonies were counted and classified according to their morphology. After a 7-day expansion, the percentage of CD34 + cells was higher with UM171 in comparison to control (UM171, 48 ±5.1% vs. DMSO, 25.3% ±6.2% [mean ±standard error] n = 5; p = 0.003). The cell surface EPCR is a reliable marker for the purification of the HSPC compartment. Therefore, we evaluated the CD34 + EPCR + subpopulation, which was increased after the UM171 treatment (UM171, 14.4 ±4.1% vs. DMSO, 1.8% ± 0.6%; n = 5; p = 0.03). Additionally, cells expanded with UM171 gave rise to more progenitor cells, as observed by the CFU assay (UM171, 142 ± 18 vs. DMSO, 94% ± 9; n = 4; p = 0.01). No significant telomere attrition was observed with the expansion. Bone marrow failure is a common manifestation in patients with telomere disorders since the telomerase complex is essential for maintaining the self-renewal capacity of the HSPC pool. Our data suggest the possible future use of UM171 to enhance ex vivo production of HSPC from TBD patients, making it feasible to use these cells in autologous transplants. These findings should improve our understanding of the effect of UM171 on these cells and help to develop protocols for scaling up its production for future therapies. Our studies reveal UM171 to be a potent molecule to expand HSPC in patients with telomeropathies. Further experiments are required to confirm these results besides the assessment of the engraftment potential of the expanded cells in a murine model.

Effect of CD34⁺ Total/Myeloid CD34⁺ Cell Progenitors and B-Lymphoid Progenitors Within the Bone Marrow Grafts on the Hematopoietic Recovery After Hematopoietic Stem Cell Transplantation.

The influence of CD34⁺ cells on transplant outcomes following hematopoietic stem cell transplantation remains controversial. A minimum of 2.0 to 2.5 million CD34⁺ cells/kg of patient weight is requested for a rapid and durable engraftment. The aim of this study was to detect the ratio of CD34+ B-lymphoid progenitors (hematogones) in bone marrow grafts and investigate their effects on hematopoietic recovery after transplant. Our study included 41 patients who received a bone marrow graft from their HLA-matched donor from 2016 through 2019. The CD34⁺ cell numbers within the graft were detected using Stem-Kit (Beckman Coulter). The ratio of CD34⁺ hematogones was determined either by their light scatter characteristics or by the detection of CD34, CD19, or CD10 coexpressing cells in a separate tube. The median number of CD34⁺ cells was 5.9 × 106/kg (0.8-14.3 × 106/kg). The CD34⁺ cells consisted of 71% (range, 35.7%-100%) and 29% (range, 5.7%-64.3%) myeloid and B-lymphoid progenitors, respectively. Percentage of CD34⁺ (P < .001) and total (P < .001) hematogones in correlation with donor age. Time of neutrophil engraftment was significantly longer (P = .039) when total infused CD34⁺ cell content was <3 × 106/kg. A remarkable population of hematogones within the CD34⁺ cell pool was detected in bone marrow grafts. Detection of the ratio of hematogones and most primitive stem cells (CD34⁺CD90⁺CD38⁻) may overall provide more information to build a better correlation between CD34⁺ cell content and the recovery of bone marrow.

OPTIMIZED PROTOCOLS OF EXPANSION OF PERIPHERAL BLOOD-DERIVED δT-CELLS FOR THE ESTABLISHMENT OF AN OFF-THE-SHELF ADVANCED CELL THERAPY AGAINST CANCER

The use of autologous T cells expressing chimeric antigen receptors (CAR) has revolutionized the treatment of hematological malignancies. However, the use of allogeneic CAR-T cells offers many advantages over autologous therapies, including the immediate availability of cryopreserved batches for infusion and the use of healthy donor cells since it is possible to obtain high amounts of fully functional cells. Moreover, this strategy also allows for the scaling up of the production of CAR-T cells resulting in cost reduction. Cells that do not express TCR αβ, such as gamma delta (γδ) T, are an alternative source to allow widespread allogeneic therapy possible. Their ability to recognize targets in an MHC-independent manner and the expression of innate immunity receptors increases the potential of using these cells in immunotherapies. Aiming to improve the effectiveness of γδ T-cell-based immunotherapies against tumors, we tested various optimization strategies. The most widely used protocol for selectively expanding Vδ2T cells relies on zoledronic acid (ZOL) stimulation of PBMCs in combination with IL-2 or/and IL-15 cytokines. Here, we tested different concentrations of IL-2 (100 and 1,000 U/mL) and IL-15 (12.5 and 100 ng/mL) with 5 μM zoledronic acid. In addition, we compared cell expansion in three defined cell culture media, OpTmizer T Cell Expansion SFM, X-vivo10, and TexMACS (supplemented with 2% AB serum). A total of twenty-four different combinations of IL-2, IL-15, and media were tested in the present study.After twelve days of culture, cells were counted and labelled with anti-CD3, anti-CD8, anti-CD4, anti-Vδ2, anti-PD1, anti-CD69, and anti-NKG2D antibodies for flow cytometry analysis. Our results demonstrated that although TexMACS medium is capable of supporting T-cell activation and expansion, it was not optimal for maximal expansion of Vδ2T cells (fold expansion 5.99 ± 1.61). However, the fold expansion was similar in OpTmizer and X-vivo10 media (fold expansion 21.38 ± 5.13 and 18.37 ± 5.42, respectively). Among the medium supplementation strategies, cultivation in OpTmizer combined with 100 U/mL IL-2 + 12.5 ng/mL IL-15 led to highest expansion index (32.4-fold). As for the activation markers of Vδ2T cells, the frequency of NKG2D+ cells was similar for all conditions tested (above 85%). However, the percentage of CD69+ cells was higher in cells cultured in TexMACS medium compared to OpTmizer and X-vivo10 media (81.6±5.3, 69±9.9, 60.2±7.2, respectively). It is known that the Programmed Cell Death-1 receptor (PD-1) plays an important role in regulating immune cell function. We examined the expression of PD-1 after twelve days of expansion. We observed lower levels of PD-1 in cells expanded in X-vivo10 medium supplemented with 1,000 U/mL IL-2 + 100 ng/mL IL-15, whereas levels of expression were up-regulated significantly when cells were cultivated in the same conditions without IL-15 (28.7% versus 70.6%). Cells expanded in either OpTimizer or TexMACS demonstrated a similar frequency to PD-1 positive cells (41±13% and 49.7%±9.5). In conclusion, we reported an optimized protocol to improve the expansion and the quality of Vδ2 T cells. These findings set the groundwork for the establishment of an off-the-shelf advanced cellular immunotherapy using this alternative population of T cells.

Potential for TCDD to induce regulatory functions in B cells as part of the mechanism for T cell suppression in EAE

Part of the mechanism by which 2,3,7.8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses immune function involves induction of regulatory T cells and suppression of effector T cells. The goal of this project was to examine whether TCDD's suppression of effector T cells was due in part to inducing B regulatory cells (Bregs). TCDD's potential to increase the percentage and/or function of CD24+CD38+ B cells was assessed in response to lipopolysaccharide (LPS) + interleukin (IL)-4 in vitro and in a mild model of experimental autoimmune encephalomyelitis (EAE) in vivo. In vitro, TCDD did not consistently increase the percentage of CD19+CD24+CD38+ cells using splenocytes, purified B cells or bone marrow (BM) cells. However, TCDD increased IL-10 in all three culture preparations, and TCDD increased the percentage of CD5+CD24+CD38+ cells producing IL-10. In EAE, TCDD did not affect the percentage of the CD24+CD38+ cell population in CD19, B220 or CD5 B cells in splenocytes (SPLC), lymph nodes (LN) nor BM cells at end-stage disease. On the other hand, TCDD increased the CD19+CD24+CD38+ percentage in the spinal cord (SC) in EAE. Moreover, TCDD-treated B cells isolated from spleens or TCDD-treated BM cells in EAE mice modestly reduced the ability of naïve effector T cells to express interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Together these data show that TCDD can induce regulatory functions in B cells, although it was not obvious simply by examining the expression of regulatory markers but by assessing function by cytokine production or mixed lymphocyte responses.

Dynamic change, influencing factors, and clinical impact of cellular components in human breast milk.

Numerous cellular components have been well demonstrated in human breast milk. However, little is known about their dynamic change, influencing factors, and potential clinical impacts on infants. Sixty and forty-five healthy mother-infant pairs were enrolled in the colostrum group and mature milk group, respectively. Participants' demographic and clinical information were collected by questionnaires, and the infants were followed up until 6 months after birth through telephone interview. Colostrum and mature milk were collected, and the percentage of various cell components were determined by flow cytometric analysis. The results showed that, the total cell numbers, and the percentages of some stem cells, including CD34+, CD117+, CD133+, CD90+, CD105+, and CD146+ cells, were different in colostrum and mature milk. Besides, participants' characteristics had influence on the cellular components. Finally, high-CD34+ cells in colostrum, as well as the high-CD133+ cells and low-CD105+ cells in mature milk were associated with a significantly increased risk of infantile eczema within their first 3 months after birth. Our data showed a dynamic change of cellular components, identified some of their influencing factors and their potential clinical impacts on infantile eczema, which helps to better understand the cellular components in human breast milk. Some stem cell markers were dynamically changed in human colostrum and mature milk. Different cellular components were shown to be influenced by different participants' characteristics. High percentage of CD34+ cells in colostrum, as well as high percentage of CD133+ cells and low percentage of CD105+ cells in mature milk, were associated with a significantly increased risk of infantile eczema within their first 3 months after birth. To our knowledge, this is the first study on the clinical impacts of stem cells on infantile diseases, which helps to give a better understanding of human breast milk.

Is hepatitis-B immunization effective during chronic liver fibrosis? Investigation of secretory and cellular immune responses on an experimental model

Objective Adults with end-stage of chronic liver diseases have lower antibody titers after hepatitis-B vaccination. We have less amount of knowledge about the effect of non-viral cause chronic liver fibrosis on vaccination. In this study, we investigated the effect of non-viral chronic liver fibrosis on hepatitis B vaccine and the effect of tetanous toxoid co-administration at the level of humoral and cellular immune responses in an experimental model. Methods Hepatitis B vaccine was administered either alone or in combination with tetanus toxoid in thioacetamide-induced fibrotic BALB/c mice. Fibrosis level was determined by Knodell scoring. Anti-HBsAg, biochemical parameters, inflammatory (IL-1β, TNF-α), and anti-inflammatory (IL-10) cytokine levels were investigated in serum samples by automated systems and ELISA; respectively. Frequencies of activated lymphocytes were determined in flow cytometer. Results Antibody titers significantly decreased after immunization of fibrotic mice. However, co-administration of toxoid significantly elevated antibody titer. The percentage of CD19+CD69+ B lymphocytes was found to be lower in vaccinated fibrotic group compared to vaccinated naive group. Simultaneous administration of toxoid significantly increased the frequencies of CD4+ and CD8+ T cells expressing CD69 and CD127. Interestingly, CD19+CD5+CD1high Breg cells were significantly reduced in the group vaccinated with hepatitis B vaccine and toxoid, simultaneously. The reduction in Breg percentage did not expose a significant decrease in the level of IL-10. Conclusion Non-viral chronic liver fibrosis causes a reduction on specific antibody level after vaccination. Reduction on Breg cell frequency may have an effect on elevation of antibody level after co-administration of tetanus toxoid.

Adaptive immunity in different CT patterns of active tuberculosis and possible variability according to patients' geographic provenience.

BackgroundIt is still unclear if low lymphocyte levels are directly related to immunological modifications induced by the TB infection or if they depend on the general pre-existing health impairment of affected patients. Our aim was to detect eventual differences in the immunological status of patients with pulmonary TB compared to an age and sex-matched group of hospitalized patients with other bacterial community-acquired pneumonia (CAP). In addition, we tried to assess an association between alterations in the peripheral lymphocyte subsets and the development of different CT patterns of active TB and to discover differences in the immunological status and in the radiological patterns of TB presentation between patients of different geographic proveniences.MethodsThis observational study included 48 patients with TB and 48 sex- and age-matched patients affected by other bacterial CAP. The presence of HIV/AIDS, other immunocompromising conditions, and confounding chronic pulmonary comorbidities was excluded. Flow cytometry was performed on all the enrolled subjects at admission, before starting the appropriate antibiotic therapy. Patients with TB also underwent a computed tomography (CT) scan.ResultsPatients with TB showed a decrease in the absolute count of all the lymphocyte subsets compared to the CAP group. Only the reduction in the percentage of CD4+ T-lymphocytes was significant, while the percentage of CD8+ T-lymphocytes was significantly increased. Patients presenting exudative forms with atypical locations of TB showed a significant reduction in the absolute count and percentage of CD19+ B-lymphocytes compared to those affected by productive TB forms with the typical location. Despite being younger, our black Sub-Saharan Africans showed a significant reduction in the CD4+ T-lymphocytes compartment and a higher prevalence of atypical and exudative forms of TB compared with white Europeans.ConclusionTuberculosis itself may alter peripheral blood lymphocyte subsets compared to other CAP. An impaired CD19+ B-lymphocyte compartment may result in an abnormal exudative response in atypical locations and a suboptimal bacterial control. Other constitutive or environmental causes may influence immunological differences found in patients with TB, particularly in case of different geographic origins. Anyhow, flow cytometry may be of great value in evaluating the immune function of these patients.

A new approach to the study of Hodgkin lymphoma by flow cytometry

Hodgkin lymphoma (HL) appears to originate from germinal centre B cells but lacks expression of most B cell markers. In contrast to non-Hodgkin B lymphomas, HL is not routinely diagnosed using flow cytometry techniques, and diagnosis is mainly based on immunohistochemical and cytomorphological pathology studies. Hodgkin and Reed-Sternberg cells are large and fragile, making them difficult to study by flow cytometry. The aim of this study was to characterise the CD71 expression pattern on CD4+ T cells from HL patients and to design a simple flow cytometry algorithm to complement the histopathological diagnosis of HL. The present study suggests the utility of a conventional staining protocol with a simple panel of seven markers (CD15, CD30, CD4, CD8, CD71, CD3, and CD45) and a well-defined analysis strategy. The proposed algorithm uses the CD71 ratio (calculated as the percentage of CD71+ CD4+ T cells divided by the percentage of CD71+ CD45+ CD3- lymphocytes), with a cut-off of 0.5 to establish diagnosis groups as suggestive (≥0.5) or not suggestive (<0.5) of HL. In HL, CD71 expression is higher on CD4+ T lymphocytes than on non-T lymphocytes. In addition, the CD4+ T cell population is increased in HL patients, with no change in amounts of CD8+ T cells. Application of the CD71 ratio algorithm yielded a sensitivity of 82% and specificity of 87%, with 84.61% of patients correctly diagnosed. Although histopathology remains the gold standard for definitive HL diagnosis, the proposed flow cytometry method provides a rapid method to guide the study that would allow a more robust and integrated diagnosis. Moreover, the procedure is easily applicable in most clinical laboratories as it does not require state-of-the-art cytometers and uses standard reagents.

Modulation of Leukocyte Subsets Mobilization in Response to Exercise by Water Immersion Recovery.

Purpose: To investigate the effect of different water immersion temperatures on the kinetics of blood markers of skeletal muscle damage and the main leukocyte subpopulations. Methods: Eleven recreationally trained young men participated in four experimental sessions consisting of unilateral eccentric knee flexion and 90 min of treadmill running at 70% of peak oxygen uptake, followed by 15 min of water immersion recovery at 15, 28 or 38°C. In the control condition participants remained seated at room temperature. Four hours after exercise recovery, participants completed a performance test. Blood samples were obtained before and immediately after exercise, after immersion, immediately before and after the performance test and 24 h after exercise. The number of leukocyte populations and the percentage of lymphocyte and monocytes subsets, as well as the serum activity of creatine kinase and aspartate aminotransferase were determined. Results: Leukocytosis and increase in blood markers of skeletal muscle damage were observed after the exercise. Magnitude effect analysis indicated that post-exercise hot-water immersion likely reduced the exercise-induced lymphocytosis and monocytosis. Despite reduced monocyte count, recovery by 38°C immersion, as well as 28°C, likely increased the percentage of non-classical monocytes in the blood. The percentage of CD25+ cells in the CD4 T cell subpopulation was possibly lower after immersion in water at 28 and 15°C. No effect of recovery by water immersion was observed for serum levels of creatine kinase and aspartate aminotransferase. Conclusions: Recovery by hot-water immersion likely attenuated the leukocytosis and increased the mobilization of non-classical monocytes induced by a single session of exercise combining resistance and endurance exercises, despite no effect of water immersion on markers of skeletal muscle damage. The monocyte response mediated by hot water immersion may lead to the improvement of the inflammatory response evoked by exercise in the skeletal muscle.

An experimental study on the effect of preoperative autologous blood donation on bone marrow hematopoiesis and hematopoietic stem cells.

To evaluate the effect of autologous blood transfusion (ABT) on hematopoietic stem cells through the observation of the changes in the number and activity of bone marrow CD34+ cells after preoperative autologous blood donation (PABD). Rabbit bone marrow specimens were collected preoperatively (T2), 6 h postoperatively (T3), and 24 h postoperatively (T4). Next, the percentages of CD34+ cells, the cell cycle, and the relative expression of telomeric DNA were measured in each group of rabbits. Peripheral blood specimens were collected before PABD (T1) and at T4 to measure reticulocytes. At T3 and T4, the percentages of CD34+ cells and the expressions of telomeric DNA were significantly higher, and the percentages of cells in the G1 phase were significantly lower in each experimental group compared with those in the blank control group (group A) (P<0.05). Compared with the surgical blood collection group (group C), the CD34+ cells and the expressions of telomeric DNA were significantly higher, and the percentages of cells in the G1 phase were significantly lower in the preoperative autologous whole blood group (group D) and the preoperative autologous blood component group (group E) (P<0.05). Compared with group D, the CD34+ cells and the expressions of telomeric DNA were significantly lower, and the percentages of cells in the G1 phase were significantly higher in group E (P<0.05). At T4, the reticulocyte percentages in the surgery group (group B) and group C were significantly higher than in group E and group D, and the reticulocyte percentages in group E were higher than in group D (P<0.05). This study's findings indicated that ABT suppressed bone marrow hematopoiesis, while autologous blood component transfusion had less of an effect than that of whole blood transfusion. Therefore, PABD blood component transfusion would be superior to autologous whole blood transfusion.

Upregulation of NKG2D ligands impairs hematopoietic stem cell function in Fanconi anemia.

Fanconi anemia (FA) is the most prevalent inherited bone marrow failure (BMF) syndrome. Nevertheless, the pathophysiological mechanisms of BMF in FA have not been fully elucidated. Since FA cells are defective in DNA repair, we hypothesized that FA hematopoietic stem and progenitor cells (HSPCs) might express DNA damage–associated stress molecules such as natural killer group 2 member D ligands (NKG2D-Ls). These ligands could then interact with the activating NKG2D receptor expressed in cytotoxic NK or CD8+ T cells, which may result in progressive HSPC depletion. Our results indeed demonstrated upregulated levels of NKG2D-Ls in cultured FA fibroblasts and T cells, and these levels were further exacerbated by mitomycin C or formaldehyde. Notably, a high proportion of BM CD34+ HSPCs from patients with FA also expressed increased levels of NKG2D-Ls, which correlated inversely with the percentage of CD34+ cells in BM. Remarkably, the reduced clonogenic potential characteristic of FA HSPCs was improved by blocking NKG2D–NKG2D-L interactions. Moreover, the in vivo blockage of these interactions in a BMF FA mouse model ameliorated the anemia in these animals. Our study demonstrates the involvement of NKG2D–NKG2D-L interactions in FA HSPC functionality, suggesting an unexpected role of the immune system in the progressive BMF that is characteristic of FA.

Effects of compound small peptides of Chinese medicine on intestinal immunity and cecal intestinal flora in CTX immunosuppressed mice.

The study was designed to explore the improvement effect of CSPCM (compound small peptide of Chinese medicine) on intestinal immunity and microflora through the treatment of different doses of CSPCM. A total of 100 male Kunming mice were weighed and divided into five groups, namely, group A (control group), group B (model group), group C (0.1 g/kg·bw CSPCM), group D (0.2 g/kg·bw CSPCM), and group E (0.4 g/kg·bw CSPCM). The use of CTX (cyclophosphamide) caused a series of negative effects: the secretion of IL-2, IL-22, TNF-α, sIgA, length of the villi, and the area of Pey's node were significantly reduced (P < 0.05); the depth of crypt and the percent of CD3+ and CD4+ cells were significantly increased (P < 0.05); the cecal flora taxa decreased; the abundance of Firmicutes and Lactobacillus increased; and the abundance of Bacteroidetes, Deferribacteres, Proteobacteria, Mucispirillum, Bacteroides, and Flexisprra decreased. The addition of CSPCM improved the secretion of cytokines and the development of intestinal villi, crypts, and Pey's node. The number of CD3+ and CD4+ cells in groups C, D, and E was significantly higher than that in group B (P < 0.05). Compared with group B, the abundance of Firmicutes in groups C, D, and E was decreased, and the Bacteroidetes, Deferribacteres, and Proteobacteria increased. The abundance of Lactobacillus decreased, while that of Mucispirillum, Bacteroides, and Flexisprra increased. It is concluded that cyclophosphamide is extremely destructive to the intestinal area and has a great negative impact on the development of the small intestine, the intestinal immune system, and the intestinal flora. The CSPCM can improve the negative effects of CTX.

KIR- Ligand Interactions in Hypertensive Disorders in Pregnancy.

HypothesisThe activity of natural killer (NK) cells is considered an important factor for the tolerance of the fetus during pregnancy. The complications of pregnancy, such as hypertensive disorders (HDP), may be therefore associated with this immune compartment.MethodsThe current study included 41 pregnant women diagnosed with HDPs (Gestational Hypertension; GH or Preeclampsia; PE) and 21 healthy women. All the patients were under continuous obstetric care during the pregnancy and labour. The number of mother-child mismatches within killer immunoglobulin-like receptors (KIRs), their ligands [MM], and missing KIR ligands [MSLs] was assessed. KIRs and their ligands were assessed with Next Generation Sequencing (NGS) and Polymerase Chain Reaction Sequence-Specific Oligonucleotide (PCR-SSO) typing. The subsets of NK cells were assessed with multicolor flow cytometry and correlated to the number of MSLs.ResultsThe number of MSLs was significantly higher in HDP patients when compared to healthy non-complicated pregnancy patients. Some MSLs, such as those with 2DS2 activating KIR, were present only in HDP patients. The percentage of CD56+CD16-CD94+ NK cells and CD56+CD16-CD279+ NK cells correlated with the number of MSLs with inhibiting KIRs only in healthy patients. In HDP patients, there was a correlation between the percentage of CD56-CD16+CD69+ NK cells and the number of MSLs with inhibiting and activating KIRs. As compared to the healthy group, the percentage of CD56+CD16-CD279+ NK cells and CD56-CD16+CD279+ NK cells were lower in HDP patients. HDP patients were also characterized by a higher percentage of CD56+CD16+perforin+ NK cells than their healthy counterparts.ConclusionsPatients with HDP were characterized by a higher number of MSLs within the KIRs receptors. It seemed that the number of MSLs in the healthy group was balanced by various receptors, such as CD94 or inhibitory CD279, expressed on NK cells. Conversely, in HDP patients the number of MSLs was associated with the activation detected as the increased level of CD69+ NK cells.

The generation of stem cell-like memory cells early after BNT162b2 vaccination is associated with durability of memory CD8+ Tcell responses.

COVID-19 vaccines elicit humoral and cellular immune responses. Durable maintenance of vaccine-induced immunity is required for long-term protection of the host. Here, we examine activation and differentiation of vaccine-induced CD8+ T cells using MHC class I (MHC-I) multimers and correlations between early differentiation and the durability of CD8+ T cell responses among healthcare workers immunized with two doses of BNT162b2. The frequency of MHC-I multimer+ cells is robustly increased by BNT162b2 but decreases 6 months post-second vaccination to 2.4%–65.6% (23.0% on average) of the peak. MHC-I multimer+ cells dominantly exhibit phenotypes of activated effector cells 1–2 weeks post-second vaccination and gradually acquire phenotypes of long-term memory cells, including stem cell-like memory T (TSCM) cells. Importantly, the frequency of TSCM cells 1–2 weeks post-second vaccination significantly correlates with the 6-month durability of CD8+ T cells, indicating that early generation of TSCM cells determines the longevity of vaccine-induced memory CD8+ T cell responses.

P459: NUC-7738 REGULATES BETA-CATENIN SIGNALLING RESULTING IN REDUCED PROLIFERATION AND SELF-RENEWAL OF AML CELLS

Background: NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine triphosphate (3’-dATP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I/II clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. We have recently shown in cell lines a potential therapeutic role for NUC-7738 in patients with acute myeloid leukemia (AML), whereby NUC-7738 induced myeloid cell differentiation and mitochondrial-mediated apoptosis. AML leukemia stem cells (LSCs) are required for the initiation and maintenance of the disease. Activation of the Wnt/β-catenin pathway is required for the survival and development of LSCs and therefore, targeting β-catenin is a potential therapeutic strategy. Aims: The aim of this study was to determine whether NUC-7738 regulates β-catenin and the expression of its target genes in AML cells Methods: AML cell lines KG1a, OCI-AML3, HL-60 and U937 were treated with NUC-7738 for 48 or 72 hours. LSCs were determined by the expression of surface markers CD34, CD38 and CD123 via flow cytometry. Self-renewal was assessed by performing the colony forming assay using MethoCult Enriched media. Cell proliferation was determined by the Alamar Blue assay. Immunoblotting was performed for β-catenin, PI3K-p110, phosphorylated AktSer473 and phosphorylated GSK3βSer9 Results: NUC-7738 reduced PI3K-p110, phosphorylated AktSer473 and phosphorylated GSK3βSer9 resulting in reduced β-catenin expression. NUC-7738 decreased the percentage of CD34+ CD38- CD123+ (LSCs) from 15% to 7% and significantly reduced the total number and size of leukemic colonies. NUC-7738 inhibited cell proliferation in U937, OCI-AML3 and HL-60 cells, with IC50’s of 1.55 µM, 7 µM and 16 µM, respectively. Summary/Conclusion: Self-renewal pathways, including Wnt/β-catenin, are key for the initiation and long-term maintenance of LSCs. Through the ability to reduce PI3K-p110, phosphorylated AktSer473, and phosphorylated GSK3βSer9 and suppress β-catenin signalling, NUC-7738 exploits a key developmental process of AML, supressing the expansion and survival of LSCs. These results indicate that targeting LSCs with NUC-7738 may offer a successful therapeutic strategy for treating patients with leukemia.

Abstract LB064: B cells synergize with T cell regulation at immune hotspots in lung squamous cell carcinoma

Abstract The interplay of immune cell subpopulations underpinning heterogeneous immune infiltration is poorly understood, hindering the establishment of robust prognostic markers and therapeutic targets. To study the prognostic implications and constitution of immune cell aggregates in lung squamous cell carcinoma, we integrated transcriptomic data and deep-learning-based spatial analysis. With the automatically identified tumor and immune cells on hematoxylin and eosin (H&amp;E) diagnostic whole slide images, we were able to map regions with lymphoid aggregates (immune hotspots - IHs) and tumor aggregates (cancer hotspots) using the Getis-Ord hotspot analysis (Getis and Ord, 1992; Nawaz et al., 2015), in 462 TCGA samples. IHs were further categorized into intratumoral (intra-IH) and peritumoral (peri-IH) based on whether they overlapped with cancer hotspots. The intra-IH score, quantifying the fraction of IHs at the cancer interface, was associated with poor overall survival independently of age, stage, and packs per year (p &amp;lt; 0.01, Hazard Ratio (HR) = 2.1 [1.3-3.2]). The increased score was coupled with upregulated transcriptional signals of B cells and T regulatory cells, and expression of B cell-related genes CXCR5, MS4A1 (CD20), CD79b. To investigate cellular compositions of IHs, we selected 10 TRACERX patients with high intra-IH score based on H&amp;E and performed immunohistochemistry on two serial sections followed by a deep learning model to locate subpopulations of T cells (CD4+FOXP3-, CD4+FOXP3+, CD8+) and B cells (CD20+CXCR5-, CD20+CXCR5+, and CD79b+). By spatially aligning these IHC images with H&amp;E, we observed higher abundances of CD20+CXCR5+ and CD79b+ B cells at peri-IHs than intra-IHs (p&amp;lt;0.01; p&amp;lt;0.05), whereas none of the T cell subtypes showed a difference in localization. Tertiary lymphoid structures (TLSs) accounted for a minor proportion of peri-IHs (5.04 +- 4.38%) and intra-IHs (0.43 +- 0.61%). Furthermore, percentages of CD20+CXCR5+ B cells were significantly higher at peri-IHs than intra-IHs when TLSs were excluded (p=0.007), suggesting that the enrichment of CD20+CXCR5+ B cells at peri-IHs was not fully explained by the presence of TLSs. To test if IHs have a role in the regulation of anti-tumor activity of T cytotoxic cells, we further measured the ratio of CD8+ to CD4+FOXP3+ T cells infiltrating into the tumor nest. The ratio at tumor regions adjacent to intra-IH was lower than the rest of the tumor nest (p&amp;lt;0.001), suggesting a suppressive effect of intra-IH on tumor-infiltrating CD8+ T cells. Taken together, our results signified a protumor role of intratumoral lymphoid aggregates beyond TLSs, which may be attributed to the spatial interplay between CD8+ T cells, Tregs and B cells. Immune hotspots can serve as a prognostic indicator and potential therapeutic target in lung squamous cell carcinoma. Citation Format: Hanyun Zhang, Khalid AbdulJabbar, David A. Moore, Ayse Akarca, Katey Enfield, Mariam Jamal-Hanjani, Shan E Ahmed Raza, Selvaraju Veeriah, Dhruva Biswas, Roberto Salgado, Nicholas McGranahan, TRACERx Consortium, John Le Quesne, Charles Swanton, Teresa Marafioti, Yinyin Yuan. B cells synergize with T cell regulation at immune hotspots in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB064.